Plenary lectures HIll1

III

PL-3 QUINONE REDUCTION IN THE BC-COMPLEX OF RB~ SPHAEROIDES. A.IL Crofts, (3. Beehmarm, Beth Hacker and R.B. Germis Program in Biophysics and Dept. of Biochemistry, University of Illinois, Urbana Redox titrations of the be-complex show a component (eyt b150) with a higher Em (150 mV) than oft bH (Em ~50 mV), which is not seen when titrations are performed in the presence of antimycin (AA). AA addition when cyt b150 (but not eyt bit) is reduced, results in oxidation ?f eyt b. •• (cytb150) Q ~ ,-*L~l-t2 Ae,~.~ AA eyt bH-. Q- (2I-I+) o oft . QH2 o eyt b.Q (or vacant) ¢:> c ~ bH. . Several strains (N223V, G°fl~0b0~,N222A, R I I 4 K ~ WI29A) with mu~tions at the Qi-site have a lower fractlon of cyt b m the Dl>o to_tin,aria a reaucoa extent d AA-indueed eyt b oxidation. In these s t r ~ s , the J~.t ror ~ I~ a~Se lower (Em 12-20 mV) than in wild-type. Conversely, m IzP~A, ana a aoun mutant K251D/D252K, which have a higher E m for eyt b-l, AA-induced oxidation and [cyt b150] were higher. The results are explainedby formation ofcyt b 150 by reversal of the left two equilibria above, and an equilibriumc~n~ant: Kea (I)150) = exp {(Em(cyt bH) + P-~n(Q/QH') - z V,m(Ql~opl));r/r~J ) ~ whereEm(¢yt b~), En~Q/QH'/) ahd Em(Q/QH2) ~ e poten.uats ot eytocrtrome I~.i, the quinone)semiqumone couple at the sate, an.d the qumone pool re.specy.ively. Computer simulations can moaet reaox taralaons or- ~ 0 ana antmaycl,n induced ox~da4ionin wild-type and mutant strains, with the different Em(c~ values as the only variable, using wild-type values for all other parameters.

Plenary lectures PL-1 - PL-13

PL-1 OXYGEN PRODUCTION IN PHOTOSYSTEM II: LESSONS FROM OXYGEN CONSUMING ENZYMES Gerald T. Babcock, Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA. The water-splitting assembly in Photosystem II comprises the photochemical hole generator, P680, the redox active tyros'me YZ, and a cluster of 4 Mn ions bound into the D1/D2 heterodimer; C~/z+ and CI- are also essential cofactors. This understanding sets the stage for addressing well-focused inquiries into the mechanism by which water oxidation occurs. Useful insight into O~ evolution can be gotten from an awareness of the mechanisms that are~sed to control oxygen chemistry by enzymes that reverse the process, i.e., that reduce 02 (or peroxide) to water. The respiratory protein, cytochrome oxidase, has been helpful in this regard, as have been radical enzymes, such as prostaglandin H synthase. The latter enzymes use metal-bound dioxygen to produce an amino acid radical that initiates catalysis by H-atom abstraction from substrate, as follows: metal/C2 ( H* radical ( H. substrate. Recent data on water oxidation suggest that PSII may operate by simply reversing the direction of the H-atom current, that is, (Mn)4 H20 H- ) YZ "It-" ) P680+. These and related analogies will be considered.(Supportedby NII-I& USDA)

PL-2 THE STRUCTURE OF THE LH2 ANTENNA COMPLEX FROM THE PURPLE NON-SULPHUR PHOTOSYNTHETIC BACTERIUM RHODOPSEUDOMONASACIDOPHILA STRAIN 10050 ~..J. Co,dell l, G. McDermott2, S.M. Prince 2, A.A. Freer2, A.M, Hawthornthwaite-Lawless3, M.Z. Papiz 3 and N.W. Isaacs2, Division of Biochemistry and Molecular Biology I and Department of Chemistry 2, University of Glasgow, Glasgow G12 8QQ and CCL Daresbury Laboratory3, Warrington, Cheshire W A 4 4AD, U.K. The crystal structure of the B800-850 antenna complex from Rps. acidophila has been determined to a resolution of 2-5A. It IS an a91~9 nonamer and looks like a cylinder or ring-doughnut. The inner wall of the complex is made up of 9 a-helices of the a-apoprotcin and the outer wall by 9 a-helices of the l~apoprotein. All of the pigments arc complexed between these two rings of helices. This talk will give the latest information on the details of this strUctur~ This work was supported by grants from B B S R C and the EU.

PL.4 REACTION CENTERS OF PURPLE BACTERIA G. Feher, University of California, San Diego, Physics Department, 0319, Drive, La Jolla, CA 92093-0319, USA Three decades of research has resulted in a detailed understanding of the reaction center (RC) of purple bacteria; it probably represents the best understood integral membrane protein to date. In this talk I shall try to give a historical overview of the work leading to our present knowledge of the structta¢ and function of the RC. Special emphasis will be placed on the three dimensional struettae as well as the declrenic structure of the primary reactants and their relations to elecla-on.transfer, including the ~melmeof the intermediate bacteriochlorophyll in the ~ step and the prob em of the preferential electron transfer along the A-branch. Electron transrerls followed by the protonation of the secondary ubiqninone accepter, l ne mechanism of this process involves several key amino acids that have been identified by site-directed mutagenesis. The secondary electron donor is a cytochrome, which in some purple bacteria, e.g. Rb. viridis forms an integral part of the RC, whereas in other species, e.g. Rb. sphaeroides is an exogenous, water soluble eyt %. The RC:cyt c2 complex of Rb. sphaeroides has been crystallized and its proposed structures will be presented. The structure of the RC from purple bacteria is believed to be similar to the (hitherto undetermined) structure of the RC of PSII from green plants. Their structures will be compared.

PL.5 PHYSIOLOGICAL RESPONSES OF PURPLE BACTERIA TO ENVIRONMENTAL PHYSICAL/CHEMICAL CHANGES Howard Crest, Biology Department, Indiana University, Bloomington, IN 47405, USA The evolution of anoxygenic photosynthetic bacteria apparently involved the acquisition of regulatory circuits that ensure maximal bioenergetic efficiency. Nonsulfur purple bacteria can obtain energy for growth either from a~aerobic photophosphorylation or aerobic respiration, and the regulation of bioenergetics in such organisms is focused on environmental control of photopigment synthesis. Light intensity and 02 tension are particularly important variables in this connection. Recent experiments have demonstrated that in addition to serving as an energy source, light also functions as a sensory signal for phototactic m o v e m e n t s of colonies of the p u r p l e bacterium Rhodost3irillum centenum (Arch. MicrobioL 165. 1 1995). In nature, photota~cis (and chemotaxis) presumably aids motile cells in positioning themselves in locales optimal for satisfying the physical and chemical requirements for growth. The development of macroscopic "blooms" of photosynthetic bacteria under natural conditions will be considered from this standpoint.

Plenary lectures II

IIII

I

I

PHYCOBILIPROTEINS: STUDIES OF BILIN ATTACHMENT.

PL-6

PL-9 ROLES OF ENVELOPE MEMBRANES IN PLASTID DEVELOPMENT AND CELL METABOLISM

A.N. Glazer, C.D.Fairchild, L.J. Jnng, and C.F. Chan, Department of Molecular and Cell Biology, Univ. of California, Berkeley, CA 94720-3206. Phycobiliproteins, unlike other proteins involved in photosynthesis, bear covalently attached chromophores, The bilin chromophores are attached through thioether bonds to cysteinyl residues, The number of bilin attachment sites on all of the phycobiliproteins of a particular cyanobacterium or red alga ranges from a minimum of 8, all carrying the same bitin, to >20, with up to three different bilins. Posttranslational modification of proteins usually involves recognition of simple linear amino acid sequence determinants. Surprisingly, this does not appear to be the case for bilin attachment to phycobiliproteins. Synechococcus sp. PCC 7002, the genes cpcE and cpcF encode a heterodimeric phycocyanin c~-subunit phycocyanobitin (PCB) lyase. In vivo, this lyase catalyzes PCB addition specifically to the phycocyanin ct-subunit. In a cpcEF mutant, addition to the other PCB attachment sites is normal. Similarly, Anabaena sp. PCC 7120 genes pecE and pecF encode a phycoerythrocyanin ct-subunit phycobiliviolin lyase, which in vivo catalyzes specifically the attachment of phycobiliviolin to the phycoerythrocyanin ccsubunit. In a pecEF deletion mutant the phycoerythrocyanin et-subunit carries PCB. Several lines of evidence indicate that phycobiliprotein bilin lyases show high selectivity rather than absolute specificity for both the polypeptide and the bilin substrate.

J. Joyard, M.A. Block, A.J. Dome, N. Rolland & R. Douce Laboratoire de Physiologie Celhilaire Vrgrtale, URA CNRS 576, Drpartement de Biologic Motrculalre et Structurale, CEA-Centre d'Etudes Nuclraire de Grenoble et Universit6 Joseph Fourier, 38054 Grenoble-cedex 9, France The role of the chloroplast envelope in plant cells is often restricted to its participation to the regulation of photosynthesis, owing to the phosphate/triose phosphate translocator, and to the control of the translocation of nuclearencoded chloroplast proteins. In fact, this membrane system has a much wider diversity of functions than usually assumed. For instance, the inner envelope membrane contains DNA-binding proteins that mediate the binding of nucleoids in developing plasfids. Envelope membranes also contain a whole set of enzymes involved in the biosynthesis of major specific thylakoid constituents (glycerolipids, pigments and prenylquinones). Electron transfer proteins and semiquinones have been recently characterized by EPR in purified envelope. Together with the presence of ravines and the lack of cytochromes, this suggest the presence of a unique electron transi~r chain in envelope membranes. In addition, chloroplast envelope membranes deliver to the cytosol numerous metabolites (not only triose phosphate but also aminoacids, fatty acids...) and also potentially regulatory molecules, such as several fatty acid derivatives synthesized in the envelope and involved in specific cellular responses to pathogen attack or wounding and/or in gene expression during embryogenesis.

PL-7

C H L O R O P L A S T C O N T R O L OF NUCLEAR GENE EXPRESSION J.C. Gray, R. Somarajah, A.A. Zabron, C.M. Duckett and M.S. Khan, Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK

The high-level expression of nuclear genes encoding photosynthesis components is dependent on a positive regulatory signal from the chloroplasts. Disruption of chloroplast function in the light with norflurazon, a carotenoid biosynthesis inhibitor, results in decreased transcription of nuclear photosynthesis genes. The chloroplast processes required for the production of the regulatory signal, or plastidic factor, have been studied in trartsgenic tobacco seedlings expressing the 13-glucnronidase (GUS) reporter gene from promoters of various photosynthesis genes. This has shown that the production of the plastidic factor requires chloroplast transcription and translation, and that photosynthesis or chlorophyll synthesis are not necessary. Analysis of deletions of the promoters of the plastocyanin (PetE) and ferredoxin-NADP oxidoreductase (PetH) genes in transgenic plants has defined regions which are responsive to the plastidic factor. Nuclei from leaves of norflurazon-treated plants, but not of green plants, contain a DNA-binding protein which interacts with TATA-proximal elements in the promoters of these genes. This suggests that the plastidic factor acts to remove a repressor from the promoters of nuclear photosynthesis genes, allowing highlevel expression.

PHOTOSYNTHESIS AND RISING CO2 CONCENTRATION

PL-10

S.P.Long 1, P.A. Davey1, P.K. Farage |, I.F. Mc/Kee1, G.Y. Nie2, C.P. Osborne l, C.A. Raines~, A. Rogers I and G.R. Hendrey~. ~Dept. of Biology, University of Essex, Colchester, CO4 3SQ, UK; adept, of Applied Science., Brookhaven National Laboratory., Upton, NY11973, USA. Photosynthesis is the primary process by which plants respond to rising CO2 concentration and is fundamental to understanding the effects of atmospheric change on both crop performance and carbon cycling in natural ecosystems. Over 1000 studies have examined the effects o f i n e r ~ CO2 concentration on the development of photosynthesis in terrestrial Cs plants. The large majority have shown loss of photosyntbetie capacity and in particular loss of Rubisco, but this may in part be an artefact of experimental design. Alternatively, this loss of Rubisco may result from a wider homeostatic adjustment to increased carbon availability mediated via gene expression, The extent and mechanisms of these responses will be examined, as will their modification by light, temperature, nitrogen supply, atmospheric pollutants, and genotype. The significance of acclimation within the leaf to photosynthesis at the whole plant and canopy level in the fidd will be examined via the large scale Free-air CO2 Enrichment (FACE) experiments.

PL-8

PL-11

REGULATION OF SUCROSE-PHOSPHATE SYNTHASE AND OTHER CYTOSOLIC ENZYMES BY REVERSIBLE PROTEIN PHOSPHORYLATION

GENETIC ENGINEERING OF PHOTOSYNTHETIC CAPABILITY UNDER TEMPERATURE AND SALINITY STRESSES

Steven C. Huber USDA/ARS and Depts. of Crop Science & Botany, N.C. State University, Raleigh, NC 27695-7631, USA

N. Mumta National Institute for Basic Biology, Okazaki 444. Japan

Phosphorylation of sucrose-phosphate synthase (SPS) is responsible for modulation of activity in response to light/dark signals and end-product accumulation. The major regulatory phosphorylation site involved has been identified as Set-158 in spinach leaves and as Ser-162 in maize leaves. Regulation of the phosphorylation status appears to involve: i) calcium; ii) metabolites; and iii) novel "coarse" control of the protein phosphatase. Manipulation of SPS expression in vivo confirms the role of this enzyme in the control of sucrose biosynthesis. Recent results suggest that phosphorylation may also control the activities of other key cytosolic enzymes, such as NADH:nitrate reductase and sucrose synthase, and thereby influence C/N balance and also sucrose synthesis/degradation.

Photosynthetic capability is limited by various stresses, and its increase by genetic manipulation will improve crop productivity. We have performed genetic manipulations to increase the tolerance of plants to various environmental conditions. By manipulating genes for fatty-acid desaturases in cyanobaeteria, we modified the level of fatty acid unsaturation of membrane lipids and, as a result, enhanced their tolerance toward chilling and freezing stresses. The kinetic analysis demonstrated that the increased unsaturation of membrane lipids at low temperature accelerates recovery of the photosystem I1 complex from photoinhibition. Glycine betaine, a compatible solute, is synthesized in some salt-tolerant plants. We have isolated, from Arthrobacter globiformis, the coda gene for choline oxidase, which converts choline into glycine betaine, and have introduced the gene into Synechococcus sp. PCC 7942. As a result, this cyanobacterium acquired the capability of growing in 400 mM NaCI, conditions under which the wild-type strain cannot grow. The codA gene was also able to confer salt tolerance to Arabidopsis.

Plenary lectures

PL-12 ATP SYNTHESIS BY ROTARY CATALYSIS: STRUCTURE OF BOVINE F1-ATPASE AT 2.8A RESOLUTION J.E. Walker, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK The structure of FI-ATPase at 2.8]~ shows that its three catalytic sites have different conformations. The presence of a central coiled-coil of o~helices suggests that the conformations could interconvert in a cyclical manner by rotation of the coiled-coil, supporting Boyer's binding change mechanism of ATP synthesis. If so, rotation must be generated by proton transport through Fo. Models for generating rotation and for coupling it ATP synthesis will be presented.

Abrahams, J.P., Leslie, A.G.W., Lutter, R. and Walker, J.E. (1994) Nature 370, 621-628.

PL-13 PRESENT STATE OF THE CRYSTAL STRUCTURE ANALYSIS OF PHOTOSYSTEM I

W.D. Schubert*, O. Klukas*, N. Kraue,*, W. $aenger*, P. Fromme, H.T. Witt • Institut f0r Kristallographie, Freie Universitit Berlin, D-14195 Berlin, Germany Max-Volmer-lnatitut, Technische Univeraittlt Berlin, D-10623 Berlin, Germany Recent progress is reported regarding the structure analysis of photosystem I by X-ray. Improvements of the 6 ~ electron density map (Nature (1993), Vol. 361, 311-326) are described in a new map at 4.5 A resolution with respect to the arrangement of the helices, antenna chlorophylls, and the electron transfer chain.

Historical s e s s i o n

Historical

session

HL-1 - HL-2

HL-I FORTY YEARS

OF PHOTOSYNTHETIC ELECTRON FLOW

A. Trebst Plant Biochemistry, Ruhr-University, Bochum Cyclic photophosphorylation was described by D.Arnon in 1954. The development of photosynthetic electron flow since will be followed in four periods. The first ten years established the principal biochemistry of cyclic, non-cyclic and pseudo-cyclic ATP formation. The major e-carriers were identified in a zigzag scheme with two photosystems. The second decade brought photosynthesis to the hight of attention of general biochemistry. Its research was instrumental for the acceptance of the chemiosmotic theory. The third ten years consolidated the field, molecular b i o l o g y crept in and was the basis for understanding p r o t e i n folding across the membrane, culminating in the X-ray structure of the bacterial reaction center. The last ten years used the new topological thinking for refined insights in the biophysics of e-flow and the structure of the membrane. Gene technology opened the field of the biogenesis, import and assembly of the thylakoid proteins and regulation of their gene expression.

HL-2 <
David Walker Robert Hill Institute, University of Sheffield,Sheffield, S 10 2TN, UK

A brief and personal excursion into the manner in which the development o f understanding of photosynthesis, over the last 45 years, impinged on this investigator. Reference will be made to the archaic technical problems encountered in establishing the role of phosphoanolpyruvic carboxylase in CAM plants, the involvement of CO2-dependent 02 evolution by isolated chloroplasts in the concept of the phosphate translocator and the effect of eytosolic phosphate on chlorophyll a fluorescence emission.

Economical s e s s i o n ES-3

Photosynthesis and global environment: is vegetation a regulator of the carbon cycle? by B. Saugier, Universit~ Paris-Sud, bat. 362, 91405 Orsay cedex France.

Economical

Atmospheric C O 2 concentration has increased by more than 2 5 % since 1800, reflecting combustion o f fossil fuel and deforestation. This increase could be limited by an enhanced storage o f carbon in biomass and soils. W e computed the CO 2 fixation by the land vegetation using satellite data, and its release by plant respiration using maps o f biomass and surface temperature, The method is still rough but has potential for improvement. On the other hand, a doubling in atmospheric C O 2 concentration leads to an enhancement o f biomass production that varies from 20*/, to more than 80% according to species and growth conditions. Can these experiments be extrapolated to the whole terrestrial biosphere? T w o data sets support an increase in global plant production in the past decades: a large increase in the width o f tree rings o f several european species in various locations, and an increase in the amplitude o f seasonal variations o f atmospheric CO 2 in the northern hemisphere.

Session

ES-1 - ES-3 (in French)

ES-I

Photosynthesis a n d r e n e w a b l e energy Gosse Ghislain (INRA/Grignon) The stored chemical energy, through photosynthesis processes, is our prime concern here, since it is this biomass which can be harvested for food, fuel, fibre or other uses. Monteith (1972) has proposed a method to study the efficiency of this solar energy conversion. In this method, a crop is considered as a spatial organisation of physical solar sensors which characterised the structure of the crop. The main advantage of this method relies on its ability to separate the effects of climate, structure of the crop, or intrinsic leaf properties on the solar energy conversion. But, this approach does not take into account all sources of energy contained or used to produce all the inputs which are now necessary in agriculture such as fossil fuels, fertilisers, pesticides... These energy costs are now well established by using a method such as life cycle analysis. The CO2 balance of a crop may be considered as a by-product of the energy balance, a specific parameter is applied for each source of energy (fossil fuel, coal, gas, nuclear electricity...). Recently, detailed analysis of the energy balance of crops used to produce biofuels have clearly illustrated advantages and limits of the energy efficiency concept. Energy and CO2 balances are now two important concepts in order to manage agriculture under environmental constraints.

ES-2 THE AGRICULTURE AND ITS FUTURE PROSPECTS G. Paillotin Institot National de la Recherche Agronomique - 147 rue de rUniversit~ 75338 PARIS Cedex 07 At the end of the seventies, many hopes were based on the direct transformation of solar energy by the photosynthetic apparatus. Agriculture and forestry, however, are always the ways leading to the most efficient use of photosynthasis. During the last fifty years, mainly thanks to the progress of genetics, the productivity of agriculture has increased considerably. So agriculture is able to answer, in a quantitative point of view, to the present and future needs of the world. However, deep inequalities exist between the countries as well on sufficiency as on production of food. In the future, agriculture will have to face diverse challenges : production requirements of cunrse, but also natural resources preservation, global cenuol of the environment as well as rural development. Building of answers well adapted to these multiple demands requires new efforts of research mainly in the biology of development, and adaptation end rational conU'ol of biotechnologies.

Symposium i

S-1-2-03 PROTEIN COMPONENTS OF C H L O R O S O M E S OF THE GREEN S U L F U R B A C T E R I U M CHLOROBIUM TEPIDUM D0rlald A. Bryant and Soohee Chung Dept. of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802 USA

Symposium 1-2

Antenna

systems: structure and function

S-1-2-01 - S-1-2-10

Chlorosomes are light-harvesting antenna complexes found in green bacteria. Although chlorosomes from Chlorobium tepidum have been shown to contain 10 different protein species with masses ranging from 6.2 to 27 kDa, the protein:Bchl c ratio of chlorosornes is much lower than that observed in other antenna systems. Using amino acid sequence information obtained from chlorosome proteins, the genes encoding five chlorosome proteins, denoted CsmA (6.2 kDa), CsmB (7.7 kDa), CsmC (14.5 kDa), CsmD (11.5 kDa), and CsmE (7.7 kDa), have been cloned and characterized. The csmA and csmE genes encode similar proteins that bear similar C-terminal sequences that are post-translationally removed to produce the mature proteins. Each of these proteins has been overproduced in Escherichia coli and purified to homogeneity; polyclonal antibodies have been raised against each of these proteins. Interposon mutagenesis of C. tepidum and C. vibrioforme is being performed to examine the role(s) of the various gene products in chlorosome structure, function, and biogenesis. Results obtained from mutagenesis of the csmA and csmC genes with either an aphH (kanamycin resistance) gene cartridge or cartridge (streptomycin resistance) will be discussed.

S-1-2-01

S-I-2-04

REGULATION OF LIGHT HARVESTING EFFICIENCY IN GREEN PLANTS

PROBLEMS

P. Horton Robert Hill Institute, Dept of Mol Biol & Biotech, University of Sheffield, P.O. Rox 594, Western Bank, Sheffield S t 0 2UH, U.K

Z=Feti_sova L V . N o v o d e r e z h k i n , A . T a i s o v a & K . M a u r i n g A.N.Belozersky I n s t i t u t e of P h y s . - C h e m . B i o l o g y of Moscow State University, Moscow 119899, Russia

Although the structure of the light harvesting system of PSII in plants is arranged for high efficiency of energy transfer to the reaction centre, it can undergo a rapid and profound change that can result in over 80% of absorbed light being dissipated as heat; this is an essential physiological mechanism which prevents damage to the thylakoid membrane by excess light and is detected as nonphotochemieal quenching of chl fluorescence, qN (Horton et al Plant Physiol [1994] 106, 415-420). This change results from structural alteration in LHCII induced synergistically by protonation of amino acid side chains and by the de-epoxidation of violaxanthin bound by the complexes (Horton et al [1991] FEBS Lett 292, 1-4). Recent work is exploring the nature of these changes: the involvement of the minor LHCII components; the identification of the H+ binding sites; the control of qN by viol/zea ratio; the changes in pigment/pigment and pigment/protein interactions associated with quenching; how more stable dissipative states of LHCII are formed under more severe stress, and the way in which the pigment and protein composition of LHCII gives rise to the physiological regulation of the capacity and dynamics of qN.

The k e y goal of the p r e s e n t w o r k is the study of d y n a m i c s of e x c i t a t i o n e n e r g y t r a n s f e r (EET) w i t h i n the c h l o r o s o m e of g r e e n bacteria. The p r o b l e m of m e c h a n i s m of E E T w i t h i n the c h l o r o s o m e is m o s t acute in v i e w of oligomeric organization of c h l o r o s o m a l pigments. The t h e o r y of E E T has b e e n d e v e l o p e d for d i f f e r e n t e x c i t o n m o d e l s of a g g r e g a t i o n of BChl c (d or e) w h i c h are c o m p a t i b l e w i t h the key spectral features found in vivo, i.e. the exciton level structure, r e v e a l e d by h o l e - b u r n i n g experiments, and p o l a r i z a t i o n of all the levels p a r a l l e l to the long axis of the chlorosome. The f u n d a m e n t a l p r o b l e m is that the d y n a m i c s of E E T from BChl c (d or e) to BChl a should be a n a l y s e d in v i e w of the fact that in a g r e e m e n t w i t h the s t a t i s t i c a l analysis of the sizes of c h l o r o s o m e s in cells, p e r f o r m e d by us w i t h e l e c t r o n microscopy, any e n s e m b l e of the c h l o r o s o m e s m u s t be c o n s i d e r e d in terms of fractal geometry.

S-1-2-02 STRUCTURE AND FUNCTION OF PLANT LIGHT-HARVESTING COMPLEX, LHC-II W. Kiihlbrandt, EMBL, Meyerhofstr. 1, D-69117 Heidelberg The structure of the light-harvesting chlomphyU a/b-protein complex (LHCI]) has been determined by electron microscopy and electron diffraction of two-dimensional crystals. LHC-II, an integral membrane protein, binds roughly half of all pigment molecules involved in plant photosynthesis. The atomic model of the complex shows the structure of three transmembrane helices, a short amphipathic helix at the membrane surface, and the associated chlorophylls and ¢arotenoids. Two helices are connected through symmetrical ion pairs which also function as chlorophyll ligands. In the centre of the monomer, two symmetrical carotenoid molecules are in van-der-Waals contact with 7 of the 12 bound chlorophylls. The arrangement of the pigments in the complex explains the photoprotcctive function of the carotenoids and helps us to understand the role of the chlorophylls in trapping and transmission of solar energy. Reference: Kiihlbrandt, W., Wang, D.N. und Fujiyoshi, Y. Atomic model of plant light-harvesting complex by electron crystallography Nature 367, 614-621 (1994)

OF

EXCITON

DYNAMICS

IN CHLOROSOME

ANTENNA

S-1-2-05 ~qERGY TRANSFER I N B A C T E R I A L A N T E N M A S AMDREACTION CENTER

S.J. Bradforth, R. Jimenez, M. Ricci, D. Jonas, T. Joo, Y. Jia, Y. Nagasawa, M. Lang, and G.R. Fleming. Dept. of Chemistry and James Franck Institute, The University of Chicago, USA. Energy transfer within the purple bacteria antenna systems, LHI and LH2 has been studied by fluorescence up-converslon. The major depolarization time scale is I00 fs and 50 fs respectively. Fluorescence from carotenoid S 2 has been time-resolved, both in vivo and in vitro, and the mechanism of carotenoid to chlorophyll energy transfer will be discussed. These studies have been complemented by photon echo measurements on B800 of LH2, and a model for proteln-plgment spectral density obtained. The ultrafast transfer of energy from the accessory chlorophyll to the special pair in reaction centers of Rhodobacter spheroides has been studied by pump-probe spectroscopy with < 20 fs resolution. The transfer time is somewhat longer than had been previously thought and is qualitatively inconsistent with a Fozster mechanism.

Symposium

S-1-2-06 LIGHT-HARVESTING-PROTEINS OF DINOFLAGELLATES: MULTIGENES AND POLYPROTEINS Ro~,er G. Hiller. Pamela M.Wrench and Frank P. Sharpies School of Biological Sciences, Macquarie University, NSW, Australia 2109 The photosynthetic Light- harvesting apparatus of Dinoflagellates comprises the water soluble Peridinin-Chiorophyll-a-Proteins (PCPs) and the intrinsic chlorophyll-a-c-carotenoproteins (LHCs). The PCPs are closely related proteins with polypeptides of 32kDa (or 2 x 16kDa) A spectrally distinct PCP (6 peridinin / 2 chlorophyll a) having an apoprotein of 34kDa has also been purified and sequenced from Amphidinium. This retains the two domain structure of the main PCP forms but the sequence is only 30% identical. Putative chlorophyll-binding histidine residues are conserved. Genomic PCR has demonstrated a number of mainform PCP genes , two of which are adjacent and separated by ~500bp. LHCs are distinct from the PCPs in size of polypeptide (19kDa) and primary sequence. Multiple forms of the membraneincorporated polypeptide are synthesised as a polyprotein which is processed to the 19kDa peptides by cleavage at single arginine residues. The above data are reviewed in relation to fight-harvesting proteins from other chromophyte algae.

ON THE DYNAMICS OF THE ENERGY AND ELECTRON TRANSFER PROCESSES IN PHOTOSYSTEM I AND II ANTENNA AND REACTION CENTER COMPLEXES

S-1-2-07

S-1-2-09 TRIMERIZATION OF LIGHT-HARVESTING COMPLEX (LHCII) Harald Paulsen, Ralf F6rster, Stephen Hobe, & Andrea Kuttkat Botanisches Institut III, Universitat M0nchen, Menzinger Str. 67, D-80638 M0nchen, Germany The major light-harvesting complex (LHCII) of photosystem II can be reconstituted in vitro in its native, trimeric form starting from the pure apoprotein, pigments, and thylakoid lipids (Hobe et al., 1994, EMBO J. 13, 3423-3429). We are interested in the structural determinants of the oligomerization of LHCII. The hydrophilic domain comprising 61 N-terminal amino acids of the LHCII apoprotein (LHCP) is dispensable for the formation of monomeric LHCII; however, trimerization of reconstituted monomers is abolished by the deletion of more than 15 amino acids on the N terminus of LHCP. More specifically, we identified a "trimerization motif" in position 16 to 21 of LHCP: Mutant LHCII monomers carrying amino acid exchanges in this domain are no longer capable of formin .g trimers in vitro. Consistently, when the same LHCP mutants are inserted into isolated thylakoids, they appear in the monomeric LHCII fraction whereas wild-type is assembled into LHCII trimers. Possible functions of the trimerization motif will be discussed.

S-I-2-I0 Molecular mechanisms responsible for the tuning of the absorption of peripheral light-harvesting proteins from purple bacteria Bruno Robert. SBPM/DBCM CEA and URA 1290 CNRS, Centre d'Etudes de Saclay, 91191 Gif sur Yvette Cedex, FRANCE.

Alfred R. Holzwarth Max-Planck-lnstitut ftir Strahlenchemie; D-45470 Miilheim a.d. Ruhr, Germany

The binding rites of many different peripheral fight-harvesting complexes (LH2) extracted from purple bacteria have been studied by FT-Raman preresonance spectroscopy, and the interactions assumed by the bacteriochlorophyll molecules have been measured in complexes exhibiting different types of absorption, namely 1) wild-type 800-820 and B 800-850 complexes 2) sitespecifically mutated 800-850 complexes exhibiting absorption transitions peaking between 835 and 820 nm and 3) natural B 800-850 complexes, the absorption of which has been shiRed to 820 nm by detergent treatment. In all these complexes, the 850 nm absorption correlates with the presence of Hbonds at the level of the 2-acetyl substituents of both the BChl a molecules responsible for the most red-shitted electronic transition. These molecules interact with residues at position 44 and 45 on the ot polypeptide, i.e. either two tyrosines (in Rb sphaeroides and Rps sulfidophilus), or a tyrosine and a tryptophane in most other bacterial species.

The dynamics of the energy and electron transfer processes in photosystem I core complexes (100 Chl/trT00) from Synechococcus sp., in the isolated light-harvesting complex II from photosystem II, in photosystem II core particles, and in isolated D1-D2 reaction center complexes by femtosecond transient absorption, picosecond time-resolved fluorescence, and kinetic modelling. The talk will emphasize the general aspects common to the kinetic and spectroscopic behaviour of these different systems.

S-1-2-08 MOLECULAR NETWORKS AND FUNNELING PROCESS OF ENERGY TRANSFER IN GREEN PHOTOSYNTHETIC BACTERIA M. Mimur91 , K. Matsuura 2, K. Shimada 2, Y. Nishimura3, I. Yamazaki3, M. Koba~cashi4, Z.-Y. Wang4 and T. Nozawa4 1Natl. Inst. Basic Biol., Okazaki 444, ZTokyo Metropoli. Univ., Hachioji 192-03, 3Hokkaido Univ., Sapporo 060, and 4Tohoku Univ., Sendal 980-77, Japan. Energy transfer processes in a green photosynthetic bacterium Chloroflexus aurantiacus were studied from structural, optical and kinetical points of view. Spectral heterogeneity was found in each of chlorosomal BChl c, baseplate and B806-866 complexes in the membranes. All the components were not involved in the main energy flow pathway to the RC. Upon excitation of BChl c, the energy flow occurred directly from the main BChl c component to the shortwavelength form of baseplate BChl a or c, and then to the B866 in the membranes. The overall transfer time was estimated to be 25 ps by the timeresolved fluorescence anisotropy spectra. The energy transfer process from the baseplate to the B806-B866 complex was not the rate-limiting step. During the energy transfer from the baseplate to B866, the plane of polarization was kept, suggesting the good alignment of components and thus a specific interaction between the baseplate and B806-866 complex. Energy transfer kinetics in intact cells were different from those in chlorosomes. Based on the time-resolved and magnetic circular dichroism (MCD) spectra, energy flow pathways and transfer mechanism will be discussed, including the energy transfer from carotenoids.

Symposium S-3-4-04 1 3 C M A S N M R v e r s u s F T I R C h a r a c t e r i z a t i o n of Q A Bindin~ sphaeroides R26 Photosynthetic RC. in Rhodobacter H.de G r o o t l , w . v a n L i e m t l , G . B o e n d e r l , p . G a s t 2 , A . H o f f 2, J. L u g t e n b u r g I. ILIC and 2Huygens Lab. P.O. Box 9502, 2300 RA Leiden, The Netherlands.

Symposium 3-4

Isotropic shifts and anisotropies for both QA carbonyls were d e t e r m i n e d w i t h site-specific 13C labeling at the 1,2,3 and 4-13C positions. The shifts of both carbonyls upon binding are negligible, w h i l e the anisotropies are only slightly different. In contrast, FTIR of RC r e c o n s t i t u t e d w i t h labelled u b i q u i n o n e - 1 0 (R. Brudler et al. EMBO J. 13, 1994, 5523) or ubiquinone-3 (J. B r e t o n et al. B i o c h e m i s t r y 33, 1994, 14378) revealed an u n u s u a l l y large d o w n f i e l d shift of 60 cm -I for a mode involving the 4-C=O stretch, revealing decrease of the 4-C=O bond order. The apparent controversy can be largely explained using an a p p r o x i m a t e d e s c r i p t i o n of the p a r a m a g n e t i c contribution to the chemical shift anisotropy where the contributions from local charge densities and from bond orders are separated. The 60 cm -I F T I R effect translates into up to 10-20 ppm shifts of tensor p r i n c i p a l components, w i t h i n experimental error of the NMR. Hence b o t h the NMB and FTIR-data can be explained in terms of a change of carbonyl bond order without affecting the charge densities at the labeled positions in the ring. This interpretation is s u p p o r t e d by H a r t r e e - F o c k calculations.

R e a c t i o n centers: purple bacteria a n d PSII 8-3-4-02

-

8-3-4-10

S-3-4-02 U L T R A F A S T I N F R A R E D AND V I S I B L E BACTERIAL REACTION CENTERS

SPECTROSCOPY

OF

W. Zinth*, T. Arlt*, H. Penzkofer*, P. Hamm*, B. Dohse + and D. Oesterhelt+ * Institut far Medizinische Optik, Universit~t Mtinchen + Max Planck Institut fur Biochemie, Martinsried The molecular processes of the primary electron transfer (ET) in reaction centers (RC) are studied for wildtype (WT) and modified RCs. Mutated RCs of Rhodopseudomon~z~ viridis are investigated, where the exchange of amino acids in the vicinity of the special pair P modifies the primary ET. For instance, the mutation of histidine L168 to tyrosine leads to a strong acceleration of the decay of P* and to a strong bleach of the absorption band of the monomeric bacteriochlorophyll B. The ratio of absorption changes in WT and mutated RC quantitatively agrees with the value expected for the stepwise model with ET via P+B- in WT and mutated RC. Data from a series of mutants of viridis are used, to estimate the reorganisation energy of the first ET step to be close to 1000cm- I. Information on even more rapid processes are obtained by femtosecond spectroscopic experiments on RC of Rhodobactersphaeroides in the mid infrared revealing a new 200 fs reaction prior to P* decay. This reaction is connected with strong absorption increases in the 1000-1500 cm -1 range. The absorption changes can be explained by a fast initial motion in the RC changing the coupling between different electronic states of P. Arguments are presented relating the 200 fs process to still unresolved questions of the primary reaction such as excitation trapping from the antenna and unidirectionality of the electron transfer.

S-3-4-03 C O N S E Q U E N C E S O F C H A N G E S IN H Y D R O G E N BONDS T O T H E BACTERIOCHLOROPHYLL DIMER ON THE OXIDATION P O T E N T I A L AND E L E C T R O N T R A N S F E R R A T E S IN R E A C T I O N C E N T E R S F R O M RHODOBACTER SPHAEROIDES J. C. Williams and J. P. Allen Dept. of Chemistry and Biochemistry & Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, A Z 85287-1604, U.S.A. The primary electron donor in the reaction center from purple bacteria is a bacteriochlorophyll dimer containing four conjugated carbonyl groups that may form hydrogen bonds with amino acid residues. In wild type reaction centers from Rhodobacter sphaeroides there is only one hydrogen bond, between His L168 and the 2-acetyl group of the A-side bacteriochlorophyll of the dimer. Mutations have been created that either break the existing hydrogen bond or introduce histidines near the other carbonyls. Spectroscopic analyses of reaction centers with these mutations confirm that hydrogen bonds can be formed between each of the four carbonyl groups and the histidine residues. The addition of each hydrogen bond is correlated with an increase in the oxidation potential of the dimer, resulting in a 355 mV range in the midpoint potential (Lin et al., 1994, Proc. Natl. Acad. Sci. USA 91, 1026510269). The resulting changes in free energy differences alter the electron transfer rates for several reactions including electron transfer from cytochrome c2 to the oxidized dimer, charge recombination from the primary quinone, and the initial forward electron transfer.

S-3 -4-05 CONTINUUM ELECTROSTATIC CALCULATIONSOF COFACTOR MIDPOINTS IN RPS. ~ AND ~ . ~ , ~ [ ~ . ~ , A ~ O N CENTERS: IMPLICATIONS FOR ELECTRON TRANSFER DRIVING FORCE AND DIRECTIONALITY. M.R. Gunner, A. Nicholls, and B. Honig *Physics Dept., City College of New York, and Dept. of Biochemistry and Molecular Biophysics, Columbia Univ. Continuum electrostatic calculations were carried out with RC structares from Rns. viridis and Rb. sohaeroides. The cost of rapidly separating charge by 18 A to form P+H" from P* in vacuum is = 1.1eV. Iftbe protein and surroundings are treated as material with e=2, appropriate at short times and low temperature, the initial dielectric relaxation stabilizes P+H" by -,-680 meV. Thus, charge separation would be uphill. However, the distribution of charges within the protein provides a static field to stabilize P+HL-. The field favors charge separation by ~-700 meV in both Ros. viridis and Rb. sDhaeroides RCs. The potential is asymmetric in both RCs so the L branch eofaetors are in regions that are 200-300 meV more positive than their M branch analogues. In RDs. viridis RCs, 43 residues contribute more than 25 meV to the field driving eh/u'ge separation and 20 and 25 residues provide this much distinction between the B's and H's respectively. These sites are predomently ionized residues. The large number of sites involved may contribute to the difficulty in charging RC directionality by site directed mutations. Supported by N.I.H. RO1-GM48726

S-3-4-06 MAGNETIC RESONANCE STUDIES OF PHOTOSYNTHETIC REACTION CENTERS: LINKS BETWEEN ELECTRONIC STRUCTURE OF THE COFACTORS AND THE ELECTRON TRANSPORT PROCESSES W. Lubitz Max-Volmer-lnstitut, Technische Univarsitiit Berlin, D-10623 Bedin, Germany The electronic structure of the cofactors in bacterial reaction centers (RCs) is investigated by EPR, ENDOR/'I'RIPLE, and 2D-ESEEM techniques, performed on the radical ions generated in the light-induced charge separation process. By these methods the hyperfine coupling constants (hfc's) with the various magnetic nuclei are determined. Assignment of the hfc's to specific molecular positions yields a map of the electron spin density distribution of the respective radical. Vary detailed information about the electronic and spatial structure of the cofactors (e.g. the pdmary donor, D +" and the quinone acceptor, QA') is obtained from EPR/ENDOR studies of RC single crystals of Rb. sphaeroides. The asymmetry of the bactedochlorophyll (BChl) dimer cation radical D +" is studied in several bactadal RCs containing BChl a. The results are compared with our recent data obtained for species containing BChl b (Rps. viridis) and BCh117 (H, chlorum, H. mobilisl. In genetically modified RCs of Rb. sphaeroides the effect of hydrogen bonds on the structure of the dimer is investigated. In addition, the effect of hydrogen bonds on the hetarodimer |BChI-BPh) is studied. The rates for charge recombination and for the cytochrome reduction of D +" are correlated with the asymmetry of dimer.

Symposium S-3-4-10

S-3-4-07 PATHWAYS

OF PROTON UPTAKE

IN BACTERIAL

REACTION

CENTERS

P. Mar6ti I, P. Sebban 2 and D. K. Hanson 3 XDept. Biophysics, JATE University Szeged, Hungary; 2Centre de Genetique Mol~culaire, CNRS, Gif/Yvette, France; 3Center Mechanistic Biology and Biotechnology, Argonne National Laboratory, Argonne, IL 60439 USA. The proton transfer pathway from the aqueous solution to the secondary quinone (QB) binding site buried within the protein matrix is proposed to involve an extended network of protonatable species either amino acid side chains or water molecules. The water molecules fill the voids between distant protonatable sites in the static protein structure but mobile water molecules (not visible in the X-ray structure) may also participate in proton delivery. The protein maintains a negative electrostatic potential near Q~. This will make the proton transfer to QB more favorable from both kinetic (acceleration) and energetic (tuning of p ~ values) points of view. Combined studies of molecular genetics and biophysics will map the network of proton delivery system to QR-

S-3-4-08 STRUCTURE OF THE OXIDIZING REDOX-COMPONENTS IN P H O T O S Y S T E M II. S P E C T R O S C O P I C I N V E S T I G A T I O N S B A S E D O N A THEORETICAL STRUCTURAL MODE

Stenbittm_ Styrin_~l~Paul van Kan1, Bangt Svensson 1, Cecilia Tommos1, Mafia Rova2, Charlotte Kullander2, Per<)lof Fredriksson2 1. Dept of Biochemistry, An'henius Laboratories, Stockholm Univ., S-106 91 Stockholm, Sweden. 2. Dept of Chemistry. Univ. of Karlstad, Box 9501, S-650 09 Karlstad. Sweden We have used computer modelling to derive a three-dimansional structural model for the innermost core of the PS II reaction canter. We have used the computer model to study the structure of the proposed chlorophyll dimer in PSII. We have fitted optical spectroscopic data to a chlorophyll structure that could be aecomedated in the structural model. In this approach, both the optical spectra and the structural model provide constraints on the results. A dimerie structure of P680 where the distance between the two Mg2+ ions in the chlorophyll pair is about 10A and the angle between the Qy transitions is about 150° fits the spuctm well. This structure is also well aecomodated in the structural model. After fitting the dimerie structure in the computer model, two residues, D1-Thr286 and D2Ser283, are found in the close vicinity of the chlorophylls, probably forming hydrogen bonds to their ester groups. We have applied site directed mutagnaesis in combination with EPR spectroscopy to verify proposals from the model in the vicinity of Tyr-D O'yrl60 on the D2 protein) and Tyr-Z O'yr-161 on the D1 protein). A central conelusion is that D2His189 probably is hydrogen bonded to Tyr-D. The correspunding residue, D1-Hisl90 is elnae to Tyr-Z. We will present spectroscopic results on Tyr-Z in several mutants close to Tyr-Z. We have also studied electron donation from exogenous mangal~se and other donors in several mutants. Altered electron donation as compared to wildtype is discussed in terms of putative metal binding sites in PSII.

S-3-4-09 CHARACTERIZATION

OF SUPRAMOLECULAR

ASSEMBLIES

D. M. T i e d e 1, P. M a r o n e 1, A. M . W a g n e r I and P. T h i y a g a r a j a n 2 1Chemistry Division D - 2 0 0 and 2Intense Pulsed N e u t r o n Source, A r g o n n e National L a b o r a t o r y , A r g o n n e , I L 60439 U S A W e are using small angle neutron scattering [SANS] to r~solve structural features o f photosynthetic s u p r a m o l e c u l a r assemblies. Results show that the Rb. sphaeroides reaction c e n t e r exists in an equilibrium b e t w e e n m o n o m e r i c a n d a g g r e g a t e d states. T h e equilibrium is strongly dependent upon the solubilizing detergent and temperature. Additions such as ethylene glycol used f o r low t e m p e r a t u r e spectroscopy and polyethlene glycol used for crystaUiTation w e r e found to induce aggregation. M e a s u r e m e n t s suggest that a g g r e g a t e s are connected to p a t h w a y s that lead to reaction center crystallization. Structures o f the different a g g r e g a t e s are being pursued by fitting S A N S data with scattering profiles calculated for reaction center a g g r e g a t e models. This a p p r o a c h is also being used to m a k e correlations b e t w e e n the structure for the reaction c e n t e r - c y t o c h r o m e c 2 electron transfer c o m p l e x in solution with electron transfer kinetics. Funded by DOE BES contract W-31-109-Eng-38 and NASA MSFC contract H-13058D.

THE

REACTION

CENTER

OF

PHOTOSYSTEM

II

N.J. v a n G o r k o m Dept of Biophysics, Huygens Lab, State University, P.O. B o x 9504, 2300 R A Leiden, The N e t h e r l a n d s T h e r e a c t i o n center of P h o t o s y s t e m II is responsible for l i g h t - i n d u c e d o x i d a t i o n of water, the u l t i m a t e electron source for the b i o s y n t h e s i s of v i r t u a l l y all organic matter. It still is a m a j o r challenge in p h o t o s y n t h e s i s r e s e a r c h to explain h o w the r e q u i r e d o x i d i z i n g p o t e n t i a l is r e a c h e d a n d c o n t a i n e d a n d h o w damage to the r e a c t i o n center itself is avoided. The k i n e t i c s of excitation- a n d e l e c t r o n t r a n s f e r in various conditions in i s o l a t e d r e a c t i o n centers a n d p h o t o s y s t e m II core complexes p r o v i d e information on these processes. T h e p r i m a r y e l e c t r o n donor, P680, is not a s t r o n g l y c o u p l e d 'special pair' like in p u r p l e bacteria, but system II p r o b a b l y does contain a small central group of p i g m e n t s h o m o l o g o u s to those in the b a c t e r i a l reaction center, all a b s o r b i n g a r o u n d 680 nm. U p o n p h o t o o x i d a t i o n the p o s i t i v e charge p r e s u m a b l y is l o c a l i z e d on the least o x i d i z i n g pigment, w h i c h m a y not always b e the same.

Symposium II

S-5-03 FERREDOXIN REDUCTION BY WILD TYPE A N D SITEDIRECTED M U T A N T S OF PSI FROM THE CYANOBACTERIUM SYNECHOCYSTIS PCC 6803 Symposium

5

P. S~tif CEA Sactay - DBCM/SBE a n d U R A CNRS 1290 91191 Gif sur Yvette Cedex, France

Reaction centers: green sulfur bacteria and PSI S-5-01

- S-5-04

S-5-01 COMPARATIVE ASPECTS OF FeS-TYPE REACTION CENTERS Gfinter HAUSKA Lehrstuhl f/ir ZeUbiologie und Pflanznephysiologie, Universit~t Regensburg, 93053 Regensburg, Germany.

S-5-04 STRUCTURAL ORGANISATION OF THE ACCEPTORS At, Fx, FA AND FB IN PHOTOSYSTEM I 0PS I) FROM EPR-STUDIES IN SOLUTION AND SINGLE CRYSTALS A. Kamtowski, A. van der Est, P. Fromme, D. Stehlik Physics Department, FU Berlin and Max-Volmer Inst, TU Berlin, Germany

A brief summary of the similarities and differences between the FeS-type reaction centers of the heterodimeric forms in photosystem 1 of chloroplasts and eyanobacteria, and of the homodimeric forms in green sulfur - and heliobacteria will be given.

S-5-02 MUTATIONAL ANALYSIS OF PHOTOSYSTEM I POLYPEPTIDES P. R. Chitnls1, Q. Xu1, V. P. C h ~ $ 1 y. S. Jungz, J. H. Golbeck2 1Division of Biology, Kansas State University, Manhattan,Kansas, USA; 2Department of Biochemistnj, Univendtyof Nebraska, Lincoln, Nebraska, USA The pigments and redox centers ofphotosystem I (PSI) are bound to the PsaA, PsaB, and PsaC polypepddes. We are studying functions of the remaining subunits by targtted mutagenesis in the cyanobacterium Synechocystissp. PCC 6803. Phenorypes of these mutants revealed many roles of these PSI subunits. (1) Interaction with r,oluble electron carriers: PsaD and PsaE are crucial for interaction of photosysrem I with fertedoxin and flavodoxin. The C-terminal domain ofPsaD (induding the K106 residue), a lysine-containing region of PsaC, and the C-terminal region of PsaE may form the reducing site on PSI, (2) Org~nl,ation of photosystem I: PsaL and PsaI are required fur obtaining PSI trimers. These proteins may form the connecting domain of PSl that could "be linked to the catalytic domain through PsaD-PsaL interaction. (3) Spatial arrangement of proteins within photosystean I: The very low mass hydrophobic polypeptides (e.g. PsaI and PsaJ) may stabilize the organization of other integtal-membtane subunits (e.g. PsaF and PtaL) in the PSI complex. (4) Protection of redox centers: PsaD and PsaE protect PSI from oxidative damage. Their absence causes enhanced degradation of PSI core when the cyanobacteria are exposed to stresses. Supported by NSF and USDA grants.

10

The kinetics of reduction of soluble ferredoxin (Fd) b y p h o t o s y s t e m I (PSI) were m e a s u r e d at r o o m temperature by flashabsorption spectroscopy in the visible region. The spectral and kinetic properties of 3 different first-order phases of ferredoxin reduction by wild type PSI will be s h o w n a n d will be discussed within a structural model of interactions between PSI and Fd. Implications for electron transfer within PSI will be also underlined. The Fd reduction process will be described in several site-directed m u t a n t s of either PSI-D or PSI-E subunits, thus allowing to identify several residues of each of these suburdts as essential for binding Fd a n d for its fast reduction by PSI.

Following a brief review on kinetic and structural properties of the transient charge-separated state P~+At" and the subsequent electron transfer step to Fx, low temperature EPR-results on P S I single crystals will be described. They yield structural information on the reduced iron sulphur centers FA and FB and the PsaC protein binding them: (i) Orientation of both g-tensors with respect to the crystal axes. (ii) Relative orientation of the g-tensors of FA" and FB', found to be in good agreement with that evaluated from the known structure of bacterial ferredoxins such as PaFd of Peptococcus aerogenes. (iii) Orientation of the PsaC unit in the P S I complex as compared with the relevant PaFd-motif fitted into the electron density map of the P S I X-ray structure at 4.5 ~. Although location and orientation of the two FeS centers is fixed, a local C2 symmetry axis prevents the assignment of FA and FB from these data. Additional information can provide this assignment as will be discussed.

Symposium

S-6-03 W H A T C A N BE L E A R N E D A B O U T T H E F U N C T I O N S OF CAROTENOIDS IN PHOTOSYNTHESIS FROM MODEL STUDIES? Symposium

Thoma~ A. Moore. Devens Gust and Ana L. Moore, Center for the Study of Early Events in Photosynthesis and Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287 - 1604.

6

Three-dimensional structures of ca_~tenoid-contsining chlorophyll-proteins permit comparison of the structure-function relationships found in model studies with the structure-function relationships in native proteins. Because the photochemistry between carotenoids and chlorophylls in proteins is/in effect, unimolecular, carotenoid pigments covalentiy linked to porpnyrin oerivatives (CPs) are appropriate models. Photochemical studies of such models have established parallels between singlet energy transfer from, and triplet energy transfer to, the carotenoid moiety. Both processes were shown to depend on electronic interactions between the chromophores which require overlap between donor and acceptor orhitals. In native proteins, this requirement is met through van der Waals contact between the carotenoid and chlorophyll pigments, as has been found in every case reported. C P s in which the number ot conjugated double bonds in the carotenoia vary n'om a to 11 iuuslrate me change from singlet oxygen sensitization by the jx~'phyfin, through singiet oxygen sensitizauun by the carotenoid, the quenching of sin._81etoxygen itself, and finally suppression of singlet oxygen sensitizauon. These moaeis mso demonstrate the transition from non quenching of the porphyrin singlet by the carotenoid to strong quenching. The well defined flow of excitation enerjgy in this series is relevant to the functions of carotenoias in pnotosyntnesis including their putative role in the control of singlet energy dissipation.

Carotenoids S-6-01

- S-6-05

S-06-01 LOW-LYING EXCITED STATES OF CAROTENOIDS ASSOCIATED WITH THE XANTHOPHYLL CYCLE AND ENERGY TRANSFER IN PHOTOSYNTHESIS Harry A. FrankJ Agnes Cua) Veeradej Chynwat, ] Andrew Young, 2 David Gosztola ~ & Michael R. Wasielewsld 3 ~University of Connecticut, Storrs, CT 06269 USA; 2Liverpool John Moores University, Liverpool L3 3AF, UK; 3Argoane National Laboratory, Argonne, IL 60439, USA The energy gap law for radiationless transitions has been used to deduce the low-lying excited state energies of several carotenoids important in photosynthetic energy transfer and involved in the xanthophyll cycle of green plants and diatoms. The data suggest that carotenoids can regtdate the flow of energy to and from chlorophyll a. Violaxanthin, which has nine conjugated double bonds, functions as an antenna pigment by transferring energy from its S~ state to that of chlorophyll a. In contrast, zeaxanthin, which has eleven conjugated double bonds, is capable of trapping excess excitation energy from chlorophyll a within the lhc of higher plants andsome algae. Also investigated are the photophysical properties o f the xanthophylls associated with the diadinoxanthin cycle in diatoms. The data show that the S, energy of diadinoxanthin lies above that of chlorophyll a, whereas the St energy of the deepoxidated derivative, diatoxanthin, lies below that of chlorophyll a. These olgservations support the hypothesis that low-lying S t states ofcarotenoids are involved in the non-photochemical quenching of excess energy in photosynthesis.

S-6-02

S-6-04 ROLE OF CAROTENOID BOUND TO THE PHOTOSYSTEM II REACTION CENTRE Alison Telfer and James Barber Photosynthesis Research Group, Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, U.K. Carotenoids normally protect photosynthetic pigment-protein complexes from the damaging effect of singiet oxygen (102), by quenching chlorophyll triplets before they can form this highly reactive species. However t-carotene bound to the isolated Photosystem II (PSII) reaction centre complex does not trap the triplet state of the chlorophyll pigments of P680. Thus illumination under aerobic conditions leads to formation of 102 (which has been detected both by its luminescence at 1270 nm and by chemical trapping) and consequently leads to inactivation of the reaction centre. Experiments with isolated PSII reaction centres, binding different levels of carotenoid, show that although t-carotene does not trap chlorophyll triplets it quenches 102 directly affording considerable protection against photodamage. We also discuss a second role for t-carotene which appears to act as a sacrificial electron donor to P680+ if electron donation from the water splitting complex is inhibited.

S-6-05

REGULATION OF CAROTENOID BIOSYNTHESIS IN PLANTS AND ALGAE

EVIDENCE FOR THE ROLE OF CBR-ZEAXANTHIN COMPLEXES IN NON-PHOTOCHEMICAL QUENCHING

J. Hirsehberc,, 1. Pecker, T. Lotan, R. Gabbay and V. Mann

Paula Braun, Shmuel Malkin and Ada Zamir Biochemistry Department, Weizmann Institute of Science, 76100 Rehovot, Israel

Department of Genetics, The Hebrew University of Jerusalem, Jerusalem 91904 Israel Carotenoids are synthesized and accumulate in plastids by the isoprenoid pathway. Genes for all the biosynthetic steps from geranylgeranyl pyrophosphate to ~-earotene have been cloned from eyanobecteria, algae and plants. The primary structures of these enzymes are conserved among organisms with oxygenic photosynthesis but distinct from those of purple bacteria and non-photosynthetic microorganisms. Each of the enzymes is a single-gene product that is functional in an autonomous manner in heterologous cells. Increased transcription of carotenoid biosynthesis genes plays a role in the induction of earotenogenesis in leaves under high light. Other environmental stresses have similar effects in algae. Transcription of the genes encoding phytoene synthase and phytoene desatumse increases in flowers and fruits, while transcription of the gene for lycopene cyclase decreases in the fruit. An Arabidopsis mutant which is sensitive to high light, was found to be impaired in xanthophyll accumulation.

Dunaliella, a unicellular green alga outstanding in its ability to withstand highly intense sunlight, provides a highly useful model to study light stress responses. A gene, cbr, cloned from Dunaliella bardawil, was preferentially expressed under light stress conditions and encoded a protein similar to higher plant Elips. Cbr is associated with a minor LHCII component and several lines of evidence point to its role as a zeaxanthin-binding protein. Zeaxanthin, the light-stress induced product of violaxanthin deepoxidation, together with the formation of ApH across the thylakoid membrane were both shown to be required for the development of non-photochemical quenching. Fluorescence measurements revealed that on prolonged exposure of D. bardawil cells to light stress, non-photochemical quenching of chlorophyll fluorescence became resistant to uncoupler. The course of resistance development resembled that of Cbr accumulation, leading to a model whereby formation of Cbr-zeaxanthin complexes enables nonphotochemical quenching to develop independently of ApH. Effects of light quality on zeaxanthin and Cbr accumulation will also be discussed.

11

Symposium

S-7-03 E L E C T R O N T R A N S F E R ON T H E ELECTRON DONOR SIDE OF PSII

Symposium

Oxygen

X.-S. Tang l, B.A. Diner 1, G.T. Babcock 2, R.D. Britt 3, G.W. Brudvig4, G.C. Dismukes 5 1 CR&D, E. I. Du Pont de Nemours & Co., 2Michigan State University, 3University of California, Davis, 4yale University, 5princeton University

7

We have been using magnetic resonance spectroscopy and site-directed mutagenesis to investigate the protein environments of redox-active tyrosines, Y z (D1-Tyrl61) and YD (D2-Tyrl60), of Photosystem II (PSI/). We have found that a histidine, D2-His189, acts as a proton acceptor to YD and that this histidine remains hydrogen bonded upon oxidation to YD'. The homologous histidine, D1-Hisl90, is from kinetic evidence probably hydrogen bonded to Yz, but does not appear to remain hydrogen bonded following formation of YZ'- YZ" appears to have more rotational degrees of freedom than YD" and to be located in an environment more accessible to solvent exchange. Saturationrecovery, a pulsed EPR technique, indicates that the non-heine iron of PSI] is equidistant (37A) from YZ• and YD', consistent with C2 symmetry of the Photosystem II reaction center. The manganese cluster responsible for water oxidation appears to be preferentially associated with polypeptide D1, breaking the C2 symmetry. Site-directed mutagenesis has shown several regions of the D 1 polypeptide to be implicated in coordination and function of the Mn cluster. Isotopic labeling and ESEEM have established imidazole nitrogen and a peptide carbonyl oxygen as ligands to the Mn cluster.

evolution

S-7-01

- S-7-05

MODELS

FOR

S-7-01 MANGANESE

Y. Frapart, E. A n x o l a b ~ h ~ r e Mallart, G. Blondin, J.-J_ G~r~rd L a b o r a t o i r e de C h i m i e I n o r g a n i q u e , LIRA CNRS 420, U n i v e r s i t ~ Parls-Sud, 91405 Orsay, France

P o l y m a n g a n e s e species have b e e n o b t a i n e d w h i c h are i n t e r e s t i n g s t r u c t u r a l a n d s p e c t r o s c o p i c m o d e l s for the o x y g e n e v o l v i n g center. T h e i r study has c o n t r i b u t e d to e s t a b l i s h that the 0EC is m a d e of two MnO2M/2 pairs. However, e l e c t r o c h e m i c a l studies d e m o n s t r a t e that n o n e of these artificial s p e c i e s is a w a t e r oxidation catalyst. The dinuclear [44] species are t h e r m o d y n a m i c a l l y a b l e (E° for the [ 4 4 ] / [ 3 4 ] ~ 1.44V versus NHE) to o x i d i z e w a t e r but t h e r e is no e f f i c i e n t p a t h w a y . T r i n u c l e a r and t e t r a n u c l e a r species (with a h i g h e r O / M n ratio) h a v e m u c h lower E ° (0.54V) w h i c h d e m o n s t r a t e s the s t a b i l i z a t i o n i n d u c e d b y oxo groups. So it is also c h e m i c a l l y sensible that M n O 2 M n p a i r s keep i n d i v i d u a l i t y in the 0EC. One m a y e n v i s i o n two tYPes of m e c h a n i s m symbolized b e l o w ...... O ,

M~@M.

$7-04 FTIR STUDIES ON THE STRUCTURE AND REACTIONS OF THE OXYGEN-EVOLVING CENTER IN PHOTOSYSTEM II

PHOTOSYNTHESIS

O'"-',,,,,Mn

Takumi Noguchi Solar Energy Research Group, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan Oxygen-evolving mechanism in PSII was investigated by means of Fourier transform infrared (FFIR) spectroscopy Flash-induced FTIR difference spectra between the S1 and $2 states (S2/SI) were measured with both normal and Ca 2+depleted PSII membranes. Upon Ca2+ depletion, coupled symmetric and asymmetric COO- stretching bands and most of amide I bands disappeared. The data indicate that 1) there exists a COO- ligand connecting Mn and Ca ions, 2) the coordination of this COO" ligand to Ca breaks upon $2 transition, 3) Ca depletion liberates this COO- ligand even from Mn, and 4) Ca is necessary for conformational changes of protein around the Mn-cluster upon $2 formation. Also, an $2/S1 FTIR spectrum measured in I320 buffer showed a large upshift of an asymmetric CO0- stretching band by about 18 cm" 1. Together with the FFIR spectra of model carboxylate compounds, this large upshift suggests the presence of a COO- ligand that is hydrogen-bonded to a water molecule bound to the Mn-cluster.

M.(.,,,a~_J O,-----Mn

S-7-02 ESE-ENDOR STUDIES OF PARAMAGNETIC OF PHOTOSYSTEM II

CENTERS

$7-05 STRUCTURAL CHARACTERIZATION OF THE MN CLUSTER IN PSII USING X-RAY ABSORPTION SPECTROSCOPY

R. David Britt, David W. Randall, M. Lane Gilchrist, Jr. • James A. Ball Department of Chemistry, University of California, Davis, CA 95616

P.J. Riggs-Gelascoa, R. Mei b, D.F. Ghanotakisc, C.F. Yocuma,b, and J.E. Penner-Hahna, Departments of Chemistrya and Biologbb The University of Michigan, Ann Arbor, MI 48109-1055 USA, and Chemistry Departmentc, University of Crete, Iraklion, Crete, Greece.

Electron Spin Echo ENDOR experiments performed on a broad radical EPR signal observed in photosystem II particles depleted of Ca 2+ indicate that this signal arises from the redox-active tyrosine Y z . The tyrosine EPR signal width is increased relative to that observed in a manganese-depleted preparation due to a magnetic interaction between the photosystem II manganese cluster and the tyrosine radical. The manganese cluster is located asymmetrically with respect to the symmetry related tyrosines Yz and YD. The distance between the tyrosine and the manganese cluster is estimated to be approximately 4.5 /~. Based on the close proximity of the Mn cluster to the redox active tyrosine Yz, we propose that this tyrosine abstracts protons from substrate water bound to the Mn cluster. In addition, SSMn ESE-ENDOR experiments are revealing new details about the electronic structure of mixed valence Mn model compounds and the Mn cluster of PS II.

Extended x-ray absorption f'me structure (EXAFS) and x-ray absorption near edge structure (XANES) spectroscopies have been used to characterize the structure of the Mn cluster in the oxygen evolving complex from PSI/. Redueed derivatives prepared using NH2OH or hydroquinone (H2Q) have dramatically different Mn local structures, consistent with their different reactivities. The structural results are consistent with a model in which two dimer sites can undergo indpendent redox reactions. The EXAFS and XANES data together suggest an average oxidation state of Mn(III)2Mn(IV)2 for state S 1 and Mn(ITl)4 for the S-1 state prepared by NH2OH incubation. The role of Ca in photosynthesis has been probed by making EXAFS and XANES measurements on samples in which the Ca(i/) has been replaced by Sr(I1), Dy(Hl), or LaOlI). These data show no change in.the 3.3 A EXAFS feature suggesting that, at least for these samples, the3.3 A distance must represent only Mn-Mn and Mn-C, and not Mn-Ca, scattering. There are small changes in the 2.7 ./k Mn-Mn distance as a function of the metal in the Ca site. These are consistent with a role for Ca in mediating hydrogen bonding to the Mn cluster.

12

Symposium S-8-03 STRUCTURAL STUDIES ON THE CYTOCHROME C O M P L E X F R O M CHLAMYDOMONAS REINHARDTII.

Symposium

J.-L. p o p o t I . B . P . C . , 11 r u e P. et M . Curie, F-75005 PARIS, France

8

T h e g r e e n unicellular alga Chlamydomonas reinhardtii is a v e r y conv e n i e n t o r g a n i s m for the e x p e r i m e n t a l study o f o x y g e n i c p h o t o s y n thesis. W e h a v e developped a protocol that yields m g a m o u n t s o f h i g h l y purified C. reinhardtii c y t o c h r o m e b6f c o m p l e x . T h e purified c o m p l e x c o m p r i s e s 7 subunits, 6 o f w h i c h are integral and one, the Rieske protein, peripheral, one c-type h e m e , two b-type heroes, a n d one m o l e c u l e o f chlorophyll a (see poster by Pierre et al.). T h e c o m p l e t e s e q u e n c e o f one o f the smallest subunits, PetX, and that o f its c D N A h a v e b e e n established (see poster by de Vitry et al.). T h e d i m e r h a s b e e n reconstituted into lipid vesicles, w h e r e it can be induced to f o r m t w o - d i m e n s i o n a l crystals. Its detergent-associated f o r m appears suitable for g r o w i n g three-dimensional crystals (see poster by M o s s e r et al.).

Cytochrome b-c complexes S-8-01

b6f

- S-8-04

S-8-01

AN ORDERED H 2 0 CHAIN IN CYTOCHROME f AS THE pSIDE H + EXIT PORT OF THE CYTOCHROME b6f COMPLEX. S. E. Martinez, D. Huang, M. Ponamarev, J. L. Smith, and W. A. Cramer Dept. of Biol. Sciences, Purdue Univ., West Lafayette, IN 47907 USA

Refinement of the atomic model for ~ e 252-residue lumen-side domain of cytochrome (cyt) f a t a resolution of 1.96A showed the presence of five internal H20 molecules, four of which define a vector-like pathway that extends 12/~ from the His25 heme ligand to within 6A of K66, part of a basic domain including K65, K58, and possibly K187 that may form a docking site for carboxylate residues of plastocyanin (PC). A triple Lys(-) mutant of cytf, K58Q, K65S, K66E, was made to test this concept for the docking site. A functional role of the H20 sites is implied by: (a) high occupancy, (b) temperature factors comparable to those of adjacent atoms, and (c) H-bond contact from main chain carbonyl oxygens and amide nitrogens and from conserved amide side chains. The Era of the soluble turnip cytfin vitro, Era7 = +0.365V, is pH-dependent with pKox = 8.0-8.5. It is proposed: (a) c y t f functions in the pathway of the H+ pump; (b) the vectorial H20 chain forms an H + channel that is the p-side exit port for H ÷ pumped by the cyt b6f complex; (c) PC carboxylates at the docking site form the H + sink for the pump, and neutralization of these carboxylates the mechanism for PC release from cytj~ (d) H + donation to cytfis mediated by the p-side domain of the Rieske FeS protein, which may consist of a 137 residue C-terminal Rieske fragment made by activation of a protease in turnip chloroplasts (supported by USDA 9301586; NIH GM-38323).

CHARGE

TRANSFER

EVENTS

S-8-04

IN T H E C Y T O C H R O M E

bcl-COMPLEXES A. Semeno_xv1, P. Rich2, K Gourovskaya 1 and N. Ugulava1 1 A N Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia and 2Glynn Research Institute, Bodmin, UK Charge transfer reactions in the cytochrome bcl-complexes has been investigated by: (i) direct electrometry and (ii) flash-spectrometry in photosynthetic bacterial chromatophores and in purified complexes from rnitochondria. The electrometrical study of chromatophores from Rhodobacter sphaeroides suggest the possible arrangement ofredox centres in the bcl-complex. Laser flash photolysis has been used to investigate the reduction of oxidized cytochrome c 1 in the mitochondrial bcl-complex by the redox dye phenazine methosulphate. Addition of stigmatellin increased the cytochrome c 1 photoreduction extent. On the basis of the data, the E m value for the FeS Rieske centre was found to be approx. 30 mV lower compared to that obtained by EPR techniques.

S-8-02 M U T A T I O N A L STUDIES OF THE S T R U C T U R E AND FUNCTION OF THE C Y T O C H R O M E B C I C O M P L E X Fevzi Daldal Department of Biology, Plant Science Institute, University of Pennsylvania. 204 Mudd Bldg. Philadelphia PA 19104-6018 The cyt bcl complex in bacteria and mitoehondria and its analog the cyt b6f complex in chloroplasts is a multi-redox, membrane enzyme important for both photosynthesis and respiration. It is a redox-driven proton pump involved in the formation of the proton motive force necessary for ATP synthesis In the facultative phototroph Rhodobacter, intensely studied as a model system for eukaryotic organelles, the cyt bcl complex is constituted of three subunits (cyt b, cyt Cl and the FeS protein) that contain four redox centers (two b hemes-bH and bL, a c heme and a [2Fe2S] cluster). These components form two quinone processing domains (Qo and Qi) where the electrogenic events and the accompanying proton uptake and release take place. Although a 3D structure for the cyt bcl complex is not yet available. yet great progress on our understanding of its structure and function has been accomplished in recent years using multidisciplinary approaches including genetics, molecular biology, biochemistry and biophysics. Recent findings related to the redox centers, the quinone binding domains and the assembly of the subunits will be presented.

13

Symposium

STRUCTURAL AND FUNCTIONAL ANALYSES OF SPINACH FERREDOXlN:THIOREDOXIN REDUCTASE Symposium 9

Peter SchOrmann Laboratoire de Biochimie v~getale, Universit6 de Neuch&tel, Rue Emile Argand 11, CH-2007 Neuch&tel, Switzerland

Electron transfer proteins

Ferredoxin:thioredoxin reductase (FTR), a Fe-S protein involved in the light regulation of carbon metabolism in oxygenic photosynthesis, catalyses the reduction of thioredoxins with light generated electrons. FTR is composed of two dissimilar subunits, a variable subunit and a catalytic subunit The variable subunit has no known catalytic function. The catalytic subunit contains eight cysteine residues. Their structural roles were examined by chemical modifications. Two N-terminal Cys seem to have no particular function. The six other cysteine residues are clustered as Cys-Pro-Cys and Cys-His-Cys motifs. Cys54 and Cys84 constitute the redox active disulfide/dithiol couple with a measured midpoint potential of -230 mV enabling it to reduce the chloroplast thioredoxins. The remaining four Cys residues ligand the [4Fe--4S] cluster. A possible involvement of the Fe-S cluster in the transfer of electrons will be discussed.

S-9-01

- S-9-04

S..9-01 MOLE~

RECC~NITIOI~ I~ ELECTRON T P ~ S F E R

PR~INS

C. C~Smez-Moreno Departamento de Bioqulmica y Biologia Molecular Universidad de Zaragoza, Spain

S-9-04

A STRUCTURAL COMPARISON OF THE INTERCHANGEABLE ELECTRON TRANSFER PROTEINS, PLASTOCYANIN AND CYTOCHROME

P r o t e i n s i n v o l v e d in e l e c t r o n t r a n s p o r t chain: exchange electrons after the formation of complexe; w h i c h are stabilised b y electrostatic interactions These protein-protein complexes provide the n e c e s s a ~ environment for the transfer of electrons between th~ two redox centres involved. The interaction of amint a c i d r e s i d u e s w i t h the c o r r e s p o n d i n g redox group: p r o d u c e d r a s t i c c h a n g e s in t h e i r r e d o x potentia~ p o i s i n g t h e m in the a d e q u a t e range for the redo: r e a c t i o n s involved. O n c e t h e c o m p l e x is forme~ certain amino acid residues could have a more direcl involvement in the process: i.e., acting as mediato: in the electron transfer reaction. Kinetic data o b t a i n e d w i t h the system consisting i~ the enzyme ferredoxin-NADP + reductase a n d ferredoxit (or flavodoxin) f r o m the c y a n o b a c t e r i u m suggest the s p e c i f i c i t y of the r e c o g n i t i o n proces~ and indicates the involvement of aromatic, as well a~ charged residues, in the electron transfer reaction.

S-9-02 NMR APPROACHES TO THE STUDY OF STRUCTURE-FUNCTION RELATIONSHIPS IN IRON-SULFUR PROTEINS: RUBREDOXIN, [2Fe-2S] FERREDOXINS, AND RIESKE PROTEIN

,l~hn L. Marklev? Bin Xiaf Young Kee Chae, ~ Hung Cheng, ~, William M. Westler, ~ Jeremie D. Pikas,~ & B~an G. Foxf ~Biochemistry Department and ~nstitate for Enzyme Researchb University of Wisconsin-Madison, Madison W153706, USA. Newer NMR methods in conjunction with stable isotope labeling (with 2H, 13C,and I~N) offer exciting approaches to structure-function studies of paramagnetic proteins. The application of multidimensional multinuclear NMR spectroscopy to iron-sulfur proteins, which are paramagnetic in both of their accessible oxidation states, and the optimization of NMR pulse sequences for rapidly-relaxing spins have made it possible to determine sequence-specific assignments for a large number of NMR signals in rubredoxins, ferredoxins, and highpotential iron proteins, including those from the uysteine residues that ligate the iron ion or iron-sulfur clusters, It is now possible to interpret the wealth of information derived from NMR parameters, such as the temperature and pH dependence of chemical shifts and the relaxation properties of the resonances that report on interactions between nuclei of the protein and unpaired electron density from the metal center. This information can be used to test theoretical descriptions of electron distribution within these molecules and to model the structures and dynamic properties of the proteins in solution. Mutagenesis of these proteins, in conjunction with NMR studies, is beginning to reveal which residues are important for cluster formation and stability arid which residues play a rote in dectrou transfer to and from redox partner proteins. We will discuss the application of these methods to investigations of Clostridium pasteurianum rubredoxin, the vegetative and heterocyst ferredoxins from Anabaena 7120, human ferreduxin, and the Pseudomonasmendocina KR 1 tmoC Rieske protein. [Supported by NSF grant MCB-9215142. NMR studies were carried out at the National Magnetic Resonance Facility at Madison which has partial support from the Biomedical Research Technology Program of the NIH (RR02301).]

14

S-9-03

C6

Cheryl A. Kerfeld, Matthew R. Redinbo, Sabeeha Merchant and T o d d O, Yeates U C L A Dept. o f C h e m . & Biochem. and Molecular Biology Institute B o x 951570, Los Angeles, C A 90095-1570, U S A In s o m e algae and cyanobacteria, the transfer o f electrons from the cytoc h r o m e b 6 f c o m p l e x to photosystem I can be accomplished b y either cytoc h r o m e c6 or plastocyanin. W e h a v e determined the crystal structures o f c y t o c h r o m e c6 and plastocyanin at high resolution f r o m the same organism, Chlamydornonas reinhardtii. The surface features o f these proteins will be c o m p a r e d in an effort to understand the structural basis for their ability to interact with the same electron transfer partners. In addition, biochemical and crystallographic data suggest that c y t o c h r o m e c6 m a y be a functional dimer. T h e evidence for oligomerization will be discussed in the context o f its possible significance for electron transfer in photosynthesis.

Symposium S-1O-03 MOLECULAR DISSECTION OF THE 8 SUBUNIT OF THE CHLOROPLAST ATP SYNTHASE Symposium

ATPase,

J. A. Cruz, V. Tabidze, C. Radkowski and Department of Biology, The Johns Hopkins University, Baltimore, MD 21218 USA

10

protons

S-10-01

and

energy

transduction

- S-1O-04

Wild-type, mutant and truncated forms of the ~ subtmit of CF 1were overexpressed as inclusion bodies in Escherichia coli. For several of the mutants, including 8-C6S, e as active as that purified from CF~ could be obtained by a dilution method. The ability of the various types of 8 to inhibit the ATPase activity of CF, deficient in ~ (CF~-E)as well as to confer to CF~(-e) the ability to block proton conductance by CF0 were tested. In most cases the two activities were affected to a similar extent by mutation or truncation. One mutant e., e-H37R is a much more effective ATPase inhibitor than proton conductance blocker, suggesting that the two activities may be uncoupled. Supported by NSF.

S-1O-O4

S-1O-01 PROTONS AND THE MECHANICS OF THE ATP-SYNTHASE

MISSING LINKS IN THE UNDERSTANDING OF F- AND V-

ATPases W. Junoe, G. Groth, H. Lill and S. Engelbrecht Universit~it Osnabrfick, Abt. Biophysik, D-49069 Osnabr0ck, Germany Proton intake, proton transfer, and proton release by the ATP synthase of chloroplasts was investigated under flashing light. Under synthesizing conditions a dichotomy between "activating" and "driving" proton flow was searched by specific inhibitors, venturicidin and tentoxin. The activating proton flow rose first and the driving one lagged behind as in consecutive reactions. Under conditions of proton slip (no added nucleotides) a kinetic disparity between proton intake (fast) and transfer (slower) was apparent. Two agents which both blocked proton slip by binding to F1 selectively inhibited different protolytic reactions, ATP blocked proton transfer but not transient proton intake, and dequalinium inhibited proton release but not transient proton intake and transfer, a selective conformational coupling between binding sites on F1 and certain protolytic reactions in Fo. The functional necessity for motion of subunit 8 relative to ( ~ ) 3 was studied with genetically engineered 8 carrying Ser-->Cys replacements at either of six positions spread over the sequence. Crosslinking of 8 with a (in one case 13) did not inhibit ATP hydrolysis by solubilized CF1 but ATP synthesis by CFoCF1. Relative flexibility of 8 seems necessary for coupling ATP synthesis to proton flow but not per se for processing nucleotides.

N. Nelson Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 U.S.A. The chloroplast H+-ATPase belongs to a large family of proton pumps that contain two subfamilies of F-ATPases and V-ATPases. Two recent developments may shed light on the possible mechanism of action of these proton pumps. First and foremost, the F-ATPase from beef heart mitochondria was crystallized and solved to a 2.8A resolution. Second, the genes encoding most of the subunits of F- and V-ATPases were cloned and sequenced. Sequencing of these genes revealed the homology and analogy among different subunits of these two subfamilies of proton pumps. While three of the subunits ct or B, 13or A and the proteolipids in F- and V-ATPases are highly homologous, the remaining subunits show no significant sequence homology. Recently we cloned the gene encoding subunit D in V-ATPase and it appears that this subunit is analogous to the V subunit of F-ATPases. The 7 subunit of F-ATPases was implicated in this mechanochemical act of energy coupling. This was nicely verified by the crystallization of F-ATPase from beef heart mitochondria. The possible involvement of subunit D of V-ATPase in the mechanochemical coupling will be discussed. We initiated a study with scintillation proximity radioassy on the high affinity nucleotide binding site.

S-10-02 ON THE CATALYTIC CYCLE OF THE H+-ATPase FROMCHLOROPLASTS Peter gr~ber, Institut for Physikalische Chemie, Universit~t Freiburg, Albertstr. 23a, 79104 Freiburg The H+-ATPase from chloroplasts is a strongly regulated enzyme. We have investigated the kinetics of proton transport coupled ATP synthesis and ATP hydrolysis. From these data a model for the catalytic cycle was proposed. The enzyme has two main conformations: in E 1 proton binding sites in CF 0 are directed to the inside, in E 2 to the outside. The catalytic cycle starts with proton binding from the inside to CF O. This is followed by random binding of AOP and phosphate to the CF I part. The enzyme changes into the conformation E2 with proton binding sites to the outside and formation of bound ATP. The protons dissociate to the outside from CFo, ATP is released and finally a change back to E 1 closes the reaction cycle. Using literature data the energatics (A GO'diagram) of the cycle is constructed under multisite and uni-site conditions. The interaction between the different nucleotide binding sites was investigated after covalent blocking of the different catalytic and non-catalytic sites. It resulted that one catalatic site has different properties as compared to the two other catalytic sites.

15

Symposium

S-II-03

ADVANCES IN THE ANALYSIS OF THE THREEDIMENSIONAL (3D) STRUCTURE OF PHOTOSYSTEM II

Robert C. For0*, Richard P. Collins*, Toby D. Flint#, Ashraf Kitmitto*, Mark F. Rosenber~, Fiona H. Shepherd #, Svetla Stoylova#:~ and Andreas Holzenburg~r-~ *Dept. Biochem. & Applied Mol. Biol., UMIST, Manchester M60 1QD, UK #Dept. Biochem. & Mol. Biol. and ~Dept. Genetics, University of Leeds, Leeds LS2 9JT, UK

S.~mt~o~inm 1 !

Organisation of the photosynthetic :lpparatus S-II-0I

The 3D organisation of photosystem II (PSII) subunits has been studied by electron microscopy of ordered 2D arrays in grana thylakoids. The studies have shown for the first time the subtle relationsh!p between structure and oxygen evolution in PSII. An intramolecular lumenal cavity (18 nm3) is formed by the arrangement of the 33-, 23-, and 17-kDa extrinsic subunits in juxtaposition with the large lumenal domains of the core chlorophyll-binding proteins CP47 and CP43. Removal of the extrinsic subunits causes the loss of the cavity and in vitro rebinding of these polypeptides restores the original structure. The locations of all the extrinsic polypeptides and of CP47 as well as CP43 have been assigned in the 3D map of PSII. Small features interpreted as lightharvesting chlorophyll a/b protein appear to surround the core of PSII in a ring composed of 8-10 monomers. On the basis of a pseudo-twofold rotational symmetry the heterodimeric core of PSI] is predicted.

- S-11-05

S-11-01 ANALYSIS OF ELECTRON TRANSFER REACTIONS IN PURPLE BACTERIA AT SUBZERO TEMPERATURES P. Joliet 1. A. Joliet I & A. Verm~glio2 ~Inst. Biologie Physico-Chimique, 13, rue P.& M. Curie 75005 Paris, France 2CEA SBC/DPVE CE Cadarache, 13108 Saint-Paul-lez-Durance, France A high-sensitivity spectrophotometric technics has been developed, adapted to measurements of light-induced spectral changes in photosynthetic material at temperatures between 35 ° and -60 °. Due to the material and structure of the cuvette, the sample stays in a supercooled state down to -11 °, in the absence of antifreeze. Electron transfer reactions have been analyzed at subzero temperatures in intact cells of Rb. sphaeroides. In liquid state, the activation energy of electron transfer reactions is constant between 20 ° and -12 ° . Transition from liquid to frozen state induces a slow down of more than a factor 10 of the electron transfer reactions between cyt c and the primary donor P. The electrogenie reactions associated with the electron transfer reactions within the cyt b/c~ complex do not display such a sharp transition and remain functional at temperature down to -25 °. This implies that the movment of ubiquinone within the membrane and that of cyt c~ between reaction centers and cyt b/cl complexes are not impaired at low temperature in a frozen state.

S- 11-02

$- 11-04

ALTERED CHARACTERISTICS OF ENERGY TRANSFER AND TRAPPING IN MUTANTS OF RttODOBACTER NPItAEROIDF~ CONTAINING A MODIFIED ANTENNA SYSTEM.

C. N. Hunter; Robert Hill Institute for Photosynthesis and Krebs Institute for Biomolecular Research, University of Sheffield, Western Bank, Sheffield SI0 2UH, U.K The bacterial photosynthetic apparatus is organised so that light energy absorbed by LH2 complexes migrates towards the core LH 1 complex, before being trapped by the reaction centre. This process has been studied in mutants containing blue-shifted LH2 complexes, but normal LHI-RC cores; these alterations maintain efficient transfer to LH1 but have affected energy equilibration so that back transfer from the LHI-RC core is minimised. Measurements of the dynamics of LH2--->LH1 energy transfer demonstrate that this process is charaetefised by a time constant of-3ps in the wild type, and that it becomes slower in the membranes containing blue-shifted LH2 complexes, where there is less spectral overlap with the LHI complexes. A model is presented for the association between LH2 and LHI complexes, based on the energy transfer data.

S=11=05

THE DOMAIN STRUCTURE AND FUNCTION OF THE THYLAKOID MEMBRANE

SUPRAMOLECULAR ORGANIZATION OF THE PHOTOSYNTHETIC CHAIN IN RHODOBACTER SPHAERO1DES

P.-A. Albertsson Dept of Biochemistry, Chemical Center, Univ. of Lund, Box 124, S-221 00 Lund, Sweden

Vermr~,lioa..AA, Barz b W, Joliet c A, Joliet c P and Oesterhelt b D

Recent work on the domin organization of the thylakoid will be reviewed and a model for the thylakoid of higher plants will be presented. The thylakoid membrane is divided into three main domains: the stroma lamellae, the grana margins and the grana core (partitions). Linear electron transport occurs in the grana while cyclic electron transport is restricted to the stroma lamellae. The model is based on the following results and considerations. There is no good candidate for a long-range mobile redox carrier between PSI] in the grana and PSI in the stroma lamellae. The lateral diffusion of plastoquinone and plastocyanin is severely restricted by macromolecular crowding in the membrane and the lumen respectively. There is an excess of 14 4- 18% chlorophyll associated with PSI over that of PSlI. This excess is assumed to be localized in the stroma lamellae where PSI drives cyclic electron transport. The model predicts a quantum requirement of about 10 quanta per oxygen molecule evolved, which is in good agreement with experimentally observed values.

16

aCEA-DPVE/LBC CE Cadarache F-13108 St Paul-lez-Durance bMax-Planck-Institute for Biochemistry 82152 Martinsried, FRG ClBPC, 13 Rue P-et-M CURIE, F-75005 Paris The s u p r a m o l e c u l a r o r g a n i z a t i o n of the p h o t o s y n t h e t i c s p h a e r o i d e s has been electron t r a n s f e r s y s t e m of Rb. investigated by absorption spectroscopy. We will show that RC, cyt c2 and b c l complex form a supercomplex both in intact cells and isolated chromatophores. Analysis of lightinduced a b s o r p t i o n changes of whole cells of a m u t a n t deleted in the PufX shows that this polypeptide is essential for the p h o t o s y n t h e s i s only w h e n the q u i n o n e pool is reduced. The putative role of PufX in flip-flop of quinone molecules in relation to the supercomplex model will be discussed.

Symposium

S-12-03 A PUTATIVE CO 2 TRANSPORTER IN S Y N E C H O C Y S T I S PCC6803 ENERGIZED BY NADPH DEHYDROGENASE-MEDIATED PS-I CYCLIC ELECTRON FLOW

Symposium

A. Katoh I, K.Lee 2, H. Fukuzawa 2, K.Ohyama 2 & T.Ouawa 1'3 1Biochem. Regulation, 3BioScience Center, Nagoya Univ, Nagoya 464-01, Japan and 2Lab of Plant Mol Biol, Dept of Agr Chem, Kyoto Univ, Kyoto 606-01, Japan

12

Alternative electron transfer pathways and regulation S-12-01

- S-12-04

The CO 2 concentrating mechanism in cyanobacteria consists of two basic components; inorganic carbon (Ci; CO 2 + HCO3- ) transport energized by light and RunisCO-containing carboxysome for efficient utilization of internal Ci. Both CO 2 and BCO3transport are driven by photosystem-i cyclic electron flow mediated by NADPH dehydrogenase. Recently we have isolated two mutants (SCI and SC2) which form very small colonies under CO2-1imited conditions (pH7.0, 60ppm CO2). Both mutants showed normal activity of HCO 3- transport but the activity of CO 2 uptake in the mutants was much lower than in the wild-type. A gene that complements both mutants was cloned and analyzed. The gene, designated cotA, showed amino-acid sequence homology with c e m A which encodes a chloroplast envelope membrane protein. There was substitution of a singlebp nucleotide in c o t A of SCI and deletion of 7-bp nucleotides in SC2. The results indicate that c o t A may encode a CO 2 transporter.

S-12-04

S-12-01 CONTROL AND ORGANIZATION OF ELECTRON TRANSPORT AND CARBON ASSIMILATION IN LEAVES

A. Laisk, Institute of Molecular and Cell Biology, University of Tartu, Riia str. 181, Tartu, EE2400, Estonia. Electron transport through PSI is coregulated by donor side oxidation, acceptor side reduction, plus variable excitation capture efficiency by the centre chlorophyll P700, as revealed from the extrapolation of the (PSI quantum yield) vs. (D830 rim, signal difference from the dark level) plots to D830=0. The basic rate-constant of plastoquinol oxidation is (6 ms) 1 , which is downregulated to (40 ms) 1 . During the dark-light induction of the carbon reduction (CR), O~ evolution exceeds CO~ uptake, to accumulate RuBP. The accum01ated RuBP causes pos-tillumination CO2 uptake, which follows the kinetics explainable by free RuBP pool and competition by free PGA and Pi. During the induction, P700 becomes oxidised gradually, in proportion with the activation of the CR. These kinetics show that equilibration of the redox potential between the acceptor sides of different PSI complexes is prevented. Possibly, the PS I acceptor side carriers form an electron channelling enzyme system that is linked to a substrate channelling enzyme system of PGA reduction and RuBP regeneration, a "photosynthetosome". Rubisco, RuBP and PGA are freely diffusible, cross-diffusion between the photosynthetosomes is restricted.

E L E C T R O N TRANSPORT TO NITROGENASE IN THE PHOTOSYNTHETIC BACTERIUM R.CAPSULATUS

O2-DEPENDENT ELECTRON FLOW FUNCTION IN PHOTOSYNTHESIS

AND ITS

REGULATORY

Ulrich Schreiber, Henning Hormann & Christian Neubauer Lehrstuhi Botanik I, Universit~t Wilrzburg, Mittlerer Dallenbergweg 64, D-97082 Wiirzburg, Germany Plants have developed special mechanisms for protection against photodamage. In recent years, primarily on the basis of chlorophyll fluorescence quenching analysis, the transthylakoidal ApH emerged as the key factor in the regulation of photosynthesis under light stress. Experiments with intact chloroplasts suggest a central role of O2-dependent electron flow (MAC, MehlerAscorbateperoxidase Cycle) in the formation of the regulatory ApH. In MAC light driven water oxidation is combined with light driven reduction of 0 2 and of transiently formed monodehydroascorbate to yield back water and ascorbate. This cycle constitutes a valve reaction without acceptor limitation, involving energy transformation at both photosystems and energy dissipation at the antenna level via ApH-related processes. Contrary to all other types of electron flow, MAC is enhanced by ApH, resulting in autocatalytic characteristics.

S-12-02

Jouanneau Y.. Hugo N., Armengaud J., Naud I., Meyer C. and Willison J. Laboratoire de Biochimie Microbienne, CEA and CNRS URA 1130, C.E.N.G., 17 rue des Martyrs, F-38054 Grenoble cedex 9, France. In Rhodobacter capsulatus, tic high energy requirements of nitrogen fixation are met by the photochemical conversion of light. The present study deals with the identification of the protein components involved in the electron transport to nitrogenase. A set of 10 genes, involved in this transport has recently been discovered, six of which, called rnfA to rnfF, may code for a membrane-bound complex containing Fe-S clusters. The rnfgenes products have been individually overproduced in E.coli using TT-based expression systems. The recombinant proteins are currently being purified and used to generate antibodies which will be helpful in the identification, localisation and characterization of the putative rnf encoded membrane-bound complex. Besides, a 214Fe-4S] ferredoxin (PdI), has been shown to serve as electron donor to nitrogenase. None of the other five ferredoxins that we have identified in R.capsulatus can functionally replace FdI. It is proposed that FdI mediates electron transfer between the rnf-encoded membrane-bound complex and nitrogenase.

17

Symposium S-13-03

Symposium

Expression prokaryotes

S-13-01

LIGHT RESPONSIVE PHYCOBILISOME BIOSYNTHESIS A. Grossman, D. Kehoe & E. Casey. Department of Plant Biology, The Carnegie Institution of Washington, 290 Panama Street, Stanford, CA 94305, USA

13

and

regulation

The biosynthesis of the phycobilisome, a light harvesting complex in the cyanobacteria and red algae, is extremely sensitive to environmental condition_s. In certain cyanobacteria light quality dramatically alters phycobilisome composition by modulating the transcription of genes enc~xting the different phycobiliprotein subunits (pigmented polypeptides of the phycobilisome). This process is called chromatic adaptation. Recently, we have used in vivo techniques to establish regions of the phycobiliprotein gene promoters that contain light-responsive elements. We have also generated and complemented mutants that exhibit aberrant chromatic adaptation. One of the genes isolated in the complementation studies encodes RcaC, a polypeptide resembling the regulator of bacterial two component regulatory systems. Site-directed mutagenesis of RcaC suggests that phosphoryladon at an N-terminal apartate residue is critical for chromatic adaptation. Analyses of the sequences that complement the different mutant strains is helping us establish the signal transduction pathway leading from photoperception to transcriptional control of the genes encoding the phycobiliprotein subunits.

of genes:

- S-13-05

S-13-01 THE ROLE OF THE PUF X PROTEIN IN PHOTOSYNTHETIC GROWTH OF RHODOBACTER SPHAEROIDES W. Barz 1, G. Venturoli2, F. Francia2, B.A. Melandri2, A. Vermrglio 3 andD.

t fur Biochemie, D-82152 Martinsried, ~University1,of1Max.Planck_Institu Bologna Dpt. of Biology, Via Irnerio 42, 40126 Bologna, Italy, 3CEA Centre d'Etudes de Cadarache, Dpt. de Physiologic Vrgrtale et Ecosyst~mes, 13108 Saint Paul lez Durance, France. The v u f X gene is essential for photoheterotrophic growth of the purple bact~-riumRhodobacter sphaeroides. It codes for a 9 kDa polypeptide which is expressed in the cell and associated with the reaction center -LH I complex. The hypothesis that it plays a critical role in facilitating the functional interaction between this complex and other components required for light-driven cyclic electron uansfer ( J.W. Farchaus, W.P. Barz, H. Griinberg and D. Oesterhelt, EMBO J. 11, 2779-2788, 1992) has now been confirmed by detailed biophysical studies on wild type, pufX deletion and revertant strains. We show that the ubiquinone/ubiquinole (Q/QH2) exchange rate is reduced approximately 500-fold by the deletion of pufX when the quinone pool is nearly completely reduced demonstrating an essential role of PufX for the access of ubiquinone to the QB site in the reaction center. The fast Q/QH2 exchange is partially restored by suppressor mutations altering the macromolecular antenna structure. These results suggest an indirect role of PufX in structurally organizing a functional photosynthetic apparatus.

S-13-04 MOLECULAR FACTORS THAT CONTROL GENE EXPRESSION dN A FILAMENTOUS CYANOBACTERIUM J. Houmard, G. Schyns, L. Jia, A. Sobczyk, S. Liotenberg, D. Campbell and N. Tandeau de Marsac Physiologie Microbienne, CNRS URA1129, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France

Calothrix sp. PCC 7504 and PCC 7601 are closely related filamentous species that undergo complementary chromatic adaptation; as major light-harvesting antennae, they synthesize phycoerythrin when grown under green light, and phycocyanin-2 under red light. Transcriptional controls operate involving specific DNA-binding proteins: RcaA, RcaB and RcaD. A nearly homogeneous preparation of RcaA has been obtained, and RcaD has been partially purified. Eleven promoters have been tested for their ability to inititate transcription in in vitro abortive runoff assays using RNA polymerase purified from cells grown under either red or green light, and with or without RcaA and RcaD. These DNA-binding proteins modify the in vitro activity of the RNA polymerases on a few specific promoters. The nature of the nitrogen source available in the growth medium also influences phycobiliprotein synthesis. A decrease in phyeoerythrin mRNAs and an increase in phycocyanin-2 transcripts are observed when ammonium instead of nitrate is used. This is reminiscent of a red light effect and leads to assume the existence of common regulatory elements.

S-13-02 LIGHT-RESPONSIVE EXPRESSION OF CYANOBACTERIAL PHOTOSYNTHESIS GENES Susan S. Golden, Rixin Li, Resham D. Kulkami, Nicholas F. Tsinoremas, and Shivanthi Anandan Dept. of Biology, Texas A&M University, College Station, TX 77843, USA The psbA and psbD genes of the cyanobacterium Synechococcus sp. strain PCC 7942 are three- and two-member multigene families, respectively, which encode the reaction center proteins of photosystem If. Each family's members respond differentially to changes in light intensity and light quality. The psbA genes respond to an increase in intensity of white light through transcriptional induction ofpsbAH and psbAlll and accelerated degradation of psbAl and psbAlllmessages. The psbDlI gene is also light-responsive, and appears to be coregulated with psbAH and psbAllI. These genes also exhibit a blue/red photoreversible response which is almost indistinguishable from the highlight response. The regulatory regions of psbAH and psbAlll include basal promoters, upstream negative elements, and sequences downstream of the promoters that are required for induction by high light. The light-responsive cis elements have the properties of enhancers, and bind specific soluble proteins. Experiments to map the stability determinants of the psbA messages implicate the untranslated leader regions in mediating accelerated degradation of the psbAl and psbAlll messages at high light. Recent data suggest that translational control, as well as known transcriptional and post-transcriptional mechanisms, are all at work in the complex regulation of these genes.

18

S-13-05 RHODOBACTER CAPSULATUS AS A MODEL PHOTOTROPH

FOR STUDIES ON NITROGEN GENE REGULATIONAND CYTOCHROME C BIOGENESIS

R. G. Kranz Biology Dept, Washington University, St. Louis, MO 63130, USA In vivo and in vitro studies on the nitrogen control circuitry in R, capsulatus have revealed some similarities and differences to circuits in other prokaryotes. Although a nitrogen-sensing 2-component system similar to enterics is present (ie. NtrBfNtrC), the mechanism of NtrCdependent activation of RNA polymerase at nitrogen-controlled promoters is different; all other NtrC-like enhancer binding proteins require the sigma 54 factor (ie. RpoN). The results indicate that this large family of prokaryotic activators is more versatile then originally envisioned. Concerning cytochromes c biogenesis, at least seven different proteins are required: HelABCDX and Cc112. Apocytochromes c, heine, and reducing and ligation components are specifically transported to the periplasmic space for assembly of c-type cytochromes. Biochemical, genetic and topological studies have been used to develop a model for the biogenesis pathway. Recent discoveries of hel and ccl homologs in mitochondrial genomes of plants and protozoa suggest that this assembly pathway is ubiquitous.

Symposiura

S-14-03 LIGHT ACTIVATED TRANSLATION OF THEpsbA mRNA

Symposium

S.P. Mayfield, A. Danon, C.B. Yohn, A. Cohen, R. Bruick, Dept. of Cell Biology, The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037 Translation of key photosynthetic proteins within the chloroplast requires nuclear encoded factors as well as portions of the 5' untranslated region (UTR) of the chloroplastic mRNAs. We have isolated a set of proteins that bind with high specificity and affinity to the 5' UTR of the psbA mRNA. Binding activity of these proteins can be modulated in vitro by both redox potential and ADP-dependent phosphorylation. Using site-directed mutagenesis of the psbA UTR we have identified RNA elements required for light activated translation. Structural analysis of the psbA UTR has shown that RNA secondary structure is an essential component of translation and protein recognition. Examination of psbA-RNAIribosome association in vivo has indicated that changes to the UTR may alter the rate of initiation complex formation. Finally we have characterized nuclear mutants deficient in translation of chloroplastic mRNAs, and identified that one of the psbA binding proteins (47 kDa) is altered in mutants which fail to translate the psbA mRNA. These data suggest that light modulated translational activation is accomplished by binding of specific proteins to the 5' UTR of chloroplastic mRNAs in response to photosynthetic production of redox potential. Binding of these proteins to the 5' UTR results in alteration to secondary structure of the RNA at the ribosome binding site which affects the rate of initiation complex formation and hence translation.

14

Expression and regulation of genes: eukaryotes S-14-01

- S-14-05

S-14-01 DISSBCTING THX RZGULATORY EL~B0~TS GEN~S BY A TRANSGINZC APP~0ACK

OF TOBACCO

PLASTID

P ~ , Lori A. Allison, Sharon Levine, Wei Wu, Ivan Kanevski and Robert Hayes. Waksman Institute, Rutgers, The State University of New Jersey, Piscataw~y, NJ 08855-0759 We have used plastid transformation to identify cis elements regulating plastid gene expression in response to light. Transcrintion: Deletion analysis of the light-responsive psbD and psbC promoters de~8onstrated different mechanisms of regulation. Sequences directly upstream of the psbD -35/-10 elements were found to enhance transcription ~200- fold, in~licating upstream activators of promoter activity. In contrast, lightinduced (-10X) transcript accumulation from the psbC prc~oter was linked to the -35/-10 core, probably mediated b y sigma-like factors. T ~ Deletion analysis indicates that light-induced translation of the rbcL n~RNA is dependent on a short (17 nt) segment of the 182 nt m R N A leader which forms a ste~-loop structure. Current experiments aim at identifying the proteins which interact with the 17 nt m R N A segment, and thereby regulate translation.

S-14-02

REGULATION OF TRANSCRIPTION DURINGCHLOROPLASTDEVELOPMENT

S. Lerbs-Mache, R. I r a t n i , L. Baeza, L. Diederich and R. Mache Laboratoire de Biologie Mol~culaire V~g~tale, Universit~ Joseph Fourier and Centre National de la Recherche Scientifique, B.P. 53, F-38041 Grenoble cedex 9, France Transcription of the plastid genome during plant development is maintained by at least two types of RNA polymerase. The nature and the exact polypeptide composition of both enzymes are s t i l l not known, as well their role in gene expression is poorly understood. Results obtained from in uiuo and x~ v ~ o studies on the transcription of the rRNA operon of spinach plastids suggest a mechanism of regulation by sequencespecific DNA-binding proteins which might interact with both types of RNA polymerase.

S-14-04 COPPER-RESPONSIVE GENE EXPRESSION DURING ADAPTATION TO COPPER-DEFICIENCY Sabeeha Merchant. Jeanette Quinn and Kent Hill University of California at Los Angeles, Los Angeles, CA 90095, USA The biosynthesis of plastocyanin and cyt e6 is regulated in a reciprocal fashion by a metal sensing system: the presence of copper promotes plastocyanin synthesis but represses cyt c6 synthesis, whereas the absence of copper prevents plastccyanin accumulation but induces cyt c6 transcription, thus assuring the availability of one or the other of these structurally distinct but functionally equivalent proteins for photosynthesis. The transcriptional response of the cyt c6-encoding gene (Cyc6) is remarkable for its high sensitivity to and selectivity for copper, and for its response range. This response is effected via two copper-responsive elements (CuREs) that lie between nucleotides -127 and -56 relative to the start site of transcription and function as activators of transcription in copper-deficient cells. Other adaptation to copper-deficiency which occur coordinately with induction of cyt c6 accumulation include induction of a copper uptake pathway (to increase the intracellular copper in response to nutritional deficiency), and induction of coproporphyrinogen oxidase expression (to increase flux through the tetrapyrrole biosynthetic pathway in response to the increased demand for heine).

S-14-05 T H E R O L E O F T H E 3' U N T R A N S L A T E D R E G I O N CHLOROPLAST RNA PROCESSING AND STABILITY

IN

Stern, D.B., Drager, R.G., Levy, H., *Suzuki, H. and *Kindle, K.L. Boyce T h o m p s o n I n s t i t u t e and , P l a n t Science Center, Cornell University, U S A The 3' termini of m o s t chloroplast m R N A s coincide with inverted repeat (IR) sequences t h a t can form stable stem/loop s t r u c t u r e s . In Chlamydomonas, we h a v e shown t h a t t h e 3' IR of atpB m R N A s t a bilizes t h e transcript both in vivo and in vitro. In addition, processing signals i m m e d i a t e l y d o w n s t r e a m of t h e IR serve as a recognition site for a n endonuclease activity t h a t processes t h e transcript. S t r a i n s harboring a deletion of t h e atpB 3' IR express approximately 10% of t h e wild-level of t h e A T P a s e ~-subunit, and are weakly photosynthetic a n d light- a n d temperature-sensitive. We h a v e described three k i n d s of s u p p r e s s o r s of t h e s e phenotypes. In the first, t h e t r u n c a t e d atpB gene is amplified, leading to wild-type overexpression of t h e 6subunit. In t h e second, a nuclear m u t a t i o n leads to stabilization of a t r a n s c r i p t lacking t h e IR. Finally, a sequence forming a n artificial s t r o n g tertiary s t r u c t u r e (poly-G) can partially s u b s t i t u t e for t h e native IR in vivo. These results s u g g e s t t h a t both R N A s t r u c t u r e and trans-acting factors play a role in t h e function of the 3' IR.

19

Symposium

S-15-16-04 CHAPERONIN-MEDIATED PROTEIN FOLDING

Symposium

A.A. Gatenbv BioProcess Deveiopment Center, DuPont, Experimental Station, P.O. Box 80328, Wilmington, DE 19880-0328, USA

15-16

Moiecuiar chaperones have a key role in cellular metabolism, and function primarily to stabilize protein folding intermediates. In the case of the chaperonin family of molecular chaperones, this stabilization is achieved by binding of non-native poiypeptides to an oligomeric structure composed of fourteen 60 kDa subunits (cpn60). The captured non-native species fail to progress to native states while sequestered by cpn60, but folding continues when the target poiypeptides are reieased by the addition of a co-chaperonin in the presence of ATP. Chaperonins are ubiquitous and have been identified in bacteria, chloropiasts and mitochondria, together with a cochaperonin that facilitates correct folding during the polypeptide discharge reaction. The co-chaperonins are composed of seven 10 kDa subunits (cpnl0), with the exception of chloroplast co-chaperonins which possess two domains fused together (cpn21). Chaperonins are known to interact with non-native features present following import into chloroplasts, and on newly-synthesized or chemically-denattared polypeptides. Whatever these features are, they are not present or inaccessible in native target proteins.

Protein translocation and assembly

S-15-16-02

-

S-15-16-09

S-15-16-02 SYNTHESIS AND CARBOXYL-TERMINAL PROCESSING OF D1 PRECURSOR PROTEIN OF THE PHOTOSYSTEM I I REACTION CENTER Kimiyuki Satoh ( D e p a r t m e n t o f B i o l o g y , Okayama U n i v e r s i t y , 700 and NIBB, O k a z a k i 444, J a p a n )

Okayama

In the damage-repair cycle of PSII reaction cent e r , s y n t h e s i s o f D1 p r o t e i n i s r e g u l a t e d by l i g h t a t the stage of translation, through signal(s) provided by p h o t o - d i s i n t e g r a t i o n of the protein under functional states. The C - t e r m i n a l e x t e n s i o n o f t h e n e w l y synthesized D1 p r e c u r s o r p r o t e i n , consisted of 8-16 amino acids, is excised immediately after translat i o n , by a p r o t e a s e p r e s e n t i n t h e l u m e n a l s p a c e o f thylakoids. For organisms furnished with the extension, this enzymatic process is absolutely required in the manifestation of oxygen-evolving function. In r e c e n t y e a r s , c o n s i d e r a b l e p r o g r e s s h a s b e e n made i n the elucidation of the enzymatic process of C-terminal cleavage. The m e c h a n i s m o f l l g h t - r e g u Z a t e d translation o f D1 p r o t e i n has also been analyzed. The p r e s e n t l e c t u r e w l l l p r o v i d e some new r e s u l t s and an o v e r v i e w o f t h e p r o g r e s s i n t h i s r e s e a r c h f i e l d .

S-15-16-05 Unexpected functional and structural diversity of ~uting/ass~bly processes for thylakoid proteins R.G. Herrmann & R.B. Kl~sgen Botanisches I n s t i t u t der Ludwig-Maximilians-UniversitBt, ger Str. 67, 80 638 Mfinchen (Munich), Germany

Menzin-

The import and assembly of c y t o s o l i c components f o r the photosynthetic machinery into the c h l o r o p l a s t require the operation of s p e c i f i c t a r g e t i n g and sorting signals f o r the c o r r e c t organ e i l e and d e s t i n a t i o n w i t h i n the organelle. The t r a n s i t peptides of nuclear-encoded p ] a s t i d p r o t e i n s , which are recognized by the appropriate t r a n s l o c a t i o n systems and subsequently removed, exh i b i t high v a r i a b i l i t y in terms of sequence and length, and f a l l into two p r i n c i p a l categories. Remarkably, the association with a given type is not c o r r e l a t e d with the p o l a r i t y nor with the l o c a t i o n of a protein w i t h i n the o r g a n e l l e / t h y l a k o i d membrane. The d i s t r i b u t i o n of f u n c t i o n a l epitopes along the polypeptide chains e x h i b i t s unexpected complexity as do the t r a n s l o c a t i o n machineries. Approx. 20 components studied use a single entrance machinery into the o r g a n e l l e , but several, mutually exclusive routes e x i s t into or across the t h y l a k o i d membrane. Consistent with gene I s o l a t i o n s , t h i s d i v e r s i t y f i n d s a phylogenetic analogy: only part of the machineries and mechanisms appear to be prokaryotic h e r i t a g e , and b i p a r t i t e " t r a n s i t peptides may have d i f f e r e n t phylogenetic o r i g i n s .

S-15-16-03

S-15-16-06

T H R E E SIGNAL PEPTIDE/SIGNAL ANCHOR PATHWAYS FOR PROTEIN TRANSPORT INTO THYLAKOID MEMBRANES.

CTPA, A NOVEL C-TERMINAL PROCESSING PROTEASE INVOLVED IN THE BIOGENESIS OF THE PHOTOSYSTEM II COMPLEX

K. Cline. R. Henry, X. Li °, N. E. Hoffman*, and J. Yuan. Hort Sci, Univ of Florida and *Dept of Plant Biol, Carnegie Inst of Wash.

Himadri B. Pakrasi 1, Sergey Shestakov 2, Ralf Oelmaller 3 end Reinhold G. Herrmann 3 1Department of Biology, Washington University, St. Louis, MO 63130, USA; 2Department of Genetics, Moscow State University, Moscow, 119899, Russia and 3Botanisches Institut der LMU, Menzinger Str. 67, 80638 M0nchen, Germany

Nuclear-encoded thylakoid proteins are imported across the plastid envelope and then transported across or into thylakoids. Analysis of transport requirements and results of precursor competition assays reveal three different pathways for thylakoid protein transport. Commitment to pathway is determined by subtle differences in targeting peptides, which resemble classical signal peptides. We recently purified a stromal protein required for transport on one pathway and showed it to be a plastid homologue of the bacterial SecA protein. Collaborative studies have identified a protein required for a second pathway as a homologue of the signal recognition particle (SRP) subunit SRP54. A third pathway is unique in that it relies solely on a trans-thylakoidal pH gradient. Current studies are aimed at identifying the pathway-specific elements of targeting peptides, in reconstituting binding of targeting peptides with pathwayspecific components, and in identifying other putative components of the plastid SRP.

20

The rapidly turned over D1 protein of the Photosystem II (PSll) reaction center complex is synthesized as a precursor protein with a carboxylterminal extension. Processing of this C-terminal extension is an important event in the biogenesis of this protein complex, since in the absence of such processing, the 4 Mn-ensemble in PSII is not formed. We have performed complementatioe analysis of a random PSII-deficient mutant strain of the cyanobacterium Synechocystis 6803 to identify ctpA, the gene encoding the C-terminal processing protease for the D1 protein. We have also used this gene as a probe to clone the homologous nuclear gene from barley. In higher plants, the expression of this geee is light-modulated and tissue-dependent. Antibodies raised against the CtpA protein overexpressed in E. coil have been used for the localization of this protein. Further genetic and biochemical characterizations of this novel protease will be discussed during this meeting.

Symposium S-15-16-07 THE PROTEIN IMPORT MACHINERY OF CHLOROPLASTS J. Soil Botanisches Institut, Universit~t Kiel, D-24098 Kiel, Germany A large number of plastid localized proteins are post"translationally imported as precursor proteins from the cytosol into the organelle. Recognition and translocation is accomplished by a subset of chloroplast envelope proteins. The outer envelope proteins OEP86, OEP75, OEP70 (an heat shock cognate 70 homologue) and OEP34 are clearly involved in the import event and can be isolated as one functionally active trenslocation unit. For t w o of these proteins oDNA clones have been obtained very recently in our laboratory, namely OEP86 and OEP34. OEP86 seems to be a precursor protein receptor, which could be regulated by GTP binding and ATP dependent phosphorylation-dephosphorylation. OEP75 is traversing the membrane in multiple I~-sheets and seems to be part of the translocation pore. OEP34 is tightly associated with OEP75 in situ. OEP34 represents a new type of GTP-binding protein, which possesses endogenous GTPase activity. Multiple GTP binding and hydrolysis cycles as well as protein phosphorylation-dephosphorylation events might therefore regulate the interaction of a precursor protein with the translocation machinery of the outer envelope, making it very distinct from the mitochondrial outer membrane system.

S-15-16-08 FUNCTIONAL DOMAINS OF THE FERREDOXIN TRANSIT Peter Weisbeek ''2, Ben de Kluijf~ "3 and Marinus pilon ''~3 'Department of Molecular Cell Biology, 2Institute of Biomembranes and ~Center for Biometubranes and Lipid Enzymology. Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Protein import into chloroplasts depends on the presence of an N-terminal tratlsit sequence. A set of 20 deletion mutants, covering the full length of the transit sequence of ferredoxin, was generated. These mutant proteins were analysed by in vitro assays for binding, uptake and processing and a subset was analyzed in vivo in transgenic Arabidopsis. This analysis indicates the existence of four domains which are involved in the different stages of translocation. Five mutant transit peptides were purified after over-expression in Escherichia coli and tested in inaport competition and in lipid monolayer assays. Specific parts of the transit sequence were found to be required for insertion into lnonolayers composed of a total lipid extract of the chloroplast outer envelope membrane. The capability to recognize the chloroplast is correlated with the capacity to insert into the lipid extract at physiologically relevant lateral pressures. This suggests an important role for transit sequence-lipid interactions ha the chloroplast protein uptake process.

S-15-16-09 BIOGENESIS OF CYTOCHROIM B 6 F COMPLEXES IN C2~/~MIrDONONRB R E I ~ H A R D T I I .

F.-A. W o l l m a n and R. Paris, France.

Kuras,

Service de Photosynth~se,

IBPC,

The recent d e v e l o p m e n t of t e c h n i q u e s for c h l o r o p l a s t gene t r a n s f o r m a t i o n by h o m o l o g o u s r e c o m b i n a t i o n in C. r e i n h a r d t i i has o p e n n e d the w a y to the study of c h l o r o p l a s t - e n c o d e d p r o t e i n s m o d i f i e d by s i t e - d i r e c t e d mutagenesis. This a p p r o a c h was u s e d to u n d e r s t a n d various aspects of the b i o g e n e s i s of c y t o c h r o m e b6f complexes, w h i c h comprise b o t h n u c l e a r and c h l o r o p l a s t - e n c o d e d subunits. We c o n c l u d e to a m u l t i - s t e p a s s e m b l y p r o c e s s w h e r e the functional o l i g o m e r i c p r o t e i n results from the f o r m a t i o n of a core complex, c o m p r i s i n g m a i n l y c h l o r o p l a s t - e n c o d e d proteins to w h i c h binds the R i e s k e p r o t e i n w h i c h is n u c l e a r encoded. The s t o i c h i o m e t r i c a s s e m b l y of the subunits in the core complex results from the i n t e r p l a y of a c o - t r a n s l a t i o n a l -or e a r l y p o s t - t r a n s l a t i o n a l - r e g u l a t i o n of c y t o c h r o m e f synthesis, w h i c h most likely involves a c y t o c h r o m e b 6 / s u I V subcomplex, and a p o s t - t r a n s l a t i o n a l r e g u l a t i o n of the a c c u m u l a t i o n of the other subunits w h i c h are d e g r a d e d w h e n not assembled. C y t o c h r o m e f b i o g e n e s i s was further c h a r a c t e r i z e d by a s i t e - d i r e c t e d m u t a g e n e s i s approach. We d i s c u s s the r e s p e c t i v e c o n t r i b u t i o n s of c y t o c h r o m e f processing, heme a t t a c h e m e n t and c a r b o x y - t e r m i n u s synthesis, to the a s s e m b l y of c y t o c h r o m e b6f complexes. In p a r t i c u l a r we o b s e r v e d that p r e a p o c y t o c h r o m e f could still b i n d heme and r e m a i n e d c o m p e t e n t for assembly.

21

Syrnposiuna S-17-03 A BRANCHED PATHWAY FOR LIGHT-DEPENDENT CHLOROPHYLL BIOSYNTHESIS DEFINED BY NADPH:PROTOCHLOROPHYLLIDE OXIDOREDUCTASES A AND B_ IN ARABIDOPSIS THALIANA

Symposium 17

SOnke Runge, Genny Frick, Ulrich Sperting, Klaus Apel & Greqory Armstronq. Department of Plant Genetics, Institute for Plant Sciences, Swiss Federal Institute of Technology (ETH), CH-8092, Zurich, Switzerland.

Biosynthesis of tetrapyrroles

Illumination releases the arrest in chlorophyll biosynthesis in etiolated angiosperm seedlings through the unique enzymatic photoreduction of protochlorophyllide (Pchlide) to chlorophyllide. This reaction, the first light-dependent step in chloroplast biogenesis, is mediated by the nuclearencoded, plastid-localized NADPN:Pchlide oxidoreductase (POR). Paradoxically, light negatively regulates POR activity and protein levels such that bulk chlorophyll accumulation during greening occurs in the virtual absence of the enzyme. We have recently identified two distinct A. thaliana POR-encoding genes that display dramatically different patterns of regulation in response to light and developmental state. PorA but not PorB mRNA disappears upon illumination. Only young seedlings detectably express Po~PA, whereas both seedlings and adult plants express PorB. These and other data lead us to propose the existence of a branched light-dependent chlorophyll biosynthesis pathway in which POR A performs a specialized function early in the greening process and POR B maintains chlorophyll levels throughout angiosperm development.

s-17-01 - s-17-06

S-17-01 BIOS~IC

ORIGIN OF OXYGENS IN BAC~J~RZOCHLOROPH~I~A

R. Ports, I. Katheder, H. Scheerf B o t e n i s c h e s Institut der U n i v e r s i t A t M~nchen, W. Schlfer, M a x - P l a c k - I n s t i t u t fur Biochemie, Martinsried Germany

Respiring, dark grown cells of Rhodobacter sphercides 2.4.1 were transferred to low oxygen t e n s i o n and irradiated with white light, in the p r e s e n c e of either H2180 or 1802. Products w e r e a n a l y z e d by m a s s spectrometryy, and by suitable chemical t r a n s f o r m a t i o n s in order to ascertain labeling at critical positions. It has b e e n shown, that all six oxygens, including in particular the one located at C-13 I, derive from water. In the c l o r o p h y l l s a and b, of oxygenic photosythesis, this atom derivez from m o l e c u l a r oxygen, as well as the 7-CHO oxygen in chlorophyll b. S i m i l a r experiments with R h o d o c o c c u s d e n i t r i f i c a n s shall be reported.

S-17-02

S-17-04 M O L E C U L A R GENETIC ANALYSIS OF Mg-TETRAPYRROLE BIOSYNTHESIS.

Carl Bauer, Indiana University, Department of Biology, Bloomington, IN 47405, USA The purple photosynthetic bacterium Rhodobacter capsulatus that synthesizes bacteriochlorophyll a has proven to be an excellent experimental organism for genetic analyses of Mg-tetrapyrrole biosynthesis (Bauer et al. 1993, J. Bact. 175:3919-3925). Sequence and mutational analysis has revealed the existence of 16 genes that are believed to code for enzymes involved in the Mg branch of the tetrapyrrole p a t h w a y . Several of these genes have been overexpressed in E. coli yielding cell-free extracts containing the predicted enzymatic activities. Transcription of bacteriochlorophyll and carotenoid biosynthesis genes are known to be repressed by high light intensity and oxygen, and a DNA binding repressor responsible for aerobic repression has recently been described (Ponnampalam et al., 1995, J. Bacteriol. In Press). Chlorophyll biosynthesis genes from cyanobacteria, algae and plants, that are homologs of R. capsulatus bacteriochlorophyll biosynthesis genes, have been cloned and sequenced.

S-17-05

LIGHT STRESS PROTEINS (ELIPs); THE INTRIGUING RELATIVES OF CAB GENE FAMILY

BLUE-LIGHT REGULATED EXPRESSION OF GENES FOR TWO E A R L Y STEPS OF CHLOROPHYLL BIOSYNTHESIS IN CHLAMYDOMONAS REINHARDTII

Iwona Adamska and Klaus Kloppstech Institut ~ r Botanik, Universit~ttHannover, Herrenhauser Str.2, 30419 Hannover, Germany

Gall L. Matters and Samuel I. Beale. Divisionof Biology and Medicine, Brown University, Providence, Rhode Island 02912 USA

Green plants respond to light stress by expression of nuclear-encoded early light-inducible proteins (ELIPs) which accumulate in the stroma lamellae of thylakoid membranes. This group of proteins appears also transiently during the early stages of greening of etiolated plants. The mature form of ELIP has three transmembrane loops and is integrated into thylakoid membranes in an orientation similar to that of light-harvesting chlorophyll protein. Despite the homology of ELIPs with all cab gene products a binding of photosynthetic pigments has been never shown for ELIPs. Using barley mutants completely or partially arrested in the pigment biosynthesis pathways we can demonstrate that the stability of ELIPs in the thylakoids depends on the level of accumulated pigments.The direct interaction of ELIPs with individual pigments was characterized using an in vitro integration system Our data indicate that the ELIPs may play a role during PS II turnover and repair by interaction with pigments

22

In light:dark synchronizedculturesof Chlamydomonas reinhardtii, the genes encoding the enzymes for two early steps of chlorophyUbiosynthesis,glutamate1-semialdehyde aminotransferase(gsa) and &aminolevulinicacid dehydratase (alad), are expressed at high levels early in the light phase,just prior to a rapid burst of chlorophyll synthesis. Inductionofgsa mRNA in synchronizedcells is totally dependent on light, whereasinduction of a/ad mRNA occurs to approximatelyone half the light-induced level even in cells kept the dankduring the light-phase, and appears to be dependent on the cell cycle or a circadian rhythm. Blue (400--480 nm) and green (490-590 nm) light, but not orange (>560 rim) or red (>610 nm) light, induce gsa mRNA and a/ad mRNA accumulation,indicating the participation of a blue-light photoreceptorsystem rather than a protochiorophyllide-or thndopsinbased photoreceptor. Light induction ofgsa mRNA accumulationis absent in a carotenoid-deficient mutant, which suggeststhat a carotenoid-containing blue light photoreceptoris involved. In the later part of the light phase, the gsa mRNA level decreases more rapidly than that of a/ad mRNA. Turnover studies indicate that the half-life of alad mRNA is twice that of gsa mRNA, which partially accounts for the more rapid decline in gsa mRNA levels after the peak of light induction is reached. Thus, differential blue-light induction and mRNA stability regulate the expressionof these two chlorophyllbiosynthetic genes. DOE and NSF support.

Symposium S-17-06 REGULATION OF CAROTENOID CONTENT AND CHLOROPLAST DEVELOPMENT BY LEVULINIC ACID Arshad Jilani & Baishnab C. Trinathv School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India The development of photosynthetic apparatus was monitored during greening of etiolated barley leaf discs in the presence of levulinic acid, an inhibitor of chlorophyll biosynthesis. Although levulinic acid is not a direct inhibitor of carotenoid biosynthesis, it resulted in a linear reduction in both chlorophyll and carotenoid contents. The chlorophyll biosynthesis seems to control the carotenoid biosyntheses. As expected, in the presence of low amounts of chlorophyll the light-harvesting chlorophyll protein complex H was not visible in comossie-stained gels in 20 mM levulinic acid-treated tissues. However, they were detected as a faint band by immunoblotting and this small amount of light-harvesting chlorophyll protein complex II induced significant amounts of cation-induced grana stacking which was monitored as an increase in the ratio of variable fluorescence/maximum fluorescence. When levulinic acid was washed from the treated leaf discs and allowed to green in its absence, the chlorophyll content, carotenoid content and the photosynthetic activities approached the control values. Levulinie acid could he used to arrest the light-induced chloroplast development at a desired phase of greening and removed by washing the leaves to restore the developmental process without any apparent toxic effect. (This research was supportrd by Department of Biotechnology, Government of India Grant BT/TF/T/02/91 to B. C. T.)

23

Symposium

S-18-03 FROM NAPHTHO- TO BENZOQUINONES - A (R)EVOLUTIONARY REORGANISATIONOF ELECTRON TRANSFER CHAINS W. Nitsehke1, D.M. Kramer1,2, A. Riedel3 & U. Liebl4 Symposium

18 1Biologic II, Univ.Freiburg/FRG;2Dept.Biophys., Univ. of Illinois/USA 3Inst.Biophysik,Unlv.Regensburg/FRG;4Dept.Biology, Univ.Philadelphia/USA

Evolution of photosynthesis S-18-01

Oxygenic photosynthesis in chloroplasts/cyanobacteria and respiration in mitochondria/purplebacteria are correlated to the presence of plastoquinone (PQ) and ubiquinone (UQ), respectively. Within the realm of the Bacteria, however, the majority of species use menaquinone (MK), i.e. a naphthoquinone-type molecule for their bioenergetic e--transport chains, both in photosynthesisand in respiration. Considering the distribution of species containing UQ/PQ- or MKbased e--transport chains within the phylogenetic tree of species (16S r-RNA), one is led to conclude that the UQ/PQ-type chains have evolved from those based on MK. Recent studies of MK-based pathways in a variety of species showedthat the significantly lower redox potential of MK as compared to that of UQ/PQ is paralleled by adjusted Em-values for almost all the remaining redox eentres, resulting in an overall similar mode of functioning for the MK- and the UQ/PQtype e--transport chains. Therefore, the basic principles of these e--transport chains have evolved only with respect to some details. The replacement of MK by UQ/PQ at some moment in evolution apparently has required strong concertation between all redox centres since no extant species bridging the gap between MK- and UQ/PQ-basedbioenergetic chains have been discovered so far.

- S-18-04

S-18-01

S-18-04

Origin and evolution of phosphorylating and non-phosphorylating glyceraldehyde-3-phosphate dehydrogenases.

EVOLUTION OF THE CHLOROPLAST GENOME IN GREEN ALGAE

Rfidiger Cefff Institute of Genetics, University of Braanschweig, D-38106 Braunschweig

M. Tunnel, E. Boudreau, S. Mayrand, C. Otis & C. Lemieux. Drpartement de biochimie, Universit6 Laval, Qurbec (Qurbec) G1K 7P4, Canada.

Glyceraldehyde-3-phosphate dehydrogenases (GAPDH) fall into three separate classes, one of which, non-phosphorylating class Ill, is a member of the aldehyde dehydrogenase superfamily with no sequence similarity to its phosphorylating counterparts, class I and class II. While class I GAPDH of photosynthesis and glycolysis is widespread in eubacteria and eukaryotes and has recently also been found in archaebacteria, the phylogenetic distributions of class II (archaebacteria) and class III (photosynthetic eukaryotes) GAPDHs seem relatively limited. Our findings suggest that eukaryotic genes encoding class I GAPDH are of eubacterial, endosymbiotic, origin and were transferred to the nucleus from the progenitors of present-day chloroplasts and mitochondria, respectively, thereby replacing preexisting GAPDH genes of the host cell. Intron conservation patterns and gene protein correlations in class I GAPDH genes further support the early origin of splieeosomal introns and their implication in the primordial assembly of GAPDH protein structure in agreement with the exon theory of genes. Literature: Cerff R. (1995). The chimaeric nature of nuclear genomes and the antiquity of introns as demonstrated by the GAPDH gene system. In: Tracing Biological Evolution in Protein and Gene Structures, Taniguchi International Symposium (ed. Mitiko Go), Elsevier Science, Amsterdam, in press.

In land plants, the chloroplast DNA (cpDNA) evolves very conservatively and is under strong contraints to retain a compact gene organization. Although the chloroplasts of green algae and land plants share a common endosymbiotic origin, the limited data available on green algal cpDNAs have revealed much more variability in size and gene order. To gain insight into the mode and tempo of cpDNA evolution in green algae, we have initiated studies on the phylogeny and chloroplast gene organization of selected taxa. We will report our phylogenetic analysis of the chloroplast large subunit rRNA gene sequences from about 65 green algae representing all of the major classes previously described, and will also present our results on the arrangement of chloroplast genes in Nephroselmis olivacea, Tetraselmis carteriiformis, and representatives of the order Chlamydomonadales. Chlamydomonads occupy the most derived lineages in the phylogeny reconstructed; their cpDNAs display considerable variability in gene order and differ from their counterparts in Nephroselmis and Tetraselmis by the absence of most of the evolutionarily primitive operons found in land plant cpDNAs.

S-18-02 PLASTID ORIGINS

C.J. Howe, Department of Biochemistry, University of Cambridge, UK. It is widely accepted that plastids arose from endosymbiosis between photosynthetic prokaryotes and non-photosynthetic hosts. However, there is still controversy over whether plastids of "red" and "brown" algae arose from the same endosymbiosis that gave rise to green plastids (i.e. have a monophyletic origin), or whether they are derived from independent endosymbioses (i.e. have a polyphyletic origin). Many phylogenetic trees based on sequence data from plastids indicate a monophyletic origin, but these analyses have been criticised on the grounds that they may have been misled by the non-random base composition of plastid genomes. I shall present analyses that endeavour to avoid this problem by using (i) datasets that may not be subjected to the same patterns of biased compositions, and (ii) the recently developed LogDet transformation (which may allow the correct phylogeny to be determined under conditions where other methods select the wrong tree),

24

Symposium S-19-20-03 R|IBI~CO: CATALYSIS AND REGItI.A'rlON T J+,!m.Andrews. qu.,aJi!le":Of~t'aemmelcl+ Colleen J Mate~. Gi'aham S. Hu,J+onand h~hn R. L~'an,:+. Riga+oh 5ch0~l el Bi+log£c,+-IScie~es. Aust++ali:mNaUr,tml IJniver~iry, (~nbcrra. !Presenl address. [}++p+~+~m+:no| t Biochemistry. University nf Nevada. Reno

Symposium 19-20 E n z y m o l o g y o f the p h o t o s y n t h e t i c metabolism S-19-20-01 - S-19-20-10

S-19-20-01 THE PHOTOSYNTHETIC CARBOXYLASES - - RUBISCO AND PEPC

Raymond Chollet Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0664, USA This symposium w i l l emphasize various aspects of the structure/ function relationships and regulation of Rubisco. However, this bifunctional stromal enzyme is n o t the only photosynthetic carboxyqase present in higher plants, but rather acts in concert with cytosolic PEP carboxylase (PEPC) to effect the net f i x a t i o n of atmospheric C02 during C, photosynthesis and CAM. In l e a f tissue of C, and CAM species, PEPC a c t i v i t y is regulated p o s t t r a n s l a t i o n a l l y and i n t e r a c t i v e l y by a l l o s t e r i c control by p o s i t i v e [glucose 6-P (G6P)] and negative (L-malate) effectors and a complex regulatory phosphorylation cycle that modulates the enzyme's s e n s i t i v i t y to L-malate and G6P. This b r i e f presentation will overview the wealth of in vitro and in vivo findings that have established the existence of a highly regulated, Ca'*-independentprotein-Ser/Thr kinase and a protein phosphatase type 2A which mediate the interconversion of PEPC between a high K,/lowK. phospho-form and a low KJhigh K. dephospho-form, thus resulting in the up-regulation of the phosphorylated target enzyme during periods of atmospheric C02 fixation under intense illumination (C,) or at night (CAM).

S-19-20-02 T H E G L Y C I N E D E C A R B O X Y L A S E S Y S T E M IN H I G H E R P L A N T S R. Deuce, J. Bourguignon, C. Cohen-Addad, D. Macherel & M. Neuburger Laboratoire de Physiologic Cellulaire VOgOtale, U R A CNRS 576, D6partement de Biologie Mol6culaire et Structurale, CEA-Centre d'Etudes NuclEaire de Grenoble et Universit6 Joseph Fourier, 38054 Grenoble-cedex 9, France The glycine decarboxylase system is present at tremendously high concentrations within the mitochondrial matrix. This complex consists o f four protein components (P-, H-, T- and L-protein) and its structural and mechanistic heart is provided by the lipoic acid-containing H-protein which undergoes a cycle o f reductive methylamination, methylamine transfer and electron transfer. Lipoic acid is attached to the H-protein via an amide linkage to the e -amino group o f a lysine residue. The X-ray crystal structures o f two forms o f the Hprotein have been determined at 2 A. The lipoate cofactor attached to Lys63 is located in the loop o f a hairpin configuration but following methylamine transfer it is pivoted to bind into a cleft at the surface o f the H-protein. The methylamine group is held by three hydrogen bonds whereas the carbon atoms of the lipoamide a n n interact through van der Waals' contacts with several hydrophobic residues. All four proteins o f the complex are encoded by unique nuclear genes, translated on cytosolic ribosomes, and imported into the mitochondrial matrix during the course o f leaf development.

We 5cek to exFlain the lule of r(llb~t+ct) d(.'llx'~2in Fefrtt~of t]Je Opell lind closed collfomlatJol15 Ihrot,+~h v,rtlch Rubl+<.co'+,~l~;lixe:,itc "~lternatesd,]ring the catal3,~icc)cle Fo!lowinE hitldlng ~F ~ :4bulost 1%bi~ph~l~phalv IRu]3P) to ++.~tbadlylat+'d~ubist¢,, .~e,:e~d dJ-.onlered Ioop¢+ ult3~icovcf the a~td~(rMr4+~('ql}t'~,tt+rir+L~lh+.treaction intffrtnt.diale.~from ~olvent OpenJllp,of the active Sll~ 1o, pi,M,+,'{rel+':~.¢ may be trJgger~'l hy cleavage of the c,+'allet.t£ooberw.+enthe tear [~ll~bphate ,~o~elics. WheiI the active site i5 (lOS,rl over a lifam'l +hal canno! I:¢ cle;~v~d, ope.ning o! Ihe actJ+,'esit~ and rclea.~¢of the ligand iu "+'elyslo,+v St+oh,~.~mpr¢~luctive ch)+t'd ct+lnpIe~es ,Ecu: whcr pct,,.ulose blsph~sl;hate is(~mer-:awe h~rn!¢dla the active xitc. whorl tile aoct~wnal inhib~tt!~2"-ca~h+~s+~arabinitol-l.pho~pbatehind.,+to the c;:rF<~mylltlo]a~t~e slt~ and when RuL+Pbrn,+'ts{o the Jlicarbam)la~ed site. We hyputbes~e ~ha: actJ~',~se,i)~imedinn) an act+re ¢onform+atioaby A'll' hy(Iml)~, biud-, ~h'+ctively m ',uch
S-19-20-04 A STRUCTURE BASED MECHANISM FOR THE REACTIONS OF RIBULOSE BISPHOSPHATE CARBOXYLASE/OXYGENASE $. Gutterid+,e 1 and J. N e w m a n 2 1Cenu-al Research and Development, DuPont Experimental Station, Wilmington D E 19880; 2Department of Biochemistry and Molecular Biophysics, Columbia University, New York NY 10032 As Rubisco catalyses the carboxylation or oxygenation of ribulose bisphosphate the enzymes activity can be viewed most conveniently as the successive shuttling o f protons between various active site residues, substrate and reaction intermediates. An assortment of high resolution models o f native and mutant enzyme has assisted in pinpointing some of the players in these movements. Both carboxylation and oxygenation require the abstraction o f the C3 proton from the bisphosphate substrate generating an enediol intermediate that is susceptible to attack by CO2 or 0 2 . Until recendy the identity of the group(s) responsible for this step was obscure. The crystal structure o f the S y n e c h o c o c c u s enzyme with the intermediate analogue 2 C A B P bound in the active site suggests that it is the essential carbamino group and metal ion that initiate the catalytic cycle. A number o f further proton transfer steps are involved in the subsequent partial reactions, and it is tempting to speculate that the carbamino group plays a similar role in these transfers as well.

S-19-20-05 CATALYTIC ROLES OF FLEXIBLE REGIONS AT THE ACTIVE SITE OF RIBULOSE-BISPHOSPHATE CARBOXYLASE/OXYGENASE (RUBISCO) Fred C. Hartman, Mark R. Harpel, Yuh-Ru Chen, Eric M. Larson, and Frank W. Latimer Protein Engineering Program, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 USA Rubisco catalysis entails several unstable intermediates. Two flexible regions, loop 6 (from the [3/(x-barrel domain) and helix B (from the N-terminal domain of the adjacent subunit) [Knight et al. (1990) J. Mol. Biol. 215, 113], at the entry to the interfacial active site may sequester and stabilize these intermediates. To explore these possibilities, we have used site-directed mutagenesis to truncate loop 6 and to substitute two active-site residues, Lys329 of loop 6 and Glu48 of helix B, which interact electrostatically when these regions close over the active site. The mutant proteins, impaired in overall catalysis, retain preferential competency in enolization of ribulose bisphosphate and in correct processing of the isolated carboxylated reaction intermediate. However, aberrant products are generated from substrate due to misprocessing and decomposition of reaction intermediates. Characterization of two of these products confirms a peroxy intermediate in normal oxygenation. Chemical rescue of debilitated mutants with homologs of the targeted residues reveals the sensitivity of the CO2/O2 specificity to the nature of side chains at positions 329 and 48. Clearly, conformationally-dynamic regions of the active site stabilize reaction intermediates and ensure proper throughput of substrates. (Supported by USDOE under contract DE-AC0584OR21400 with Martin Marietta Energy Systems, Inc.)

25

Symposium

S-19-20-06 REGULATION OF PEP CARBOXYKINASE ACTIVITY IN PLANTS Robert P. Walker and Richard C. Leegoed, Robert Hill h~dto~ and Departmentof Animal and Plant Sciences, Universityof Sheffield, Sheffield, S10 2TN, UK In plants, phosphoenolpyruvate carboxykinase (PCK) catalyses the conversion of oxaloacetate to PEP which is a key step in photosynthesisin some C4 and CAM plants. We know virtually nothing about the regulation of PCK activity in plants. An enigma ennceming PCK has been the relative lack of regulatory, properties of the purified enzyme which would explain its regulator}, role in gluconengenesis, or the light-dark regulation of the enzyme in C4 plants or CAM plants. For example, in CAM plants PEP earboxylase (active at night) and PCK (active during the day) are both eytosolic. Such regulation could be achieved by phosphorylafion. Incubationof the purified cucumber enzyme with 7-32p-ATP and either PEP-carboxylase kinase or mammaliancAMP-dependent protein kinase led to labelling of the enzymein a part of the molecule separate from the active site. This was reversed by inonbation with protein phosphatase2A. Cucumbercotyledons supplied with 32pi contained a heavily labelled polypeptide whichwas confirmedas PCK by immuanprecipitation. Labelling of PCK by 32pi in darkened cotyledons was reversed by illumination or by supplying sucrose in the dark. PCK is also phosphorylated in the C4 grass, Urochloa panicoides. The phosphorylationand the regulator}, properties of PCK in leaves of C3, C4 and CAM plants will be discussed.

S-19-20-09 A STUDY OF THE CATALYTIC AND REGULATORY SITES OF THE ENZYMES OF STARCH SYNTHESIS Jack Preiss, M. Ballicora, Y. Fu, J. Sheng, Y. y. Chamg, H. Guan, T. Kuriki H. Cao, K. Funane and D. Stewart, Dept. of Biochemistry, Michigan State University, East Lansing, MI 48824, USA Chemical modification and site-directed mutagenesis studies of the starch biosynthetic enzymes, ADP-glucose pyrophosphorylase (ADPGlc PPase; EC 2.7.7.27) and branching enzyme (BE; EC 2.4.1.18) have identified essential amino acids and peptide domains needed for catalysis and regulation. Lys residues, K382 and 419 are necessary for the binding of the allosteric activator, 3-P-glycerate of the Anabaena PCC7120 ADPGIe PPase. The sequences surrounding these residues are highly conserved in the higher plant ADPGlc PPase large and small subunits.Via chemical modification, the equivalent K419 residue has been located as K440 in the small subunit and the equivalent residue, K382, located in the large subunit of the spinach leaf ADPGlc PPase. Expression of the cDNAs encoding the higher plant two subunits in Eschen'chia coli has enabled us to determine that the small subunit plays the major role in catalysis and the large subunit in regulation. BE is considered to be part of the c~-amylase group of enzymes. Consistent with this are chemical modification and site-directed mutagenesis studies that show that His, Arg and Glu/Asp residues are essential for maintenance of branching enzyme activities.

S-19-20-07 IDENTIFICATION OF THE RESIDUES INVOLVED IN LIGHTACTIVATION AND CATALYTIC ACTIVITY OF NADP-MALATE DEHYDROGENASE M. Mi~iniac-Maslow. E. Issakidis, M. Lemaire, P. Decottignies, M. Saarinen* & I-P. Jacquot URA CNRS 1128. Institut de Biotechnologie des Plantes. B,% 630. Universit6 de Paris-Sud 91405 Orsay Cedex France and * Uppsala Biomedical Center, P.O. Box 590-$751 24, Uppsala, Sweden Chloroplastic NADP-malate dehydrogenase is activated by light through thiol/disulfide interchange with thioredoxin reduced via the photosynthetic electron transfer chain. Site-directed mutagenesis experiments performed with a cDNA coding for the sorghum leaf enzyme allowed the identification of two different regulatory disulfides per subunit: one N-terminal, the other, Cterminal. Biochemical evidence and molecular modeling suggest that the activation rate is limited by a slow conformational change at the active site. The regulatory cysteines are not involved in catalysis. The active site of the enzyme consists of histidine and aspartate residues, strictly conserved among all the malate dehydrogenases. A cysteine is located close to the active site. It has no catalytic function but is responsible for the sensitivity of the enzyme to thiol reagents.

S-19-20-08 CHARACTERISTICS

OF THE

INTERACTION

BETWEEN

RUBI$CO

AND RUBISCO

ACTI~FASE. A r c h i e R. Portis, Jr. I, Brian Esau I, Eric M. Larson I, Genhai Zhu ~, Chris J. Chastain 2, Carolyn M. O'Brien 2, & Robert J. Spreitzer 2, IPhotosynthesis Research Unit, USDA/ARS, Urbana, IL 61801; ZDepartment of Biochemistry, University of Nebraska, Lincoln, NE 68583, USA. Rubisco activase catalyzes the activation of the inactive, substrate-bound form of Rubisco. However, activation is severely impaired with heterologous combinations of these proteins when one of them is from tobacco. Comparison of the known sequences of the proteins identified several residues in each that are relatively unique in tobacco and which m a y be responsible. In Rubisco activase several of these are clustered at the C-terminus and in Rubisco they are widely dispersed in the primary sequence but clustered adjacent to the active site in the holoenzyme. Therefore the basis for the observed specificity in the interaction between the proteins is being approached by: (1) analysis of Rubisco activase chimeras composed of combinations of the spinach and tobacco proteins and (2) analysis of Rubisco isolated from Chlamydomonas transformants with site specific replacements of the suspected large subunit residues with those occurring in tobacco.

26

S-19-20-10

RUBISCO AND THE CHLOROPLAST CHAPERONIN H. Roy, A. Hubbs, and M. Gilson Department of Biology Rensselaer Polytechnic Institute Troy, New York 12180-3590, USA

In pea (P. sativum) chloroplast extracts supplied with radioactive amino acid and an ATP regenerating system, newly synthesized L subunits of Rubisco bind to the chloroplast chaperonin Cpnt0. In the absence of S subunits at low KCI, a distinct complex forms which appears to be an aggregate of L subunits. When KCI and S subunits are added, even in the absence of ATP, this complex disaggregates and appears to serve as a reservoir for the assembly of Rubisco hoioenzyme. We have begun experiments with in vitro transcription and translation of the L subunit in E. coli extracts using a plasmid containing the L subunit gene from maize and the bla gene. In this system the newly synthesized L subunit binds to the E. coli groEL chaperonin. When the electrophoretieally distinct chloroplast chaperonin is added to this system, radioactive proteins bind to it. This reaction seems more pronounced when the maize L subunit gene is present in the plasmid than when it is not. We hope to use this system to test whether the chloroplast Cpnt0 will support the in vitro assembly of Rubisco.

Symposiuxn

S-21-03 CARBON CONTROL OF NITRATE ASSIMILATION

Symposium

C. Lara Inst. Bioqufmica Vegetal y Fotosfntesis. Univ. de Sevilla-CSIC. Apdo. 1113, E-41080 Sevilla, Spain

21

Integration of the C,N,S metabolisms S-21-01

- S-21-05

A general phenomenon observed in microalgae and higher plant leaves is that nitrate cannot be assimilated unless CO2 fixation is operative or a carbohydrate source is available. This feature has been considered as a proof of the energy dependence of nitrate assimilation upon carbohydrate oxidation. However, evidence is now available indicating that i) nitrate assimilation uses photosynthetically generated assimilatory power; and, ii) the carbon dependence of nitrate assimilation is related to the control of initial regulatory steps of the process, namely, nitrate transport activity in cyanobacteria and nitrate reductase activity in plant leaves. Whereas nitrate transport activity in cyanobacteria is subjected to both a feed-back negative nitrogen control and a positive carbon control, nitrate reductase activity in plant leaves does not seem to be sensitive to nitrogen and exhibit a dependence upon CO2 fixation which is the basis for its light-dark activation-inactivation behaviour. A comparative discussion on the carbon control of these two systems will be presented.

Financed by DGICYT (PB91-0611) and PAl (gr. 3101)

INTEGRATION OF NITRATE IN PLANT METABOLISM

S-21-01

Michel Caboche, Patrice Cr~t6, Anne Krapp, Laureut Nussaume, Alberto Quesada, Christian Meyer. Emmanuelle Pigaglio, Hoai-Nam Truong, Thdr6se Moureanx and Franqoise Ved~le. Laboratoire de biologie cellulaire, INRA Versailles, France. The nitrate assimilatory pathway has been the matter of intensive genetic analysis in N. plumbaginifolia. Molecular studies on the expression Nia and Nii genes coding for nitrate and nitrite reductases respectively, and for a putative nitrate transporter are under way. Analysis of the regulation of the pathway has shown evidence for the involvement of nitrate, light and/or sucrose, and reduced nitrogen in the regulation. A Nia mutant was transformed with a gene conferring the constitutive expression of a nitrate reductase eDNA sequence carrying a N-terrmnal deletion involving sequences upstream the MoCo domain of the enzyme. In the obtained transgenic plants, NR activity was restored and allowed normal growth. However, the post transcriptional regulation of this enzyme by light was abolished in the trausformants. The modification of regulation of the nitrate assimilatory pathway by constitutive expression of a nitrate reductase or a nitrite reductase gene was investigated. A constitutive expression of nitrate reduetnse led to a decreased storage of nitrate in vegetative tissues, and can be envisaged as a possible approach to the improvement of the nutritional characteristics of crops.

S-21-02

BIOSYNTHESIS OF B R A N C H E D - C H A I N A M I N O ACIDS IN PLANTS: STRUCTURE AND FUNCTION OF ACETOHYDROXY ACID ISOMEROREDUCTASE

V. Biou 1, E. Pebay-Peyroula 1, C. Cohen-Addad 1, F. Vives 2, D. Job2, R. Douce2 & IL Dumas2 llnstitut de Biologie Structurale, 41 Rue des Martyrs, 38027 Grenoble, France. 2Unit~ Mixte CNRS/Rhfne Poulenc Agrochimie, 14-20 Rue Pierre Baizet, 69263 Lyon, France. Acetohydroxy acid isomeroreductase is involved in the biosynthetic pathway leading in plants and micro-organisms to branched-chain amino acids. The enzyme catalyses a two-step reaction in which an acetohydroxy acid substrate (2-acetolacte or 2-aceto-2-hydroxybutyrate) is converted via an alkyl migration and a NADPH catalysed reduction to a dihydroxy acid product (respectively 2,3-dihydroxy-3-isovalerate and 2,3-dihydroxy-3methylvalerate). With the aim to understand the molecular basis of this complex reaction, structural studies were carried out on the chloroplastic spinach enzyme overexpressed in Escherichia coll. Mutagenesis experiments disclosed two magnesium binding sites involved in the two steps of the reaction. Also, the three-dimensional structure of the enzyme co-crystallized with Mg2+, NADPH and competitive inhibitors such as N-hydroxy-Nisopropyloxamate (IpOHA) or 2-dimethylphosoPhinoyl-2-hydroxy acetic acid (Hoe 704) was resolved at a resolution of 2.4 A.

S-21-04 CYSTEINE BIOSYNTHESIS AS A SULFUR A S S I M I L A T I O N P A T H W A Y IN PLANTS: MOLECULAR AND B I O C H E M I C A L APPROACH Kazuki SAITO Faculty of Pharmaceutical Sciences, Laboratory of Molecular Biology and Biotechnology in Research Center of Medicinal Resources, Chiba University, Inage-ku, Chiba 263, Japan Cysteine biosynthesis is the major assimilation pathway of sulfur in plants. We have isolated three cDNAs (cysA, B and C) which encode three isoforms of cysteine symhase (CSase) localized in cytoplasm, chloroplasts and mitochondria, respectively, from spinach. CysA and C were expressed in green and etiolated plants; whereas eysB was expressed primarily in green plants. Transgenic tobacco plants, integrated with the constructs for 35S-cysA, 35S-pea transit peptide-cysA and 35S-antisense cysA, were obtained and analyzed for modulation of cysteine biosynthesis in response to various sulfur stress. The results indicated that over-accumulated foreign cysteine synthase in chloroplasts could enhance biosynthetic flow of cysteine. The cDNAs encoding serine acetyltransferase (SATase) were isolated from plants. The activity of recombinant SATase was inhibited by L-cysteine, the end product of the pathway, in an allosteric manner at a low concentration, indicating a regulatory role of SATase. The recombinant CSase and SATase formed a multi-enzyme complex.

S-21-05

INTERACTION OF CARBON AND NITROGEN METABOLISM IN PHOTOSYNTHETIC CELLS: CLUE FROM UNICELLULAR ALGAE.

David H. Turpin and Heather C. Huppe, Department of Biology, Queen's University, Kingston Ontario, K7L 3N6 CANADA Unicellular algae grown under nitrogen limitation respond rapidly and uniformly to activate carbon respiration and modify photosynthetic carbon fixation during primary nitrogen assimilation. Combining physiological and biochemical measurements, we have examined the changes which occur during the onset of nitrogen assimilation to gain clues about the role of nitrogen assimilation in controlling carbon metabolism. The carbon required to support the formation of amino acids from inorganic nitrogen is partially supplied by either a breakdown of starch or recent photosynthate depending upon the rate of nitrogen assimilation and the carbon status of the ceUs. To assimilate nitrogen, there is a requirement for anapleurotic carbon fixation via the PEP carboxylase to supply the carbon skeletons for amino acid synthesis. During nitrate assimilation, the extra energy required to reduce nitrate to ammonia results in the initial role of carbon respiration to participate in the supply of electrons. This occurs through an activation of glucose 6-phosphate dehydrogenase and the oxidative pentose phosphate pathway. The regulatory properties of these two enzymes as involved in the integration and control of carbon and nitrogen metabolism will be discussed. Worksupportedby the NationalScienceand EngineeringCouncilof Canada

27

Symposium

S-22-04 ENGINEERING OXIDATIVE STRESS TOLERANCE IN CHLOROPLASTS D. Inz~ ~, L. Slooten2 & M. Van MontaguI ILaboratorium voor Genetica, Universiteit G e n t , K.L. Ledeganckstraat 35, B-gooo Gent, Belgium; 2Laboratorium voor Biofysica, Vrije Universiteit Brussel, B-lOSO Brussel, Belgium

Symposium 22

Numerous environmental stress conditions produce an excess amount of active oxygen species, such as superoxide (0~), hydrogen peroxide (H2Oz) and hydroxy radicals (OH.) potentially leading to inhibition of photosynthesis and destruction of the photosynthetic apparatus. Our major aim is to understand the mechanisms used by chloroplasts to l i m i t the production or the damaging effect of the AOS and to use anti-oxidant enzymes for improving stress tolerance of chloroplasts. Transgenic tobacco plants overproducing a Mn superoxide dismutase (MnSOD) are considerable more tolerant to paraquat-induced oxidative stress. Overproduction of FeSOD, endogenously present in chloroplasts, provides an e v e n larger protection to oxidative damage. Overproduction of a cytosolic ascorbate peroxidase (Apx) in the chloroplasts protects the photosynthetic apparatus to elevated levels of H20~produced by an inhibition of catalases by 3-aminot r i a z o l . Because SOD converts O~ into H~Oz, we expect that overproduction of Apx alongside SOD in chloroplasts will provide a synergistic protection.

Photoinhibition

S-22-02 - S-22-05

S-22-02 THE EFFECT OF UVB RADIATION (280o315nm) ON PHOTOSYSTEM H OF HIGHER PLANTS, GREEN ALGAE AND CYANOBACTERIA R. Barbato Dipartimento di Biologia, Universit~i di Padova, Via Trieste 75, 3513 Padova, Italy The effect of UVB radiation (25 I.tE.m-2.s-1) on Photosystem II of higher plants, green algae and cyanobacteria has been investigated by means of a number of different methods Specific breakdown of the reaction centre D1 protein and dismantling of a number of PSII centers has been observed as a consequence of UVB irradiation. D 1 protein is cleaved giving rise to a main C-terminal fragment of about 20 kDa, suggesting that the cleavage site is located in the second transmembrane loop or neaby it. Selective washing procedures able to block oxygen evolution by different mechanism as well as studies with mutants carrying different lesions, such as Scenedesmus LF1, which lacks most of bound manganese and Synechocystis PCC6803 with the psbO gene inactivated, which shows abnormal cycling of the S-states, strongly suggest the donor side as the target of UVB radiation.

S-22-03 Molecular basis of photoinhibition L Barber Photosynthesis Research Group, Biochemistry Department, Imperial College of Science, Technology & Medicine, London SW7 2AY, UK The rapid turnover of the D1 protein is clearly an expression of the requirement of photosystem two (PSII) to repair and protect itself from photoinactivation. Studies using in vitro systems have identified two pathways by which this protein can be damaged, one associated with impairment of the donor side and the other with acceptor side inhibition. Moreover, in vitro studies have shown that the degradation of the D1 protein does not involve a direct photo-cleavage but results from a photochemically induced conformational change which provides a 'triggered' state. Fast Atom Bombardment and Electrospray Mass Spectrometry is being used to identify the nature of this triggered state and has revealed how extensive oxidation of the D1 protein occurs as a result of illumination. It is therefore possible that such oxidations provide a signal which, in vivo, gives rise to selective degradation and turnover of the D1 protein. This turnover rate is faster in vivo than the in vitro degradation rate as also shown by comparison of in vivo systems when chloramphenicol is added to prevent D1 protein synthesis or when specific mutations have been made.

28

S-22-05 DYNAMIC REGULATION OF psbA and psbD IN BARLEY John E. Mullet. Minkyun Kim, Daryl Mofishige, Arnold DuBell, Paul Ulanch, Lutz Eichacker and Wolfhart Rudiger. Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843 and Institute of Botany, University of Munich, Germany. The chloroplast genes psbA and psbD encode the pSII reaction center proteins D1 and D2. Accumulation of D1 and D2 is regulated at the level of transcription, translation and protein turnover. For example, DI and D2 accumulation depends in part on accumulation of chlorophyll. Ribosomes translating DI, CP43 and CP47 pause at discrete points during synthesis of the chlorophyll apoproteins. Ribosome pausing is correlated with co-translational binding to and stabilization of these proteins. Translation of D1 is also regulated in response to light. Experiments that document the binding sites of two RNA binding proteins that may participate in translation regniation will be presented. Transcription of psbD is activated by blue light from a special light responsive promoter. A combination of in vitro transcription assays, gel shift and DNase I footprinting assays have been used to identify the slructure of this promoter and the location of proteins that regulate promoter usage. A model describing how blue light regulates psbD transcription will be described.

Symposium

S-23'03

CARBON DIOXIDE FIXATION: STOMATAL AND NON-STOMATAL LIMITATION IN DROUGHT-STRESSED NICOTIANA TABACUM L. CULTIVARS.

S y m p o s i u m 23

GH.J. Kr0ger & L. van Rensburg Department of Plant and Soil Sciences, Potchefstronm University, Potehefstrocm, South Africa.

Water deficiency and salt stress S-23-01

Linear resistance analysis suggests large effects of stomata] closure on photosynthesis during drought stress which implicates a synchronous decrease in CO2 diffusion capacity and leaf water potential: i.e. intercellular CO2 concentration remains constant. Our studies on tobacco cultivars with different drought tolerance, however revealed that in all caltivars mesophyU limitation exceeded stomatal limitation in drought stress-induced impairment of photosyntbesis due to an increase in intercellular CO2 concentration as the carboxylation efficiency decreased relatively more than the stomatal resistance with a decrease in leaf water potential. This effect was less pronounced in the drought tolerant caltivars. The basis for differential drought tolerance among cultivars may be their capability to maintain a higher assimilation rate possibly due to maintenance of a higher carboxylation efficiency and thus resistance to decrease in water use efficiency. Upon rewatefing of the test plants all photosynthetic parameters monitored, recovered without lag, but at differential rates being significantly faster in the drought tolerant enltivars.

- S-23-04

BIOCHEMICALMECHANISMS FOR SALINITY STRESS TOLERANCE

2520,

S-23-01

S-23-04 GENE EXPRESSION AND SIGNAL TRANSDUCTION PLANTS UNDER WATER STRESS

Hans I. Bohnert. Manabu Ishitani, Urea Rani Kamasani, Arun Lahiri Majumder 1, Donald E. Nelson, Gerald Rammesmayer, Shigehiro Yamada2 and Richard G. Jensen. Department of Biochemistry, The University of Arizona, Tucson, AZ 85721, U.S.A.; present addresses: 1Bose Institute, Calcutta, India; 2japan Tobacco, Iwata, Japan. Salt stress elicits a large number of changes in gene expression and biochemical pathways. In glycophytic plants most of these reactions are consequences of the stressed state, rather than adaptive mechanisms that eliminate or reduce the effect of the stress. We study mechanisms in a halotolerant species, ice plant, compare its reactions to stress with those of glycophytes and use transgenes to mimic halophyte functions in salt-sensitive plants. The focus writ be on four complex biochemical pathways that are essential for growth maintenance and osmotic adaptation under salt stress conditions, and on genes for essential components of these pathways: inositol biosynthesis, activated methyl cycle, polyol biosynthesis and components of the ion uptake machinery. Reactions catalyzed by these pathways will he viewed in a whole plant context. After transfer to a glycophyte, no single biochemical component will confer salinity tolerance comparable to that of the ice plant, yet synergistic, additive increases of tolerance seem possible after multiple gene transfer. (Supported by DOE, USDA-NRI; JSAS, and The Rockefener Foundation).

IN A R A B I D O P S I S

Kazuo Shinozaki I , Kazuko Yamaguchi-Shinozaki 1,2, Takeshi Urao 1,2, Takeshi Mizoguchi 1, Takashi Hirayama 1, Tomohiro Kiyosue I , Takeshi Katagiri I & Masa-aki Noji I 1Lab of Plant Mol Biol, The Inst of Phys and Chem Res (RIKEN), 2JIRCAS, Ministry of Agriculture, Forestry and Fisheries, Tsukuba 305 Japan. Under water-deficit conditions various genes are induced and thought to function in protecting cells from dehydration. To understand the molecular process of signal transduction from initial water-stress signal to gene expression, we cloned 25 cDNAs (named RD or ERD) for genes that are induced by water stress by differential screening. We precisely analyzed the rd29A promoter and identified a novel cisacting element containing 9 bp, TACCGACAT (DRE, Dehydration Responsive Element), which is involved in the gene expression under drought, low temperature and high salt conditions but does not function in ABA-responsive slow expression of rd29A. There seem to be more than three independent signal transduction pathways under water-stress; two are ABA dependent and one is ABA independent. We have shown that a transcription factor myb homologue, Atmyb2, two genes encoding calcium dependent protein kinase, ATCDPK1, 2, and one gane for phospholipase C, AtPLC1, are induced by water stress. Moreover, three genes for protein kinases involved in the MAP kinase cascade (MAP kinase, MAPKKK and ribosomal $6 kinase) are induced by drought, high salt and low temperature. These observations suggest that the MAP kinase cascade and the IP3-calcium cascade may function in the signal trensduction pathways under water stress conditions. These inducible factors probably amplify the efficiency of stress signal transdcution.

S-23-02 I N T E R A C T I O N O F L I G H T A N D D E S I C C A T I O N IN T H E R E S U R R E C T I O N P L A N T CRATEROSTIGMA PLANTAGINEUM D. Bartels, J. Alamillo, C. Bockel, G. Bemacchia, F. Salamini Max-Planck-Institut fiir Z0chtungsforschung, Carl-von-Linn6-Weg 10, D-50829 K61n, Germany

Leaves and ABA-treated callus from the resurrection plant Craterostigraa plantagineum tolerate extreme dehydration. The desiccation process is characterized by the expression o f a specific set o f genes. The expression o f these genes is regulated by A B A and/or desiccation. Light has a strong positive effect on the transcript level o f several desiccation-related genes. Although desiccation leads to changes in the composition o f photosynthetic pigments, chloroplastic structures and chlorophyll are retained during the drying treatment. Photosynthesis related gene expression is down-regulated during dehydration and transcripts start to accumulate again during rehydration. - Special attention is given to desiccation induced gene products which are localized in the chloroplasts. One o f these gene products is thought to be a general light stress protein which is supposed to be involved in protective mechanisms o f the photosynthetic apparatus. Some evidence will be discussed.

29

Symposium

S-24-03 LOW TEMPERATURE INTERRUPTS CIRCADIAN REGULATION OF TRANSCRIPTIONAL ACTIVITY IN CHILLING-SENSITIVEPLANTS

Symposium

Donald R. Oft 1,z, Tamara JonesZ;1Photosyn Research Unit, USDA/ARS & ZDept. of Plant Biology, University of Illinois, Urbana, IL 61801 USA

24

In chilling-sensitive species such as tomato, low temperature stalls the endogenous rhythm controlling transcription of certain nuclear-encoded genes causing the synthesis of the corresponding transcripts and proteins to be temporally mistimed when the plant is rewarmed. We have shown that the activity of sucrose phosphate synthase (SPS), a key enzyme controlling the sucrose biosynthetic pathway, has both a diurnal and circadian rhythm in tomato, and that these rhythms are delayed by chilling treatments. We have further demonstrated that the SPS protein level does not vary and is unaffected by short term low temperature episodes that cause shifts in the pattern of SPS activity. Using specific kinase and phosphatase inhibitors we have shown that the daily pattern of SPS activity is the result of phosphorylation and dephosphorylation of the protein. The sensitivity of the pattern in SPS activity to specific inhibitors of transcription and translation revealed expression of the gene coding for SPS phosphatase is responsible for the diurnal and circadian rhythm in SPS activity. Our findings suggest that the "inappropriate" phosphorylation state caused by the low temperatureinduced mistiming of the transcription of SPS-phosphatase underlies the chilling-sensitivity of photosynthesis in tomato.

Temperature stress S-24-01

- S-24-04

FACTORS ASSOCIATED WITH LOW PHOTOSYNTHETIC ACTIVITY IN MAIZE LEAVES AT LOW GROWTH TEMPERATURES

S-24-01

S-24-04 COLD AFFECTS PARTITIONING: DOES PARTITIONING AFFECT PHOTOSYNTHESIS?

Nell R. Baker1, Michael J. Fryer~, Kevin Oxborough ~, Gui-ying Nie 1, Elizabeth J. Robertson2, Rachel M. Leech2, Donald R. Oft3; 1Dept. Biology, University of Essex, Colchester CO4 3SQ, UK; 2Dept. Biology, University of York, York YO1 5DD, UK; SDept. Plant Biology and USDA/Agricultural Research Service, University of Illinois, Urbana, IL 61801-3838, USA.

C.J.Pollock I, J.F. Farrar 2, A.L. WintersI, J. Gallagher t and A.J. CairnsI. i Institute of Grassland and Environmental Research, Aberystwyth SY23 3EB, Wales, UK; 2 Dept. Biological Sciences, University of North Wales, L1_57 2UW, Bangor, Wales, UK.

Low growth temperatures restrict chloroplast development and prevent attainment of photosynthetic competence in maize leaves. The effects of low temperatures on chloroplast development are complex. Although the number and structure of chloroplasts are not modified, the levels of all thylakoid proteins and photosynthetic pigments are reduced and some chloroplast-encoded gene products are markedly under represented relative to nuclear-encoded proteins in the membranes. These factors contribute to the depressed photosynthetic capacity of the leaves. Immunocytology has demonstrated that the effects on thylakoid proteins are heterogeneous with adjacent mesophyll cells often responding quite differently. The low lightuse efficiency for CO2 assimilation is primarily attributable to zeaxanthinrelated quenching of absorbed excitation energy at PSII. Despite the photoprotection afforded by this quenching, a significant and persistent depression of photosynthetic efficiency results from chill-induced inhibition of the rate of repair of damaged PSII reaction cantres.

In chilling-tolerant temperate Gramineae, cold reduces demand for fixed carbon and leads to a rapid increase in leaf sucrose content. Subsequently, the synthesis of fructans (polymers of fructose based upon sucrose) is initiated, and the content of sucrose stabilises. This product repartitioning is reversible, and is also observed under a range of other environmental treatments which differentially alter sink demand and photceynthate supply. The hypothesis we propose is that sucrose content (assimilate abundance) regulates, at the level of gene expression, the capacity of grass leaves to repartition photosynthate. Cold is only one of the many environmental variables which alter sucrose contents. In C~ Gramineae, feedback of increased assimilates through to reduced photosynthesis is less common, although in other species, key photosynthetic genes are down-regulated by elevated sucrose. The reasons for this difference will be discussed.

S-24-02

THE PHOTOCHEMICAL ACTIVITY OF LEAVES UNDER TEMPERATURE STRESS M. Havaux DPVE, CEA, CE Cadarache, F-I 3108 Saint-Paul-lez-Durance, France The photosynthetic electron transport chain is vulnerable to high temperatures: in potato leaves, photosystem (PS) II and linear electron transport start to be inhibited at around 38"C. This threshold temperature is influenced by various environmental factors usually associated with heat in the field, such as water stress and light. This talk will deal with the shortterm adaptive responses of the photosystems to heat stress in the light. The PSI and PSI! activities have been studied in vivo using non-invasive methods including chlorophyll fluorometry, kinetic spectrophotometry and photoacoustics. Moderately elevated temperatures trigger the rapid conversion of PSII from its "normal" (heat- and light-sensitive) state to a resistant state. This phenomenon will be described and a possible molecular mechanism for the rapid adjustment of the PSII thermostability will be presented. In heat-injured leaves, PSI can compensate the interruption of the PSIl-driven electron transport by catalyzing an alternative electron flow, the characteristics and physiological significance of which will be described.

30

Symposium

S-25-03 PLASMODESMATA & MACROMOLECULAR TRAFFICKING: ROLE IN PARTITIONING & A I . J J ~ A T I O N OF PHOTOSYNTHATE

Symposium 25

~ , S. Balachandran t, S. Wolf2 & A.A. Olesinski2 IPiant Biology, University of California, Davis, CA 95616, USA; ~)ept. of Vegetable Crops, Hebrew University of Jerusalem, Rehovot, ISRAEL.

Intra- and inter-cellular exchanges

Plasmodesmata form an important pathway for cell-to-cell transport and communication in plants. The formation and complex structure of primary and secondary plasmodesmata will be discussed in terms of the creation of specialized cytoplasmic microchannels that permit the coordination of physiological and developmental processes at the tissue, organ and whole-plant level. Our studies have established that this coordination likely involves cell-tocell trafficking of proteins and/or nucleic acids. This novel aspect of piasmodesmal biology will be discussed in terms of our molecular and cellular studies on viral and endogenous plasmodesmal movement (transport) proteins. This information will then be discussed in terms of the hypothesis that plasmodesmal macromolecular trafficking plays an important role in determining the manner in which recently fixed carbon is allocated to various sink tissues within the plant (Balachandran et al. (1995) Plant Cell & Environ In Press; Olesinski et al. (1995) Planta In Press).

S-25-01

- S-25-04

Photosynthesis and pH-regulation in leaves

S-25-01

U. Heber, Julius-von-Sachs-Institute, Univ. Wiirzburg, Mittlerer Dallenbergweg 64, D-97082 Wiirzburg, Germany The activity o f several enzymes o f the Calvin cycle is known to be pHdependent. Also, photosynthetic electron transport is pH-controlled. However, although CO 2 is a potential acid, photosynthesis proceeds at high rates in leaves o f several plant species at CO2 concentrations as high as 20 % in air. This cannot be explained on the basis of chloroplast buffering. It is also not due to diffusion limitation by stomata] closure. Rather, cytoplasmic acidification by CO2 triggers energy-dependent proton pumping into leaf cells vacuoles. Counter-transport of cations permits bicarbonate accumulation in cytoplasmic compartments reestablishing a chloroplast pH favorable for photosynthesis after the initial acidification caused by a transition from air levels of CO2 to very high CO 2 concentrations has been replaced by an alkalinization response.

S-25-04 INTRA- AND INTER-CELLULAR EXCHANGES VIA ENI~PLASMIC RETICULUM Y.V.Gan~ei Komarov Botanical Institute, Prof. Popov S1xeet2, 197376 St-Petersbmg,Russia Key role of photosynthesis in the establishment of endoplasmic membrane system (ER) and the evolu~c~,y transfer from procaryo~c to eucayotic organizatiun is a theoretical proposal. Acconting to this proposal the ER is a main Uunsportsystem for symplasmic dism'bution of phomassinn~eg Six independent evidences are demonsWated:1) The changes of sugax content in the leaf alx~plastin response to the changes in photosynthate level in the leaf under blockage of photosynthesis by darkening or pho~ynthate export by petiole chilling in sympla,mdc and apoplasmic species respectively. 2) Ixr,alizatm of the osmotic reaotions in mesophyll and phloem cells under the same treamamts in the same species. 3) ~ i T a t i o n of ER in leaf ~ n s in ~ to ~ o ~ or ~ c h a ~ of photosynthate level in the leaf. 4) Locallmfon ofplastids and mitochondria inside ELL5)Functional changes of ER from rod to tube in pla.mmdesma~ 6) Visualization of intra- and inter-celkdar continuity of the ER by mining.

S-25-02 TRANSPORT

P R O C E S S E S IN P L A N T CELLS

Ulf-Ingo Fl/igge, Botanisches Institut der Universitat zu K61n, Gyrhofstr. 15, D-50931 KOln, Germany. We are interested in the characterization and cloning of metabolite translocators that are involved in the transport of photoassimilates in source- and sink-tissues. Chloroplast from C4-plants cells contain a phosphate translocator (PT) that, in contrast to the translocator from C 3plants, also transports phosphoenolpyruvate, the substrate for phosphoenolpyruvate carboxylase. Repression of the cI~ severely alters diurnal carbon patitioning between leaves and sink tissues. Nongreen plastids of heterotrophic tissues (e.g. maize kernels, cauliflower buds) are carbohydrate-importing organelles that rely on the import of hexosephosphates (and ATP) as precursors for starch biosynthesis. Transport measurements with intact organelles or reconstituted tissues from different plants suggest that this transport is mediated by a phosphate translocator that transport also hexosephosphates. We have cloned a PT from cauliflower bud plastids that is different from its chloroplast counterpart.

31

Symposium S-26-03 Control of stomatal conductance by hydraulic and chemical messages from roots. F. Tardieu INRA, Laboratoire d'Ecophysiologie des Plantes sous Stress Environnementaux, 2 place Viala, F34060 Montpellier Cedex 1 France

S y m p o s i u m 26

Considerable experimental evidence has suggested an important role for chemical signals originating from roots in the stomatal behaviour of droughted plants. We have attempted to develop a predictive approach of stomatal conductance (gs) in drying soil under fluctuating evaporative demand. In the majority of cases, the concentration of ABA in the xylem (xylem [ABA]) behaved as a baseline signal, depending on soil water status only and largely independent of evaporative demand. Stomatal response to this signal differed among two groups of plants. In a first case (isohydric behaviour, e.g. maize), gs depends on both xylem ABA and leaf water status. As a consequence, gs decreases with evaporative demand, and plants maintain daytime leaf water status almost constant, regardless of soil water status. In a second case (anisohydric behavieur, e.g. sunflower), gs mainly depends on xylem [ABA], and is almost unaffected by leaf water status and changes in evaporative demand. In this 'anisohydric' behaviour, leaf water status cannot be maintained, and therefore appears to be in good correlation with stomatal conductance in spite of the absence of controlling effect.

Diffusion of CO 2 to the chloroplast S-26-01

- S-26-04

S-26-04

S-26-01 A GUARD CELL ANION CHANNEL WITH FUNCTIONAL PROPERTIES OF A C O 2 SENSOR Rainer Hedrich1, Irene Marten1, Gabi Lohse1, Petra Dietrichl,Heike Winter2, Gertrud Lohaus2 and Hans-Walter Heldt2 linstitut ~ Biophysik, Universit~itHarmover,Herrenh~userstr.2, 30419 Harmover,Germany 2Institut for Biochemie der Pflanze, Universit~t G6ttingen, Untere KarspOle2, 37073 G6ttingen, Germany Malate changes in the intracellular concentrationprovide part of the osmotic motor to guard cells. Since alterations in the malate concentrationreport on the photosynthetic c~0aeityon one side and stomatsl action on the other, we proved whether the extracellolarmalate level represents an indicator for changes in the ambient CO2 concentrationand a key regulator of ion transport in guard cells. Here we demonstrate that alterations in the ambient CO2 level modify the extracelinLarmalate concentrationof Viciafaba leaves. Elevated external malate caused stomatal closure in a concentrationdependent manner (Kmm~ = 0.3 re_M).Slight variations in the external malate concentration strongly regulate the voltage-.depea~lent properties of GCAC1, an anion-release channel in the plasma membrane of guard cells. Superfnsion of guard cell protoplasts with malate levels in the physiological range (Kmmal = 0.4 raM) caused the voltage-gate to shift towards the resting potential of the cell activating GCAC1. In the absence of extracellular chloride the plasma membrane lacked anion conductance until the addition of malate induced channel opening. We thus conclude that feedback-regularionof volume and turgor with respect to the ambient CO2 concentration via malate-sensitive anion channels may provide a CO2 sensor to guard cells.

S-26-02 CARBONIC ANHYDRABE E X P R E S S I O N I N ~

PLANTS

J.R. Coleman l, N. MaJeau 2, and J.P. Fett 3 IDept. of Botany, University of Toronto, Toronto,, Canada M5S 3B2; 2Friedrich Miescher Inst., Basel, Switzerland; ~ept. Biological Sciences, Dartmouth College, Hanover NH 03755 USA carbonic anhydrase (CA) is an abundant, soluble enzyme of the chloroplast stroma and yet its role in ~ photosynthesis is still uncertain. By speeding the acquisition of CO2/HCO 3 equilibrium, CA is thought to facilitate diffusion of C i species across the chloroplast envelope and through the stroma to the site of carboxylation. Catalysis of dehydration in close proximity to Rubisco may improve the rate of CO 2 supply. We have examined the regulation of expression of CA in pea and Arabidopsis and have used antisense and over-expression in transgenic tobacco in an attempt to determine a role for this enzyme in photosynthesis. Severe antisense suppression of CA activity has little impact on photosynthetic CO 2 fixation under ambient conditions but the plant may respond by increasing conductance to enhance intracellular CO 2 levels. Atmospheric levels of CO 2 also appear to regulate CA expression by modification of transcript abundance. High levels of CO 2 appear to repress CA expression whereas low levels of CO 2 result in enhanced expression. In pea, co2-regulated CA expression appears to be co-ordinated with modification of Rubisco exprsssion. The association of CA with other Calvin cycle enzymes was examined in an attempt to establish a functional relationship between CA and Rubisco.

32

TWO

RESISTANCBS

WATER

I.

AND

TO

MZSOPHYLL

T e r a s h i m a I, M.

C02 CBLL

DIFFUSION:

LBAF-SURFACE

WALLS

I s h i b a s h i 2 & K.

Hikosaka 2

I I n s t B i o l Sci, U n i v of T s u k u b a , T s u k u b a , J a p a n ; 2 D e p t of P l a n t Sci; G r a d s c h o o l of T o k y o , H o n g o , Tokyo, 113 J a p a n

I b a r a k i , 305 Sci, U n i v of

Leaf-surface water which causes partial stomatal c l o s u r e a n d o c c l u s i o n of s t o m a t a l p o r e s is a p o t e n t resistance to CO2 d i f f u s i o n . A f t e r 2 4 - h r a i n t r e a t m e n t (12 h l i g h t / 12 h dark), p h o t o s y n t h e t i c rates measured at s a t u r a t i n g l i g h t in the a b s e n c e of s u r f a c e w a t e r a r e 50 to 60% of the o r i g i n a l r a t e s ( I s h i b a s h i & Terashima, Plant, Cell & Environ, 18, A p r i l issue, 1995). Surprisingly, the d e c r e a s e in p h o t o s y n t h e t i c c a p a c i t y is f u l l y e x p l a i n e d b y the d e c r e a s e in R u b i s c o content (See t h e p o s t e r b y I s h i b a s h i et al.). In the s e c o n d p a r t , e f f e c t s of m e s o p h y l l a n a t o m y on C O 2 d i f f u s i o n in t h e i n t e r c e l l u l a r s p a c e s a n d a c r o s s mesophyll cell walls are considered. If the p o r o s i t y of c e l l w a l l s is less t h a n 0.i, l o w p h o t o s y n t h e t i c r a t e s in e v e r - g r e e n l e a v e s w i t h t h i c k c e l l w a l l s c a n be mostly explained by large wall resistance.

Syxnposium S-27-04 PHOTOSYNTHETIC CHARACTERIZATION OF CHLOROPLASTIC PBPASE ANT|SENSE PLANTS Joachim Fisahn, Jens Kolhuanu, Wolfgang Bilger, Lotlmr Willmitzer Institut flit Genbiologische Forschung, 14195 Berlin, lhnestr. 63

Symposium 27

Transgenie potato and tobacco plants were regenerated that exhibited a progressive inhibition in the activity of the chluroplastic fructose-1,6-bisphosphatase. Chlorophyll fluorescence measurements were performed on these plants under a wide range of light and temperature regimes. The nonphotechemieal quenching of the FBPase antisense plants usually exceeded that of the wild type plants. Correlated with the increase in the nonphotochemieal quenching of chlorophyll fluorescence was an increase in the pool size of the xanthophyll cycle pigments of the potato plant lines. Gas exchange measurements confirmed that the CO 2 uptake rates were reduced within the FBPase antisense plants. Furthermore, the water use efficiency declined within the antissnse plants. CO 2 compensation points remained uneffected in comparison to the wild type. Results of respiration and light saturation experimentswill be presented. Ultrastructural analysis revealed that the number of smaller grana stack was significantly increased within the transgenic plants. The results obtained on the FBPase antisense plants will compared to some results obtained on sucrose transporter antisenso plants.

Carbon partitioning and productivity S-27-02 - S-27-04

S-27-02 MEMBRANE

CONTROL

OF

ASSIMILATE

TRANSPORT

S. Delrot. A. Bourbouloux, M. Noubahni & S. Sakr Lab. Plant Physiology & Biochemistry, URA CNRS 574, 25 rue du F a u b o u r g St-Cyprien, F-86000 Poitiers, France The m a s s - f l o w t r a n s l o c a t i o n of assimilates in the p h l o e m largely d e p e n d s on the activity of transporters w h i c h m e d i a t e sugar/H ~ and amino acid/H* transport across the p l a s m a m e m b r a n e of various cells located in the source and in the sink organs. D u r i n g their life, crop plants may u n d e r g o a v a r i e t y of m e c h a n i c a l w o u n d i n g s (grazing, pruning, w o u n d i n g by animals, insects or fungi). We have studied the local and the remote effects of various m e c h a n i c a l treatments on the e x p r e s s i o n and a c t i v i t y of the p l a s m a membrane B÷-ATPase, and of the sugars and amino a c i d s / H + transporters in sugar beet and in b r o a d bean leaves. The treatments u s e d include cutting, peeling, crushing; the a c t i v i t y of the A T P a s e and transporters was m e a s u r e d w i t h p u r i f i e d p l a s m a m e m b r a n e vesicles; the amount of A T P a s e was m e a s u r e d by B L I S A w i t h ATPase antibodies; the amounts of A T P a s e and t r a n s p o r t e r s transcripts w e r e m o n i t o r e d by N o r t h e r n blot. The data s h o w that m e c h a n i c a l w o u n d i n g modifies, w i t h i n a few hours, the transcription, the t r a n s l a t i o n and the a c t i v i t y of the H÷-ATPase and of the transporters. The o b s e r v e d changes, w h i c h d e p e n d on the type of m e c h a n i c a l treatment used, indicate the e x i s t e n c e of transcriptional and p o s t - t r a n s l a t i o n a l controls of these enzymes.

S-27-03 SUCROSE SYNTHESIS, TEMPERATURE, AND PLANT YIELD

Thomas D. Sharkev, Marianne M. Laporte, and Ban 3, J. ]~4Joallef Department of Botany, University of Wisconsin, Madison, WI 53706 USA The synthesis of sucrose and other end products of photosynthesis is more temperature sensitive than is photosynthesis. Therefore, control of photosynthesis is oRen dominated by sucrose synthesis between 15 and 20°C. This phenomenon can be observed as a loss of O2 (and CO2) response in C3 plants. We have used oxygen sensitivity as an indicator for sucrose and end product synthesis capacity limiting photosynthesis. We have measured the oxygen response of photosynthesis in plants where the capacity for sucrose synthesis has been altered by mutation or genetic engineering of enzymes in the sucrose synthesis pathway. Enhanced sucrose synthesis enhanced the responsiveness to 02 and CO2 while decreased sucrose synthesis reduced the responsiveness to these gases. Leaf level partitioning was measured for many of these plants. In cytoselic FBPase deficient mutants sucrnse synthesis was reduced while transgenio plants with enhanced SPS partitioned more photosynthate to sucrose. The acclimation to growth in elevated CO2 was dependent on sucrose synthesis capacity. Yield was niten but not always enhanced by increasing the capacity for sucrose synthesis. Yield enhancement was usually caused by a change in whole plant partitioning (harvest index). We speculate that leaf level partitioning (starch vs. sucrose) may affect plant level partitioning (frnitvs. leaves). We sospect and are nowtesting whethcr some ofthevuriobilityinyield enhancement was related to temperature during growth of the crops.

33

Symposium

S-28-03 INFLUENCE OF B L U E LIGHT, UV-B RADIATION AND TIDAL PHASING ON SEAWEED PHOTOSYNTHESIS IN SUBLITTORAL COASTAL ECOSYSTEMS

Symposium

Matthew J. Drinq I'2, Ralner Schmid ~ & Klaus LHning 2 ISchool of Biology and Biochemistry, Queen's University, Belfast BT7 INN, Northern Ireland, U.K.; 2Biologische Anstalt Helgoland, D-27498 Helgoland, Germany

28

Photosynthesis and ecosystems productivity S-28-01

Blue light stimulates the light-saturated photosynthesis of brown algae (but not of red or green algae or seagrasses) but the sensitivity of this response to blue light is so high that the rapid attenuation of blue light in coastal waters is rarely sufficient to inhibit productivity in the sublittoral zone. One hour of UV-B radiation results in prolonged reductions of variable fluorescence in delicate sublittoral red algae (e.g. Delesseria, Plocamium), but other sublittoral species with thicker thalli (e.g. Laminaria, Chondrus), which contribute more to ecosystem productivity, show little sign of damage until after 24 h exposure. Tidal phasing probably has a greater influence on productivity than either of these factors because the penetration of light through seawater is poorer during spring tides. If the high waters of spring tides occur around midday, therefore, much of the sublittoral zone is in virtual darkness for 3-4 days per fortnight.

- S-28-04

S-28-01 MARINE CYANOBACTERIAL VIRUSES: CHARACTERISATION, MOLECULAR RECOGNITION. TO WHAT EXTENT DO THEY CONTROL PICOPLANKTON PRODUCTIVITY? W.H. Wilson, N.J. Fuller, N.H. Mann and N.G. Cart, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL. Examination of lyric cyanophages active against oceanic phyeoerythrin-containing Synechoeoccus sp are predominately members of the Cyanomyoviridae genus. The relationship between nutrient status of host organism and effectiveness of viral infection has been investigated. Southern and sequence analysis has revealed a sequence common to all strains examined and which has homology to the gene coding for a capsid protein of bacteriophage T4. Oligonucleotide probes are being developed which will allow PCR analysis of natural populations and hence an estimate of the role of viruses in cyanobacterial productivity.

S-28-02 PUMP-DURING-PROBE TECHNIQUE FOR MEASUREMENT OF PHOTOSYNTHETIC CHARACTERISTICS OF ALGAL CELLS IN FLOW CYTOMETRY AND MICROFLUOROMETRY

R.J. Olson, A.M. Chekalvuk, H. M. Sosik, and M.YU. Gorbunov (*) Woods Hole Oceanographic Institution, (*) Moscow State University We developed a pump-during-probe technique based on time-resolved measurements of the increase in chlorophyll (Chl) fluorescence induced in a cell by a rectangular light pulse of 10 to 100 Ixs duration. Information about the quantum yield of PS II photochemistry and the functional absorption cross-section of PS II is retrieved; the reoxidation time of primary electron acceptor QA can also be estimated. Implementation of this approach, using both analog and photon counting techniques, is described for flow cytometry and microfluorometry. The results of measurements on several species of phytoplankton (including the smallest ones, prochlorophytes, at 0.7 lain) are presented. Estimates of quantum yield obtained by averaging data from many individual cells were well correlated with bulk measurements of DCMU-enhanced Chl fluorescence. The potential of the pump-during-probe technique for both laboratory and field studies of phytoplankton photosynthesis is discussed.

34

S-28-04 VARIATIONS IN PHOTOCHEMICAL ENERGY CONVERSION EFFICIENCY IN OCEANIC PHYTOPLANKTON: SCALING FROM REACTION CENTERS TO THE GLOBAL OCEAN P. Falkowski, M. Behrenfeld & Z. Kolber Oceanographic and Atmospheric Sciences Division, Brookhaven National Laboratory, I South Technology Street, Upton, NY 119735000, USA The development of high sensitivity, fast-repetition-rate fluorometers (Kolber et al., Nature 371:145, 1994) has permitted rapid, real-time measurements of the quantum efficiency of photochemistry and the effective absorption cross section of PSII in natural phytoplankton. In most regions of the oligotrophic open ocean, quantum efficieneies are extremely low, ranging from ca. 0.2 to 0.35 at the surface and increasing nonlinearly with depth to the nutricline. The change in efficiency is mirrored by a change in the effective absorption cross section. Low temperature fluorescence excitation/emission spectra, and western blots of DI and CP43, reveal that decreases in photochemical yields are due primarily to a decline in trap density. Photochemical yields of reaction center density and absorption cross sections are governed by physical features of ocean circulation, which influences the distribution of inorganic nutrients. Circulation patterns on scales of 100s to 1000s of kilometers reveal the coupling between large-scale physical processes and the molecular dynamics of PSII in natural phytoplankton.

Symposium

S-29-03 ACCLIMATION OF PHOTOSYNTHESIS AND THE RESPONSE C A R B O N B A L A N C E T O R I S I N G A T M O S P H E R I C C O 2.

Symposium

Bert G. Drake and James Jacob Smithsonian Environmental Research Edgewater, Maryland, 21037. U S A

29

Center,

OF ECOSYSTEM

P.O.

Box

28,

Acclimation of p h o t o s y n t h e s i s in leaves and canopies to rising a t m o s p h e r i c CO 2 c o n c e n t r a t i o n was studied in a b r a c k i s h wetland, a deciduous u n d e r s t o r y and a scrub-oak ecosystem. A l t h o u g h e l e v a t e d C02 increased carbohydrate, reduced R u b i s c o and soluble p r o t e i n concentrations, and reduced p h o t o s y n t h e t i c c a p a c i t y in leaves of the dominant species in each ecosystem, the rate of p h o t o s y n t h e s i s of intact leaves was h i g h e r in e l e v a t e d C02 than in normal ambient c o n c e n t r a t i o n b y 20-100%. In the w e t l a n d community, d o m i n a t e d b y the C 3 sedge, Scirpus olneyi, e c o s y s t e m gas exchange m e a s u r m e n t s showed that e l e v a t e d CO 2 s t i m u l a t e d net e c o s y s t e m carbon a c c u m u l a t i o n (NEC; 25-120%) throughout eight years of treatment in open top chambers in the field. The relative s t i m u l a t i o n of NEC was i n v e r s e l y c o r r e l a t e d w i t h total e c o s y s t e m carbon a c c u m u l a t i o n and w i t h total p h o t o s y n t h e t i c p h o t o n flux suggesting that the major effect of rising atmospheric C02 on e c o s y s t e m carbon b a l a n c e o c c u r e d t h r o u g h the relief of environmental stress.

Photosynthesis: global aspects S-29-01

- S-29-06

ELEVATEDATMOSPHERICCO2:A RETURN TO THE PAST?

UNSATURATED RUBISCO - SATURATED GROWTH? ECOSYSTEMS IN A C02-ENRICHED WORLD

Geor,,e Bowes Dept of Botany, University of Florida, Gainesville, FL 32611, USA

Ch. KOrner, Institute of Botany, University of Basel, Switzerland

When plants made the transition to land atmospheric [CO2] was up to 16-fold higher than today; since then it has declined to low values. Modern plants exhibit photosynthetic adaptations to cope with the low [CO2]/[O2] ratio: high specificity and low KIn(CO2) of rubisco; pathways to recapture photorespiratory C and N; CO 2 concentrating mechanisms in some species; and improved stomatal regulation. Since the Last Glacial Maximum, atmospheric [CO2] has doubled, but it still does not saturate 95% of species. A further doubling could reduce photorespiration, dark respiration and stomatal conductance, while enhancing WUE, the C/N ratio, and growth modulator effects; but, these "efficiency effects" are not always realized. Acclimation can down-regulate the A / C i curve, to optimize carbon acquisition with its utilization. Plants with high sink capacity, such as crop and competitive-strategy species, have the greatest response to CO2-enrichment, and are not down-regulated. Among submersed species, the growth of CO2-asers is enhanced, but HCO3"-users show minimal response. Increases in [CO2] will likely alter the distribution and competitive interactions among species. It is uncertain how plants will readapt, as opposed to acclimate, to the rise in CO 2. Supported in part by U S D A / N R I C G Photosynthesis and Respiration Program.

S-29-02 FEEDBACKS

ON PHOTOSYNTHESISAND

GROWTH

IN ELEVATED

S-29-04

S-29-01

CO 2

F.A. ~azzaz Dept. of Organismic & E v o l u t i o n a r y Biology, H a r v a r d University, 16 D i v i n i t y Avenue, Cambridge M a s s a c h u s e t t s 02138 U.S.A. CO 2 c o n c e n t r a t i o n in the atmosphere is increasing, causing global temperature rise. CO 2 also has a direct rise effect on p l a n t s : p l a n t s grown in an e l e v a t e d C02 environment have h i g h e r p h o t o s y n t h e t i c rates than plants grown in ambient C02. Many studies however indicate enhancement can decline with time. D o w n - r e g u l a t i o n m a y be caused by some or all of several factorst e.g. reduction in the expression of the large subunit of Rubisco. In tobacco down-regulation after long exposure to e l e v a t e d CO 2 occurred in w i l d type plants that have high initial Rubisco concentration and activity. In contrast, in transgenic lines that were deficient in Rubisco, there was a slight up-regulation. Sink strength seems to play a m a j o r role in this down-regulation. Plants grown in a high CO 2 e n v i r o n m e n t usually produce leaf litter e n r i c h e d with carbon and lignin relative to plants grown in ambient CO2. AS a result the d e c o m p o s i t i o n rate of the leaves p r o d u c e d under high CO 2 may be reduced, leading to reduced availability of nutrients to plants. D o w n - r e g u l a t i o n may cause less response among ecosystems to elevated CO 2 than originally assumed.

In this paper I will discuss homoeostatic trends of photosynthetic capacity at community level and on a global scale. Feedbacks at the ecosystem level will be illustrated to further contribute to the surprising similarities of primary production of the major global biota, if the active growth period rather than a full year is taken as a reference. Over the past 20 000 years atmospheric C02 levels have doubled and are likely to double again within the next 100 years. Leaf photosynthesis in most C3 species still appears very responsive to COs enrichment, both under horticultural and under natural growth conditions. Yet, in most natural terrestrial systems biomass production has not been found to be very responsive to C02 fertilization, in contrast to findings from agricultural research. How can we explain this discrepancy? What is the fate of the additional carbon assimilated under elevated C02? A brief review of recent experimental findings in complex ecosystems under natural or near natural growth conditions will illustrate our current understanding of this biogeochemically quite important matter.

S-29-05 L O O K I N G F O R T H E MISSING CARBON SINK IN T R O P I C A L AND BOREAL FORESTS P.G. Jarvis, J.B. Moncrieff, J. Grace and J. Massheder Institute of Ecology and Resource Management, University of Edinburgh, Darwin Building, Mayfield Road, Edinburgh EH9 3JU. Best estimates of the global carbon balance indicate that of the c a 6 Gt/a of carbon of anthropogenic origin that is put into the atmosphere each year, about 2 Gt/a cannot be accounted for. Inverse modelling of the global network flask sample data for [CO2] and 13C in CO 2 suggest that major sinks for this C may lie in the tropical forests of mid-latitudes and the boreal forests of northern latitudes. One way to address the question directly as to whether these forests are sources or sinks for C is to go there and measure the net CO 2 exchange flux continuously for extended periods. Teams from Edinburgh University have been making such measurements using newly developed eddy covariance techniques with the well-developed EddySol software in Cameroon, Brazil, Canada and Northern Europe. The methods used will be briefly summarised and data will be shown from both tropical and boreal forest stands that demonstrate that both are functioning today as C sinks. Problems of scaling up from stand scale to landscape and regional scales will be addressed.

35

Symposium S-29-06 GLOBAL CHANGE OF PHOTOSYNTHESIS, INTERACTIONWITH NITROGEN. A.H.C.M. Schapendonk 1, F. Miglietta 2 and J. Goudriaan 3 1 AB-DLO, P.O. Box 14, 6700AA Wageningen, The Netherlands. 2 CNR-IATA, Piazza delle Cascine 18, 50144-Florence, Italy.

3 AgriculturalUniversity, DepartmentofTPE,.P.O. Box430,6700AK Wageningen, The Netherlands. The instantaneous response of leaf photosynthesis to a change in temperature or carbon dioxide concentration strongly depends on plant species, plant nutrient status and plant water status, Whether the initial leaf response can be maintained is partly determined by the strength of a negative feedback through sink limitation and morphology. On a canopy scale, the leaf response may or may not be translated into an enhanced formation of new leaf area. Extending the time scale even further, differential effects on species competition and effects on foot-soil interaction will influence vegetation composition in various ways, that have little to do with photosynthesis. In this study, the present vegetation cover is assumed as given. Published scenario's for climatic change are used to estimate their effects on gross photosynthesis and on net primary production for arable land and grasslands within pre-set limits of nitrogen availability.

36

Poster

P-1-2-003 LINEAR POLARIZATION SPECTROSCOPY OF COMPLEX MOLECULAR SYSTEMS WITH INTERNAL ENERGY TRANSFER Poster

session

A.A. Demidov & D.L. Andrews School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, UK

1-2

Antenna systems: structure and function

P-I-2-001

- P-1-2-085

In this report we present results of our investigations on the polarization spectroscopy of molecular complexes excited by linearly polarized light. The complexes considered consist of a limited number of molecules (less than a hundred). Within each complex the molecules (or chromophores) are rigidly positioned and oriented, and there is internal energy transfer between them. These molecules have broad and strongly overlapped spectra. We have derived theoretical formulae for determination of the degree of fluorescence polarization and absorption anisotropy at particular wavelengths of excitation and with either fluorescence detection or absorption probing. Two specific cases, relating to steady-state and 8-pulse excitation, were considered in detail. The derived formulae depend on the rates of energy transfer, spectroscopic and structural parameters, and can in particular be employed for the analysis of the latter. Three well-known photosynthetic molecular complexes were considered: Cphycocyanin, allophycocyanin and the bacteriochlomphyll Bg00-B850 complex.

P-1-2-004

P-I-2-001 TEMPERATURE DEPENDENCE OF FLUORESCENCE QUANTUM YIELD IN PHOTOSYSTEM II CORE PARTICLES

SYNTHETIC METALLOCHLORIN AGGREGATES AS MODELS FOR SUPRAMOLECULAR ANTENNA COMPLEXES IN CHLOROSOMES OF GREEN PHOTOSYNTHETIC BACTERIA

Jan P. Dekker, Aukje S. Hassoldt, Marloes Groot and Rienk van Grondelle Dept. of Physics and Astronomy, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands

Amakawa, 1 A. R. Holzwarth 2 & K. Schaffner 2 ~Department,1ofM.Bioscience and Biotechaology, Faculty of Science and

The absolute quantum yield of chlorophyll fluorescence upon excitation with weak continuous 610 nm light was measured as a function of temperature in intact PS II core preparations. The data reveal a moderate increase of the quantum yield upon cooling from 298 K (~ 0.012) to 77 K (~ 0.025) and a more pronounced increase upon further cooling to 4 K (- 0.09). The peak maximum of the main emission band gradually shifts from 681 nm at 298 K to 686 nm at 77 K. At temperatures at and below 77 K a second emission band appears that peaks at 694 nm at 77 K and gradually blue-shifts to 690 nm at 4 K. The shape of the emission spectrum at 4 K is identical to the spectrum observed for the isolated CP47 complex, but in this complex the redshifl of the peak maximum and the strong decrease of the quantum yield upon raising the temperature to 77 K are not observed. The data are interpreted to mean that at 4 K a large part of the excitation energy is lost in the core antenna. Upon raising the temperature the possibility of energy transfer to P680 increases, especially for those complexes with relatively blue-absorbing low-energy antenna chlorophylls. The data suggest that the 77 K F695 emission band of PS II arises from a minor fraction of the complexes, i.e., only from those containing the red-most part of the inhomogeneous distribution of long-wavelength core antenna chlorophylls.

Engineering, Ritsumeikan University, Kusatsu, Shiga 525-77, Japan; 2MaxPlanck-lnstitut fiir Strahlenchemie, D-45413 Miilheim an der Ruhr, Germany Zinc and magnesium chlorins with a hydroxymethyl group at the 3 l-position were synthesized. The molecules self-aggregated in l%(v/v)tetrahydrofuran and hexane (solution) and in thin films (solid state) to form oligomers absorbing at longer wavelengths than the monomeric form. Visible and circular dichroism spectra of several zinc chlorins showed that the requirements for the aggregation are central zinc, 31-hydroxyl, and 131-keto carbonyl groups in the molecule. Some assignments of the vibration bands measured in the solid film by means of infrared spectroscopy indicated that the oligomers have two characteristic structural features: i) 5-coordinated metallochlorin macrocycle with an axial ligand, and ii) special hydrogen-bonds, C=O---H(X)O...M (= Zn and Mg). A comparison of the spectra of the in-vivo and in-vitro aggregates suggests that the supramolecular structure of the bacteriochlorophylls-c aggregates in extramembranous antenna complexes (chlorosomes) of green photosynthetic bacteria is similar to those of the artificial oligomers, and that self-aggregation is the predominant organization principle in the natural antenna system.

P-1-2-005

P-1-2-002 (SUB)PICOSECOND POLARIZED TRANSIENT ABSORPTION MEASUREMENTS ON MONOMERIC AND TRIMERIC LIGllTIIARVESTING COMPLEX II FROM GREEN PLANTS Foske J. Kleima, H. Matthieu Visser, Erwin J. G. Peterman, Florentine Calkocn, Rienk van Grondelle and Herbert van Amerongen Dept. of Physics and Astronomy, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands We have performed (sub)picosecond transient absorption measurements on LHC Ii. A white-light continuum was used to probe the samples. After wavelength selection, part of the continuum was amplified and used to selectively excite either monomeric or trimeric LHC lI. Polarized data were used to discriminate between inter- and intramonomer energy transfer. The temperature dependence was studied in the range 4-293 K.

AN INVISTIGATXON OF THE LHC FAMILY OF GE~ES H O M O S P O R O U S F E R N , Ceratopteris Richardii

AND

PROTEINS

IN THE

O. Oberschmidt I, S. Kim 2, J.M. M c G r a t h 2, M.M. Jansco 2, R. Bassi 3,4, B. Piechulla I & ~. Picherskv 2, iInstitute of Plant Biochemistry, University of Gottingen, 37073 Gottingen Germany, 2Biology Department, U n i v e r s i t y of Michigan, Ann Arbor MI 48109 USA, 3Biology Department, U n i v e r s i t y of Padova, Padova Italy, 4present address: University of Verona, Verona Italy We investigated the composition of the light-harvesting complexes (LHCs) I and II in a homosporous fern species, Ceratopteris richardii. We found several distinct LHCII and LHCI chlorophyll a/b-binding proteins in this fern, and w e also report the isolation and characterization of three Lhcb cDNA clones. These Lhcb genes were expressed in both gametophytic and sporophytic tissues. Their steady-state mRNA levels increased during the morning until a p p r o x i m a t e l y noon, and decreased thereafter. Under constant darkness these oscillations continued for several days. We thus concluded that each of the three genes is under the control of the circadian clock.

37

Poster

P-1-2-009

P-1-2-006 B800 S U B S T I T U T I O N OF T H E B800-850 LIGHT H A R V E S T I N G COMPLEX OF RHODOPSEUDOMONAS ACIDOPHILA 10050

GELHAYE El., McDERMOTT GI., UCKER B.2. SCHEER H.2 and COGDELL R1. l: Institute of Biomedical and Life Sciences,University of Glasgow,Glasgow, G12 8 QQ, U.K. 2: Botanishes Institut der Universitat Munchen, 8000 Munchen, Deutschland. The light harvesting a p p a r a t u s of purple photosynthetic bacteria is comprised of discrete pigment-protein complexes which contain non covalently b o u n d bacteriochlorophyll (Bchl) a n d carotenoid molecules. W e h a v e d e v e l o p e d a m e t h o d , u s i n g the d e t e r g e n t Triton BG, to exchange the B800 Bchl in the B800-850 antenna complex from Rps. acidophila strain 10050. Exchange h a s b e e n achieved with Bchl's modified in both the esteryfying alcohol a n d the bacteriochlorin ring. T h e a b s o r p t i o n , C D , a n d fluorescence e m i s s i o n a n d excitation properties of the complex with different Bchl's present in the B800 b i n d i n g site will be presented. This work w a s s u p p o r t e d by grants from the BBSRC, E.U. and the British Council.

PROJECTION MAP OF THE LIGHT-HARVESTING COMPLEX I FROM RttODOSPIRILLUM RUBRUM

8.5

A

Simonc Karrasch 1'2, Per A. Bullough2, Robin Ghosh3 IM.E MOiler Institute for Microscopic Structural Biology, Biozentrum, Klingelbergstr. 70, CH-4056 Basel, Switzerland, 2MRC Laboratory of Molecular Biology, Hills Rd., Cambridge CB2 2QH, U. K., and 3Laboratory of Bioenergetics, University of Geneva, C h des Embrouchis, 10, CH-1254 Jussy-Luillier/GE, Switzerland Two-dimensional crystals from the light-harvesting complex I (LHI) of the purple non-sulphur bacterium Rhodospirillum rubrum have been reconstituted from detergent-solubilized protein complexes Frozenhydrated samples have been analyzed by electron microscopy. The crystals diffract beyond 8 ~, and a projection map was calculated to 8.5 A. The projection map shows 16 subunits in a 116 .& diameter ring with a 68 A hole in the centre. These dimensions are sufficient to incorporate a reaction centre in vivo. Within each subunit, density for the Qt and 13 polypeptide chains is clearly resolved and the density for the bacteriochlorophylls can be assigned. The experimentally determined structure contradicts models of the LH I presented so far.

P-1-2-007 R E G U L A T I O N O F T H E E X C I T O N D E N S I T Y IN P H O T O SYNTHETIC ANTENNA SYSTEM: "CRUISE CONTROL" W. I. Gruszecki a , A. Srivastava b , M. Matula a , Z. Krupa c , R. J. Strasser b a) D e p a r t m e n t o f Biophysics and c) Department o f Plant Physiology, M a r i a C u r i e - S k t o d o w s k a University, Lublin, Poland; b) Laboratory o f Bioenergetics, University o f Geneva, Switzerland A hypothesis is presented in which the exciton density in photosynthetic antenna s y s t e m is down-regulated to maintaha a constant low level. A simple model is considered in which the exciton density decreases due to the trapping by reaction centers, light and heat emission. Three kind o f experiments are presented to support the model: O 2 evolution, heat and light emission from leaves illuminated with increasing light intensity. Possible physiological and physical m e c h a n i s m s responsible for the regulation o b s e r v e d are discussed.

P=I-2-008

P-I-2-010 OF TI~ ~ ' k L A ' n O N Qt sn'E ~,~m/it~ats o r Tm~ ~

O~"I~w~ma~'r ~ e/r cotvnvJ~ eq ~

"lO Q) AND ~

~

, L. Cheng2, G. Garab 1, J.F. Anen2 of Plant Biology, Biological Research Center, Szeged, Hungary, and 2Department of Plant Cell Biology, University of Lund, Sweden It has been well established that the cytochrome b6/f complex plays a key role in the activation of the ldnase(s) phosphorylating LHCII and other phosphoproteins of the thylakoid membranes. However, the molecular mechanism and the exact site of the activation remains to be clarified. In this work we carried out a systematic investigation of the effect of Qo site inhibitors (DBMIB, stigmateUin, bromanil) and Qi site inhibitors (HQNO, MOA-stilbene) of the cyt b6/f complex on the phosphorylation of the seven most intensely phosphorylated proteins, LHCII, D1/D2 and the 56, 43, 20, 18 and 9 kDa polypeptides. Our analysis suggests that the phosphoproteins belong to three main groups: LHCII and the 56 kDa protein appear significantly more sensitive to DBMIB and stigmatellin than D1/D2 and the 43, 20 and 9 kDa phosphoproteins, whereas the pbesphorylation of the 18 kDa polypeptide can hardly be inhibited. Qi site inhibitors have no inhibitory effect on thykakoid protein phosphorylation. We propose that the activation site of the phosphorylation is located between the Qo and Qi site of the cyt b6/f complex.

P-I-2-011

LOW

INTENSITY TRANSIENT ABSORPTION STUDY ON THE LIGHT HARVESTING CHL A/B COMPLEX LHC-II

ENERGY TRANSFER IN THE LH 2 COMPLEX OF RB SPHAEROIDES STUDIED BY SUB PICOSECOND ABSORPTION SPECTROSCOPY

M.Huckel,C.Mullineaux2, P.Horton2 and A,R. Holzwarth 1 ~Max-Planck-Institut for Strablenchemie, MUlheim a.d.Ruhr, Germany, 2Robert-Hill-Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, UK.

R. Monshouwerl, I. Ortiz de Zarate2, F. van Mourik 1, R. Picorel 2 and R. van Grondelle l 1 : Dept. of Physics and Astronomy, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands 2 : Dept. of plant nutrition, Estaci6n Experimental de Aula Dei (CSIC), Apartado 202, 50080- Zaragoza, Spain.

The excited state kinetics in trimeric preparations of the LHC-II antenna complex isolated from photosystem II (spinach) has been measured. Ultrafast energy transfer between close-lying Chl b and Cbl a molecules has been characterized by two-colour femtoseeond pump-probe experiments. Two components with zero-crossing amplitudes dominate in the decay associated absorption difference spectra upon preferential Chl b excitation at 650 nm. Due to the high pigment content of the complex (221 Chl a and >15 Chl b) it is necessary to characterize the threshold for singlet-singlet-annihilation. The intensity dependence of the transient absorption signal is compared with simulations including annihilation processes. Improved temporal and dynamic resolution compared to earlier experiments allow an aproach to annihilation-free kinetics. The results of this study suggest, that the dominant energy transfer rates are apparently increased under low excitation intensity conditions as compared to the rates reported so far.

The energy transfer properties of the LH-2 antenna complex of the purple bacterium Rb. sphaeroides were measured as a function of wavelength and temperature.The measurements show that transfer from the B800 pool to the B850 pool takes place with a higher rate than reported so far. A study of the power dependence indicates that this is probably due to the low power densities that are used in our experiments. The decay times measured in the red flank of the B800 band increase from about 0.6 ps at roomtemperature to 1.2 ps at 77K. The decay of the B800* state is strongly dependent on the wavelength. The decay time is constant above 800 nm and speeds up rapidly below 800 nm. This is in agreement with hole burning measurements at 4K, and was ascribed to the contribution of inter band transfer in the blue part of the B800 band. We find that using powers above 5W/cm2 (a laser beam of about 0.5 mW focused with a 15 cm lens) strongly decreases the transfer rate. This effect is irreversible, and thus suggest permanent changes rather than annihilation to be the cause.

38

Poster P-1-2-015

P-1-2-012

TRIMERIC LHC II IN PLANTS LACKING 16: it IN PHOSPHATIDYLGLYCEROL

EXCITATION LOCALIZATION IN SPECTRALLY INHOMOGENEOUS LIGHT-HARVESTING ANTENNA

E. Selstam I & M. Krol 2

T. Pullerits, S. Hess, M. Chachisvilis & V. Sundstrtm Chemical Dynamics and Spectroscopy, Lurid University, P.O. Box 124, 22100 Lund, Sweden

IDept of Plant Physiology, Ume~. university, S-901 87 Umeh, Sweden; 2Dept of Plant Science, University of Western Ontario, London, Canada N6A 5B7 The fatty acid composition of phosphatidyl glycerol (PG) from seven species of orchids were investigated, six of these lack 16:lt. Separation of chlorophyll protein complexes on "green gel" showed that trimers of LHC II are present after mild solubilisation of all orchid thylakoids. However the stability of the LHC II complexes are correlated to the fatty acid composition of PG. LHC II trimers of Platant6ra (16: It) were more resistant to SDS solubilisation than LHC II trimers of Goodydra (16:0). Low temperature fluorescence emission spectra of the isolated LHC II trimers from wheat (16:1 t, Platant6ra (16: It), Goody6ra (16:0) and Calante (16:0) were almost similar. These results show that presence of PG 16:lt is not necessary for the formation of LHC II trimers. PG-16:lt might stabilise the trimers better than PG-16:0.

We describe excitation dynamics in the antenna of purple bacteria as incoherent Ftrster hopping in the system of spectrally inhomogeneous (and heterogeneous) pigment protein complexes. It is known that in this case excitation can be trapped by the most red pigments of the inhomogeneous distribution (ID). We have found that in special conditions (low temperature, weak electron-phonon coupling, narrow frequency distribution of protein phonons which are bound to optical transition) excitation can be also localized in the blue edge of the ID of pigments. Femtosecond pump-probe experiments give additional support to the theoretical prediction. Our result has direct implication to the possible fundamental role of the different forms of electron-phonon coupling in distinct transfer steps in the antenna. The result has a general nature and is relevant to whatever spectrally inhomogeneous system where excitation transfer takes place.

P-1-2-016

P-1-2-013 SPECTRAL HOLE BURNING AS OF CHLOROSOMAL ANTENNA

A TEST

FOR

EXCITON

MODELS

[=~2X2~£~

LHC AGGREGATION IN INTACT LEAVES OF TOBACCO PLANTS STRESSED BY CO 2 STARVATION. P. ~>iffel and F. Vficha

& Z.Fetisova A.N.Belozersky Institute of Phys.-Chem. Biology of Moscow State University, Moscow 119899, Russia

Inst. Plant Mol. Biol., Acad. Sci. Czech Rep., Brani~ovskd 31, 370 05 ~eskd Bud6jovice, Czech Republic

The theory of absorption and hole-burning spectra in chlorosomal antenna of green bacteria was developed. Spectra were calculated using the standard approach to exciton-phonon problem in molecular crystals. It was demonstrated that none of the models proposed earlier (single/double chain m o d e l ~ and tubular ones of BChl c,d or e self-aggregated oligomers) exhibits the in v i v o exciton level structure revealed by hole burning study. We proposed the alternative models of several exciton-coupled chains where aggregation in a chain results from direct BChI-BChl interactions, whereas the spatial arrangement of the chains is determined by another type of interaction. Only such a models are compatible with key spectral features found in vivo, i.e. the exciton level structure with intensive higher levels and polarization of all the levels parallel to the long axis of the chlorosome.

Plantlets of tobacco (Nicotiana tabacum L.) cultivated in glass bottles were stressed by CO 2 depletion. CO 2 starvation resulted in photodamage of both photosystems, and in formation of aggregates of the light-harvesting complex (LHC). The LHC aggregates were detected by characteristic emission bands at 681 and at 697-699 nm. A shoulder at 691-693 nm was formed in absorption spectra upon LHC aggregation. Fluorescence of the LHC aggregates was reversibly quenched upon irradiation with actinic light. Nevertheless, the pronounced increase of the 697-699 nm emission was observed only after complete disappearance of variable fluorescence and it coincided with rapid decline of chlorophyll content in leaves. No zeaxanthin was found in leaves and capacity of zeaxanthin cycle declined during the stress. Therefore, the LHC aggregation occurring under CO 2 starvation seemed to be related to photodamage of photosynthetic apparatus rather than to nonphotochemical quenching of fluorescence.

P-1-2-014

P-1-2-017

HOLE-BURNING STUDY OF Fe-LIMITED AND Fe-REPLETED CELLS OF D U N A L I E L I J I T E R T I O L E C T A

ROLE Or TI~LAKOID LIPmS ON THE MACROORGANIZATIONAND THE LIGItT~DUCED REV]EaSmLE S T R U ~ OIANGES IN LHCH

iT. Polivk~L tJ.Pgen~ik, Ip.Kroh, 1D.Engst, 20. Pr~il, sP.G. Falkowski ,tj. Hfila

I. Simidiiev 1, V. Barzda t, I. Horvlith 2, G. Garab 1 lInstitute of Plant Biology, and 2Institute of Biochemistry, Biological Research Center, Szeged, Hungary

1Department of Chemical Physics, Faculty of Mathematics and Physics, Charles University, Ke Karlovu 3, 121 16 Prague 2, Czech Republic; 2Institute of Microbiology, Czech Academy of Sciences, 379 81 T~ebofi, Czech Republic, SOceanographic and Atmospheric Sciences Division, Brookhaven National Laboratory, Upton, New York 11973, U . S A The results of hole-burning experiments on Fe-limited and Fe-repleted whole cells of marine algae D. tertiolecta are reported. These experiments revealed that the iron-limited condition during the growing of D. tertiolecta affects spectral characteristics and excited energy transfer (EET) in core antenna proteins. The low temperature (T = 4.2 K) fluorescence spectra exhibit differences between both types in the spectral region 680-695 nm, The holeburning experiments show no influence of iron-limited condition on EET near 687 nm (Tt ~ 10 ps for both types) but significant influence on EET at 682 nm (694 nm): Tt ~ 14 ps (8 ps) for Fe-limited and Tl ~ 7 ps (4 ps) for Fe-replcted cells.

It has been shown earlier that the pigmeet-protein complexes in granal thylakoid membranes are assembled into chiral macroaggregates, the organization of which is facilitated by the light-harvesting chiorophyll a/b protein complex (LHCII). Purified LHCII has also been shown to form chiral macroaggregates. As reveaied by circular dichroism (CD), chloroplast thylakoid membranes are capable to undergo light-induced reversible structural changes. In this work, by investigating the electron microscopy and CD of LHCII macroaggregates, we show that thylakoid lipids and especially lipid acyl chains play a crucial role in the formation of two- and three-dimensional macroaggregates of LHCII. We also show that lamellar type aggregates possess light-induced reversible ACD, the amplitude of which can be enhanced significantly by the addition of thylakoid lipids; different lipids facilitate the formation of different structures and ACD kinetic patterns. It is concluded that thylakoid lipids play a significant role in the structure and function of LHCII macroaggregates.

39

Poster

P-1-2-018 ENERGY TRANSFER, TRAPPING AND DETRAPPING IN THE PHOTOSYNTHETIC PURPLE BACTERIUM RHODOSPIRILLL~t RUBRUM KOu T h , m ~ ' m 1, viny SundatrOm2 and Arvi Freiberg 1 lInstitute of Physics, Estonian Academy of Sciences, EE2400 Tartu, Estonia 2Department of Chemical Physics, Lund University. Chemistry. Center, Box 124, S-22100 Lurid, Sweden The processes of energy migration in the light-harvesting antenna (LHA), trapping of the energy by. the reaction center (RC) and detrapping of the energ'¢ from the RC in the photosynthetic purple bacterium Rhodospirillum rubrum were investigated by means of time-resolved absorption and fluorescence spectroscopy in the temperature interval from room temperature to liquid nitrogen temperature..at low temperature spectral inhomogeneity of LHA pigments favors the excitation energy to b¢ concentrated in a few ps in the redmost side of the antenna absorption band and then trapped by the RC with a time constant of about 40 ps. The temperatUre dependence of the trapping time is weak. At room temperature quite a noticeable amount of excitation energy (ca 25 %) escapes from the RC back to the antenna before the primary charge separation takes place. With the lowering of temperature the efficiency of detrapping diminishes, being only a few per cents at liquid nitrogen temperature.

P-1-2-019

P-1-2-021 TWO TYPES OF PHOTOSYSTEM 1 AND PECULIARITIES OF THEIR INTERACTION WITH MEMBRANE PHOSPHOPROTEINS S.M.Kochubey Institute of Plant Physiology and Genetics, National Academy of Sciences, 31/17 Vasilkovskaya str., Kiev 252022, Ukraine When studying thylakoids and PS1 (Photosystem 1) particles two types of PSI posessing different abilities to interact with phosphoproteins were revealed. Excitation spectra of fluorescence detected at 735 and 720 nm display an alternative increase of a.c.s. (absorption crosssection) for F735 and F720 PSI antennae induced by protein phosphorylation. An increase of only the first or only the second was observed respectively for maize thylakoids from L346 inbred line and pea thylakoids from plants grown under normal conditions of irradiance or temperature. A.c.s. of both F735 and F720 was found to be increased in maize thylakoids from F7 line and pea thylakoids from plants grown under unfavorable conditions. Enhancement of only non-cyclic or only cyclic electron flows was observed for the variants when a.c.s, of only F735 or only F720 becomes increased. Thus it was assumed that in thylakoid membranes there are two PS1 complexes that posess F735 and F720 antennae and are included in non-cyclic and cyclic electron flows.

P-1-2-022

THE POLYPEPTIDE AND CAROTENOID COMPOSITION OF LHCH IN DIGITALIS PURPUREA IN RELATION TO NONPHOTOCHEMICAL QUENCHING.

COMPETITION BETWEEN EXTRA-MEMBRANE FRAGMENT OF LHCII AND ITS PARENTAL PROTEIN IN THYLAKOID MEMBRANES.

J.I. Webster t, A.J. Young2 and P. Horton 1. 1Robert Hill Institute, University of Sheffield, PO Box 594, Firth Court, Western Bank, Sheffield, Sl0 2UH, UK; 2School of Biological and Earth Sciences, John Moores University, Liverpool, UK.

+Sty_s D *Stancek M., and +Allen J.F. +Plant Cell Biology, Lund University, Box 7007, S-220 07 Lund, Sweden. *Department of Molecular Genetics, Comenius University, Mlynsk~i dolina B2, SK-842 15 Bratislava, Slovakia Competition between a fragment of the extra-membrane domain of LHCII and its parental protein was used for evaluation of formation of protein complexes and analysis of phosphorylation-dependent adaptation mechanisms in the thylakoid phosphoprotein pea thylakoid membranes. The phosphorylated N-terminal fragment competes with membrane phosphoproteins in phosphatase reaction. Both phosphorylated and non-phosphorylated LHCII fragments compete with LHCII for its binding sites during cation controlled segregation of PSI and PSII. Non-phosphorylated LHCII fragments prevent efficient quenching of energy captured by free antenna complex. The effects of the peptides on thylakoid kinase activity may be explained by combination of specific and unspecific mechanisms. The specific and unspecific effects of binding of positively charged peptides to membrane surface will be discussed and put in frame of recent physico-chemical evaluations of related model systems.

Digitalis purpurea has been shown to have a large capacity for energydependent non-photochemical quenching (qE). This capacity for quenching is also much larger in high tight grown plants than in plants grown under a lower light. But what determines the capacity for quenching in a plant? Carotenoid analysis of Digitalis grown under low, intermediate and high light conditions show that there is no change in the xanthophyU pool cycle size in relation to the total carotenoid content, whereas polypeptide analysis indicates that there may be a reduction in the amount of the 27 kDa polypeptide in the LHCIIb, as previously reported by Andersson and coworkers. We propose that in Digitalis purpurea the major factor governing the capacity for quenching is structural and that the xanthophyll cycle pool size only plays a small role, if any.

P-1-2-020 THI 8 ~ A/B-PRO~

0~GJ~qIZATION OF T mm ~ LIGHT-Na%RVEST~ CHLOROPHYLL ~ OF PHOTOSTSTIM II (LRCZI) ~ STt~Tm~ BY A NOVEL, V E R T I C 2 L L - B E D I~R4-DI~4ATURING I S ~ C FOCUSinG.

G.Jackowski A d a m M i c k i e w i c z University, Department of A i , N i e p o d l e g l o ~ c i 14, 61-713 Poznafi, Poland

Plant

Physiology,

The s o l u b i l i z a t i o n of LHCII with d o d e c y l - ~ - D - m a l t o s i d e followed b y a v e r t i c a l - b e d n o n - d e n a t u r i n g i s o e l e c t r i c focusing in a p o l y a c r y l a m i d e gel containing the same detergent a l l o w e d the purification of LHCII into its subcomplexes with a high p e r f o r m a n c e and without any d i s r u p t i o n of p i g m e n t - p r o t e i n in teractlon. LHCII i s o l a t e d from carnation and b a r l e y leaves y i e l d e d three and five subcomplexes, respectively, c h a r a c t e r i z e d by t r i m e r i c arrangement. The trimers focused in the pH range of 4.35-4.52 (carnation) and 4.02 - 4.24(barley). The s p e c t r o s c o p i c data d e m o n s t r a t e d a close s i m i l a r i t y among LHCII subcomplexes c o n c e r n i n g their chlorophyll content and organization. LHCII of b o t h species were r e s o l v e d into two apoproteins i.e. 27 and 26 kDa (carnation) or 26.5 and 25.5 kDa (barley). The apoproteins w e r e s e p a r a t e d by d e n a t u r i n g IEF into i-i0 pI isoforms covering 4.2-5.9 pH range with each subcormplex containing distinct subsets of pl isoforms.

40

P-1-2-023 PHOSPHORYLATED PHOTOSYSTEM I ANTENNA PROTEINS IN BARLEY J. Knoetzel, D.U. Meyer & L.H. Grimme Institute for Cell Biology, Biochemistry and Biotechnology, University of Bremen, Leobener Str./NW II, 28334 Bremen, Germany Phosphorylation of protein subunits and fight-harvesting proteins of photosystem II (PSII) provides the basis for responses of the photosynthetic apparatus to light and redox changes. Reaction centre or light-harvesting proteins of PSI are not known to be phosphorylated. Barley PSI has four antenna proteins of 21, 22, 23 and 25 kDa encoded for by the genes Lhca 4, 1, 2 and 3, respectively (Knoetzel et al., 1992, Eur J Biochem 206, 209-215). These fight-harvesting proteins of PSI-200 preparations were tested with antibodies against phosphorylated threonin and phosphorylated serin showing that all LHCA-proteins were phosphorylated. Isolated LHCA-proteins reacted with both antibodies indicating multiple phosphorylation sites on each antenna protein. Cleavage of the proteins with endoproteinase Lys-C was used to identify stroma-exposed phosphorylated anaino acid residues.

Poster

P-1-2-027

P-1-2-024 CHLORINA MUTANTS OF BARLEY LACKING SPECIFIC LHCAPROTEINS

SEPARATION OF PHOTOCHEMICAL AND PLASTOQUINONE QUENCHING OF FLUORESCENCE IN LEAVES

R BoBmann t, S. Jansson 2 & J. Knoetzel l

David M. Kramer], Giorgio Di Marco2 & Francesco Loreto2

lInsfitute for Cell Biology, Biochemistry and Bioteehnology, University of Bremen, Leobener Str./NW II, 28334 Bremen, Germany; 2Dept of Plant Physiology, UmeJt University, 90187 Ume/t, Sweden

Chlorina mutants of barley were identified on the basis of chlorophyll deficiency and by low temperature fluorescence emission spectroscopy. They are valuable in detenninin~g the function of light-harvesting proteins (Simpson et al., 1985, Carlsberg Res Commun 50, 223-238). Thylakoid membrane polypeptides from 19 mutants were tested with antibodies against the four PSI antenna proteins LHCA 1, 2, 3 and 4 with the aim of finding mutants which lack individual fight-harvesting proteins. Three mutants with increased amplitude of the 720 nm component of the 77K fluorescence emission spectra arising from PSI centres lacking LHCI were found to lack specific LHCA-proteins. Chlorina-f2 ~°] lacks LHCA 2, the 23 kDa-LHCI-680B-protein. The mutants clo- 12* and clo- T M lack the gene products of Lhcal and Lhca4, the LHCI-730-dimer.

tDepartment of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 U.S.A. 2CNR-Istitutodi Biochhnica ed Ecoflsiologia Vegetali, Roma, Italy. We measured the rise in fluorescence upon closure ofphntosystem II (PSII) reaction centers with 800 ms pulses of white light or saturating single-ttmaoverxenon flashes. The saturating pulses reduced both the plastoquinune (PQ) pool and QA, giving the parameters Fm or Fro', while the xenon flashes reduced QAwithout changing the redox state of the PQ pool. The rise in fluorescence was lower when induced by the xenon flash than by the saturating pulse. This difference was most likely due to direct (nun-photochemical) quenching by oxidized plastoquinone (ch~) since, in leaves infiltrated with DCMU, where PQ pool reduction was blocked, the saturating pulses and xenon flashes induced the same fluorescence rise. In the dark, where the PQ pool is oxidized, ~ e accounted for 40-50% of the measured saturation pulse-induced rise. With progressively more intense illamination, quenching due to Ch~ diminished. With over 1000/~mol m':s-~, the saturation pulse induced change was almost entirely due to reduction of Q^ indicating that the PQ pool was predominantly reduced. Removal of the electron sinks (CO2 and 02) also decreased Od~,indicating a net reduction of the PQ pool. A comparison of the photochemical efficiencies estimated by the two techniques suggests that the saturating pulse technique overestimates quantum efficiancies by about 20% when the PQ pool is oxidized (i.e., when quantum efficiency is high).

P-1-2-028

P-1-2-025 THE THREE-DIMENSIONAL STRUCTURE OF ALLOPHYCOCYANIN K. Breic, R. Ficner, S. Steinbacher & R. Huber Max-P]anck-Institut fiir Biocbemie, Abt. Strukturforschung, Am Klopferspitz 18a, 82152 Mardnsried, Germany The X-ray crystal structure of allophycocyanin (APC) from the cyanobacterium Spirulina platensis has been solved recently. The three-dimensional structure of the APC is similar to other known phycobiliproteins (Schirmer et al., (1987), JMB, 196, 677-695; Duerring et al., (1991), JMB, 217, 577-592; Ficner et aL, (1992), JMB, 228, 935-950). The major differences in the molecular structure of the subunits arise from deletions and insertions of segments involved in protein-chromophore interactions. In C-phycocyanin (C-PC) the absorption spectra of the monomers and the trimers do not differ markedly (near 620 nm). In A-PC, the monomer spectrum is very similar to that of monomeric C-PC, but the spectrum of the trimer is significantly different. The trimeric form has a sharp m a x i m u m near 650 rim. The exciton interactions between the neighbouring chromophores (MacColl et al., (1981), Arch.Biochem.Biophys., 208, 42-48) or changed chromophore conformation (Murakami et al., (1981), J.Biochem., 89, 79-86) were proposed as the cause for the 650 nm absorption maximum in APC trimers. The comparison of the chromophore binding regions of C-PC and APC indicates that a significant conformational change occurs in the ct84 chromophore, probably due to the different protein environment of the ct and neighbouring 13-subunit. This could be the reason for the red-shifted absorption maxima of APC trimers.

R O O M T E M P E R A T U R E I N V E S T I G A T I O N O F T H E LH2 ANTENNA O F R H O D O B A C T E R SPHAEROIDES BY N O N - L I N E A R P O L A R I Z A T I O N S P E C T R O S C O P Y IN T H E F R E Q U E N C Y D O M A I N (NLPF) ~ l , Scheer2

B. Voigd, J. Ehlert I, D. Leupold 1, M. Bandilla2, B. l~lcker2 and H.

Max-Born-Institute of Nonlinear Optics and Short-Pulse Spectroscopy, D-12474 Berlin, Germany; 2 Institute of Botany, Ludwig-Maximilians University D-80638 Mtinchen, Germany NLPF band shapes of the bacterial peripheral antenna LH2 have been investigated at room temperature in the spectral regions of both the 800 nm as well as the 850 nm absorption band. The samples included chromatophores of Rb. sphaeroides of the isolated pigment-protein complex B800-850, and of complexes in which bacteriochlorophyU at the B800 site has been replaced by modified pigments. The NLPF signals reveal heterogeneous broadening (subbands) of both bands and routes of energy transfer among them. The homogeneous and inhomogeneous width of each subband has been determined. The nature of these subbands (individual "forms" or exciton components) will be discussed in relation to recent X-ray data of Cogdell's group.

P-1-2-026

P-I-2-029

L O W T E M P E R A T U R E ABSORPTION, F L U O R E S C E N C E AND H O L E B U R N I N G S P E C T R O S C O P Y O F A L G A E A F F E C T E D BY UV-B

COMPARISON OF BACTERIOCHLOROPHYLL BINDING REQUIREMENTS IN THE CORE UGHT-HARVESTING COMPLEXES OF RHODOSPIRILLUM RUBRUM AND RHODOBACTER SPHAEROIDES

STRESS. D. Engst, T. Kroh, T. Polivka, J. P§en6ik,*L. Nedbal, *0. Pr~iil and J. I-Ifila Department of Chemical Physics, Faculty of Mathematics and Physics, Charles University, Ke Kariovu 3, 12t 16, Czech Republic, Prague 2, *Institute o f Microbiology, Czech Academy o f Sciences, 379 81 Tfebofi, Czech Republic The effects of short-term increased UV/B irradiance on excitation energy transfer within the light harvesting antenna of whole cells of green alga Scenedesmus obliqus were studied by means of low temperature absorption (A), fluorescence (17) and hole-burning spectroscopy. No significant changes in A or F (at 30OK) were observed in UV-B stressed algae. On the other hand, UV-B caused a relative increase in F at 4,2 K. This emission originates from PSII antennae. Detailed information about excited energy rate constants k ~ r and pigment-protein interactions were obtained from the widths o f resonant holes and ratio between area o f resonant (zero phonon) and phonon holes in persistent spectral hole burning experiments.

P.S. Parkes-Loach1, C.M. Davis1, C. Cook1, K.A. Meadows~, M. Bandilla2, H. Scheer2 & P.A. Loach 1. 1Department of Biochemistry, Molecular Biology & Cell Biology, Northwestem University, 2153 Sheridan Rd., Evanston, IL 60208, USA; 2Botanisches Institute, UniversifiR Munchen, Msnzinger Str. 67, D800O Munchan 19, Germany. Structural requirements of bacteriochlorophyll (BChl) for formation of the core light-harvesting complex (LH1) and its structural subunit were examined by reconstitution with BChl analogs and the o~-and ~-polypeptides of R. rubrum and of Rb. sphaeroides. Similar results were obtained with both sets of o~and ~-polypeptides and most of the BChl analogs, indicating the similarity of the BChl binding sites in the two bacteria. These systems showed the following requirements for formation of both the subunit complex and LH1 : (1) Mg or an analog containing a metal of similar size and coordination chemistry (e.g., Zn, Cd, Cd, Ni), (2) a bacteriochiodn oxidation state of the macrocyclic ring, (3) C132-carbomethoxy group, and (4) an intact ring V. C3LvinylBChl a and C132-OH BChl a, however, behaved differently with the two sets of polypeptides. These results will be discussed in terms of sources of stabilization of the complex and possible binding groups in the protein.

41

Poster P-1-2-030

P-1-2-033

MINIMAL STRUCTURAL REQUIREMENTS FOR FORMATION OF THE SUBUNIT COMPLEX OF LH1 OF PHOTOSYNTHETIC BACTERIA

The major light-harvesting chl binding protein ofProchlorococcus marirrus is similar to CP43', a chl binding protein induced by iron-depletion in cyanobaeteria

K.A. Meadows, J. W. Kehoe and P.A. Loach. Department of Biochemisitry, Molecular Biology & Cell Biology, Northwestern University, 2153 Sheridan Rd., Evanston, IL 60208, USA.

Julie La Roche1, Frederic Partensky2 & Paul Falkowski~ 'Dept. of Applied Science, Brookhaven National Laboratory, Upton NY USA 11973, 2Station Biologique de Roscoff, BP74, 29682 Roseoff, France

Isolation and reconstitution of the fundamental subunit complex, also called B820, from the core light-harvesting complexes (LH1) of 4 different photosynthetic bactaria have enabled a systematic probing of the structural elements responsible for the characteristic properties of bactedochlorophyll. Several approaches have been ultilized to shorten the native c¢- and I~polypeptides from both the N-terminus and C-terminus in order to evaluate minimal length requirements for formation of the subunit and LH1 complexes. These modifications have included chemical cleavage at the AspPro bond in ct-polypeptides, specific endoproteinase cleavage at the carboxyl side of Lys and Glu residues, carboxypeptidase cleavage of the C-terminal and penultimate C-terminal residue of the I~-polypeptide of Rb. sphaeroides, and chemical syntheses of vadous polypeptides from 16 to 31 amino acids in length reproducing the C-terminal end of the I~-polypeptide of Rb. sphaeroides. From these studies, a single polypeptJde of 30 amino acids is sufficient to reproduce the formation of a subunit-like complex with a KA~,c¢ equal to that of the intact ~-polypeptide of Rb. sphaeroides.

P-1-2-031 HYDROPHOBIC MODIFICATION OF ANTENNA CHLOROPHYLL IN CHLOROBIUM DURING GROWTH WITH ACETYLENE John Ormerod I

P-1-2-034 THE CHL a/b ANTENNA FROM PROCHLOROPHYTES IS RELATED TO THE IRON-STRESS INDUCED CHL a ANTENNA (lsiA) FROM CYANOBACTERIA

& Niels-Ulrik Frigaard 2

~Biology Dept., Oslo l~niversity, Oslo, Norway; 21nstitute of Biochemistry, Odense University, Odense , Denmark. Bacteriochlorophyll (BChl) d in chlorosomes is believed to be organised as aggregates stabilised by scaffold polypeptides. Low light leads to limited formation of hydrophobic side chains on C8 of BChl d, from ethyl up to neopentyl, presumably allowing denser aggregates and better light absorption at low intensities. We have used acetylene to analyse this phenomenon. This gas specifically inhibits formation of BChl d and chloros~nes yet cultures with acetylene grow just as last as the controls at moderate light intensities. The small amount of BChl d present (5% of control) is very hydrophobic and we believe that this may be connected with higher energy transfer efficiency to the reaction centers. This possibility will be tested by fluorescence measurements.

P-1-2-032 THE SIZE OF THE PHOTOSYNTHETIC UNIT IN PURPLEBACTERIA Otristof Francke and Jan Amesz Department of Biophysics, Huygens Laboratory, University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands Pigment analysis was performed by means of normal phase HPLC on a number of species of purple bacteria that contain core antenna only. In species containing hacteriochlorophyll (BChl) ae small amounts of BChl aoo were found, and presumably also of BChl a esterified with dihydro and tetrahydro geranyl-geraniol. The becteriocldorophyll/bacteriopheophytin ratio indicated that in the bacteria a cunstant number of 25 + 3 antenna BChis is present per reaction centre. This number strongly supports the current model in which the core antenna consists of 12 a-J3 heterodimers surrounding the reaction centre. Determination of the/n vivo extinction coefficient of BChl in the core-reaction centre complex yielded a value of circa 150 mM"l cm"1 for BChl a as well as for BChl b containing species.

42

Prochloroccocus marinus, a minute marine photosynthetic organism, is the most recently discovered prochlorophyte. Prochlorophytes, also comprising Prochloron and Prochlorothrix, differ from cyanobacteria by the absence of phyeobilisomes and the presence ofchl a/b binding protein complexes. These differences in lightharvesting systems are difficult to reconcile with molecular phylogenetie data which suggests that the three prochlorophytes have emerged independently within the cyanobaeterial lineage. However, N-terminal sequences from light-harvesting proteins ofP. marinus showed 80% identity with that of CP43', a ehl-binding protein encoded by the isiA gene which is induced by iron-depletion in cyanobacteria. Using degenerate primers, we have amplified a DNA fragment which has high homology with isiA genes. This was used as a probe to clone genomic DNA fragments encoding isiAfflke genes from two strains ofP. marinus. Similarity between the primary amino acid sequences of the light-harvesting complex from P. marinus and CP43' from cyanobacteria will be discussed.

G.W.M. van der Staav l, A. Ducret 2, R. Aebersold 2, R. Li 3, S.S. Golden 3, R.G. Hiller 4, P.M. Wrench4, A.W.D. Larkum 5 and B.R. Green 1 1Botany Dept., Univ.of British Columbia; Vancouver, B.C., Canada; 2Molecular Biotechnology Dept., Univ.of Washington, Seattle, WA, USA; 3Biology Dept. Texas A&M Univ., College Station, TX, USA; 4 Biologicgl Sciences, Macquarie Univ., Sydney, Australia; 5 Biological Sciences, Univ. of Sydney, Sydney, Australia Tryptic fragments of Chl a/b antenna polypeptides from the prochlorophytes Prochlorothrix and Prochloron showed sequence homology to the product of the isiA gene of cyanobacteria, which encodes the polypeptide of an iron-stress inducible Chl a antenna complex (CP43'). A Prochlorothrix genomic DNA clone was found to contain two non-identical/siA like genes plus the N-terminus of a third. This suggests that the different proteins of the Chl a/b antenna in Prochlorothrix are encoded by a gene family. Since isiA shows sequence relatedness to psbC, the apoprotein of CP43, it appears that proteins binding only Chl a and those binding Chls a and b can be members of the same gene family and may have had a common ancestor.

P-1-2-035 THERMAL BROADENING OF THE LHCII ABSORPTION SPECTRUM ANALYSED BY SUB-BANDS DECOMPOSITION. Giusevve Zucchelli. Robert C. Jennings, Flavio M. Garlaschi, Laura Finzi Centro CNR Biologia Cellulare • Molecolare Piante, Dipartimento di Biologia, via Celoria 26, 20133 Milano, Italy. The absorption spectra of the major fight harvesting chlorophyll protein complex of PSII, LHCII, have been measured in the temperature range 70300K and analysed by numerical sub-bands decomposition. Six different subbands have been found in the wavelength range 655-684nm and the thermal evolution of each sub-band has been analysed to obtain information about the reorganisation energy of the chromophores. The results indicate that all the subbands have an essentially harmonic behaviour with a reorganisation energy in the range 7-15 cm"1 and an inhomogeneous contribution of between 110-180 cm"1. Thus, if a mean low frequency bath of around 20 cm1 is assumed, the coupling is between 0.35-0.75 for the chla forms. The full width at half maximum of the sub-bands is about 10nm at room temperature, a significant sharpening with respect to the width of the absorption band of chla in solution (~ 20 ran). Another interesting feature is the presence of a significant long wavelength sub-band (~ 684nm) with a contribution that changes reversibly with temperature.

Poster P-1-2-039

P-1-2-036 ~NEMICALLY IS3[ATED PSI-200

I~j'rLTr~RATI~ STEADYSTATE ~

IN

Ro~erta

Croce ÷, Flavio Garlaschi ÷ , Giuseppe Zucchelli + , B a s ~ , Rahert Jemnings ÷ . * Dipartimento di Biologia e Cemtro di Studio del CI~R, Univemsita' di Milano, via Celoria 26, Milano, Italy. * Biotecnologie, Universita' di Vercr~, Strada Le Grazie, verona, Italy.

PSI cctltaining both core and periperal anterana complexes %~s prepared from maize using octyl gluccside. Steady state ahsozpticm and fluorescemce spectra were determ/ned between 70K-300K in cctlditictls in which uncoupled chlorophylls were abeent. It is d~cDstrated by calculations using the Stepanov relation that approximately 90% of the steady state fluorescence at room temoerature derives from red spectral foIlnS absorbing beyond 695rln. Sclae spectroscopic characteristics of these red forms are discussed.

ORGANIZATION OF PHOTOSYSTEM I ANTENNA t~. S~rv~ri. G. Malatinszky & P. Nyitrai Department of Plant Physiology, E6tv6s University, tt-1445, Budapest, P.O.B 330, Hungary Photosystem I (PSI)particles were isolated from maize mesophyll and in purer form from 5undle sheath thylakoids by mild Deriphat PAGE: (i}complete PSI containing core, light-harvestingcomplex [{LHCI} and a complex built of 27 k D a polypeptides; {ii)P S r c o m p o s e d of core a n d L H C I or (iii}27 k D a co_mpl-ex,respectively; and (iv} PSI core complex. T h e long wavelength fluorescence emission m a x i m u m shifted f r o m 730-3 n m (i, ii), to 720-5 n m (iii)and to 715-20 n m (iv). Stronger solubilizalion increased the a m o u n t of smaller complexes but did not changed their composition. Second-dimensionsl mild P A G E of complete PSI separated PSI reaction centre, 27 k D a species and LHCI730 mainly in an oligomeric state, and LHCI-680 mainly in a m o n o m e r i c state. The 27 k D a complex of PSI did not contain any 25 k D a polypeptide, emitted fluorescence at 680 n m {of m u c h higher intensity in isolated than in complexed form}, and s h o w e d a higher maximurn/650 n m fluorescence excitation ratio than its LHCII counterpart. A s both LHCI- and 27 kDa-contRinln8 PSI were isolated it w a s concluded that both types of antenna were connected directly to the PSI core. OTKA {T-5503}andEEC {CIPACT9302O21gjanlsam greatlyacknowledged.

P-1-2-040

P-1-2-037 ~

A B S O I ~ - C ~ ANALYSIS OF ~

DI/D2/CYI~559

CC~w_EX Roberta Cattaneo, Giuseppe Zucchelli, Flavio Garlaschi, Laura Finzi, ~ Jerm/ngs. Dipaztimmat~ ~ o ~ - I - o ~ - - - e O m t r o di St~qio del C~R, /hiversita' di Milano, via Celoria 26, Milano, Italy. Absorption spectra of the Dl/D2/cytb559 complex were measured between 80K-300K. All spectra were analysed in terms of a linear cumbinaticn of gaussian bands and the tb-rm~l ~ data interpreted in tenvs of a model in which the spectrum of each pigment site is broadened by (a) an ~ ccmlxlmmt due to linear electron-~xl3on couplip~ to a low frequency protein vibration and (b) an inhcmDgelae(x~ , temperature insensitive, cxll~x3nent. In this way seven Qy absorption bands were found. The optical ~ t i o n energy (S ) for all accessory pi@ms~ts and pheophytin is between 15 - 20cm-I while that for P680 is about 40cm-i. The inhcmogeD~ously b _rcada~ed line width is in the range i00 - 140cm-i. At room temperature the overall half hand widths for accessory pigments is 10ran-12nm (210 250cm-a ) while that for P680 is near 16rim.

PEPTIDE SEQUENCES OF MAIZE CP24 AND CP26, THE TWO MAJOR ZEAXANTHIN-BINDING PROTEIN OF PHOTOSYSTEM II, AND THE SEQUENCES OF THEIR GENES P. Dainese 1, E. Ber~antino 1, S. Sechi2, R. Bassi 3 and E. Pichersky4 1Dipartimento di Biologia, Universit/t di Padova, Italy; 2Laboratory of Experimental Carcinogenesis, NCI, NIH, Bethesda, USA; FacoltA di Scienze, Universit/t di Verona, Italy; 4Natural Science Building, Ann Arbor, University of Michigan, USA. The heterogeneity of the photosystem II (PSII) chlorophyll a/b-binding (CAB) proteins was shown both on the basis of their organization and function within the PSII unit, by biochemical and molecular genetic studies. Particularly in respect to the minor antennae CP24, CP26 and CP29, in many species the correspondence of a given Lhc (Light harvesting complex) gene to its product is still unclear. We report the partial sequencing of CP24 and CP26, the two major zeaxanthin-containing complexes (Bassi et al., 1993, Eur. J. Biochem. 204, 297-303), purified in their native pigment-binding form from maize chloroplasts. We also report the isolation and sequencing of cDNAs encoding the CP24 and CP26 apoproteins, establishing the identities of these polypeptides and their genes in Zea mays.

P-1-2-038 PURIFICATION AND CRYSTAIJJT.ATION OF THE LIGHTHARVESTING COMPLEX FROM THE ALGA MANTONIELLA SQUAMATA (PRASINOPHYCEAE) C. Welte, R. Nickel and A. Wild Institute of General Botany of the Johannes Gutenberg-University, D-55099 Mainz, Germany The light-harvesting complex of Mantoniella squamata was chosen for our attempt to obtain more detailed information of the structures involved in solar energy absorption and the energy transfer to the photochemical reaction center. The light-harvesting complex could be isolated by density centrifugation and further purified by applying anion-exchange and molecular-sieve chromatography. Purity and stability of the complex were proved by denaturing and non-denaturing polyacrylamide-gel electrophoresis, as well as spectroscopic measurements. Molecular-mass determination gave us evidence for trimeric organization of the purified native complex. As investigated by electron microscopy, our purification procedure resulted in mixed protein-detergent micelles, small and homogeneous, the ideal material to start crystallization trials. Within several weeks crystals of different shape appeared.

P-1-2-041 A B P E C T R O S C O P I C I N V Z S T Z G A T I O N OF A G G I ~ G A T I O N OF CHLOROPHYLL • I N S O L U T I O N

D.M.G~zdaru1, L . C . P e t c u , E . M . F r a n k e n 2 IFaculty of Physics, B u c h a r e s t University, Po Box MG-II, 76900 B u c h a r e s t - M ~ g u r e l e , Romania; 2Dept. of Biophysics, Huygens Laboratory, U n i v e r s i t y of Leiden, 2300 RA Leiden, The N e t h e r l a n d s A b s o r p t i o n , c i r c u l a r d i c h r o i s m and a b s o r p t i o n d i f f e r e n c e spectra are r e p o r t e d for a s o l u t i o n of c h l o r o p h y l l - a 10-4M, in 3 - m e t h y l p e n t a n . The a b s o r p t i o n and c i r c u l a r dic h r o i s m spectra at room t e m p e r a t u r e show the p r e s e n c e of the d i m e r i c c h l o r o p h y l l - a at 679 nm. The a b s o r p t i o n spectra at low t e m p e r a t u r e s show the e x i s t e n c e of two aggregates of chlorophyll-a, at 689 nm and 709 nm. These two a g g r e g a t e s are a n h y d r o u s dimer and h y d r a t e d dimer. Flashd i f f e r e n c e s p e c t r u m of c h l o r o p h y l l - a 10-4M in 3-methylp e n t a n at 77 K can be i n t e r p r e t e d as a "sum" of the spectra of two t r a n s i e n t species. The l i f e t i m e of the t r a n s i e n t specie was d e t e r m i n a t e d to be 1,36 ms over the w a v e l e n g t h range of 500-800 nm. This result implies that the species giving a b s o r p t i o n d i f f e r e n c e s p e c t r u m have p r a c t i c a l l y the same lifetime.

43

Poster ii

P-1-2-042

THE LIGHT-HARVESTING CHLOROPHYLL a/b-BINDING (LHC) PROTEIN COMPOSITION OF PHOTOSYSTEM (PS) I (7./~ AND PS II a / ~

W. H. J, Westerhuis 1, C. N. Hunter 2 and R. A. Niederman 1 IDept of Molecular Biology and Biochemistry, Rutgers, the State University of New Jersey, Piseataway NJ, 08854, USA; 2Dept of Molecular Biology and Binlechnohigy, University of Sheffield, Western Bank. She~[fieldSt0 2UH, LTK

Ulrika Nystr6rn 1, Hreinn Stefausson2, Louie Wollenberger2, Petter Gustafsson 1, Per-Ake Albertsson2, Stefan Jansson 1 1Dept of Plant Physiology, University of Umeti, 901 87 Ume~t, Sweden 2Dept of Biochemistry, University of Lund, Box 124, 221 (30 Lund, Sweden

In previous work from our laboratory, we proposed that spectral heterogeneity of the LHI absorption band is a consequence of excitonic coupling of BChls, arranged in curvi-linear arrays. This was based on the observation that the spectral properties of detergent-sohibilized B875 complexes of R. sphaeroides change monotonically with decreasing oligomerizatiun state, whereby the consecutive removal of single ~13 units was accompanied by minor absorption blue shifts and pronounced alterations in fluorescence polarization spectra. In a structural model, thought to account for these erects, BChls were organized into two separate concentric circular arrays of coupled chromopbores, liganded to
PS II centres with different antenna size (PS Ilct and PS II~) are found in the appressed and non-appressed regions of the thylakoid, respectively. It has recently been found that a similar heterogeneity also is present among PS I centres. It has however not been established which of the 10 LHC proteins that differ in amount between the different kinds of centres. We have used our collection of monospecific antibodies to determine the level of LHC protein in PSI and PS II centres found in the three thylakoid compartments grana, grana margins and stroma-exposed thylakoids by quantitative western blotting using a phosphoimager.

P-1-2-043 S.Y. Yang Shanghai Institute of Plant Physiology, Academia Siaica, 300 Fenglin Road, Shanghai 200032, China

A.Dudkowiak1, C.Francke2, A.Planner~, R.Cegielski ~, I.Hany~,I, J.Amesz2 & D.Fra, ckowiak ~ ~Molecular Physics Laboratory, Institute of Physics, Poznafi University of Technology, Piotrowo 3, 60-965 Poznati, Poland,2Department of Biophysics, Huygens Laboratory, University of Leiden, P.O.Box 9504, 2300 RA Leiden, The Netherlands

The difference,s between chlorophyll a-dioxane aggregates [(Chla-dio),"l and chlorophyll a-water aggregates ~(Chla-H~O),] were observed not only in absorption spectra but also in electric and photochemical properties. The (Chia-dio). carrying negative charge, migrated toward anode during electroplating, while the (Chia-H20)o carrying positive charge, migrated toward cathode. The electropotential of the membrane of the (ChlaH20 ), produced by electroplatthg ascended under illumination, but that of the (Chla-

The spectroscopic features of monomeric R[E,E] bacteriochlorophyll (BChl) c and its pheophytin in a nematic liquid crystal matrix were investigated. The orientation of the pigments in the crystal was studied by means of linear dichroism and fluorescence polarization. A model of BChl c orientation in the liquid crystal is proposed and the angles between the transition moments and the orientation axis were determined. The interaction between the BChl c molecules and the nematic liquid crystal changed upon hydration of the pigment or removal of the central Mg atom. Delayed luminescence, in the/xs range, from both pigments was observed in chloroform solution as well as in the crystals. This delayed emission is believed to be due to pigment ionization and delayed charge recombination.

HzO), descened. The potential difference between these membranes could reach about 100 inV. When a small quantity of dinxane is added into the hexane suspension of the (Chla-HzO)~, the (Chla-HaO). changed into the (Chla-dio)o. If the (Chla-HzO)= is illuminated prior to the addition of dioxane, light emission could be detected during the transform processes. The intensity and duration of light emission is stronger and longer than when acetone was added as the deaggregator.

1)-1-2-044 B&CTBRZOCBLOROPBYLL

COMBZBATOBXJkL

LH2

N'JT&IITS

OF

ABD

CAROTEEOZD

RHODOBACPRR

BZIDZ~G

ZN

CAPSULATU8

Q. Hu I , 5. Delagrave 2 , D. C. Youvan 2 and R. A. Niederman I 1Mol. Biol. & Biochem. Dept., Rutgers univ., Piscatawsy, NJ, USA; 2pale Alto Instit. for Mol. Med. Mountain View, CA, USA. The peripheral (LE2) antenna complex of R. capsulatus, has served as a model protein for combinatorial mutagenesis. When the LH2-~ polypeptide was mutagenized at residues on the same face of the u-helical span as the BChl liganding His, mutants were isolated that formed a pseudo LHI antenna (pLHI) with a red-shlfted B850 band. Two of these strains also had greatly reduced carotenoid levels, which largely accounted for the red shift, since a pLH1 complex can be obtained by carotenoid extraction; however, a carotenoid-less pLnl complex is not for~ed in ~ t a n t s lacking carotenoids, and the assembly of a pLHI c o ~ l e x in these strains demonstrates that through compensatory residue changes, combinatorial mutagenesis resulted in expression of an antenna protein that is nor~ally unstable. optimized mutagenesis of the six C-terminal residues of the LH2-~ polypeptide yielded two mutants in which the conserved PW residue pair was inverted to WP, resulting in the formation of a BChl-depleted LH2 complex with an -10-nm blue shift of the B850 peak. Further spectroscopic studies of these mutant strains are currently in progress.

44

P-1-2-046 SPECTRAL PROPERTIES OF MONOMERIC BACTERIOCHLOROPHYLL C IN NEMATIC LIQUID CRYSTAL

"/'we KINDSOF CHLOROPHYLLA POLYMER

JI.LTBBBD

P-1-2-045 SPECTRAL HETEROGENEITy OF LIGHT-HARVESTING COMPLEXES PRODUCED BY EXCITONIC INTERAf~I'IONSAMONGBCHL.SIN (~IRVEDLINEARARRAYS

P-1-2-047 OLIGOMERIC STATES OF THE LHI AND LHII ANTENNA FROM RUBRIVlVAX GELATINOSUS IN DETERGENT SOLUTIONS. V. Jirsakova, J.L. Ranck and F. Reiss-Husson Centre de G6ndtique Mol0culaira, B;~t.24, CNRS, 91198 Gif-surYvette, France Molecular weights of purified LHI and LHII complexes from Rv. gelatinosus were determined by analytical centrifugation. In the presence of dodecyldimethyiamine oxide LH|I complex is an heptamer composed of 7 (od~/3Bchla/lcarotenoid) units, and does not bind a large amount of detergent (about 0.2 g per g protein). In decanoylsucrose solution LHI tends to aggregate during centrifugation ; t h e smallest oligomer is a dodecamer of 12 ((~/~/2Bchla/2hydroxyspheroidene) units, with a high amount of bound detergent (2.3 g par g protein). Electron microscope observations confirmed the different shapes of the antenna/detergent complexes : globular for LHII, annular for LHI.

Poster

P-1-2-051

P-1-2-048 LIGHT-HARVESTING COMPLEXES FROM A BROWN ALGA: BIOCHEMICAL AND MOLECULAR STUDY Caron L., Douady D., Rousseau B., Quinet-Szely M. and Berkaloff C.,URA CNRS 1810, Ecole Normale Sup6rieure, 46 rue d'Ulm, 75230 Paris Cedex 05 Solubilization by non-ionic detergents of bulk LHC from Lam/nar/a sacchar~ allowed discrete pigmented fractions to be separated by DEAE-ion-exchange chromatography or isoelectric focusing. The pigment composition of these bands has been determined by reversed-phase HPLC and their polypeptide components analysed on denaturing PAGE.In some of them, fluorescence excitation spectroscopy demonstrated good energy transfer from Chl c and fucoxanthin to Chl a.Therefore, as in green plants, the native LH particles proved to be heterogeneous and consisting of at least three different Chl a/c/fucoxanthinbinding proteins.One gene from the multigenic family coding for the LHC polypeptides has been cloned from a nuclear DNA bank, and entirely sequenced. It exhibited a high homology with the Fcp genes from the diatom P. tricornutum, and a more limited homology with Lhc genes from green plants. The deduced amino-acid sequence allowed to tentatively predict, as in green plants, three membrane spanning regions, with amino-acid conservation at positions involved in the stabilization of the structure and in the binding ofChl a. The parts of the molecule involved in the binding of Chl c and fucoxanthin remain to elucidate.

CRYSTALLIZATION OF PHYCOBILIPROTEINS OF THE CRYPTOMONAD ALGAE USING DETERGENTS AND U N U S U A L A D D I T I V E S M. Becker and R. Huber, Struktufforschung, Max-PlanckInstitut fiir Biochcmie, 82152 Martinsried, Germany

Phycobiliproteins from 3 different species of cryptomonad algae have been crystallized under a wide variety of conditions~ The crystals were typically poorly ordered. H o w e v e r , crystals of phycoerythrin 545 of R h o d o m o n a s lens that were well-ordered in 3 - d i m e n s i o n s could be obtained under 2 different conditions, which included the detergent, lauryldimethylaminoxide, as w e l l as the additive propionamide in one case, or the ion Cs+ in the other. They show diffraction to at least 3.0 A resolution, and appear suitable for structural analysis. The crystals s h o w intense, strongly-polarized fluorescence, suggesting that energy transfer in the crystals is highly efficient.

P-1-2-052

P-1-2-049 DECREASE OF THE PS I ANTENNA COMPLEX WITH INCREASING GROWTH IRRADIANCE IN PORPHYRIDIUM CRUENTUM Shi Tan t, Gregory R. Wolfe~, Francis X. Cunningham~, Jr, & Elisabeth GantP tDept. Plant Biol.and MAES, Univ. of Maryland, College Park. MD 20742, USA; ='Dept. Botany, Arizona State Univ., Tempe, AZ 85287, USA Thylakoids isolated from cells ofP. cruentum exhibit an increased PSI activity on a Chl basis with increasinggrowth irradiance, even though the stoichiometry of PS I and II in such cells shows little change. PS I activity was 20% greater in cells acclimated at 280 ttmol • m"2"s"1(VHL) than in cells acclimated at 10 ttmol • m"2s"t (LL), indicating a change in the light absorbance capacity of PS I. Isolated PS I holocomplexes from VHL cells contained 132 q- 9 Chl/P700 and those from LL cells had 165 =t=4 Chl/P700. PSI holocomplexes on SDS-PAGE showed a notable decline in three (19.5, 21 and 22 kDa) of six LHC I polypeptides in VHLcomplexes relative to LL-complexes. We suggest that the decrease in PSI Chl antenna size observed with increasing irradiance is attributable to changes occurring in the LHC I-antenna complex. Evidence for a Chl-binding antenna complex associated with PS II core complexes is lacking at this point. LHC If-type polypeptides were not observed in functionally active PS II preparations, nor did we detect polypeptides by immunocross-reactivity with LHC II specific antisera.

MOLECULAR ASSEMBLIES OF LIGHT-HARVESTING POLYPEPTIDES / PORPHYRIN DERIVATIVES AS AN ARTIFICIAL MODEL FOR THE LIGHT.HARVESTING POLYPEPTIDES COMPLEX OF PHOTOSYNTHETIC BACTERIA Kouii lida, ~ Kciji Yamashita, I Mamoru Nango, I and Paul A. Loach* °Nagoya Municipal Industrial Research Institute, Atsuta-ku, Nagoya 456. ~Department of Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466, JAPAN. *Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston IL 60208-3500, USA To study the minimal structural requirements for formation of the subunit and core Hght-harvesting complex (I.,H) of photosynthetic bacteria, molecular assemblies of the ct- and [3- polypeptides of the LH from K rubrum with mesoporphyrin derivatives in octylglycopyranoside (0(3) wcr¢ examined. The formation of the LH was evaluated by using absorbance and circular dichruism spectroscopies. The spectroscopic properties of the polypeptides complexes generated using zinc mesnporphyrin dimer which is covalently linked through amino acid residues were analogous to those obtained using the LH polypeptides and bacteriochlorophyll a in OG (1). Thus the zinc mesoporphyrin dimer is useful to study on the model for formation of the LH of photosynthetic bacteria. (1) K. A. Meadows, et. al., Biochemistry, 34, 1559-1574 (1995).

P-1-2-053

P-1-2-050 THEORETICALANALYSISOF FEMTOSECONDCOHERENTNUCLEAR MOTION IN PHOTOSYNTHErlCREACTIONCENTERS

2D CRYSTALLIZATION AND STRUCTURE DETERMINATION OF LHII FROM Rhv. sulfidophUum

Eugenijns Gaizauskas and Leonas Valkunas. Vilnius University, Sauletekio al 10, 2054 Vilnins and Institute of Physics, Gostauto 12, 2600 Vilnius, Lithuania

G. Montoya, M. Cyrklaff & L Sinnin~ EMBL, Meyerhofstr. 1, D-69012 Heidelberg, Germany

Theoretical analysis of femtosecond pump-probe spectroscopy of the reaction centers by taking into account an additional aspect of the coherent light and matter interaction is presented in the given report. This fact that the primary electron donor is a dimer is also taken into consideration. For this reason, the importance of two-photonresonanceenhanced wave-mixingprocessesas to be exploited to the field of femtosecond spectroscopyof dimers is indicated. Two specific questions are discussed. The first one is whether or not two-photonresonance provides any modification to the measured signal oscillations in pump-probeexperiments.The second one concerns interplay of oscillator signal features induced by coherent one- and two-photon resonant nonstationary electronic polarisation with those caused by nuclear motion, which can serve for the visualisation of the coherent nuclear dynamics. The analysis is applied to the femtosecond experiments of bacterial reaction centers (M.H.Voset al. Nature 363 (1993) 320-325).

Light harvesting complex II (LHII) from the purple bacterium Rhodovulum (Rhvo) sulfidophilum has been isolated using a dodecyl maltoside based solubilization procedure. The results from native gels, analytical nitracentrifugation and gelfiltratiou columns all suggest that LHII is an octamer. Based on observations in initial 3D crystallization trials, 2D crystallization has been done using microdialysis, which resulted in 2D crystals of the tubular and vesicular type. The plane group is p42t2 with a=b-~157 A. The crystals diffract to about 18 A in negative stain and to about 4A using cryogenic temperatures. A projection map calculated at 18 A resolution shows already the ring like arrangement of the protein, similar to hexadecamefic LHI from Rhodospirillum rubrum (Karraach et al, 1995, EMBO J. 14, 631-638) and nonameric LHII from Rhodopseudomonas acidophila (McDermott et al, 1995, Nature 374, 517-521). The size of the particle obtained in this study agrees best with an octamer. A higher resolution structure is underway. The questions that still remain to be answered are whether the oligomefie state of LHII in the isolated ring-like particle is dependent on the organism or on the solubilization and crystallization procedure, and finally how LHII is organized in the native membrane.

45

Poster Ill II

I

I

Ill I llll

VIBRATIONAL SPECTROSCOPY OF PHYCOERYTHRIN

P-1-2-057 THE PIGMENT-PROTEIN INTERACTIONS OF SOME UNUSUAL LIGltT-HAREVESTING ANTENNAE : A RAMAN STUDY.

M. Debreczeny & B. Szalontai Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, P.O.B. 521, H-6701 Szeged, Hungary

A__~Gall1, V. Yurkov 2, R.J.Cogdell 3 and B. Robert l

P-I-2-054

Phycoerythrin (PE) the shortest wavelength absorbing component of phycobilisomes in cyanobacteria has not yet been investigated by Raman spectroscopy, while detailed vibrational analysis of the other two light-harvesting pigment, phycocyanin (PC) and allophycocyanin (APC) is available. However, the difference in the conjugated electron system makes the comparison of these spectra valuable for the band assignments. Here we present the UV-resonance Raman (UV-RR), surface-enhanced resonance Raman (SERR) and Fourier transform Raman (FTR) spectra of PE in its different states of association. The difficulties caused by the extremely strong fluorescence of PE in the visible region could be overcome by SERR spectroscopy. The uncertainties of the SERR technique could be checked by FTR spectroscopy which provided a still resonance enhanced Raman spectrum. The SERR and FTR spectra were rather similar, the considerable differences are between the spectra excited in UV and in the visible transition of PE. The spectral differences are analysed in connection with the band assigrunents of PC and APC. It is shown that the association between PE monomers is different from the other two phycobiliproteins.

P-1=2-055 EQUILIBRATION OF EXCITONS AND DOUBLY EXCITED LIGHT HARVESTING COMPLEX OF PHOTOSYSTEM lI T. Schrttter, T. Renger, J. Voigt Institute of Physics, Humboldt-University Berlin, D-10099 Berlin, Germany To gain information about the exciton migration and relaxation in LHC lI the spectral distribution of exeitons was investigated with pump-probe experiments using two dye lasers (pulse duration 10 ns, spectral bandwidth less than 0.01 ran). The relative probe beam transmission as a function of probe beam wavelength in the Qy absorption region of Chl a and Chl b shows a course nearly independent frtm the pump beam wavelength in this region. This finding predicts a Boltzmann-like distribution of exeitons with a population maximum near 680 tun corresponding to the energetic level of the reaction centre. A simulation of the measured relative probe beam transmission is not possible without considering optical transitions from singly to doubly excited states of LHC II monomers occurring under the high pump intensities used. By means of structure data of LHC II singly and doubly excited states as well as the oscillator strengths of their transitions were calculated. The hamiltonian of 12 coupled singiet transitions of chlorophyll molecules was dingonalized using dipol-dipol approximation to describe the interaction of the pigments. We have got a good agreement with the experimental data.

P-1-2-056 THE EFFECT OF pH ON LHCII A.A.Pasca!. A.V.Ruban, P.Horton & A.J.Young* Rohen Hill Institute, Dept of Molecular Biolo~ & Biotechnology, P.O.Box 594, Universityof Sheffield, SheffieldS10 2UH, U.K. Schoolof Biological & Earth Sciences, Liverpool John Moores University,ByromStreet, LiverpoolL3 3AF, U.K. Aggregation of the chlorophyll-a/b light-harvesting complex of photosystem II (LHCII) results in dramatic changes in spectral properties. Similar organisational changes in rive (on a smaller scale) may be responsible for the down-regolation of light-harvesting efficiency in strong light conditions Chigh-energy-state" quenching of chlorophyll fluorescence, qE; Horton et al., FEBS Legs (1991), 292 1-4). We report the following observations which relate LHCII aggregation in vitro with pH: 1) Solubilisatien at pH 7.80 results in more complete disaggregation than at pH 5.00. 2) A titration of the H+-induced quenching of LHCII fluorescence (oecuring via limited aggregation) shows very close similarity to that observed in thylakoids, which itself mimics q~ (Rees et aL, Photosynth. Res. (1992), 31 11-19), in terms of the pKa, the effect of inhibitors, etc. 3) Detergent-induced disaggregation of the complex results in release of protons, the stoichiometry being in the region of 25 H+/polypeptide. In the former two cases zeaxanthin-containing complexes showed a greater tendency to aggregate than violaxanthin-containing ones, consistent with the xanthophyll cycle pigments exerting their influence on qE according to their differing polarities (Ruban et al. (1993), Photochem. Photobiol. 21 229-234).

46

1DBCM/CEA and LIRA 1290/CNRS, C~tre d'Etudes de Saelay, F-91191 Gif sur Yvette Cedex, France. 2Inst. for Biolosie H, Mikrobiolosie, Albert-LudwigUniversit~t, Freiburg, SchlinzlestraBe 1, I)-79104 Freiburg, ~ . 3Davidsou Building, I.B.L.S., University of Glasgow, Glasgow G12 8QQ, Scotland. In the fight of the recent x-ray structure of the peripheral fight-harvesting antennae (Bg00-850) from Rhodopseudomonas acidophila the usefulness of Raman spectroscopy in predicting the pigment-protein interections in these complexes has been vindicated. In this presentation we further investigate the pigment-protein interactions of other peripheral antennae. This has been achieved by isolating the spectroscopically unusual pigme~t-proteln complexes from Chromatimn purpuratum, Erythromicrobium ramosum, Roseococcus thia~ulfatophilus and Rp. palustris. Using the Rp, acidophila B800-850 model as point of reference their absorption characteristics have been compared with their predicted level of H-bonding.

P-1-2-058 A kinetie study of the reasso¢iation of the antenna complex of Rhodospirillum rubrum in detergent solution. James S t u r ~ and Bruno Robert. SBPM/DBCM CEA and URA 1290 CNRS, Centre d~Etudes de Saelay, 91191 Gif sur Yvette Cedex, FRANCE. We have used the aggregation of the polypeptides of the core light-harvesting complex fromRhodospirillum rubrum to investigate the assembly of these complexes, and the forces that drive this assembly. In this presentation we will discuss our recent work based on an analysis of the kinetics of the transitions between the B777 and B820 forms of this antenna. In the light e r a global analysis of the results of these experiments we have developed a kinetic model that descn'bes both the kinetics and concentration dependance of the spectral changes. It is hoped that this approach coupled with our previous thermodynamic measurements, and the recently published antenna structure will allow an actuate description of the association reaction pathway and the elucidation of the molecular origin of the forces that drive the assembly o f the B820 form of this light-harvesting complex from the dissociated B777 form.

P.1-2.059 INFLUENCE OF PIGMENT MODIFICATIONS ON ASSEMBLY OF THE LIGHT HARVESTING COMPLEX OF RHODOSPIRII~UM RUBRUM A, NAvek¢:,2, H. Scheer t, B. RObel't2 ~¢ J. Sturgis2 lBotanisches Institut, Universitit Miinchen, Germany; 2SBPM/DBCM, CE de Saclay, Gif-sur-Yvette, France The specific functions of proteins derive from their three-dimensional structure. While the energetics of the folding and association of globular proteins is reasonably well understood, the nature of the interactions that stabilize the structure of membrane proteins remain obscure. We are investigating the assembly of the light harvesting antennae of the photosynthetic purple bacterium Rhodospirillum rubram as a model system. In order to elucidate the role of the bound bacteriochlorophyll molecules in the stabilization of the complex we have studied the effect of exchanging the bound pigments with chemically modified pigments. We have characterised the complexes with altered pigments using a variety of electronic and vibrational spectroscopies in order to clarify the roles of the altered groups in the structure and assembly of the antenna complex. In particular we have measured the importance of the pigment in determining the standard enthalpy change associated with the formation of the B820 subunit form of the antenna.

Poster

P-1-2-060

P-1-2-063

EXCITON INTERACTION AND SPECTRAL BROADENING IN PIGMENT-PROTEIN COMPLEXES: THE USE OF SPECTRAL MOMENTS FOR THE INTERPRETATION OF CIRCULAR DICHROISM SPECTRA

OXYGEN-DEPENDENT BACTERIOCHLOROPHYLL FLUORESCENCE IN CHLOROBIUM SPECIES

Oscar J. G. Somsen, Rienk van Grondelle and Herbert van Ameron~,en Dept. of Physics and Astronomy, Vrije Universiteit, De Bcelelaan 1081, 1081 HV Amsterdam, The Netherlands Absorption and circular-dichroism (CD) spectra of pigment-protein complexes depend on pigment-pigment and pigment-protein interactions. Pigment-pigment interactions for instance lead to exciton splitting whereas pigment-protein interactions lead to the shifting and broadening of absorption and CD bands. The spectral effects caused by pigment-protein interactions are not well understood and complicate the interpretation of CD spectra. We demonstrate that with the use of spectral moments information can be extracted from the CD spectra that is independent of pigment-protein interactions. It will be demonstrated that the variability in the CD spectra of chlorosomes from green bacteria can be explained by small variations in the cylindrical organization of the BChl c aggregates.

TRANSGENIC TOBACCO PLANTS EXPRESSING RECOMBINANT LHCII

~ , M. Miller and R. P. Cox. Institute of Biochemistry, Odense University, Campusvej 55, 5230 Odense, Denmark The photosynthetic green sulfur bacterium Chlorobium is strictly anaerobic but appears to survive oxygen exposure without having superoxide dismutase or catalase activity. Under anaerobic conditions the bacteria show high fluorescence from bacteriochlorophylls (BChl). This fluorescence is quenched within a few minutes when oxygen is introduced. Oxygen is consumed by the bacteria, probably by spontaneous reactions with intracellular reductants. When oxygen is removed, fluorescence is restored in a few minutes in the light. We have investigated the relationship between fluorescence, oxygen concentration, and oxygen uptake in BChl c containing C. tepdium and BChl e containing C. phaeobacteroides grown in continuous culture. The fluorescence quenching might involve a decreased conversion of light energy to chemical energy. This would decrease the reduction of molecular oxygen to harmful oxygen species.

P-1-2-061

P-1-2-064 PIGMENT ORGANIZATION IN CHLOROSOMES - THE ANTENNA SYSTEM OF GREEN BACTERIA. RE-ORGANIZATION OF BACTERIOCHLOROPHYLL c FOLLOWING DISRUPTION WITH HEXANOL.

plalf Flachmann and Warner K0hlbrandt, European Molecular Biology Laboratory, Meyerhofstr.1, D - 69117 Heidelberg The structure of the abundant light-harvesting chlorophyll a/b protein complex (LHCII), which is associated with photosystam II, has been determined at 3.4 A resolution. To experimentally test the model and to understand the energy transfer from LHCII to the reactions centres, we created trensgenic plants expressing recombinant proteins.These proteins, attached to an affinity tag of six histindines, accumulate in the thylakoid membrane and copurify with the endogenous trimeric LHCII. Native LHCII is separated from those trimers which contain at least one recombinant monomers by Ni-chelate chromatography. Trlmers, recombinant for one, two or threemonomers, are eluted, dissociated into monomers, rechromatogrephed and reassociated into fully recombinant trimars. These trimars crystallize under conditions very similar to those found for the native trimers. We are in the process of collecting electron diffraction patterns at high resolution.

M. Miller I, R.P. Cox 1, M. Mimuro2 and K. Matsuura 3 ~Institute of Biochemistry, Odense University, Odense, Denmark National Institute for Basic Biology, Okazaki, Japan 3Department of Biology, Tokyo Metropolitan University, Tokyo, Japan It is now generally agreed that the bacteriochlorophyll (BChl) molecules in the chlorosomes of green photosynthetic bacteria are organized as supramolecular aggregates of chromophores with minimal influence from proteins. These aggregates absorb at longer wavelengths (740 nm) than monomeric BChl (670 nm). Compounds such as n-hexanol reversibly disrupt these aggregates, causing the absorption spectrum to change to that of the monomers. We have investigated the organization of these aggregates in isolated chlorosomes from the green sulfur bacterium Chlorobiurn tepidum by studying the kinetics of the re-organization of BChl c following dilution of hexanol-treated chlorosomes with aqueous buffer, using both stoppedflow spectrophotometry with a diode-array detector and circular dichroism spectroscopy. The results indicated that the Bchl c molecules within the chlorosomes can exist in two types of aggregate with different absorption maxima and CD signals.

P-1-2-062 BACTERIO~ROPHYI~ CHLOROBIUM TEPIDUM

C HOMOLOG DISTRIBUTION

IN

P-I-2-065 Pg E F F E C T ON T H E S U P R A M O L E C U L A R A S S E M B L I E S OF B A C T E R I O C H L O R O P H Y L ~ C A G G R E G A T E S IN A Q U E O U S O R G A N I C M E D I A

K. Uehara I T. Ishii I & M. Mimuro 2

] ~ , f , ~ g a a i d , R.P. Cox & M. Miller. Institute of Biochemistry, Odense University, Campusvej 55, DK-5230 Odense M, Denmark.

IDepartment of A p p l i e d Bioscience, Institute of A d v a n c e d S c i e n c e and Technology, U n i v e r s i t y of O s a k a Prefecture, i-2 G a k u e n c h o , Sakai 593, Japan; ~National Institute of Basic Biology, Myodaiji, Okazaki 444, Japan

The m~in light harvest~ ploment fmmd in the groin sulfur bacterium Chlorobium tepidum, BChl c is composed of different homologs m.inly varying in the substituents on the porphyrin ring. The importance of the composition and the nature of these homologs was investigated. Cb. tepidum was grown in continuous culture under light-limited conditions, (light intensities between 5 ttmol m"2s"1 to 100 ~mol m"2s"1) at a dilution rate of 0.04 ht . Pigments from whole cells were analyzed by revexse-phaseHPLC and idevfifled by mass spectrometry. BChl c was found to consist of mixture of different homologs. 8-ethyl- 12-ethyl farnesyl BChl c and 8-propyl- 12-ethyl famesyl BChl e accounted for most of the BCId c in Cb. tepidum. In order to change the homotog distribution long chain alcohols were added to the growth mediu~ This resulted in replacement of the esterifying alcohol in 35% of the BCId c. Attempts will be made to determine the spatial distn'oution of the various BChl c homologs within the chlorosomes.

Formation of a r t i f i c i a l a g g r e g a t e s of b a c t e r i o c h l o r o p h y l l c (BChl c) in a q u e o u s organic m e d i a was studied from the viewpoint--of pH The 6 % o r g a n i c solvent showed the largest pH effect, effect. i r r e s p e c t i v e of o r g a n i c s o l v e n t s (THF, DMSO, MeOH and acetone). Two BChl c homologs, [E,E]BChl c F and [P,E]BChl ~F, showed different--aggregation b e h a v i o r ~ n d e r the low pH c o n d i t i o n (pH 4.49), both h o m o l o g s showed a higher aggregate, as i n d i c a t e d by a large r e d - s h i f t of the a b s o r p t i o n maximum, while the latter was more red-shifted. Under a high pH c o n d i t i o n (pH 9.18), f o r m a t i o n of a higher order a g g r e g a t e was s u p p r e s s e d p r e s u m a b l y by a ligation of Mg a t o m w i t h solvent m o l e c u l e s or OH- ion, even though a lower d e g r e e of a g g r e g a t i o n was observed. These s u g g e s t e d that a g g r e g a t e units in a low degree a s s e m b l e t~ a higher ordsr a g g r e g a t e by the h y d r o g e n b o n d i n g b e t w e e n 13 -keto C=O and C3~-OH g r o u p w h i c h further c o o r d i n a t e s to Mg a t o m in another BChl ~ molecule. Under a low pH c o n d i t i o n (pH 4.49), p h e o p h y t i n i z a t i o n of BChl c was i n h i b i t e d even after 1 h i n c u b a t i o n at r o o m temperature. This indicates that Mg atoms in higher order a g g r e g a t e s was p r o t e c t e d from the e x c h a n g e of a proton, which s u p p o r t s the above m e n t i o n e d structure of higher order aggregates.

47

Poster

P-1-2-066 IN

VITRO

RECONSTITUTION

WITH

PIGMENTS

OF

MAIZE

PHOTOSYSTEM H ANTENNA CP29. E. Ginffra, D. Cugini, K Cr0ce & R. Bassi Universi~ di Verona - Facol~ di Scianze MM FF NN - Istituto Policatledra. Strada Le Grazie - 37134 Verona - Italy. CP29 is one of the minor chlorophyll a/b binding protein of green plants photosystem II. It has been localized in an intermediate position between the chlorophyll a binding core and the major antenna complex LHClI, according to its involvement in at least two regulative mechanisms in excitation energy distribution: (i) it has ben shown to be one of the major xantophyll cycle binding proteins and (ii) to be reversibly phosphorylated in photoinhibitory conditions. In order to investigate the physiologically relevant characteristics of the molecule, the CP29 eDNA has been overexpressed in E. coli and the recombinant apoprotein reconstituted with the thylakoid pigments. The recostituted protein has ~ i c and biochemical characteristics similar to the native complex and is suitable for biophysical analysis. Both ehlorephyll a, chlorophyll b and xantephylls are required for the assembly of a stable complex; however, CP29 seems to be less selective than LHCII with respect to pigment composition and ratios in the reconstitution mixture.

P-1-2-067 EVIDENCE FOR INCREASED SENSITIZATION OF P S I BY LHC H

UPON TRANSITION TO STATE 2 R. Delosmet, J. Olive2 & F.A. Wollmant

JInstitut de Biologic Physicochimique, 13 rue Pierre et Marie Curie, 75005 Paris; rlostitut Jacques Monod, Universit6 Paris VII, 2 place Jussian, 75251 Paris codex 05, France

Chlamydomonas reinhardtii undergoes extensive antenna reorganization upon state transitions in vice. About 50 % fluorescence quenching is commonly observed upon state 1 to state 2 transition, which seems much larger than what has been reported for higher plant chloroplasts. The molecular basis for state transitions is a reversible association of PSII with antenna proteins. The latter tend to dissociate from PSII in their phosphorylated state (state 2). We suspected that phospbo-CP26 and phospho-CP29, which are produced upon transition to state 2 ia C. reinhardtii but not in higher plants, might be responsible for these extensive changes. However, by immunocytochemistry, we found no evidence for a lateral displacement of CP26 from PSII to PSI regions upon transition to state 2, whereas LHCII displacement was observed. Photoaconatie spectroscopy demonstrated that about 2/3 of LHCII disconnects front PSII upon transition to state 2. Moreover, this mobile LHCII then connects efficiently with PSI. Mutants lacking eytochrome bef complex did not show such antenna reorganization, in agreement with previous reports that eyt bef mutants do not undergo state transitions. Mutants lacking PSI still show increased LHCH phosphorylation and some LHCII lateral migration upon transition to state 2. However, photoacoustie spectroscopy suggests that all antenna proteins of these mutants remain connected with the PSII pigment bed in both states.

P-1-2-068 CRYSTALLISATION OF THE LIGHT HARVESTING C O M P L E X LH2 (B800-850) F R O M R U B R I V I V A X GELATINOSU$. I.B. A r e l l a n o , E. H a l l o r e n & R. J. Cogdell. B i o c h e m i s t r y Dept., U n i v e r s i t y of G l a s g o w , G l a s g o w G12 8 Q Q UK. To u n d e r s t a n d the m o l e c u l a r details of the l i g h t h a r v e s t i n g p r o c e s s in p u r p l e b a c t e r i a , LH2 c o m p l e x f r o m Rubrivivax gelatinosus w a s p u r i f i e d a n d c r y s t a l l i s e d w i t h t h e a i m of u s i n g X - r a y c r y s t a l l o g r a p h y to d e t e r m i n e its s t r u c t u r e . The p h o t o s y n t h e t i c membranes were solubilized with the detergent lauryldimethylamine-N-oxide and loaded onto a sucrose gradient. The L H 2 b a n d s w e r e p o o l e d a n d a p p l i e d o n t o a S u p e r d e x 200 m o l e c u l a r s i e v e c o l u m n . T h o s e fractions w h i c h s h o w e d a r a t i o of the a b s o r b a n c e at 8 5 0 n m ( d u e to b a c t e r i o c h l o r o p h y l l a) to t h a t at 2 7 0 n m ( d u e to a r o m a t i c a m i n o a d d s ) h i g h e r t h a n 3.0 w e r e collected for c r y s t a l l i s a t i o n trials. T h e s e trials w e r e c a r r i e d o u t v a r y i n g the p H , the t y p e a n d c o n c e n t r a t i o n of the d e t e r g e n t s , the a m p h i p h i l e s a n d the p r e c i p i t a n t s . Details of the best c o n d i t i o n s a n d the v a r i o u s c r y s t a l t y p e s p r o d u c e d w i l l b e p r e s e n t e d i n the poster. This w o r k w a s s u p p o r t e d b y a g r a n t from the EC a n d the BBSRC.

48

P-1-2-069 F E M T O S E C O N D E N E R G Y T R A N S F E R KINETICS IN I N T A C T C H L O R O S O M E S AND B C H L c A G G R E G A T E S F R O M G R E E N PHOTOSYNTHETIC BACTERIA S. Savikhin 1, P. I. van Noort 2, Y. Zhu 2, R. E. Blankenship2, and W. S. Struve 1 lDepartment of Chemistry, Iowa State University, Ames, IA 50010, USA; 2Department of Chemistry and Biochemistry and Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, AZ 85287, USA Femtosecond pump-probe spectroscopy has been used to compare the electronic energy transfer kinetics of intact chlorosomes and BChl c - lipid aggregates from the green photosynthetic bacteria Chloroflexus aurantiacus and Chlorobium tepidum. There are close parallels between the BChl c anisotropy kinetics, coherent oscillations, and femtosecond spectral evolution of the intact chlorosomes and their corresponding aggregates. Hence, the internal BChl c energy transfer functions in the protein-free aggregates appear to replicate those in the intact chlorosomes. However, the residual pump-probe anisotropies at long times suggest that BChl c Qy transition moments in the reconstituted aggregates show more long-range disorder than in the intact ch]orosomes.

P-1-2-070 On the dependence o f L H C I I fluorescence lifetimes on temperature

M. Seydack, H. Redlin and J. Voigt Institute of Physics, Humboldt-University Berlin, D-10099 Berlin, Germany Measurements of the fluorescence kinetics with time resolution down to the ps range provide information on lifetimes of excited states. To uniquely establish a kinetic model describing the processes of energy transfer and relaxation in the light harvesting complex, however, such data are not sufficient. In our poster we present additional information resulting from measurements of the temperature dependence. For the first time we investigated the fluorescence kinetics of disaggregated LHC II trimers in the broad temperature range from 300 K down to 16 K. At all temperatures the decay courses obtained by time correlated single photon counting with 50 ps instrument response function can well be approximated by a sum of fuur exponentials. Both the lifetimes and the amplitudes of the two ns-eomponents show a distinct temperature dependence. The lifetimes increase with temperature falling from RT to 80 K by 60% and 200 % respectively and decline slightly below 80 K.

P-1-2-071 NON-LINEAR POLARIZATION SPECTROSCOPY OF LIGHTHARVESTING COMPLEX H: ABSORPTION BAND SUBS T R U C T U R E AND ULTRA-FAST E X C I T O N DYNAMICS H. Lokstein I , D. Leupold 2, F. Nowak 2, B. Voigt & P. Hoffmannl lInstitut ftir Biologie, Humboldt-Universitttt, Unter den Linden 6, D-10099 Berlin, Germany; 2Max-Bom-Institut, PF 1107, D-12474 Berlin, Germany Spectral substructure and ultrafast excitation dynamics have been investigated in the chlorophyl (Chl) a and b Qy region of isolated monomeric, trimerie and macro-aggregated pea (Pisunt sativum L.) fight-harvesting complex H (LHC II). The feasibility of Nonlinear Polarization Spectroscopy in the frequency domain (NLPF) - a novel in photosynthesis research laser spectroscopic technique - to determine population relaxation (T1) and dephasing (T2) times as well as the complex spectral substructure in the Qy band and the mode(s) of absorption band-broadening at physiological temperatures is demonstrated. Of particular interest is the differentiated participation of the "Chl-forms" in energy transfer in the various aggregation states. T2-values are in the range of a few 10 Is. Inhomogeneous broadening (if significant at all) does not exceed the homogeneous widths of the subbands. The impfications for energy transfer mechanism(s) are discussed.

Poster

P-1-2-075

P-1-2-072 NONPHOTOCHEMICAL QUENCHING OF CHLOROPHYLL FLUORESCENCE IN LEAVES: INFLUENCE OF PHOTOSYSTEM H ANTENNA SIZE AND VIOLAXANTHIN DE-EPOXIDATION Heiko Hartel & Heiko Lokstein Institut ~ r Biologie/Lehrstuhi ftir Pflanzenphysiolngie, Humboldi-Universitat zu Berlin, Unter den Linden 6, D-10099 Berlin, Germany The ability to develop nonphotochemical chlorophyll (Chl) fluorescence quenching (NPQ) was studied in barley (Hordeum vulgate) leaves comprising a step-wise reduced antenna size o f photosystem II (PS II). In parallel, xanthophyll-cycle kinetics were followed to ascertain the role o f violaxanthin (V)-de-epoxidation in this process. Proportions o f overall NPQ arise from (i) the peripheral fight-harvesting complex ofPS II (LHC II), (li) the inner LI-IC H antenna, and (lii) the core complex o f PS II. The presence o f Chl a/b-binding (CAB)-proteins influences both the availability o f V for de-epoxidation as well as the kinetics o f V conversion into antheraxanthin (A) and zeaxanthin (Z). Regardless o f both an accelerated and increased A and Z accumulation NPQ was step-wise reduced in parallel with antenna size. The NPQ level obtained after treatment o f leaves with the V-de-epoxidase inhibitor dithiothreitol was almost identical in the presence and complete absence o f LHC II. Thus, xanthophyll cycle-mediated structural changes within the CAB-proteins render the basis for the development o f the major proportion o f NPQ.

REGULATION OF COMPLEMENTARY CHROMATIC ADAPTATION (CCA) IN CYANOBACTERIA: INVOLVEMENT OF A RETINALPROTEIN (RHODOPSIN) AS PRIMARY PHOTORECEPTOR ? Jeroen H. Geerdink, Andrea Haker, Hans Balke, Hendrik Schubert, Wouter D. Hoff, Hans C.P. Matthijs, Klaas J. Hellingwerf and Lunc R. Mur Department of Microbiology, University of Amsterdam Nieuwe Achtergracht 127, 1018 WS Amsterdam. The primary receptor used in sensing of the light-colour in CCA is still unknown. Indications have been found that retinal might be the ehromophor in the photoreceptor involved. Retinal could be isolated from Fremyella diplosiphon. Nicotine, a known inhibitor of autogenic retinal synthesis, was shown to inhibit chromatic adaptation. Addition of synthetic retinal reconstituted nicotine inhibited adaptation. To ehidicate the exact role of retinal, additional adaptation studies will be shown, using retinal and retinalanalogs at different concentrations, and various light-switches. The formation of a retinal-protein complex has been monitored from spectral changes during mixing of synthetic retinal and -analogs with differently pretreated cells and cell extracts, and by use of radiolabeled retinal in reconstitution followed by resolution of the rhodopsin via SDS-PAGE.

P-1=2=076

P-1-2-073 QUENCHING OF CHLOROPHYLL FLUORESCENCE IN THE CHLOROPHYLL A/B BINDING PROTEINS OF PHOTOSYSTEM II

MINOR

A.V. Ruban ~, A.J. Young ~ & P. Horton ~ I Robert Hill Institute, Dept of Mol Biol & Biotech, University of Sheffield, P.O. Box 594, Sheffield $ I 0 2UH, U.K.;2School of Biological and Earth Sciences, Liverpool John Moores University, Byrom Street, Liverpool, U.K. An in vitro model is described for the nonphotochemical dissipation of absorbed excitation energy, qN, the physiological process that protects plants from photoinhibition; LHCII from spinach leaves exhibit strong pHstimulated quenching of chlorophyll fluorescence when they are diluted to a low detergent concentration (Ruban et al [1994], BBA 1186, 123-127); this quenching shares many features observed for qN in vivo and similar behaviour was shown by all LHCII types. The two minor complexes, LHCIla (CP29) and LHCIIc (CP26), showed the strongest quenching which was also inhibited by DCCD. The carotenoids violaxanthin and zeaxanthin, whose interconversion is known to control qN in vivo, cause inhibition and stimulation of quenching respectively. The results of this study are consistent with the suggestion that these minor pigment-proteins have an important role in qN, and that protonation and viol/zea interconversion synergistically control qE, as originally suggested in our LHCII model for qN (Horton et al [1991] FEBS Letters 292, I-4).

P-1-2-074 DCCD BINDS TO LUMEN-EXPOSED GLUTAMATE RESIDUF~ IN LHCH¢ Robin G. Waiters, Peter Horton and Alexander V. Ruban Robert Hill Institute, University of Sheffield, UK Binding of dicyclohexylcarbodiimide (DCCD) to the minor light-harvesting complexes LHCIIa and LHCIIc specifically inhibits the protective dissipation of excess absorbed light energy. LHCIIc from spinach has been labelled with [t4C]-DCCD and the apoprotein digested with trypsin or cyanogen bromide; microsequencing of the resulting labelled fragments has localised two DCCD-binding sites to the lumen-exposed regions of LHCIIc. Analysis of the counts released during sequencing shows that two glutamate residues are labelled - those corresponding to Elm and E21s in tomato. By homology with the published structure for LHCIIb, El10 is beyond the Cterminal end of the "B" transmembrane helix and E.2ts is on the amphiphilic "D" helix, both sites being potentially exposed to the lumen. These residues may therefore be involved in the detection of the lumen pH, which is the primary signal responsible for inducing energy dissipation. These residues are not present in LHCIIa, which has conserved glycine and asparagine residues at the corresponding positions. A similar approach is being used to identify the DCCD-binding site(s) on LHCIIa.

ENERGY TRA~NSFER AND A.NTENNA ORGANIZATION IN TILE PURPLE NON.SULFUR BACTERIUM RIIODOPSEUDOMONAS

CRYPTOIACI'L~. H. Kramer ~, ( i I)emum 1, A.T. Oardmer ~. R.J. Cogdell "+, C Ft,mcke ~, T.J. AaJl~tl~ ~ ar'~d J. Amesz I Dep~Unvnt of Binphysics. Umversity of leiden, P.O. Box 9504, 2300 RA Leiden, the Ne,,herlan~ and ~ Depaxmmn: of Bo~an'~. Umversity of GFa~gow, G12 8QQ, Glasgov,, U.K Different growth conditions of Rhodopseudomom2~ cryptot~c:~vrest:h~d in. tha+tsc cultures whi~h di_fl~rcd in the ¢ompo~ition of the peripheral antenna (B800 820, B800-850 or a .u.axtum of both). Vaaiabk fluorescen~.-emeasur:ment~ iqdieat::d energy a'an~fer from B880 to B800.850, but nor to B800-820. Sing,let singlct a.nrdhitut.:on m¢,a.su,','ee'~e~tsrevealed d.~main sizes for 13~(1 of 40 to 70 hacte6ochlurnphyl!s correspondh~g to oae or two photosynthc~c units only. These resnhs art: explained by '+ model for Rps. cryptolactls in wMeh each B880 unit is associated with its own complement of B800-850 withou~ an ext=nsive intercont,ecling fretwork. Time resolved transient absorption measurements show.:A trapping rams by open and closed reacnoo ccu~us el (50 ps)' and (200 ps) t, respectively. Time cons|ant': of energy transfer from B800850 to 138~0 or from B800-820 to o~¢r complexes were 12-15 ps.

P-1-2-077 Protonation or eheadeal mod~teafion of a His re~dne efidts complete blue shift of LH2 800-rim baeteriodflorophyll J.M. Ramfrez & C. Fern(mdez-Cabrera Centre de Investigaciones Biol6gicas, Veh~zquez 144, 28006 Madrid, Spain

In the light-harvesting protein 2 (I.H2) of purple photosynthetic bacteria, two populations of bacteriochlorophyll (Bchl) exhibit ballds near 800 and 850 nm respectively, while the band of the free pigment is located near 770 nm. The 800-am Bchl (cytoplasmic) domain of the Rhodobacter capsu/atus LH2 protein was selectively unfolded at high urea concentrations and, in the unfolded state, the 800-ore Bchl band shifted to 770 nm at low pH's. The par(, of the transition ranged between 6 and 7.5, depending on urea concentration. This suggests that the protonation of a His residue was involved in the spectral shift. The conclusion is supported by the additional observation that diethylpyrocarbonate, a His-modifying reagent, inhibited at basic pH's the reversal of the acid-elicited blue shift. Since the protein contains a single His residue in the cytoplasmic region, #His20, the present results are in agreement with the participation of that residue in 800-nm Bchl binding.

49

Poster

P-1-2-081

P-1-2-078

I N ~ STRUCTURAL FLEXEBIIJTY OF CHIRAL MACROAGGREGATES OF CHLOROPHYLL A/B COMPIJEXF~. LIGE[T-INI~CI~ ~ L ~ STRUCTURAL CHANGES ASSOCIATED WITH ENERGY DISSIPATION

GENETIC MANIPULATION OF Lhcb4, A GENE ENCODING ONE OF THE MINOR LIGHT-HARVESTING COMPLEXES, IN ARABIDOPSIS THALIANA

Q. Garab, V. Barzda, A. Istokovics and I. Simidjiev Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged, P. O. Box 521, H-6701, Hungary

C.A.McAuley 1, T.A.Dyer 2 and P.Horton 1. 1Robert Hill Institute, University of Sheffield, Sheffield, S10 2UH, UK. 2Cambridge Laboratory, John Innes Centre, Norwich, NR4 7UJ, UK

In chloroplasts when exposed to excess light a protective mechanism of energy dissipation is activated. The relatively slow response time (about 1 rain) of the regulation upon turning on and off the intense illumination suggests the involvement of-reversible structural changes. By the aid of circular dichroism (CD) measurements we identified changes in the chiral macroorganization of PS2 particles and show that they are associated with fluorescence (F) changes characteristic to an increased energy-dissipation. Further, we show that lamellar macroaggregates of purified LHCII also exhibit light-induced ACD and AF which closely resemble those in thylakoid membranes. The lightintensity-dependent structural flexibilty of LHCII-containing macroaggregates is proposed to be responsible for the regulation of the photophysicai pathways in the antenna of granal chloroplasts. The underlying physical mechanism is most likely the long-range coupling of chromophores in densely packed macroaggregates with chiral order.

Lhcb4 encodes LHCIIa, one of the minor light-harvesting complexes associated with Photosystem II. The precise function of LHCIIa is, at present, unknown, but it is speculated to have a role in linking the bulk LHCIlb complexes to the reaction centre. However, LHCIIa also binds DCCD, Ca2+ and a relatively large amount of xanihophyll cycle carotenoids in comparison with the bulk LHCII, which may be indicative of a role in energy dissipation. In an attempt to understand the function of LHCIIa, the gene Lhcb4 is being manipulated using antisense and overexpression techniques, with a view to altering the amount of LHCIIa protein present. Arabidopsis thaliana is being transformed with vectors containing the Lhcb4 gune either in a 5'-3' or 3'-5' orientation. Plants containing altered amounts of LHCIIa will be characterised.

P-1-2-079

P-1-2-082

EVIDENCE FOR LONG RANGE ENI~GY MIGRATION IN THE MACROAGGREGATI~ OF LIGHT HARVESWINGCHLOROIqIYI~ A/B PIGMENT PROTEIN COMPLEXES

FEATURES AND FUNCTIONS OF MINOR CHLOROPHYLL A/B-BINDING PROTEINS IN LIVERWORT (MARCHANTIA POLYMORPHA)

V. Barzda t'2, G. Garab 1, V. Gulbinas2 and L. Valkunas2 1Institute of Plant Biology, Biological Research Center, Szeged, Hungary. 2Institute of Physics, Vilnius, Lithuania. We investigated the picosecond transient absorhance kinetics under sihgletsinglet annihilation conditions and the steady-state spectroscopic features (absorbanco, circular dichroism and low temperature fluorescence spectra) in large, 3-dimensional, long-ranged chiral, so-called psi-type aggregates of the light harvesting chlorophyll arc complexes (LHCII) isolated from chloroplasts, and its form of small aggregates. We show that the macroorganizational parameters significantly influence the energy migration pathways in the aggregates. In small aggregates excitation energy migration can be characterized by a percolation type of excitation migration in a small cluster of chromophores, whereas in macroaggregates the annihilation occurs in large structures with an estimated excitation migration radius of at least 640/~. This shows that psi-type aggregates of LHCH constitute a structural basis for long-range migration of the excitation energy.

P-1-2-080 LIGHT-HARVESTING CHLOROPHYLL A/B-BINDING PROTEIN INSERTED INTO THYLAKOIDS BINDS PIGMENTS AND IS ASSEMBLEDINTO TRIMERIC LIGHT-HARVESTING COMPLEX A. Kuttkat & H. Paulsen Botanisches Institut Ill der Universit~t, Menzinger Strage 67, D8 0 6 3 8 M0nchen, Germany The light-harvesting chlorophyll a/b-binding protein (LHCP) is largely protected against protaase (except for about 1 kD on the N terminus) in the thylakoid membrane. This protease resistance, which is often used to assay successful insertion of LHCP into isolated thylakoids in vitro, is exhibited by trimeric LHCII but not by monomeric LHCII in which about 5 kD on the N terminus of LHCP are cleaved off by protease. When a mutant version of LHCP which is unable to trimerize in an in-vitro reconstitution assay is inserted into isolated thylakoids, it gives rise only to the shorter protease digestion product indicative of monomeric LHCII. It is concluded that more of the N-terminal domain of LHCP is shielded in trimeric than in monomeric LHCll. Upon insertion of LHCP into isolated thylakoids at least part of the protein spontaneously binds pigments to form LHCII which then is assembled in trimers. The dependence of the protease sensitivity of thylakoid-inserted LHCP from the oligomerization state of the newly formed LHCII justifies caution when using a protease assay to verify successful insertion of LHCP into the membrane.

50

R. Kilian & C. SchOfer, Lehrstuhl Pflanzenphyslologie, UniversitOt Bayreuth, 95440 Bayreuth, Germany The minor chlorophyll a/b-binding proteins of angiosperms have been studied intensively during the past few years, partly because they appear to have a protective function during light stress. Only few studies considered the occurrence and functions of such proteins in lower plants. In this context liverwort is of special interest because it represents in many respects the transition state between lower and higher plants. Our earlier studies have shown that M. polymorpha cells react to light stress with a pronounced increase in xanthophyll cycle pigments by de novo synthesis. Three minor chlorophyll a/b-bindlng proteins could be detected which reacted with antibodies against LHCIIa, LHCIIc and LHCIId. Their electrophoretic and immunological properties as well as their pigment contents were studied in control cells and in high light treated cells. As compared to higher plants the minor chlorophyll a/b-binding proteins In M, polymorpha have a lower apparent molecular weight, a higher isoelectrlc point, and a stronger cross reactivlty with LHCI antibodies. Considerable amounts of xanthophyll cycle pigments are bound to the proteins, and we got indications that the xanthophyll/protein ratio increases during prolonged high light stress of the celts.

P-1-2-083 Resonance Raman studies on LHC2 complexes from higher plants Truong, K., Pascal, A., Horton 1, P & Bruno Robert. SBPM/DBCM CEA and URA 1290 CNRS, Centre d~tudes de Saclay, 91191 Gif sur Yvette Cedex, FRANCE. 1 : Robert Hill Institute, Department of Molecular Biology and Biotechnology, University of Sbeffidd, PO Box 594, Sheffield $10 2UH, UK Resonance Raman experiments at various wavelengths have been performed on IMC2b and minor, LHC2 a, c and d complexes from spinach. By using excitations favouring the contributions of Chl b molecules in these complexes, the interaction states of their formyl groups could be determined in the 16251660 cm-1 range. In this region, RR spectra of minor fight-harvesting complexes are considerably simpfified, in fair agreement with the most recent Chl b stoichiometries determined for these proteins. Whereas in LHC2b more than half of these groups are strongly H-bonded, vibrating at 1630 cm-1, this frequency is not observed in spectra of the minor complexes and all of these contain a strong 1640 cm-1 band, accounting for medium-strength interaction between the formyl groups present and the protein.

Poster

P-1-2-084 Optical excitation and relaxation of two-exciton states in light harvesting pigment-protein complexes of purple bacteria H. Redlin ~. Th. Renger', B. N. Korvatovsid 2, V. Z. Paschenko 2 and J. Voigt I Institute of Physics, Humboldt-University Berlin, D 10099 Berlin, Germany 2 Faculty of Biology, Moscow State University, Moscow 119 899, Russia Energy transfer and relaxation in antennae systems of photosynthetic organisms are current problems being studied at present as well experimentally by ps and sub-ps laser spectroscopy as theoretically by quantum mechanical model calculations basing on new structural data. In this paper both methods are applied to explore two-axciton excitations in the fight harvesting complex of purple bacteria that can be expected according to the model of strong dipole-dipole interaction between pigments. The absorption difference measured at 590 nm in a pump-probe experiment at high pump intensities (up to l0 .2° photons/era = *pulse) shows a transient bleaching in a time range of l0 to 30 ps which can be related to the two exciton states. The amplitude of the bleaching depends on the pump intensity in a non-linear way. The experimental findings will be compared with a model of a dimer of strongly coupled pigments computed in the framework of density matrix formalism. In the model energy transfer and relaxation can be described in an appropriate way by including exciton-phonon interaction and the interaction with a thermal bath.

P-1-2-085 STRUCTURAL INFORMATION ON LHCII AS OBTAINED FROM EXC1TON CALCULATIONS AND POLARIZED SPECTROSCOPY. Demat GiJlen~b, Rienk van Grondelle a and Herbert van Amerongen a aDepartmcnt of Physics and Astronomy, Vrije Universiteit, Amsterdam, The Netherlands and bDepartment of Physics, METU, Ankara, Turkey. The 3.4 A ° resolution structure of LHCII, the major light-harvesting complex of the green plants, reveals a very densely packed arrangement of chlorophylls and carotenoids [1]. This structural aspect and the spectroscopic results [2] both indicate strong chlorophyll interactions (excitonic) and fast energy transfer. The two current sources of unc,ertainRy of the structure -- the identities of the chlorophyUs and the directions of the transition dipole moments -- lead to a large number of possible configurations for addressing any question that relates to the objective of structure-function relationship determination. It will be discussed how the combination of exciton calculations and polarized spectroscopy can substantially decrease the numbe~ of possibilities. Ill K ~ b r a n d t W., Wang D.N. and Fujiyoshi Y., Nature 367, 614-621 (1984). [2] Nussberger S., et al., Biochemistry, 14775 - 14783, 1994 and the references therein.

51

Poster P-3-4-003 DIRECT

AND

PHOTODYNAMIC

INDIRECT

EFFECTS HERBICIDE, ON

OF 2.2'-BIPYRIDYL, A PHOTOSYNTHETIC APPARATUS

A. M o s t o w s k a . M. S i e d l e c k a & E. P a r y s Dept. of Plant Physiology II, W a r s a w University, Krakowskie Przedmiescie 26/28,00-927 Warsaw, Poland.

Poster session 3-4

T h e e f f e c t o f l o w (1-5 mM) d o s e s o f 2 . 2 ' - b i p y r i d y l (2.2'-B) on the development of c h l o r o p l a s t s and of higher (5-30 mM) doses on ultrastructure of already differentiated chloroplasts, pigment content, CO2 exchange and primary photochemistry o f P S II i n p e a seedlings was examined. 2.2'-B caused inhibition of greening in etiolated and subsequently illuminated pea seedlings. Green pea plants were morphologically resistant t o 5 m M o f 2 . 2 ' - B b u t i0 a n d 30 m M 2 . 2 ' - B with illumination caused loss of turgot and photooxidative damages. 2.2'-B caused two groups of reactions: rapid - directly after exposure to light, measured by changes of chlorophyll fluorescence and of photosynthetic activity rate, and slow consisting of destructive changes in mesophyll cell ultrastructure and a decrease of pigment content. We consider PS II as a primary target of 2.2'-B action and all other dysfunctions as a consequence o f t h e PS I I d a m a g e .

Reaction centers: purple bacteria and PSII P-3-4-001 - P3-4-128

P-3-4-001 NEN CONCEPT OF EXCITATION PURPLE BACTERIA. A. Yu. B o r i s o v MOSCON S t a t e U n i v e r s i t y ,

TRAPPING

Bldg "A",

IN

REACTION

CENTERS

OF

119899 Moscow, R u s s i a .

A new model o f t h e purple bacteria reaction center (RE) i s developed. It represents a charge transfer state of t h e RC s p e c i a l p a i r a c c o m p a n i e d by t h e di-polaron ~hich arises in s e v e r a l h u n d r e d s o f f e m t o s e c o n d s and p r e c e d e s t h e ~ e l l - k n o ~ n ~-state. T h i s new s t a t e m a n i f e s t s t h e p r i m a r y e x c i t a t i o n trapping. The model c l a i m s t o a c c o u n t f o r a number of findings still w a i t i n g f o r an e x p l a n a t i o n : a) r e c e n t d a t a on a s m a l l excitation p o r t i o n which e s c a p e s f r o m t h e e×cited RC s p e c i a l b) t h e r e a s o n why i n purple p a i r back t o a n t e n n a B C h l s ; b a c t e r i a t h e second P-PGOO-BPH-Q b r u n c h o f RC i s n o t a c t i v e ; c ) t h e " r e d " s h i f t of t h e c o r e BChl a b s o r p t i o n peaks r e l a t i v e to those in their c o r r e s p o n d i n g RCs; d) why t h e primary e l e c t r o n d o n o r i s n o t a monomer b u t a p a i r of BChls; e) t h e r e a s o n why t h e f l u o r e s c e n c e s p e c t r a of t h e RC s p e c i a l p a ~ r s a r e enormously red-shifted relative to their absorption spectra. The model was t e s t e d w i t h a s p e c i a l l y d e s i g n e d PC-program and a l l k i n e t i c and e n e r g y d a t a o b t a i n e d proved to be in full accord with the experimental results still being published.

P-3-4-004 NOVEL ETHYLENE-BRIDGeD SIDE-TO-aIDE PORPHYRIN OF THE PHOTOSYNTHETIC ',SPECIAL P A I R "

DIM~RAS

V.S.Chirvony I, M.Chachisvilis 2, A.M.Shulga I, V. Sundstr0m 2 iInst of Molecular & Atomic Physics, 70 F. Skaryna Ave, Minsk, 220072, Belarus; 2Dept of Phys Chemistry, Ume& University, S-90187, Ume~, Sweden Until now, only strongly-coupled sandwich face-to-face metalporphyrin dimers of the type MP 2 (M=lanthanide or actinide ion, P-porphyrin) have been found to exhibit optical properties analogous to the ones of the "special pair". Unexpectedly, very similar spectral properties have been found by us for the sideto-side porphyrin dimer trans-l,2-bis(mesooctaethylporphyrinyl) ethene {tbis=OEP). The observed unusual properties include: (i) additional absorption bands in the 470-500 nm and 600-900 nm regions; (2) broadband and considerably Stokes-shifted fluorescence in the 750-1000 nm region; (3) very fast (5-8 ps) excited electronic state deactivation, the lifetime being strongly dependent on solvent viscosity. We suggest that a contmon p-conjugation through the ethylene bridge is the reason of such an unusual behaviour of the tbis=OEP. Evidences in favor of this suggestion as well as a possible contribution of the charge separation state to the near-IR absorption and emission bands are discussed.

P-3-4-005

P-3-4-002 CHLOROPHYLLa/ORGANIC S~MICONDUCTOR FOR PHOTOINDUCED CHARGE SEPARATION

JUNCTION - POSSIBLE IN REACTION CENTERS

Laura Tu~ulea & S. Antohe Faculty of Physics, Bucharest University, Bucharest - Magurele, 76900 Romania

MODEL

P.O.Box MG-II,

Two-layer photovoltaic cells ITO/Chla/TPyP/A1 have been prepared. The p-n heterojunction was obtained by vacuum deposition of TPyP (n-type semiconductor) thin layer on top of electrodeposited Chla (p-type semiconductor) thin layer. The barrier formed at Chla/TPyP interface is responsible for the observed dark (J-U) characteristics and photovoltaic phenomena. The electrical and photovoltaic properties, especially the action spectra of the cells, are strongly dependent on thickness and morphology of Chla and TPyP layers. The cell photoresponse (photovoltage, photocurrent, power conversion) is mainly determined by the Chla absorbing species forming the thin layer (i.e red peaks at 680 nm or 740 nm on film absorption spectra). The cell behavior, under illumination, suggests that the singlet excitons are the precursors of the charge carriers and that the spacecharge region is at Chla/TPyP interface. Only those excitons created in the barrier region or having diffused into it contribute to the cell photoresponse.

52

A MODEL

NEW PHENOLIC INHIBITORS OF ELECTRON TRANSFER IN PHOTOSYSTEM II Klirnov V.V., 1 Zharmukhamedov S.K., 1 AIIskhverdiev S.I., 1 Kolobanova L.P., 2 Baskakov Yu.A. 2 1Institute of Soil Science and Photosynthesis, Russian Academy of Sciences, Pushehino, Moscow Region; 21nsitute of Chemical Protection of Plants, Moscow

A n e w group of highly-efficient inhibitors (with the inhibitory constant near 5 0 - 7 0 nM) of Pail reactions - hydroxyperfluoroisopropyldinitrobenzole derivatives have been revealed. Their inhibitory effect is based on the redox interaction w i t h Pail reaction centre components leading to a rapid charge recombination between Pheo- and P 6 8 0 + (or Z + ) . The conclusion is d r a w n from the comparison of the effect of these inhibitors on the variable fluorescence, photoreduction of pheophytin, photooxidation of P 6 8 0 and o x y g e n evolution in chloroplasts, PSII membranes and isolated D 1 / D 2 / c y t b 5 5 9 complex.

Poster P-3-4-006 P H O T O I N D U C E D E L E C T R O N T R A N S F E R IN Rb, SPHAEROIDES R-26 REACTION CENTERS WITH BACTERIOPHEOPHYTINS R E P L A C E D BY P H E O P H Y T I N a M O L E C U L E S A.Ya. Shkurovatov. I.I. Proskuryakov & V.A. Shuvalov Institute of Soil Science and Photosynthesis RAS, Pushchino, Moscow region 142292, Russian Federation Electron transfer properties of pheophyfin a - modified reaction centers (RCs) from Rb. sphaeroides R-26 are investigated at room and low temperatures by optical and ESR specuvsonpy. Modified RCs preserve the ability for photoinduced electron transfer fi'om the primary electron donor P to the primary quinoue acceptor QA at 293 K and 80 K with the quantum efficiency of 80% at 293 K. The intermediary state w r displays at 293 K a recombination time constant of 1.5 ns, and the electron transfer from I to QA is characterized by a time constant of 540 ps. At 10 K the ESR signal of triplet P, detected in the modified RCs, possesses electron spin polarization and zero-field splitting parameters similar to those in non-treated RCs.

P-3-4-007 A N E W M U T A T I O N IN P U F L G E N E R E S P O N S I B L E FOR TERBUTRYN R E S I S T A N C E P H E N O T Y P E IN R u b r i v i v a x

gelatinosus. S. Ouchane, M. Picaud & C. Astier. CNRS, Centre de Genetique Moleculaire Photosynthese Bacterienue bat 24, Avenue de la Terrasse 91198 Gif sur Yvette CEDEX FRANCE

Rx.gelatinosus is a facultative phototrophic non-sulfur bacteria belonging to theft subclass of the purple bacteria. A terbutryn-resistant mutant of Rx gelatinosus has been isolated and characterized. Increase in resistance levels to terbutryn (300 fold), atrazine (10 fold) and o-phenanthroline (3 fold) were observed for the mutanLcompured to the wild type. Sequence analysis of the mutant revealed a new mutation in thepujL geue coding for the L subunit of the reaction center (RC) at codon 192 leading to an amino acid substitution from Gly in the wild type to Asp in the mutant. This substitution is located in the D helix of the L subunit, suggesting an interaction betwen terhutryn and this part of the polypeptide in the RC of Rx. gelatinosus. This is the first report of a mutation affecting the D helix in purple bacteria. Furthermore Rx. gelatinosus wild type is highly sensitive to o-phenanthroline compared to other purple bacteria. Sequence comparison of L suhunit from 5 purple bacteria in which o-phenanthroline sensitivity was measured suggests that Ser L226 might be responsible of this phenotype.

P-3-4-008 Long Range Electrostatic Effects in Bacterial Reaction Centers J. D. Delcroix 1, P. Sehbanl~ M. Schiffer 2, P. Marbti 3 and D. K. Hanson 2 1 C.G.M., CNRS, Gif, France; 2 Cente¢ for Mechanistic Biol. and Biotech., Argonne National Lab., II., USA; 3 Institute of Biophysics, Joz~ef Attila University, Szeged, Hungary The bacterial photosynthetic reaction center is one of only two protontransporting transmembraue proteins whose three-dimensionai structure is known to atomic resolution. We have measured the proton uptake stoichiometries and kinetics, different electron transfer reactions and the charge recombination rates in reaction centers from different strains from Rhodobacter caps, latus. These strains are selected (suppressors) or constructed (mutants) from the initial photoincompetent double mutant L212Glu/L213Asp -> Ala/Ala. L217Arg -> Cys, MSAsn -> Asp, L231Arg -> Leu and M43Asu -> Asp compensating additional mutations all situated at more than 10 .~ from the secondary quinone accepter (QB), restore the photosynthetic growth. Our data suggest a main role for the electrostatic potential imposed by the protein in the QB environment and/or in the proton pathway from the cytoplasm to QB. The role of this negative potential would be to increase the pKs of the different groups involved in proton transport and delivery to QB and also to increase the local proton concentration near QB 1,2. 1 Maroti P., D.K. Hanson, L. Baciou, M. Schiffer, P. Sebban (1994) PNAS USA 91, 5617. 2 Sebban P., P. Maroti, M. Schiffex,D. K. Hanson (1995) Biochemistry. This work was supported by North AtlanticTreaty OrganizationGrant CRG920725 and National Science Foundation- Centre Nationalde la RechercheScientificGrantCDP900350.

P-3-4-009 n B a c t e r i a l R e a c t i o n C e n t e r s L 2 1 2 G l u I n t e r a c t s w i t h QA" a t 17

Distance J. Miksovska 1, P. Sebban 1, M. Schiffer 2, D. K. Hanson 2 a n d P. M a r b t i 3 1 Centre de G6n6tique Mol6.culaire, CNRS, (]if, France 2 Center for Mechanistic Biol. and Blotech., Argonne National Lab., II., USA 3 Institute of Biophysics, Jozsef Attila University of Szcgnd, Hungary

In Rhodobacter capsulatus reaction centers (RCs), we have measured the pH dependencies of the proton uptake steichiometries by the QA" and QB" states in three gcactically modified strains which lack L212GIu: the photo incompetent double mutant, L212Glu/L213Asp -> Ala/Ala and two photocompetent derivatives L212GIu -> Ala and L212GIu/L213Asp -> Ala/Ala + M43Asu ->Asp. In contrast to the wild type, the RCs from the three modified strains fail to take up protons above pH 9 upon QA" formation. This suggests long range (direct or indirect) interaction between L212GIu and QA" (17 .~ distant) possibly involving protonation induced rearrangements subsequent to QA" formation. Such long distance connection between two parts of the protein is confirmed by experiments achieved in reaction centers from the same strains, where QA was replaced by low potential quiuene. This work was supportedby North AtlanticTreatyOrganizationGrantCRG920725 and National Science Foundation - Centre Nationalde la RechercheScientificGrant CDP900350. J.M. was supported by the french Minist~edes AffairesEtrang/:res(B.G.F.).

P-3-4-010 LIPID ENVIRO~8~ENTAFFECTS THE HERBICIDE C~RTERS ISOLATED FROM PURPLE BACTERIA

SEnSITIVITY

OF REACTION

L . N a c ~ x, S z . G e d e ~ , J.Tandori I, I . D e ~ , E.Fodor 2, T.Farkas 2 and P.Mar6ti I IJATE University, D e p a r t m e n t of Biophysics, Szeged, POB.655, H6701, 2BRC D e p a r t m e n t of Biochemistry, Szeged, POB.251, H-6701, Hungary Photosynthetic reaction centers (RC) from Rhodobacter sphaeroides R-26 and h e r b i c i d e resistant R/89 strains w e r e r e c o n s t i t u t e d in liposomes c o n s t i t u t e d from p h o s p h o l i p i d (PL) of b o t h species. The free energies for s t a b i l i z a t i o n of P~QAQI- state with respect to P*QA~Qa in detergents, liposomes and c h r o m a t o p h o r e s w e r e found -60, -69 and -85 meV for R-26 and 0, 5 and -46 m e V for R/89, respectively. Herbicide (terbutrine) s e n s i t i v i t y of R-26 was d e p e n d e n t on the lipid c o m p o s i t i o n of the liposomes. The Is0 values were 0.6 pM and 2.0 pM if the RCs of s t r a i n R-26 were r e c o n s t i t u t e d in liposomes of PLs from R-26 a n d R/89, respectively. However, the s e n s i t i v i t y of R/89 RC-s against t e r b u t r i n e was not a f f e c t e d s i g n i f i c a n t l y b y the p h o s p h o l i p i d environment. The Is0 values for RCs of R/89 were found to be 200 pM and 300 ~M in cases of r e c o n s t i t u t i o n in v e s i c l e s of PLs from R/89 and R-26, respectively.

P-3-4-0 1 1 S I T E - S P E C I F I C MUTAGENESIS AT HISTIDINE 118 OF T H E PHOTOSYSTEM H D1 PROTEIN OF CHLAMFDOMONA$

REINHARD Tll Ronald S. Hutchison & Richard T. Sayre, Departments o f Plant Biology and Biochemistry, The Ohio State University, 2021 Coffey Rd., Columbus, OH 43210 USA Symmetry related histidine residues are located at positions 118 in the B transmembrane spans of the DI and D2 proteins. These residues have been proposed to coordinate chlorophyll monomers which may be analogous to the accessory chlorophylls of the bacterial reaction center (Schelvis et al., 1994). In order to test this hypothesis, we have replaced HIIS-D1 with a leucine or arginine residue. Mutations at H l 1 8 result in an inability to evolve oxygen and reduce DCPIP, an enhanced sensitivity of the DI protein to turnover in the light end to accumulation in the dark, a loss o f the ability to accumulate the TyrD radical in dark grown cells exposed to light. These results indicate that the mutagenesis of H l l g - D 1 disrupts the functional and possible sUuctural integrity o f the PSII reaction center;, results that are consistent with its possible participation in coordination o f an accessory chlorophyll. We will also report on pebD mutegenesis.

53

Poster

P-3-4-015

P-3-4-012 PSII CORE PHOSPHORYLATION HETEROGENEITY IN VIVO AND THE REGULATION OF D1 PROTEIN TURNOVER

PHOTOREGULATION HETERODIMER

Bettina Geiken 1, Alessandra Cona 1, Autar Mattoo2 and Maria T. Giardi 1 qBEV-CNR, Via Salaria kin. 29.3, 00016 Monterotondo Scalo, Italy 2Plant Molecular Biology Laboratory, USDA, Beltsville, MD 20705, USA

I. Booi_i. M. Swegle, S. Dube 1, M. Edelman and A. K. Mattoo Plant Molecular Biol. Lab., USDA/ARS, Beltsville, MD 20705, and 1Center for Agricultural Biotechnology, UMCP, MD 20742, USA

The influence of phosphorylation on the turnover of the D1 protein was analysed by radiolabeling spinach and pea leaves in vivo with 32p_or 33p_ orthophosphate and 35S-methionine. Photosystem II phosphorylation heterogeneity, previously observed in vitro (Giardi et al. BBRC,176,1298, 1991; Physiol Plant,92,171,1994), was demonstrated in vivo. Four PSII core populations with different levels of phosphorylation on D1, D2, CP43, the psbHgene product were isolated. After a short pulse with 3SS-metin the light, the population containing non-phosphorylated D1 was the first to incorporate radioactivity. The most phosphorylated populations were labeled in a longterm experiment and were shown to generate a breakdown fragment of phosphorylated D1 (D1 *). Conditionsthat modulate phosphorylation levels, synthesis or assembly of PSll core proteins were studied for their effect on D1 turnover. The results suggestthat core phosphorylation and D1 turnover are strongly correlated.

Light is intimately associated with the dynamics of photosystem II (PS-II) reaction center proteins, D1 and D2. D1-D2 heterodimer is assembled on stroma lamellae and translocated to grana where it undergoes reversible phosphorylation in the light• The role of phosphorylation is still a matter of conjecture although it is a committed step in D1 turnover. The D1 kinase is being purified and characterized in order to better understand functional significance of D1 phosphorylation. D1-D2 heterodimer also share the property of being a target of ultraviolet (UV)-B irradiation damage in Spirodela ( Marcel Jansen: 1994, Ph.D. Thesis, Weizmann Inst, Israel). We have tested this further by studying D1-D2 metabolism in UVB-sensitive (CNS) and UVB-tolerant (Williams) soybean cultivars. In both cultivars, D1 and D2 have similar turn over rates in visible light but undergo higher rates of degradation under UVB irradiation in CNS versus Williams. UVB effect was more pronounced in a background of visible light. These data show that D2 protein, which is normally a stable protein, is induced to degrade under mixtures of UVB and visible irradiation, suggesting that the DI-D2 heterodimer of PSII is a protein target of environmentally relevant UVBirradiation damage to crop plants.

OF D1-D2 P S - I I R E A C T I O N

P-3-4-016

P-3-4-013 PSII CORE PHOSPHORYLATION HETEROGENEITY

M.T. Giardi 1, T. Kucera1, J.-M. Briantais2 and M. Hodges2 IBEV-CNR, Via Salaria kin. 29.3, 00016 Monterotondo Scalo, Italy 2Lab. d'Ecologie V~g~tale, Universit~ Paris-Sud91405, Orsay-cedex, France The heterogeneous nature of photosystem II core phosphorylation has previously been reported (Giardi et al. Plant Physiol 100, 1948, 1992; Planta 190, 1993). The pattern of four phosphorylated PSII core populations in the grana regions appears to be ubiquitous in higher plants. However, a mutant of wheat that shows monophasic room-temperature photoreduction of Q^, as measured by chlorophyll fluorescence induction in the presence and absence of DCMU and by fluorescence upon flash illumination in intact leaves, also lacks the usual distribution of phosphorylated PSII core populations. In this mutant, the whole PSII core population pattern is changed, probably due to altered threonine kinase activity which leads to the absence of light-induced phosphorylation of CP43 and D2 proteins. The results in correlation to previous experiments in vivo, support the idea that the functional heterogeneity observed by fluorescence is in part related to the PSII protein phosphorylation in the grana.

P-3-4-014

CENTER

INFLUENCE OF PROTEIN HYDROGEN BONDS MODIFICATION ON PICOSECOND ENERGY AND ELECTRON TRANSFER IN RB, SPHAEROIDES REACTION CENTERS. V.Z.Pasehenko, P.P.Knox, V.V.Gorokhov, A.B.Rubin Dept. of Biopbys. Faculty of Biol. Moscow 119899 Moscow, Russia

State

University,

The kinetics of the energy transfer from BPheo* to P870 and charge separation between P870 and BPheo were investigated in RC after partially substituting water by the organic solvent DMSO. Excitation was performed at 560 nm (BFheo) and 590 nm (P870). Absorption changes were registered at 665 nm. In control samples it was found that the decay kinetics of BPheo* and P870 ~s two-exponential w~th t 1 = 10 ps and t 2 ~ 5 ps. These components were attributed to the energy transfer from BPheo to P870 (10 ps) and to the charge separation process (5 ps). Aider substitution of water by DMSO t 1 and t 2 increased to 15 and 10 ps, respectively. Experimental results are explained by means of relaxation of pigments and the ion-radical pair as well as by reorientation of hydrogen bonds. Kinetic models for the interpretation of the experimental data with special emphasis on the pigment-protein interactions will be given.

P-3-4-017

TARGETED RANDOM MUTAGENESIS OF THE D2 PROTEIN OF PHOTOSYSTEM I I .

HETEROGENEOUS CHARGE SEPARATION RATES IN MEMBRANE BOUND MUTANT REACTION CENTERS.

Svctlana Ermakova 1), Zhenbao yul,*), S. Shestakov 2), and Wim Vermaas 1) l)Department of Botany, Tempe AZ 85287-1601, USA; 2)Department of Genetics, Moscow State University, Moscow 119899, Russia; *)permanent address: Institute of Botany, Academia Sinica, Beijing, P.R. China.

L. Beckman§, M. Jones:~, I. van Stokkum§, F. van Mourik§, P. McGlynn:~, R. Visschers#,N. Hunter:~ & R. van Grondelle§

To identify important residues in the D2 protein, we have chosen to randomly mutagenize specific regions of psbDI (coding for D2) and to select for mutations that lead to impaired PS II function. To do so, the psbDIC operon with its flanking regions was cloned from Synechocystis sp. PCC 6803 into pUC 118. Regions of psbDI to be mutagenized were exposed within this plasmid as single-stranded DNA loops. Treatment with sodium bisulfite, which reacts specifically with single-stranded DNA regions, introduces mutations virtually exclusively into this ss-loop, leaving the rest of the plasmid intact. The mntagenized plasmids were then transformed into a Synechocystis 6803 strain that lacked psbDI and psbDIl. A total of 20 single and multiple Synechocystis 6803 psbDImutants with impaired photoantotrophic capacity were isolated and characterized thus far using this approach. Among them are mutants with altered QA binding niche, impaired donor side of PS II and others with different functional alterations in PS II. Functional characterization of these D2 mutants will be presented.

54

§Dept. of Physics and Astronomy, #Dept. of Biology, Free Universityof Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands. :~Dept. of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2UH, U.K. Stimulated emission decay's are reported of antenna deficient membrane bound

Rb. sphaeroidesRCs bearing site-directed mutations on the M210 and the L 181

positions. The stimulated emission decay's were always found to be multi-exponential, in Wild-Type RCs (3.6ps, 12ps, ratio 80:20) the multi-exponentiality is similar to values previously reported for isolated RCs. However contrary to previous results on isolated mutant RCs (Jia et al. (1993) J. Phys. Chem. 97, p. 13180), we did not observe an enhanced multiexponentiality in the slower membrane bound mutant RCs. The decays have been analyzed in terms of a distribution of decay rates, stemming from a distribution in one or more parameters important in describing electron transfer (AG, V, ~,). We could not relate the multiexponentiality solely to a distribution in the free energy gap AG, as was done for isolated RCs (Jia et al. 1993). Furthermore we report a "dynamic blue shift" of the stimulated emission spectrum in time for membrane bound WT RCs. This is indicative for a relation between kinetic- and spectral-heterogeneity.

Poster P-3-4-021

P-3-4-018 ANALYSIS O F T H E P S I I - R E A C T I O N C E N T E R AND ITS C O M P L E X W I T H CP47 USING G E L F I L T R A T I O N Camiel Eijckelhoff, Henny van Roon and Jan P. Dekker Dept. of Physics and Astronomy, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands The use of FPLC-gelfiltration is a good way of judging the purity of PSI/Reaction Centers. Though the peaks of the CP47-RC complex, the PSILRC and the CP47 protein in the chromatogram partly overlap, the composition and purity of a peak could be determined by plotting the ratio of absorbances at 416 and 435 nm in the chromatogram. The different components all have characterisic ratios. Even complete spectra at different times in the chromatogram could be studied because a Diode Array Detector is"~Jsed for detection. During checks of different CP47-RC complex preparations it was found that this complex exists as both a monomer and a dimer. Gelfiltration was also used to prepare ultra pure RC's on an analytical scale. Using gelfiltration in combination with sucrose density gradients, PSII-RC's have been purified which were prepared using the very mild LiC104/DM-treatment. In this way CP47-RC complex and CP47 contamination could be dealt with. The goal is to compare the RC's isolated in this mild way with the RC's isolated with Triton X-100.

P-3-4-019 E N E R G Y T R A N S F E R AND T R A P P I N G IN P H O T O S Y S T E M I I C O R E PARTICLES WITH CLOSED REACTION CENTERS

P R I M A R Y R E A C T I O N S IN B A C T E R I A L DYNAMICS O F R E A C T I O N CENTERS ELECTRON ACCEPTORS

PHOTOSYNTIIESIS: WITII MODIFIED

H.I luber, T.N~igele, S.Biesdorf, J.Wachtveitl and W. Zinth lnstitut ffir Medizinische Optik, University of Munich, Germany Femtosecond pump and probe pulses were used to investigate the primary electron transfer steps in bacterial reaction centers (RCs) at various temperatures. In the last years, the model of a stepwise electron transfer along the active pigment branch via the monomeric bacteriochlorophyll BA to the bactedopheophytin H A could be established. According to this reaction scheme, the reaction dynamics should be strongly dependent on changes in the energetics at the positions B A or H A In this work, the native chromophore bacteriopheophytin was replaced by different pheophytin- or bacteriopheophytin-type molecules, where functional groups were selectively modified. It can be shown, that the changes in reaction kinetics of the modified reaction centers are well explained by the molecular modifications. The 3-acetyl group in these pigments was found to be a major determinant for the tuning of the redox potential allowing the fast and efficient electron transfer reaction, while the hydrogenation state of ring 11 is responsible for the absorption properties of the steady state spectra.

P-3-4-022 INHIBITION OF THE ELECTRON TRANSPORT IN PSII BY Cu2+

J.P.M. Schelvis, M. Germano. T.J. Aartsma and H.J. van Gorkom Department of Biophysics, Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands

Caroline Je~,ersch61d 1, Juan B. Arellano2, Wolfgang P. SchrOder I , Mathilde Baron 2 & Stenbj0m Styring 1. 1Dept. of Biocbemistry, Arrheniuslaboratories , S t o c k h o l m University, S-106 91 Stockholm, SWEDEN. 2Plant Biochemistry Dept., Est. Experimental de1 Zaidin, CSIC, 18008 Granada, SPAIN.

Picosecond absorbance changes in the Qy absorption region were measured on Photosystem II core particles with closed reaction centers by the one-colour pump-probe method. The induced absorption changes are well described by three components with lifetimes of 21+6 ps, 80 to 200 ps, 1.5 ns, in addition to a non-decaying component. The 1.5 ns lifetime component is assigned to recombination of the primary radical pair in equilibrium with the excited state. Since the lifetime of the intermediate component depends on the excitation wavelength and its spectrum differs from that of the 21 ps component, a rapid equilibration of the excitations over the whole antenna is excluded. The 21 ps component and the intermediate component are discussed in terms of energy transfer and trapping processes.

At elevated concentrations, Cu2+ exerts a toxic effect on photosynthetic activity by specifically inhibiting photosystem II (PSI]) function. Arguments for location(s) of Cu2+-binding-site(s) on either the donorside or the acceptorside or on both sides of PSII have been put forward. Here, results on the effects of Cu2+ binding on the donorside of PSII are presented: 1) Cu2+ induces a loss of the 16, 23 and 33kDa extrinsic proteins and of two Mnions. 2) The oxidation of TyrZ is blocked measured as a loss of the EPRsignal II fast. 3) A new light induced featureless radical (g=2.0028 and linewidth 8:k0.5G) is observed. 4) After EDTA-washes to remove Cu2+ TyrZ can be oxidised again but the radical is still formed. The effects of Cu2+ will be discussed in terms of a donorside block inducing alternative pathways for the electron transport in PSII.

P-3-4-020 SUB-PS TRANSIENT-ABSORPTION M E A S U R E M E N T S ON T H E P H O T O S Y S T E M II R E A C T I O N C E N T E R B E T W E E N RT AND 4 K Made-Louise Groot, Matthieu Visser, Frank van Mourik, Ivo van Stokkum, Jan Dekker and Rienk van Grondelle Dept. of Physics and Astronomy, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands We have measured absorption difference spectra of PS ]I RC's between 4 K and RT on the sub-ps to sub-ns timescale upon excitation at 670 nm, 680 or 688 nm with a spectral width of the pump pulse of 5 nm fwhm. Four to five lifetimes were found: ~ 500 fs, - lps, ~ 10 ps, 20-100 ps and a ns component, with different decay-associated spectra. The lifetimes are dependent on excitation wavelength and temperature. The results will be discussed in the context of a model for the RC dynamics proposed before [1]. From the observed Tdependence and spectra of the 10-ps and the 20 - 100-ps components we conclude that they are due to energy transfer processes. We therefore assign the ~ 1 ps-lifetime to the (intrinsic) charge separation rate. Furthermore, polarized measurents will be discussed within the framework of the same model. [1] M.-L. Groot, E.J.G Peterman, P.J.M. van Kan, I.H.M. van Stokkum, J.P. Dekker, R. van Grondelle. Biophys. J. 67, 1994, p. 318-330

P-3-4-023 DYNAMIC STUDIES ON THE PRIMARY PHOTOCHEMICAL REACTION IN THE ISOLATED PHOTOSYSTEM II REACTION CENTER BY TIME.RESOLVED ABSORPTION AND FLUORESCENCE SPECTROSCOPY Jian-Min Hou Ting~Yun Kuang, Zhen-Bao Yu, Chong-Qin Tang, Kun-Yun Yang, Pei-Song Tang Lab. of Photosynthesis, Inst. of Botany, Academia Sinica, Beijing 100044, P. R. China Tong Ye, Yan Cui, Yao-Dong Chert, Shui-Cai Wang, Xun Hou State Key Lab, of Transient Optics Technology, Xi'an Inst. of Optics and Precision Mechanics, Academia Sinica, Xi'an 710068, P. R. China in spite of the primary photochemical reaction of the isolated Photosystem II (PSII) reaction canter (RC) had been studied using time-resolved ~ i ¢ and other techniques by a number of groups, the time constant of primary charge separation and energy transfer in the PSII reaction center are =till subject to discussion, In this paper we investigated the dynamic of the primely photochemical reaction in the PSll RC using time-resolved absorption and fluorescence spectroscopy. The results showed: (1). In the time range of nano~mccx~ and subnanosecond four components of lifetime were obtained, in which 350 ps component may be attributed to the energy transfer assoaated with ~-carotene molecule. (2). In the time range of picosecond and subpicosecond several components were resolved. A 3 ps component, which is obtained fl'om both the femtcaecond absorption and the fluorescence measurements, may be related to the primary charge separation.

55

Poster

P-3-4-027

P-3-4-024 ENERGY TRANSFER AND TRAPPING IN PURPLE SULFUR BACTERIA John T.M. Kennis. Thijs J. Aartsma and Jan Amesz Dept. of Biophysics, University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands. Energy transfer and trapping processes in chromatophores of the pmple sulfur bacteria Chronu~/mn vlnosum and C. tep/dwn were studied by means of picosecond transient absorl~on spectroscopy. In C. vinosum, energy uapping in the core antenna by open resctiun ~ (RCs) occurred in 50 ps. In C. tep~wn, the decay in the core was multl-exponential, with a major contribution from a 140 ps componeat and minor contributions from 40 ps and 530 ps components. It was establisbed that the 140 ps componeat is due to energy h~tt,,ping by open RCs. This trapping time is slow as compared with other purple bacteria, which is very likely caused by the fact that the core antenna is te.d-shifted by 18 nm with ~ p e c t to the RC. C. tep/dwn ccatalns only ofie peripheral complex, B800-850. Our results indicated two time constants for energy transfer from the peripheral to the core cc*aplex, 10 ps and 30 ps, suggesting non-uniform distances between the peripheral and core complexes. C. vlnosum has both B800-820 and B800-850. Energy transfer from B800-850 to cote occurred in 7 and 30 ps. Energy transfer from B800-820 to B800-850 was significantly faster. Thus, transfer rates from B800-850 to the cote in the bacteria studied are quite similar, This may seem remarkable as the cote antenna in C. tepidura absorbs at the unusually long wavelength of 918 nm, hut it is calculated that the overlap integral for energy transfer from B800-850 to the core is not dramatically less than in C. vinosum.

L PROTEIN S U P P O R T E D Q U I N O N E E X C H A N G E AT QA SITE IN P S I I C O R E C O M P L E X S. Ozawa, H. Hoshida & Y. Toyoshima Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-nihonmatsu-cho, Sakyo-ku, Kyoto 606-01. Japan We previously reported that QA function ~as restored in thc PQ-9 depleted PSII core complex by simultaneous reconstitution ~ith PQ-9 and L protein (FEBS Lett. 354:113-116 (1994)). In this work, several exogenous quinones were reconstituted to the PQ-9-depleted PSII with helps of L protein and a lipid, digaractosyldiglyceride. The QA activity was determined by measuring the photoreduction rate of DCPlP in the presence of DPC and light-induced VI'IR difference spectra. The QA activities of PSIIs reconstituted with UQ-2, UQ-10 and PQ-9 were about 10% of that of the original PSII and significant difference was not observed among three quinones. When the reconstitution was carried out in the presence of L protein, however, the activity recovered to 30% of the original level with UQ-2 and UQ- 10 and it was enhanced up to 45% of the original level with PQ-9. Interestingly neither PQ-2 nor menaquinone-4 work as Q:v These results strongly suggest that L protein is involved in the regulation of electron transler at Q \ site in PSII.

P-3-4-025 H Y D R O G E N - B O N D I N G EFFECT O N EPR SPECTRA OF 15C-LABELED UBISEMIQU1NONES IN V I T R O T.N.K.roDachevaq J . R a a p 2, J.Lugtenburg 2, A.J.Hoff 3 mChemist-ry Dept., U d m u r t State University, Izhevsk, Russia; 2Chemistry Dept., Leiden University, Leiden, The Netherlands; aBiophysics Dept., Leiden University, Leiden, The Netherlands One o f the experimental approaches for probing the native binding sites o f quinones in bacterial reaction centers (RCs) is E P R / E N D O R studies of RCs reconstituted with m3C-labeled quinones, since maC hyperfine ( h 0 couplings are k n o w n to be sensitive to the environment. In order to interpret the o b t a i n e d results, the environmental effect on E P R spectra o f a n i o n radicals generated from specifically maC-labeled ubiquinones-0 and 10 in different models was studied. It was found that the magnitude and the sign o f carbonyl maC h f couplings indicate the strong sensitivity to the h y d r o g e n - b o n d i n g interactions with the solvent molecules. Using a c o m b i n a t i o n o f electrochemical and EPR techniques the influence o f solvent composition and p H value on ubisemiquinones f o r m a t i o n and solvatation was examined.

P-3-4-028 THE ilema~-->Met REACTION CENTRES

MUTATION IMPAIRS THE FUNCTION OF RHODOBACTER SPHAEROIDES

J. Tandori, L° N a g y and P. Mar6ti Dept. of Biophysics, J6zsef A t t i l a University, E g y e t e m u. 2, H u n g a r y

OF

QUINONES

IN

H-6722 Szeged,

The b i n d i n g and function of the secondary quinone acceptor (QB) of the m u t a n t (R/89) were c h a r a c t e r i z e d b y m e a s u r i n g e l e c t r o n t r a n s f e r k i n e t i c s (charge recombination) and multiple turnover (cytochrome o x i d a t i o n and semiquinone oscillation) assays and light i n d u c e d p r o t o n uptake by the reaction centre (RC). It s h o w e d i n c r e a s e d resistance to interquinone electron transfer inhibitors of o - p h e n a n t h r o l i n e (resistance factor, RF=10) and t e r b u t r y n e (RF=55) in UQQ r e c o n s t i t u t e d isolated RCs and in c h r o m a t o p h o r e s (RF=85). The free energy for s t a b i l i z a t i o n of QAQa- w i t h respect to QA-Qs was found AG°=-60 m e V and 0 meV in r e a c t i o n centers and AG°=-85 meV and -46 m e V in c h r o m a t o p h o r e s of R-26 and R/89 strains, respectively. The d i s s o c i a t i o n constant of the quinone UQ o and semiquinone UQ o" in reaction centers from b o t h strains showed sound and different p H dependence. The o b s e r v e d changes in binding and redox p r o p e r t i e s of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the o x i d i z e d and r e d u c e d states of Qs.

P-3-4-026 TOPOLOGY OF CHROMOPHORES AND COFACTORS IN PSH AS DERIVED FROM THE LOCAL ELECTROCHROMIC BANDSHIFTS.

P-3-4-029 P H O T O S Y S T E M 11 R E A C T I O N C E N T E R S AT 77K STUDIED BY SUBP I C O S E C O N D T R A N S I E N T SPECTROSCOPY.

D. Cherenanov 1, M. Haumann 2, W. Junge 2 and A. Mulkidjanian2 1A.N.Frurnkin Institute of Electrochemistry, Moscow, 117071, Russia, 2Abt. Biophysik, Universi~t Osnabr0ck, D-49069 Osnabrfick, Germany

H. Matthieu Visser, Marie-Louise Groot, Frank van Mourik, Ivo H.M. van Stokkum, Jan P. Dekker and Rienk van Grondelle. Dept. of Physics and Astronomy, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.

Three electrochromic difference spectra which reflected the deposition of (1) a negative charge on the primary quinone acceptor QA, (2) a positive charge on Tyr-161 of subunit D1 and (3) a positive charge on the manganese cluster, respectively, were obtained with PSll-core particles from pea and deconvoluted. Data were adequately fitted by four components which we assigned to the two chlorophyll a molecules of the primary donor P and to the accessory chlorophyll a and pheophytin a on D1. From our and other data we suggest an arrangement of pigments and redox cofactors in PSH that differs from current models as shaped along bacterial reaction centers but is better compatible with the extremely positive redox potential of P+. We found that: 1) P is formed from two orthogonally oriented chlorophyll a molecules; 2) the accessory chlorophyll a is oriented perpendicularly to the membrane with ring V pointing to the stromal side and is presumably attached to H i s - l l 8 of D1; 3) pheophytin fl is also perpendicular to the membrane but unlike its bacterial counterpart its ring V is pointing to the lumen; 4) the manganese cluster is located not in a row with Yz and P.

56

We present transient absorption data obtained on photosystem 11reaction centers at liquid nitrogen temperature (77K). Using well-defined sub-ps excitation pulses with a FWHM of 5 nm at either 671,681 or 688 nm, different spectral forms in the PS 11 RC were excited, using a magic angle configuration. The influence of this selective excitation on the early-time spectra is quite evident. Moreover, at 77K the kinetic scheme for the dynamics is simplified compared to room temperature due to significant slowing down of 'uphill' energy transfer. We have analysed these data using multi-response analysis and find several time constants; 671 nm excitation: 0.5 ps, 14 and ~1.8 ns, 681 nm excitation: 0.2 ps, 1.0 ps, 0.10 ns and ~1.2 ns and for 688 nm excitation: 0.9 ps, 75 ps and ~ 5ns. Furthermore we present data collected using parallel and perpendicular polarisation for pump and probe pulses, with excitation at 671 nm. Using a thermodynamic model consisting of several spectral forms connected by energy transfer obeying detailed balance, we conclude that the data are best described by charge separation occurring with a 1.0 ps time constant and energy transfer processes which take 14 ps 'downhill' from ~675 nm to 682 nm and 75 ps 'uphill'.

Poster

P-3-4-030

P-3-4-033

Time-resolved EPR spectroscopy of the Triplet State of the Primary Eletron Donor P680 in Photosystem II. Paul J.M. van Kan 1, Bengt Svensson 1, Ivo H.M. van Stokkum 2 and Stenbj6rn Styring 1 1Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S- 106 91 Stockholm, Sweden. 2Faculty of Physics and Astronomy, Free University, De Boelaan 1081, 1081 HV Amsterdam, The Netherlands. The triplet state of the primary electron donor P680 in Photosystem II is formed by charge recombination and has a characteristic AEEAAE spinpolarised EPR spectrum. Both EPR spectrum and electron spin-polarisation decay kinetics depend on orientation, temperature and the conditions of formation of this triplet state. We will present a detailed study on the parameters that determine the formation and polarisation decay of 3p680 in intact PS II membranes. Results from time-resolved EPR spectroscopy have shown that the electron spin-polarisation decay kinetics are temperature dependent between 4 and 40 K. Current investigations focus on the changes in the yield and spin-polarisation of 3p680 that are observed when charge recombination occurs in various conditions in Photosystem II, including conditions of photo-inhibition.

P-3-4-031 E L E C T R O N T R A N S F E R F R O M QA" TO P÷ : EFFECTS OF AG° AND T E M P E R A T U R E O N R A T E AND REORGANIZATION E N E R G Y

J. M. Ortega 1'3,X. Lin2, J. Williams2, J.P. Allen2 and P. Mathis I 1 - DBCM-SBE, CEA-Saclay, 91191 Gif-sar-Yvette Cedex, France ; 2 - Dept. of Chemistry (Center for the Study of Early Events in Photosynthesis), ASU, Tempe AZ 85287-1604, USA ; 3 - permanent address : Inst. Bioquimica Vegetal y Fotusintesis, Univ. de Sevilla- CSIC, 41080 Seville, Spain The kinetics of electron transfer from Q^" to P+ have been studied versus temperature in isolated RC of Rb. sphaeroides using a series of mutants where the Em of P was varied fi'om 410 mV tu 765 mV (Linet ~ PNAS, 1994, 91, 10265) so that the reaction AG° varied accordingly. In all cases the kinetics accelerate when T is decreased (with a rather sudden effect around 230 K ; there is no effect of T below 100 K), but the effect is stronger when AG° is small. The reorganization energy was estimated to decrease by several hundred meV from 295 K to 10 K. This decrease of 3. can account for the increase in rate at low temperature of the electron transfer from QA"to P÷. The fit of in k versus AG° at several temperatures according to Marcus theory gives estimates of vibrational modes which are coupled to electron transfer. The data can be fit using both high-fiequency modes (= 1,000 crn"1) and low-fi'equency modes (~ 100 cm'l).

P-3-4-032

E~C~ROCH~MIC

B~D-SHI~

~

CA~NOID

[NRHODOPSEUDOMONAS VIRIDIS

M. H a r ~ 1 , C . C . Moser 2 and P.L. Dutton 2 INIBH, AIST, MITI, 1-1Higashi, Tsukuba, Ibaraki JAPAN(present address) 2 j o h n s o n R e s e a r c h 305, Foundationof Molecular Biophysics, University of Pennsylvania, BS01 Richards Building, 37th & Hamilton Walk, Philadelphia PA 1 0 1 0 4 , USA We m a d e the electro-deposited film (E.D. film) of chromatophore membrane from purple Rhodopseudomonas photosynthetic bacterium sandwitched between two electrodes to viridis investigate the electrochromic band-shift (Stark-shift) of carotenoid molecule. The E.D. film showed the Stark-shift w h e n we a p p l i e d the potential to the film. The potential-induced spectrum is similar to the first derivative spectrum of the absolute spectrum of E.D. film. The amplitude of the band-shift is linear to square of applied potential. The E.D. film in a relatively dry state showed the continuou ight--induced spectrum which also resemblesSthe irst derivative spectrum of the absorption spectrum whereas chromatophore suspension did not show such spectrum. This is the first report that clearly shows potential-induced electrochromic band-shift of carotenoid in Rps. viridis chromatophore.

~

P-3-4-034 T H E L O W PH FORM OF R E D O X - A C T I V E C O M P O N E N T S IN THE DONOR SIDE OF PHOTOSYSTEM II STUDIED BY PULSED EPR A N D E N D O R . H. Mino 1, A.V. Astashkin t, A. Kawamori 1, T. Ono2 and Y. Inou¢2 ~'~y of Science, Kwansei Gakuin University, Uegahara 1-1-155, Nishinomiya 662, Japan. 2Solar Energy Research Group, The Institute of Physical & Chemical Research, Wako, 351-01, Japan Tyr.Z÷ can be trapped in PS II with oxygen evolution inhibited. The EPR lineshape of trapped Tyr.Z' in Tris-treated PSII changes with pH. Pulsed ENDOR spectra of Tyr.Z* show existence of two molecular structures of Tyr.Z+; high and low pH forms. The high pH form of Tyr.Z + shows a similar EPR lineshape as that of Tyr.D ÷, while the low pH one shows less overall width. The two forms interconvert between pH 6.0 and pH 6.5. This change may be caused by modification of surroundings of Tyr.Z+, probably by dissociation of a histidine residue nearby. Microwave power dependence of ESE amplitude shows that the 160G wide signal ($3' signal) in Ca-depleted sample has the spin 1, which is ascn'bed to an electron-electron dipole coupling. The $3' signal species is suggested to be located between P680 and the OEC. The $3' signal intensity increases at lower pH, which suggests the signal has a relevance to the low-pH form of Tyr.Z*.

P-3-4-035

R E C O N S T I T U T I O N O F T H E P L A S T O Q U I N O N E POOL OF INSIDE OUT V E S I C L E S , PSII M E M B R A N E F R A G M E N T S AND CORE COMPLEXES FROM HIGHER PLANTS

E S E E M A N D P U L S E D E N D O R S T U D I E S OF P R I M A R Y A C C E P T O R Q U I N O N E IN P H O T O S Y S T E M S II.

J. Kurreck~ A. G. Seeliger, F. Reifarth, M. Karge, G. Renger Technical University Berlin, StraBe des 17. Juni 135, 10623 Berlin, Germany

A. Kawamori 1, H• Mino I , A.V. Astashkin 1, S. Kuroiwa 2 and K. Akabori 2, 1Faculty of Science, Kwansci Gakuin University, Nishinomiya 662; 2Faculty of Integrated Arts and Science, Hiroshima University, Higashi Hiroshima 724, Japan.

The acceptur pool in standard preparations of inside-out vesicles and PSII membrane fragments is limited to 2-3 plastoquinunes and the Qa-site of core complex preparations is severely modified. Plastuquinane-9, isolated by HPLC from spinach, was now used to reconstitute the plastoquiaone pool of these preparations. After a 15 rain sonication the aeceptor capacity, estimated by measuring flash induced changes of the fluorescence yield, increased to a value of about 12 electrons corresponding to 6 plastoqniunnes in inside out vesicles and PSI:[ membrane fragments. The kinetics of electron transfer from QA"to QB(Oa-) were hardly affected. As a consequence of the enlarged plastoquinane pool the number of the typical period four oscillations of the flash induced oxygen yield, measured with a Joliottype electrode, increased from 2 to 4. In core complexes the functionality of the Qa-site was also improved by plnstoqninane-reconstitution. Measurements of flash induced changes of the fluorescence yield induced by a single laser flash in dark adapted samples revealed a marked increase of both, the extent and the rate constant of the fast decay kinetics. However, the kinetics Of Q^" renxidation still differ from those of PSII membrane fragments, probably due to modifications of the QB site by 13-dodecyl-mnltosidetreatment during the isolation of PS II core particles. Other qumones tested in this study were not suitable for reconstitution. A more detailed analysis suggests that structural constraints dominate the efficiency of a particular qninone molecule. The structure of the head group seems to be more important than the length of the hydrophobic tail.

The electron spin echo envelope modulation (ESEEM) and pulsed ENDOR spectroscopies arc used to larobe the nearest surroundings of the primary • • "2+ 2+ • aceeptor qumone, QA, m Fe -depleted, Zn -substituted and NaCN-treated preparations of plant PSIL The ESEEM data show that the structure of the binding site of Q^ in Zn-substituted PSII is similar to that determined for the bacterial photosystem by the X-ray crystallography, with the amino nitrogen in the imidazole ring of histidine and the peptide nitrogenof another aminoacid residue (probably, alanine 261 of D2 protein) coupled to the QA oxygens through the H-bonds. In NaCN-treated PSII one histidine is displaced from the coordination sphere of F 2+ and only the coupling to the peptide nitrogen is found• Also in Fe-depleted PSII most of the quinones lose their binding to histidin¢.

57

Poster P-3-4-036 HIGH RATES OF PHOTOSYSTEM II ELECTRON FLOW REQUIRE BOUND BICARBONATE O.J. de Vos ~, J.J.$. van RQn~en 1 & Govindjee 2 ~Dept of Plant Physiology, Agricultural University, Arboretumlaan 4, 6703 BW Wageningen, The Netherlands; 2Dept of Plant Biology, University of Illinois, Urbana, IL 61801, USA Thylakoids were isolated from peas and maize and treated as described by Jursinic and Stemler (Biochim. Biophys. Acta 1098: 359-367, 1992). CO 2 release was measured with an open infra-red gas analyzing system (IRGA). Pea and maize thylakoid suspensions (in 10 mM NaCI and 50 mM Na phosphate, pH 6.4) , that had been depleted of C02 without any inhibitor, but had retained high rates of electron flow, showed the release of 1,04 ± 0.10 (pea) and 0 . 5 4 ± 0.04 (maize) C02 per PSII reaction center upon the addition of 100 mM formate. Our data contradict the negative results of Jursinic and Stemler (1992) on CO2 release and reject their " e m p t y site" hypothesis. We support the essential activator model of bicarbonate for PSII. This research was supported by an NSF grant (91-16838, supplement 1994).

P-3-4-037 EXCITED STATEDYNAMICSIN PHOTOSYNTHETICPIGMENT-PRO'EEIN COMPLEXES STUDIED WITH ACCUMULATED PHOTON ECHOES.

P-3-4-039 SITE-DIRECTED MUTAGENESIS OF A BICARBONATE BINDING SITE ARGININE 269 OF THE DI PROTEIN IN CHLAMYDOMONAS REINHA RD TIL R. Hutchison, I J. Xiong, 2 R. SayreJ Govindjee, 2 IDepts. of Biochemistry and Plant Biology, Ohio State University, 2021 Coffey Rd., Columbus, OH 43210, 2 Dept. of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA It has been suggested that a HCO3" binding site is near the PSII QA-Fe-QB niche. Several arginine residues near this site have been proposed to be HCO3" ligands. To investigate this hypothesis, R269 on the DI protein of C. reinhardtii was changed into Gly (R269G). The mutant (1) is unable to grow photosynthetically or evolve oxygen, (2) its PSII reaction center is intact based on western blot analysis and 77°K Chl florescence emission when cells are dark, but not light grown, (3) its electron transfer rate from QA to the plastoquinone pool is reduced, (4) R269G binds [14C]-terhutryn poorly, and (5) the formate-enhanced g=1.82 EPR signal is greatly reduced indicating that the QA-Fe'+ state does not accumulate in the mutant. These ohservations suggest that the R269G-D1 mutation dramatically alters the architecture and function of PSII consistent with R269 possibly functioning to bind HCO3".

P-3-4-040 CONTROL

OVZRELECTRON

TRANSFER

INANOX~GENIC

PHOTOSYRTBEgIS

W.Crielaard, B.J. van Rotterdam, H.V. Westerhoff & K.J. Hellingwerf E.C. Slater Institute for Biochemistry and Microbiology, versity of Amsterdam, The Netherlands

R. J. W. Louwe and T. J. Amlsma Department of Biol~ysics, University of Leiden, P.O. Box 9504, 2300 RA Leiden, the Nefbeda~ds Accumulated photon echo experiments were used to study the mechanismof onergy transferm photosyntheticpigment-protein complexes.Measurementsperformedon the BCbl a complex of green sulphur bacteria (FMO-complex) at low temperatures (1.4 K-30 K) reveal extremely fast photon echo decays at sho~t-wavelengthexcitation in the Qy band to slow (several hundreds of picoseconds) photon echo decays at the longest wavelengths.The individual echo decays are multiphastc,due to contributions of differem states absorbing at the same wavele~4F~tw t ~ the Qy-manJfold and are explained in terms of phonon assisted wJaxadon to lower lying energy levels. The temperatme dependence of the different time constants cm~ be fitted with a combination of a power law and an exponential activation of higher energy levels. Experiments performed cm the ChI a/b plgmont-po3teincomplex of plants (LHC H) at 1.4 K display similar photon echo decays rangingfrom sub-picoseconddecays upon excitation at 670 nm to decays of several hundreds of plcosecondsat 682 nm. At least five different time constants can be discerned in this spectral region.

A thermodynamic and kinetic mathematical model describing light-driven cyclic electron transport in solubilized and reconstituted (Rhodobacter sphaeroides) reaction centers, supplemented with ubiquinon-0 and cytochrome c2, was developed to investigate the relation between electron transport and proton translocation. Analysis of the mechanism of cytochrome c reduction by ubiquinol-0, in our experimental set-up, showed that the reduction via the quinol anion [QH-] has the highest flux control coefficient, allowing the incorporation of 4 new rate constants in the mathematical model (kqc=8.17Xl02; k~q=lO.3XlO4;kqqe=l.2XlO 9 and kceq=4.5Xl0-2). The predictive value of the 12-state model was shown using donorrecombination transients, which revealed refined constants for light induced cytochrome c oxidation, Q/QH2-exchange at the Q~-site of the reaction center and recorabination from QB. Using the model it was not only possible to estimate but also to shift control to (different transitions in) the reaction center.

P-3-4-038 THE COUPLINGOF LIGHT-INDUCEDELECTRONTRANSFERAND PROTONUPTAKE: REFINED CRYSTAL STRUCTURES OF MODIFIED PHOTOSYNTHETIC REACTION CENTRES FROM RHODOPSEUDOMONAS VlRIDIS C. Roy D. Lancaster and Hadmut Michel, Max-Pianck-lnstituteof Biophysics, Dept. of Molecular Membrane Biology, D-60528 Frankfurt am Main, Germany. In the reaction centre (RC), the coupling of electron transfer and proton uptake from the cytoplasm takes place at the binding site of the secondary quinone, QBIn the original structure of the RC from Rhodopseudomonas (Rp.) viridis (Deissnhofer, F.pp, Sinning & Michel, 1995, J. Mol. Biol. 246, 429-457), the Qe site was poody defined because it was only 30% occupied in the RC crystals. Recently, quinone reconstitution expedments have yielded crystals with full quinone occupancy of the QBsite. Subsequent data acquisition, processing, and refinement has led to a weft-defined QB site model. In addition, the complex of the RC with stigmatellin, a chmmone-type electron transfer inhibitor, may serve as a model for the binding of a monoprotonated, doubly reduced intermediate, QsH'. These two structures have led to a better understanding of Qa binding and especially of the role of the residue Ser L223. Also, the structure of a Os-deplsted reaction centre will be discussed. The Os site is also a well-established site of triazine helbicide action. High quality data collected recently on RC complexes with atrazine and two chiral atrazine derivatives has enabled us to dascdbe the exact nature of triazine binding and its effect on the structure of the protein. It has also been possible to improve the original model in regions other than the Qs site, e.g. the model of binding of the primary quinone. Q,, and the structure of the carotenoid.

58

Uni-

PHOSPHORYLATION OF PSII POLYPEPTIDES DEGRADATION OF THE DI PROTEIN

P-3-4-041 PREVENTS

Volker Ebbert and Doris Godde. Lehrstuhl fib"Biochemie der Pflenzen, RuhrUnlversititt, D-44780 Bochum, Germany Phosphorylation of thyiskoid proteins is known to regulate photosynthetic electron transport by affecting the antenna size of PSI[. Recently, evidence has been shown that it is also involved in the rapid turnover of the D1 protein of the PSII reaction center. Using intact chloroplasts, we could show by labelling with either 14C leucine or 321'i that the PSH polypeptides became maximally phosphorylated at moderate light intensities, which do not induce photoinhibition. Thus, phosphorylntion of D1 protein is no consequence of photoinhibition. No exchange of phosphate groups esterified to the D1 protein can be observed in the light. Once the D1 protein was maximally phosphorylated, newly synthesized DI protein could not be phosphorylated. Obviously, there was no interchange between the phosphot~ted and the nonphosphorylated populations of the D1 protein. The phosphorylation of the D1 results in a stabilization against proteolytic degradation and only the nonphosphorylated form of Dl protein can be degraded. As a result the D1 protein once it is phosphorylated does not take part in the rapid turnover. The physiological significance of this result is discussed.

Poster P-3-4-045

P-3-4-042 COMPLEX FORMATION AND ELECTRON TRANSFER BETWEEN CYTOCHROME C2 AND REACTION CENTERS OF RHODOBACTER SPHAEROIDES Friedel Drenner, Pierre Dorlet & Paul Mathis Section de Bioenerg&ique / DBCM, CEA Saclay, 91191 Gif-sur-Yvette, France In the purple bacterium Rhodobacter sphaeroides a soluble eytochrome (cyt) c2 reduces the photooxidized primary donor, P+. This electron donation stabilizes the charge separation in the reaction center (RC) and can serve as a model system to study factors that govern interprotein electron transfer. Characterization of covalent RC-cyt complexes obtained by zero-length crosslinking yields the following results. Cyt ~ is cross-linked to the M-subunit of the RC. 80% of the RC show a flashinduced P+ reduction by covalantly bound cyt c2 in two kinetic phases with halftimes of 700ns and 50#s. The 700ns-phase, corresponding to that in the noncovalent complex, is favored when P and cyt are reduced during the crosslinking whereas oxidizing conditions predominantly produce the 50#s-phase. Amplitudes of the two kinetic phases of P+ reduction show almost pH-independent midpoint redox potentials between pH 5 and 10 whereas the midpoint potential of free cyt c2 is affected by at least two protooable groups within this pH-range 0aettigrew, Meyer, Bartsch & Kamen, 1975, Biochim. Biophys. Acta 430, 197-208). The effect of temperature on electron transfer is similar to that observed for P+ reduction by a dissociation equilibrium of soluble cyt c2 (Venturoli, Mallardi & Mathis, 1993, Biochemistry 32, 13245-13253). Below -40°C, electron transfer within the complex is impaired. Orientation of the heme plane within the covalent complexes has been studied by EPR spectroscopy.

PICOSEC~3~D SEPARATION CENTERS OF

Dieter

FLUORESCENCE STUDIES OF TEE PRIMART CEARGE AND RADICAL PAIR RELAXATION IN REACTION PURPLE BACTERIA

Dorra,

M a r c G. M ~ l l e r

und Alfred

R. H o l z w a r t h

Max-Planck-Institut f~r Strahlenchemie Stiftstr.34-36, D-45470 M~lheim/Ruhr, Germany F l u o r e s c e n c e of the e x c i t e d p r i m a r y e l e c t r o n d o n o r P decays with at least four time c o n s t a n t s in the 3 ps - 1 ns range. This so far not understood n o n e x p o n e n t i a l b e h a v i o u r c o u l d be a s s i g n e d e i t h e r to heterogenity in p r i m a r y c h a r g e s e p a r a t i o n or r a d i c a l pair recombination. To d i s t i n g u i s h b e t w e e n these two basic models, the temperature dependence of fluorescence kinetics is an i m p o r t a n t information. F l u o r e s c e n c e d e c a y s w i t h a time r e s o l u t i o n of 2-3 ps of various bacterial reaction centers have been measured at t e m p e r a t u r e s b e t w e e n 77 a n d 300 K. T h e d a t a w i l l b e d i s c u s s e d in terms of d i f f e r e n t k i n e t i c schemes.

P-3-4-046

P-3-4-043 SELECTIVE EXTRACTION OF PIGMENTS FROM THE PS II REACTION CENTER

T H E W A T E R C~L~IN A N D REACTION CENTRE FROM

~ 1 , 2 , Masami K o b a y a s h i 3, KimiyukJ S a t o h l , 2 ( 1Natl, Inst. Basic Biol., Okazaki, J a p a n , 2 O k a y a m a Univ., O k a y a m a , J a p a n , 3 T s u k u b a University, T s u k u b a , J a p a n )

G. Fritzsch, U. E r m l e r & H. M i c h e l ~ax-Planck-lnstitut for Biophysik, H e i n r i c h - H o f f m a n n Sir. 7, 60528 F r a n k f u r t a.M., G e r m a n y

T y p i c a l p r e p a r a t i o n s of t h e PS II r e a c t i o n c e n t e r so f a r a n a l y z e d c a n b e classified i n t o t w o t y p e s , in t e r m s o f p i g m e n t s t o i c h i o m e t r y . In o n e t y p e , t h e Chl a to P h e o a r a t i o is two, c o n s i s t e n t w i t h its purple bacterial counterpart. The other type of preparation, which s e e m s to b e m o r e n a t i v e t h a n t h e o t h e r , c o n t a i n s two a d d i t i o n a l Chl a a n d o n e a d d i t i o n a l [~-Car, s u g g e s t i n g a selective p i g m e n t r e l e a s e f r o m t h e c o m p l e x . In t h e p r e s e n t s t u d y s e l e c t i v e e x t r a c t i o n o f p i g m e n t s f r o m t h e i s o l a t e d PS II r e a c t i o n c e n t e r h a s b e e n a t t e m p t e d either by solvent-treatment(ether) or by detergentt r e a t m e n t ( T r i t o n X-IO0). By t r e a t i n g t h e i s o l a t e d PS II r e a c t i o n c e n t e r c o m p l e x (Chl a: !~-Car: P h e o r a t i o , 6:2:2) w i t h e t h e r , t h e p i g m e n t c o n t e n t w a s r e d u c e d t o a b o u t 7 0 96 f o r Chl a a n d 5 0 ( d r y e t h e r ) to 0 96 (50-10096 w a t e r s a t u r a t e d e t h e r ) f o r 15-Car p e r t w o Pheo a. By t r e a t i n g t h e c o m p l e x w i t h T r i t o n X-IO0 at 0 . 1 - 1 . 0 96, t h e [~-Car content was reduced from two to one per reaction center (per p h o t o a c t i v e P h e o a). T h e f u n c t i o n o f " a d d i t i o n a l " p i g m e n t s in t h e PS II r e a c t i o n c e n t e r will be d i s c u s s e d b a s e d o n t h e s e o b s e r v a t i o n s .

OTHER STRUCTURAL RB. SPHAEROIDES

ASPECTS

C. FogeP, S. Grzybek 2, R. HienerwadeP, M. Y. Okamura4, M. L. Paddock 4, J. Breton 3, E. Nabedryk 3 and W. Mantele 2 qnstitut ~ r Biophysik, Universitat Freiburg, Albertstr. 23, 79104 Freiburg, Germany; 2Institut fiir Physikalische Chemic, Universit~it Erlangen, Egerlandstr. 3, 91058 Edangen, Germany; 3SBE/DBCM, CEN Saclay, 91191 Gif-surYvette C6dex, France; 4Department o f Physics, University o f California San Diego, La Jolla, California 92093-0319, USA. Time resolved infrared (IR) and steady-state Fourier transform infrared (FTIR) spectroscopy has been used to study the coupling of proton and electron transfer in photosynthetic reaction centers (RC). In Rb. sphaeroides RC, proton uptake by the residue Glu I.,212 in the Qa pocket upon QA'Q8 ~-~ QAQn" electron transfer was recently demonstrated [Hienerwadel et aL, 1995, Biochemistry 34, 2832-43]. Since studies using site-directed mutants have shown the influence o f other protonatable residues, we have analysed the steady-state Qn'/Qn FTIR difference spectra and the transient IR signals o f several mutants in the QB site as a function o f pH. In the 1680-1760cm, 1 region, the variation o f the intensity o f several modes with pH and their absence or presence in mutants leads us to propose assignments for the groups involved in a cluster ofionizable residues around QB.

THE

~" m e a n s of t r i g o n a l c r y s t a l s the s t r u c t u r e of the r e a c t i o n c e n t r e (RC) f r o m Bb. sphaeroides has b e e n reinvestigated. Most of the earlier reported d i f f e r e n c e s in c o f a c t o r b i n d i n g b e t w e e n the RCs f r o m Rps. viridis and Rb. sphaeroides cannot be reconfirmed. (Ermler, Fritzsch, Buchanan & Michel, 1994, Structure, 2 925-936). S i g n i f i c a n t d i f f e r e n c e s between the Rps. viridis and Rb. sphaeroides s:ructures are found at ring V of the DA bacteriochlorophyll a n d at the s e c o n d a r y q u i n o n e Q~. A w a t e r chain w i t h a l e n g t h of 23 A e x t e n d s f r o m the s e c o n d a r y q u i n o n e to the c y t o p l a s m i c side of the RC. It is built up of 14 w a t e r m o l e c u l e s . This chain suggests a pathway for the p r o < o n a t i o n of Q m T h e s t r u c t u r e s of m u t a t e d resp. c h e m i c a l l y m o d i f i e d RCs and of the R26 s t r a i n s h o w c h a r a c t e r i s t i c d i f f e r e n c e s w i t h respect to the w i l d t y p e structure.

P-3-4-047

P-3 -4-044 TIME RESOLVED INFRARED AND STEADY-STATE FOURIERTRANSFORM-INFRARED SPECTROSCOPY OF NATIVE AND M U T A N T R E A C T I O N C E N T E R S O F RHODOBACTER SPAEROIDES

OF

I n v e s t i g a t i o n of the R e c o m b i n a t i o n F l u o r e s c e n c e in PS 11

B. Hilhnann t , I. Moya 2 and E Schlodder fiir Biophys. Chemic, TU-Bcrlin, Str. des 17. Juni 135, 10623 Berlin, 2Lab. pour l'Utilisation du Rayonnement Electromagn6tique, B~t 209-D, Centre Universtaire Paris-Sud, 91405 Orsay Cedex

~r-Institut

When the electron transfer to QA is blocked in PS lI charge recombination during the lifetime of P680+Ph¢o - can regenerate P680 , which equilibrates with the antenna pigments, and gives rise to long-lived recombination fluorescence. We have analysed the fluorescence decay kinetics of highly purified PS II core complexes from Synechococcus as a function of the rcdox state of QA and of the temperature between 20 and 300 K using single photon counting technique with ps time resolution. At room temperature, measuring on a time scale up 1o 320 ns, we could resolve

long lived fluorescence (x ~ l0 ns) in the presence of singly reduced QA (yield ~ l0 %). When the temperature is lowered components with lifetimes up to 140 ns are observed which is in accordance with the decay of P680+Pheo - measured by absorbancn difference spectroscopy. The recombination fluorescence decreases below 150 K but is still detectable even at T ~ 40 K, indicating a r e m a r k a b l y s m a l l energy gap between P680" and the radical pair at low temperature. A 5 ns component with an emission maximum at 691 nm is measured at T g 70 K and is attributed to antenna chlorophylls absorbing at longer w a v e l e n g t h s than P680.

59

Poster

P-3-4-048 TEMPERATURE DEPENDENCE STUDIES OF ELECTRON AND ENERGY TRANSFER IN PHOTOSYSTEM H REACTION CENTRES S.Mcrry, J.R.Durrant, J.Barber, G.Porter, D.Klug. Departments of Biochemistry, Centre for Photomolecular Sciences, Imperial College, London. Energy and primary electron transfer in Photosystem ILl(PSII) reaction eentres isolated from higher plants was studied as a function of temperature. These processes are monitored using transient absorption spectroscopy with femtosecond time resolution.The ptimary electron transfer from P680, the primary electron donor, to Pheophytin (Pheo), the primary electron accoptor, yields the radical pair P680+Pheo". At 293K, this charge separation reaction is eharacterised by a 21 ps time constant. Prior to the radical pair formation the excitation energy equilibrates in the PSI] reaction centre with a time constant of 100 fs. D a t a ~ be presented on the temperature dependence of the energy transfer equilibrium and the primary electron transfer reaction in PSII.

P-3-4-049

P-3-4-051 L PROTEIN, OVER-EXPRESSED IN E. C O L I , RESTORES NORMAL FUNCTIONING OF QA IN DI/D2/CP47/Cytb-SS9/I/ (14) PHOTOSYSTEM 11 CORE COMPLEX. T. Kobayashi, S. Ozawa, T. Shiina & Y. Toy'oshima Graduate School of Human and Environmental studies, Kyoto University', Yoshida-nihonnmatsu-cho, Sakyo-ku, Kyoto 606-0 l, Japan We prcviousl3 revealed by the reconstitution experiments that L protein is involved in normal functioning of Qx in PSII complex. (FEBS Lett. 354:113116 (1994)). To understand the actual role of the L protein on the electron transfer at Q\ site, we planned to carry out the reconstitution experiments with the various mutants of L protein pr~xluced in E. coli . Hcrc we report thc establishment of an over-expression system of I protein in E.coli to be used lbr the reconstitution of the Q\ function. L protein was produced as a fusion protein with maltose binding protein. After purification of the fusion protein by affinity chromatography, maltose binding protein of N-terminal side was digested. The resulting L protein was submitted to in vitro rcconstitution and lbund to recover the QA function in isolated D1/D2/CP47/Cytb-559/I/(H) PSII reaction center core complex. The effects of mutation of L protein on the Q.x function will be presented and discussed in relation to the regulatton of electron transli~r at Q,~ site in PSII.

P-3-4-052

MODELS OF P680 AND THE PRIMARY PHOTOCHEMISTRY OF THE PHOTOSYSTEM TWO REACTION CENTRE.

INTERACTION OF PLASTOQUINONE DERIVATIVES WITH THE QB SITE

James R. Durrant*. Stephen Merry, Stefan L S Kwat", Rienk van Grondelle~, George Porter', James Barber" and Jan P. Dekker ~ and David R. Klug" *Centre for Photomolecular Sciences, Department of Biochemistry, Imperial College, London, SW7 2AY, U K , and tDepartment of Physies and Astronomy and Institute for Molecular Biological Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.

H. Koikc, K. Yoncyama*, Y. Kashino and K. Satoh Dcpt. Life Science, Fac. Sci., Himcji Inst. Tcch., Harima Science Gardcn City, Hyogo 678-12, Japan; *Weed Center, Utsunomiya University, Tochigi 321, Japan

In this poster, we first of all consider an appropriate deseriptzon of the ehlorin excited states within the PS I] reaction centre. We suggest that exction interactions between PS II reaction centre pigments are likely to result in delocalised excited states. These exciton states are delocalised over a "multimer" of several reaction centre pigments including both the primary electron donor (P680) and pheophytin electron aeceptor. This exeiton model is then included in kinetic modelling of the PS II reaction centre. We consider possible kinetic models of charge separation in PS II which account for the experimental observations of a biphasic charge separation (25% 3ps and 75% 21 ps).

P-3-4-050

Plastoquinone-9 (PQg) mediates electron transfer between photosystem II (PS If) and cytochrome b6/f complex. PQ9 is bound to quinone-binding pocket, the QB site, and accepts electrons from a primary stable electron aeceptor, QA. However, a detailed reaction mechanism or the interaction between QB pocket and PQ9 is still unclear. Here, we measured the binding affinities of externally added plastoquinone derivatives (PQ0 PQ3) at the QB site in PS II membrane fragments prepared from spinach. The affinities of PQ derivatives to the QB site increased with the length of the isoprene chain. The turnover rate of PQ at the site, on the other hand, became lower with longer side chain. Estimated mechanism of interaction between the QB pocket and plastoquinone molecule will be discussed.

P-3-4-053 ISOLATION OF PHOTOSYSTEM H CORE COMPLEXES FROM A

PS II PRIMARYPHOTOCHEMISTRYIN WILD TYPE AND Dl-130 MUTANTS OF SYNECHOCYSTIS 6803.

CYANOBACTERIUM WITHOUT USING COLUMN CHROMATOGRAPHY

L.B. Criorgi, PJ. Nixon, S. Merry, JR. Durrant, D.M. Joseph,J. Barber, G. Porter & D.R. Klug Photosynthesis& Molecular DynamicsResearchGroups, Centre for Photomolecular Sciences, Departmentof Biochenustry, ImperialCollege, l_~nden, 8W7 2AY, UK

H. Egashira, K. Kashino. H. Koike & K. Satoh Dept. Life Science. Fac. Sci., Himeji Inst. Teoh. Harima Science Garden City, Ako, Hyogo 678-12, Japan

We have comparedthe primary photochemistryof PS II reaction centre preparations isolated from wild type Synechocystis 6803 and Dl-130 mutantsof Synechocystls to that of higher plains. D 1-130 is a glutamine m wild type Synechocystis and a glutamate in higher plants; it lies closeto the binding site of the photoactivepheophytin. We have studied mutants in whichthe residue at this positic~has been changedto a glutamate (as in higherplants) or a leucine. We find that when P680 is photoselectivelyexcited, charge separation is associatedprimarily with a ~ 21-25 ps time constantin PS II isolated from higherplants, Synechocystis and the Dl-130 mutantsof Synechocys~s. The radical pair spectrumfor wild type Synechocystis is differentto that of higher plants, in a way that is unusual and unexpected. What is most striking,however,is that this changecan be reversedby mutating the Synechocystis Dl-130 residue so that it is the same as in higherplants.

60

Oxygen-evolving photosystm ~ core complexes so far reported seemed to have mdified 0B sites. Here we will report a method to isolate and purify photosystum E particles from the thermophilic cyanobacterium, Syneohococcus vu/canus, without using coiu,m chrumatography. The thylakoid membranes were washed with a solution contains 0 . 6 % tritonX-|O0 and photosyst~ ~ particles were extracted from the men~branes with n-heptyl- B -D-thioglusoside, Photosyste~ ~ particles purified by sucrose density gradient centrifugation and ammonium sulfate fractionation had almost no phycebiliproteins, had some new subunit polypeptides and showed high rates of oxygen evolution.

Poster P-3-4-054 INTERACTIONS OF CHLOR- OR METHYL- SUBSTITUTED BENZOQUlNONES WITH THE QI3-SITE OF PHOTOSYSTEM "il REACTION CENTER. Y. Kashino, M. Yamashita, H. Koike and K. Satoh Dept. Life Science, Fac. Sci., Himeji Inst. Tech., Harima Science Garden City, Hyogo 678-12, Japan For the understanding of the structure and function of the Qe domain in photosystem II (PS II), it is very important to clanfy the precise behavior of plastoquinone in this domain. In the present study, we investigated the reduction kinetics of CI- or methyl- substituted benzoqulnones in spinach PS II membranes (BBYs) or heptylthioglucoside(HTG)-extracted PS II core particles. Affinities of the QB site for the substituted benzoquinones were estimated. The values of affinity and turn-over rates of the quinones in this site thus obtained fitted very well to those values which we reported for Synechococcus vu/canus PS II particles, showing reliability of these values and usefulness of the HTG-PS II core preparation.

P-3-4-055 MECHANISM OF PROTON-COUPLED ELECTRON TRANSFER TO Qa IN REACTION CENTERS FROM Rb. sphaeroides M. S. Graige, M. L. Paddock, S. H. Rongey, G. Feher and M. Y. Okamura. Dept. of Physics, University of California San Diego, La Jolla, C A 92093-0319 The reduction of the secondary quinone QB in bacterial reaction centers (RC) involves an unresolved sequence of electron and pr+oton transfer steos in the second electron transfer reaction Q ~ Q ~ + H ~ QAQBI-I(k~.). In order to elucidate the mechanism of this reaction we substitotecl various low potential quinones in the QA site while retaining Ql0 in the QB site. These substitutions should change the elementary electron transfer rates wi.'thout changing the proton transfer rates. M.casurements were made on natave RCs and mutant RCs with reduced k ~ due to mutations near QB. In native RCs, k ~ was found to increase with increasing driving force for electron transfer with a slope consistent with Marcus theory. A sLmilar d~endence was observed for EQ(H173) RCs. However, in DN(L213) RCs, was found to be independent of driving force. These results indicate: I) In native RCs, electron transfer is the rate limiting step. 2) In EQ(H173) RCs, the reduced k ~ is not due to rate limiting proton transfer since electron transfer remains the rate limiting step. 3) In DN(L213) RCs, slow proton transfer becomes the rate limiting step. Work supported by NIH and NSF.

P-3-4-056 THE ROLE OF AMINO ACID RESIDUES NEAR QB IN PROTON TRANSFER IN REACTION CENTERS FROM Rb. sphaeroides. ML. Paddock S.H Rongey, G. Feher & M.Y. Okamura, Physics Dept., UCSD, La Julia, CA 92093 Bacterial reaction centers (RCs) convert light into chemical energy through the double reduction and protonation of a bound quinone molecule, Qa. Site-directed mutagenesis of several amino acid residues near Qs has been used to probe the proton-coupled electron transfer kAn(2) (QA-QB" + H+ QAQBH'). The following new result2s were obtained. 1) Replacement of SerL223 with Gly maintains a fast kAs(), in contrast to the dramatic reduction in k:,B(2) observed when Ser is replaced with Ale. It is proposed that a water molecule may functionally substitute for the missing hydroxyl group of the Ser in the Ser-L223 ~ Gly mutant RCs. 2) Replacement of GIu-H173 with Gin results in a 20-fold decrease in kAB(2) (pH 7.5). The slower observed rate, however, is not due to a rate-limiting proton transfer step (see abstract by Graige et eL), but must be due to a decrease in the pK~ of the secondary semiquinone (QA-QBH) or a reduced rate of electron transfer. 3) The double mutant Asp-L213 --~ Glu/GIu-L212 ---r Asp is photosynthetically competent despite the fact that the Asp-L213 ---)Glu single mutant is not. It is proposed that compensating changes have occured to restore the electrostatic environment near QB to a functional state. Work supported by NSF and NIH

P-3-4-057 THE REACTION CENTER:CYTOCHROME C 2 COMPLEX FROM Rhodobacter sphaeroides: CO-CRYSTALLIZATION AND CHARACTERIZATION N AdiL H L. Axelrod, P. Beroza, S. H. Rongey, M. Y. Okamura and G. Feher, Dept. of Physics, Univ. of Calif, San Diego, La Jolla CA 92093 USA The reaction center (RC) and cytochrome c2 (cyt c2) of Rhodobacler ~phaeroides form a transient complex. The RC:cyt c2 complex was studied by a number of methods: i) The complex was crystallized with a maximum cyt c2:RC molar ratio of 1:3. The crystals diffract to a resolution of Y5A Aa model of the RC:cyt c2 complex was determined from analysis of the X-ray data and biochemical criteria. Electron transfer within the crystal from reduced cyt c2 to D÷ (the photooxidized primary donor of the RC) occurred in l~ts, the same time as found for the complex in solution, ii) Docking calculations based on inter-protein electrostatic complementation identified low energy docked complexes consistent with the binding site identified in the co-crystal, iii) Three RC aspartic acid residues were changed to lysines by site directed mutagenesis. The binding affinity of cyt c2 to these mutant RCs was reduced to the greatest extent when the mutation was near the position of the cyt c2 according to the co-crystal model. Work supported by NIH and NSF

P-3-4-058 THE ASSOCIATION OF DIFFERENT DETERGENTS WITH THE REACTION CENTER OF RB. SPHAEROIDES R26 AND RPS. VIRIDIS. P.W. Heanelrijk, AJ. Huff, H.L van Gorkom and P. Gast Deparlmemtof Biophysics, Huygens Laboratory. PO Box 9504, 2300 RA Leiden, The Netherlands Detergent-free reaction center (RC) proteins from the photosynthetic bacterium Rhodopseudomonas viridis and Rhodobacter sphaeroides R26 were obtained using Bio-Beads SM-2. With these RCs, the amount of detergent molecdes associated with the protein was measured for a large number of detergents. The amount of detergent molecules associated with the RCs increased by a factor 2.5 at pH 6.0 relative to pH 8.0, and decreased by a factor of two for various detergents in the presence of 5% 1,2,3 heptanetriol, supporting the notion that crystallization of the RCs is promoted by increasing the number of protein-protein contacts. The absorption band at 865 nm of the primary donor in the RC shifts to 846 nm when the detergent is removed from the RC; upon re-solubilization with various detergents this band shifts back to 865 nm. In 30% of the detergentfree RCs the secondary electron transfer from QA to Qa was inhibited. After re-addition of detergent the secondary electron transport was completely or partly restored, depending on the type of detergent.

P-3-4-059 THE STRUCTURE OF THE PHOTOSYSTEM II REACTION CENTRE DERIVED BY MOLECULAR MODELLING Bengt Svensson1 Catherine Etchebest2, Pierre Tuffery2, Jeremy Smith3, Paul van Ken1 & Stenbj6m Styring1 1Dept. of Biochemistry, ArrheniusLab., StockholmUniversity,S-106 91 Stockholm, Sweden; 2Lab. de Biochimie Th6orique, IBPC, 13 rue Pierre et Marie Curie, F-75005 Pads, France; 3SBPM, DBCM, CE-Saclay, F-91191 Gif-sur-Yvette,France The photosystem II (PSII) reaction centre is composed of the D1 and D2 proteins, which show a considerable homology with the structurally known bacterial reaction centre. This homology has ~iilowed us building a structural model for important parts of the D f/D2 heterodimer. The computational techniques applied incorporate statistical data on side-chain rotameric states and describe interactions within the protein using the CHARMM energy function. Detailed information about local structures is given for the environments of the redox components: TyrD, Tyrz, the pheophytins and the acceptor side Fe2+. Using exiton interaction calculations in combination with the protein modelling approach we have assessed the pigment positions including the structure of P680. P680 is proposed to be a chlorophyll dimer, but with a weaker excitonic coupling in comparison with the purple bacteria. In the model we explain this b y a larger distance and a sIightly different orientation between the chlorophylls. Hydrogen bonds between the protein and the chlorophylls are important for bindingand also functional properties of P680. The present model for the core of the PSII reaction centre accommodates all experimental results available and is directly verified for the regions around TyrD, Tyrz and the redox active pheophytin. From the model we have made functional predictions and hypotheses with the aim to design new experiments and theoretical studies.

61

Poster P-3-4-060 S T R U C T U R E AND F U N C T I O N O F T H E T Y R O S I N E - Z R A D I C A L I N P H O T O S Y S T E M II. C. Tommosl, X.-S. Tang 2, K. Warncke 3, C. Hoganson 3, S. Styring], J. McCracken 3, B. A. Diner 2, G. T. Babcock 3 ' Dept. of Biochemistry, Stockholm University, 106 91 Stockholm, Sweden; 2Central research and Development Dept., E. I. duPont de Nemours Co., Wilmington, DE 19880-0173 USA; 3 Dept. of Chemistry, Michigan State University, East Lansing, M148824 USA. Photosystem II (PSI/) catalyzes the light-driven oxidation of water to molecular oxygen. The water-splitting reactions are driven by the light-induced oxidation of the primary donor, P6s0, and catalyzed by the (Mn)4-cluster. The electron transfer between P680÷ and the (Mn)4-cluster is mediated by Tyrosine-Z (Yz), Y161 of the D1 reaction center protein. Yz has been trapped in its radical form by low temperatures illumination of Mn-depleted PSII core complexes prepared from a mutant stain of Synechocystis 6803 lacking YD, the second redox-active tyrosyl residue of PSI]. A complete characterization of the hyperfine structure of Yz" has been obtained by using a combination of isotopic labeling and multiple magnetic-resonance techniques. The analysis render evidence of considerable mobility of the radical, and show that Yz" is not involved in a well-ordered hydrogen-bonding interaction. By comparing these results with the situation for YD', the roles of biologically occurring amino-acid radicals in general, and recent data on the water-splitting process in PSII, we suggest that Yz acts as a H-atom transfer cofactor in PSI/. Within this model, Yz" abstract H-atoms from substrate water bound to the (Mn)4-cluster on each S-state transition.

P-3-4-063 EPR, ENDOR AND ESEEM INVESTIGATION OF THE ELECTRON

ACCEPTER RADICAL ANION QA" IN PHOTOSYSTEM II (PS II) REACTION CENTRES F. MacMillan ], J. Kurreck 1, N. Adir 2, F. Lendzian 1, H. K~8 ], F. Reifarth I, G. Renger I and W. Lubitz l. 1 Max-Volmer-Instittu ffir Biophysikalische und PhysikalischeChemie, Technische Universit~it Berlin, D- 10623 Berlin, Deutschland. 2 Department of Physics, University of California, San Diego, La Jolla, CA 92093-0319, USA. The g-tensor and hyperfme structure of QA'" is studied by Q-band EPR and X-band ENDOR. For these investigations it is necessary to remove the strong magnetic exchange coupling between the high spin Fez+ and QA" in PS II membrane fragments. This was achieved by either removal of the iron using lithium perchlorate and ophenanthroline or by cyanide treatment which leads to a low spin S=0 ground state of Fe2+. In a smaller PS II core preparation the QA'" is found to be decunpled from the high spin Fe2÷ without additional treatment. The hyperfme structure and the g-tensor of QA'" in these three preparations is compared with that of the isolated plastoquinone-9 anion radical in organic solvents. In addition, laN-hyperfine couplings from a neighbouring amino acid residue presumably hydrogen bonded to Qa'" have been observed using ESEEM spectroscopy. Differences in the electronic structure and hydrogen bonding of QA'" in these PS II preparations are discussed and the results are compared with those of QA'" in bacterial reaction centres.

P-3-4-061 H

DEPENDENT SPECTROSCOPIC PROPERTIES OF THE ROSINE D R A D I C A L IN MUTANTS O F T H E D2 P R O T E I N O F P H O T O S Y S T E M H. Davidsson. L. 1 Wermaas, W. 2 and Styring, S. t ÂDept. of Biochemistry, Arrhenius Lab., Stockholm University, Sweden. 2Dept. of Botany, Arizona State Univ., Tempe, Arizona 85287-1601 USA. In photosystem II TyrD (D2-Tyrl60) is probably hydrogen bonded to D2His 189 (Svensson et al, EMBO J. 9, 2051-2059, 1990). Four mutants in the D2 protein (His189Leu,Tyr,Gln and Gin 164Leu) in the close vicinity of TyrD have been found to have an altered EPR signal of TyrD+. In the Histidine mutants, the dominating spectral form is a narrow (linewidth 10G) featureless radical with a high g-value (g=2.0052) (Tommos et al, Biochemistry 32, 5436-5441, 1993). Interestlingy, in two histidine mutants His189Leu and His189Tyr we have observed a g-value shift between 2.0035 and 2.0050. In experiments where the g-shift was studied at different pH's, light conditions and temperatures we have observed that all three parameters affect the g-value shift. The g-shift occurs faster at a high pH (~7.5) and very slowly at low pH (~5.5), the g-shift also occurs faster in the dark than in the light and in higher temperature (~30°C) the g-shift accelerates significant. We suggest that this gshift depends on whether the tyrosyl phenolic group is protonated or not. There exists a correlation between the g-value and the protonation state of a tyrosyl radical, a low g-value indicates a protonated state and a high g-value a deprotonated state (Dixon et al, J. Chem. See., Faraday Transactions II, 72,1221-1230, 1976). Our results indicate that similar shifts in the protonation state of TyrD might occur also in vivo.

P-3-4-062 BIOCHEMICAL AND EPR CHARACTERISATION OF Zn(ll) SUBSTITUTED REACTION CENTRES FROM RHODOBACTER SPHAEROIDES AND

RHODOSPEUDOMONAS VIRIDIS. A. Gardiner F. Lendzian, R. Bitti, M. Kuhn & W. Lubitz.

Max-Vo/mer-lnstitut for Biophysikalische und Physikalische Chemie, Technische Universit~t Berlin, Straf3e des 17. Juni 135, 10623 Berlin, Germany. Two approaches have been developed for the substitution of the high spin Fe(ll) for diamagnetic Zn(ll) in the bacterial reaction centre (RC).This makes the investigation of the quinone anion radicals by EPR possible, These are (i) chemical exchange, involving reversible dissociation of the H-subunit, and (ii) biosynthetic replacement whereby the cells are grown in high Zn/Iow Fe media. Rhodobacter (Rb.) sphaeroides wild type ZnRCs have been produced by both methods. In addition ZnRCs were obtained biosynthetically from the mutant HC(M266) which contain up to 95% Zn. These methods were applied also to Rhodopseudomonas (Rps.) viridis. Chemical exchange has not yet been successful, however biosynthetic replacement resulted in RCs with a Zn(ll) content of 10 _+ 5%. The biochemical and spectroscopic (absorption, recombination kinetics, Q-band EPR & spin polarisation studies) characterisation of the different RC preparations is detailed.

62

P-3-4-064 FBNTOaBcoND SPBC~GScoPY OF Pe IZ IRB~CTZON C I ~ ! S. Greenfield 1, M. Waa£elewski 1, GovindJee 2, &

NaN IEISlgILTS 3

~ g o n n e National Laboratory, Aronne, IL 60439, USA; University of Illinois, Urbane, IL 61801, OSA~ 3National Renewable Energy Laboratory, Golden, CO 80401, USA We have reexamined rapid transient absorption processes in isolated PSII reaction center (RC) complexes. Pump-probe (magic angle I kinetic spectroscopy at 150 fs resolution employed a Tisapphlre-laser-driven optical parameter amplifier system. Data were obtained at a much greater S-to-N ratio, lower pulse energy, and more selective wavelength excitation than used earlier (PS,I~ 91, 8999, 1994). Time evolution of the absorption changes e x c i t e d in the 665-685 nm range (FWHM, 5 nm I and probed at 545 nm (Pheo reduction) or 738 (stimulated emission) showed Ts on the order of 1-3 ps, 10-23 ps, and 40-100 ps. At 685 nm excitation, the r e s p e c t i v e amplitudes were 35%, 40%, and 25S (545 nm) and 20%, 50%, and 30% (738 nm). When Pheo was reduced before the experhnent, the amplitude of the i-3 ps component was diminished c o m p a r e d to the other components. These new data reaffirm our c o n c l u s i o n that the primary charge separated state (P680+Phmo -) forms in 1-3 ps, and the longer components are associated with energy transfer phenomena in the RC preparations.

P-3-4-065 ULTRAFAST SPECTROSCOPIC STUDIES OF ELECTRON TRANSFER IN PHOTOSYSTEM II. B. Donovan, L. Walker II, C.F. Yocum, and RJ. Sension Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109-1055

Recent measurements of the primary electron transfer event in isolated reaction centers of photosystem II have led to disparate interpretations for the mechanism of photoinduced charge separation in this system. Previous time-resolved measurements have been interpreted in terms of a ca. 3 to 20 ps time scales for charge separation. In an effort to cast some additional light on the problem, we have systematically addressed the influence of sample preparation and excitation intensity on the observed kinetic signals. The number of time components observed, as well as their magnitudes and spectra are very strongly influenced by the intensity of the excitation pulse. The kinetic signals and saturation effects observed are also found to be influenced by the method of sample separation, with particular sensitivity to small amounts of CP47 contamination. Some signals which appear to be linear at moderately high excitation intensities are found to be dependent upon multiple pigment excitations in individual reaction centers.

Poster

P-3-4-066 An investigation of the role of the accessory bacteriochlorophylls in the reaction centre from Rhodobacter sphaeroides

Soiedel, D., Ridge, J.P. and Jones, M.R. Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, $10 2UH. The photoreaction centre from the purple bacterium Rhodobacter sphaeroides has three protein subunits that encase four molecules of bacteriochlorophyll and two molecules of bacteriopheophytin. The central magnesium of all four of the bacteriochlorophyll molecules is five co-ordinated, with the fifth (axial) ligand being provided by a histidine residue. It has been established that mutagenesis of the axial histidines of the special pair of bacteriochlorophylls that constitute the primary donor of electrons can lead to the replacement of the bacteriochlorophyll by a bacteriopheophytin. In this report we examine the effects of mutagenesis of the axial ligands to the monomeric bacteriochlorophylls at the accessory positions on the active and inactive pigment branches.

P-3-4-069

Light InducedChangesin PhotosyntheticReactionCenter Detected by SynchrotronRadiation Chong-Ci Li (BeijingAgriculturalUniversity) Wei Wang(National Laboratoryof Synchrotronradiation)

Absorption spectra or CD (circular dichroism) spectra obtained from ordinary spectrophotometer or spectropolarimeterare limited to wavelength longer than about 190nm. Because wavelength shorter than 190nm, the absorption of air increases quickly with the decreasing of wavelength. Furthermore, we have no convenient strong light source with wavelength under 190nm. But spectra of this shorter wavelength range are very important and useful in measuring and determining conformational change of Amino acids; proteins; pigment-protein complexesetc. In a synchrotron, light energy are emitted from and transmitted in high vacumm space, and it is strong enough for some spectral measuring work We built a device on a radiation beam from < Beijing Electron Positron Collider >,and obtaineda light beam with wavelengthdown to about 165nm. Using this device, we obtained absoption and CD spectra of pigment-protein complexes extracted from reaction center of photosynthesis. Spectra obtained from dark adapted pigment-preteincomplex and after it was illuminatedby actinic light were different. Comparing these spectra we found light induced conformational changes of the pigment-protein.

P-3-4-067 STRUCTURE AND PROTEIN BINDING INTERACTIONS OF PRIMARY DONOR OF CHLOROFLEXUS AURANTIACUS,

P-3-4-070 Time-resolved and steady state spectroscopy of membrane-bound mutated reaction centres from Rhodobacter sphaeroides

A. Ivancich 1, R. Feick2, A. Ertlmaier2 and T.A. Mattioli 1 1 Section de Biophysique des Protrines et des Membranes, DBCM, CEA and URA CNRS 1290. CE-Saclay. 91191 Gif-sur-Yvette, France; 2MaxPlanck Institut for Biochemie, D-8033 Martinsried, Germany.

Rid,qe, J.P., Beekman, L.M.P.I", van Grondelle, R.I" and Jones, MR. Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, $10 2UH and 1" Department of Physics and Astronomy, Free University of Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands

Chloroflexus aurantiacus is a thermophilic, green photosynthetic bacterium which represents an interesting species from the evolutionary point of view. Moreover, while belonging to the family of Chloroflexaceae (green bacterium) the reaction center (RC) fi'om C. aurantiacus exhibits many similarities with that of purple bacteria, such as Rb. sphaeroides. In the present work, we report near-infrared Fourier transform Raman spectra excited at 1064 nm of both reduced and oxidized states of the primary donor (P) from C. aurantiacus. From the analysis of the carbonyl region, the hydrogen-bonding pattern of P as well as the detailed protein interactions with it are deduced. The implications of these structural features on the redox midpoint potential of the primary donor are also discussed.

P-3-4-068

MAGNETIC CIRCULARDICHROISMSPECTRA OF PSI[ REACTION CENTER COMPLEX DI/D2/Cyt b559 Chong-Oin Tram, Kun-Yun Yang, Ting-Yan Kuang & Pei-Son8 Tang Lab. of Photosynthesis, Institute ofBoteny, Acdemia Siniea, Beijmg 100044. P. R. China Zi-wei Shea and Yan-dao Gong State Key Laborato~ of Biomembraneand MembraneBiotechnology, Tsmg-Hua University, Beijing 100084, P.R. China The magnetic circular diehroism (MCD) specmma is reported for isolated PSiI reaction center complex (RC). The MCD spectrum ofPS II-RC has characters of standard chlorophyll a (Chl a) and Pheophytin a (Pheo a), Companng with absorption spectnun, MCD spectrum of PSI1 RC shows no the signals of [~carotene. The components of Q band are more clearly discermble in the MCD spectrum of PSII RC than those in absorption spectrum: Qx(l-0),Qx(0-0) and Qy(1-O) transitions appea~ as three negative peaks. Qy(O-O)transition is composed of three peaks at 660 nm, 667 nm and 680 nm. By the method of photoinhl'bition, we relate them to free Chl a, accessory. Chl a and PSII prmaary_ electron donor P680. Therefore, we suggest the MCD technique as a powerfid method m the research of PSII RC.

The purple photosynthetic bacterium Rhodobacter sphaeroides strain RCO1 has the reaction centre as the sole pigment protein complex of the bacterial inner membrane. Strains of this type allow study of the effects of mutagenesis on light-driven transmembrane electron transfer whilst the reaction centre is still in the natural membrane. In this report we examine the steady state and timeresolved spectroscopic properties of a number of membrane-bound reaction centres with mutations in amino acid residues that lie in the vicinity of the cofactors on the active branch of pigments. We discuss the relevance of our findings to proposed mechanisms for light-driven electron transfer in the bacterial reaction centre.

P-3-4-071 INTERRUPTION OF THE WATER CHAIN IN THE REACTION CENTER FROM RB. S P H A E R O I D E S REDUCES THE RATES OF THE PROTON UPTAKE AND OF THE SECOND ELECTRON TRANSFER TO QB Laura Baciou and Hartmut Michel (Max-Planck-Institut Frankfurt/Main, Germany)

ffir Biophysik,

In the reaction center from Rb. sphaeroides, the possible role of the chain of bound water-molecules in proton transfer from the solution to the secondary quinone (QB) was investigated by site-directed mutagenesis and flash-induced absorbance spectroscopy. At about 9A from QB, ProL209 situated along the water chain was changed into the aromatic residues Phe and Tyr in order to interrupt the chain. In the ProL209->Tyr mutant, the lack of changes in the P+QB" recombination and the first electron transfer rates (kAs0)) indicates that the mutation does not lead to large structural changes. In the ProL209->Phe mutant, knBO) is 7-fold decreased but follows the pH dependence of the wild type. This observation may reflect some slight structural modification in this mutant and/or rearrangement of the cluster of charged residues close to the L209 position. The major effect of the mutations observed is a concommitant decrease of the rates of the second electron transfer, kAa(2), and of the associated proton uptake, more pronounced above pH 8. Our results indicate that the mutations have specifically altered the pathway of proton transfer to QB. We propose that in the wild type the chain of bound water molecules provides the most efficient pathway for the proton transfer from cytoplasm to the close vicinity of QB.

63

Poster m

P-3-4-075

P-3-4-072 ELECTRON TRANSFER AND TRIPLET STATE FORMATION IN PHEOPHYTIN A - MODIFIED REACTION CENTERS FROM RHODOBACTER SPHAEROIDES (R-26) AT LOW TEMPERATURES E.M. Franken' & A. Ya. Shkuropatov 2 & P. Gasd & A.J. Hoff ~ ~Dept of Biophysics, University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands; 21nstitute of Soil Science and Photosynthesis, Russian Acadamy of Sciences, Pushchino, Moscow region 142292, Russian Federation Similar to native reaction centers (RCs), RCs Of modified Rb. sphaeroides (R-26), in which banteriopheophytin a is replaced by plant pheophytin a, are photoehemically active down to 10 K. The yield of the P*Q, state in modified RCs is lower than that in native RCs and nearly independent of temperature. The triplet state of P is detected optically in modified RCs with pre-reduced Q,. Comparison of the absorption difference spectra for P+Q," and 3p in native and modified RCs suggests that the structure of P and its interactions with the accessory bacteriochlorophyll are not changed dramatically by the modification procedure. A lower yield of P+Q," in modified RCs is probably due to a shorter lifetime of the intermediate state P*I both at room and at low temperature.

D Y N A M I C EFFECTS ON SPECTRA O F THE P H O T O S Y N T H E T I C REACTION CENTER Bruno KallebritmI and Sven Larsson 2 ]Department of Biochemistry and Biophysics, GOteborg University and Chalmers University of Technology, 413 90 G0teborg; 2Department of Physical Chemistry, Chalmers University of Technology, 412 96 GOteborg Theoretical investigations of the photosynthetic reaction center have been made much easier thanks to the erystallographieaily solved structures of the bacterial reaction center. The reaction center is a very fine tuned system where the first excited state and the charge transfer state lie very dose to each other in energy. So far, no satisfactory theoretical explanation for the very high rate of the primary charge separation has been given. Including the dynamics in the system by means of molecular dynamics might shed some light on the mechanisms important for this very fast electron transfer. We have performed molecular dynamics on relevant ehromophores in the reaction center and sampled coordinate sets at certain time intervals to include the dynmnies of the system. We will present some results on calculations with CNDO/S where the input data are obtained by molecular dynamics

P-3-4-073 ELECTROSTATIC INFLUENCES OF GLU M236 ON QB FUNCTION IN PHOTOSYNTHETIC REACTION CENTERS FROM RB. SPHAEROIDES

E. Takah~i and C.A. Wraight Dept. of Plant Biology & Program in Biophysics, Univ. of lllinois, Urbana, IL 61801, USA The now high-resolution structure of the Rb. sphnoroidesRC (E."mleret al. (1995) Structure 2: 925) provides many details not seen in previously published sU'octeres.These include water molecules within the RC that form a continuous, hydrogan-bended chain from the proteinwatea"interface to the QB binding site, and which could piny a role in the delivery of protons to QB. Umn8 s i ~ e c ~ d mutsgeaesis, we have altexedseveral residues winch can be seen to interact with molecules of the water chain. One of these, GluM236, was altered to Gin, Lea, Gly and Asp. The pH dependence of the fLrStelectron transfer equilibrium in the alkali~ pH region was shifted from a pK of 0.9 in the Wt to -7.5 in the GIwM236-->Gly, Leu and Gin mutants. No shift was detected in the Asp mutant. Similm"p/f shifts were found in the pH dependences of the rate of the fast electron tnmsfer. However, no si~iflcant difference in the pH depondenoesof the second eleclron transfer rate were ~ for any of the four mutant.% relative to the Wt, although the Asp mutant rates were slightly fastm'. Thus, the four mutants do not impart drastic effects on RC function, inoludingproton delivery capabilities. However, GluM236 does influence the QB" semiqinnone stability, through the pK that controls the high pH behavior. This was originally attributed to GluL212 (Paddock et al. (1988) PNAS 86:6602; Takahashi & Wralght (1992) Biochemistry 31:855), but infra-red spectroscopy, indicating the ionization slate of this residue, has not supported this atlribution, at least not in a simple fashion (Hienerwadel et al. (1995) Biochemistry 34:2832). It is likely that interactions between several re,~ddnoscontribute to an "aplxtrent"pK in this alkaline region and GinM236 may be a significant contributor to this behavior. Supported by NSF and USDA.

P-3-4-076 SXTE-DIRECTED MUTKGENESIS OF THE psb& GENE TEE GREEN IkLGAE, C H L I & M Y D O H O N A S REINHARDTII.

IN

Y. T a k a h a s h i , K. U t s u m i , A. H a t a n o , Y. Y a m a m o t o Satoh D e p t o f B i o l , F a c of Sci, O k a y a m a U n i v e r s i t y , T s u s h i m a - n a k a , O k a y a m a , 700, J A P A N

& K.

We have generated several Chlamydomonas psbA transformants in which a specific amino acid s u b s t i t u t i o n is i n t r o d u c e d i n t o D1. F i r s t , S e r 3 4 5 a t +I p o s i t i o n o f c l e a v a g e s i t e of t h e p r e c u r s o r DI (pD1) w a s c h a n g e d t o Gly, Cys, V a l a n d P h e t o i n v e s t i g a t e t h e i r e f f e c t s o n t h e p r o c e s s i n g of pD1. P u l s e - c h a s e l a b e l i n g e x p e r i m e n t s of D1 d e m o n s t r a t e d t h a t a l l t r a n s f o r m a n t s p r o c e s s e d pD1 as e f f i c i e n t l y as w i l d t y p e , s u g g e s t i n g t h a t t h e r e s i d u e a t t h i s p o s i t i o n is n o t e s s e n t i a l t o t h i s r e a c t i o n . S e c o n d , T h r a n d S e r at 286, 291 a n d 292 p o s i t i o n s t h a t a r e p r o p o s e d t o b e i n v o l v e d in h y d r o g e n - b o n d w i t h P 6 8 0 were substituted by Ala or Leu. The resulting transformants showed normal PSII activity, indicating that these residues are not indispensable to PSII activity.

P-3-4-074 I S O L A T I O N O F O X Y G E N E V O L V I N G PSH P A R T I C L E S AND PSH REACTION CENTRES FROM THE FILAblENTOUS C Y A N O B A C F E R I U M Spirulina platensi~ Eva ~etlikovh t, Petr Bud~i~ 1 and Danube Sofrov~t 2 l Institute of Microbiology, Academy of Sciences oftha Czech Republic, 37981 Tgebofi; 2 Department of Biochemistry, Faculty of Sciences, Charles University, Albertov 2 0 3 0 , 1 2 840, Praha 2; Czech Republic Thylakoid membranes were isolated from Spirulina cells using mechanical disintegration with glass beads at 4"C. Extraction of the membranes with dodecylmaltoside (leA) resulted in high yields of active oxygen evolving PSII particles; LDAO, SB 12 and Triton X-100 appeared less appropriate (in the order given). The particles were purified in a sucrose gradient (20 to 40 %) and a green layer at 35 eA sucrose was loaded on an immobilized metal (Cu z+) Sepharose column for a chromatographic reaction center isolation. The washing procedure that yielded earlier good results with PSII core particles from Synechococcus had to be modified. Octylgiucoside appeared suitable for stripping offthe CP43 and CP47, while DM provided an efficient elution of the reaction center. Spectral and functional characteristics of the RC will be presented in the poster.

64

P-3-4-077 THE EFFECTS OF THE Q-~)OL REDUCTION STATE ON ELECTRON FLOW TRRGGGII THE R H O D O B R ~ z ' ~ S]PHREROIDZ$ R E A C T I O N CENTRE

B.J. van Rotterdam, W. Crielaard, R.W. Visschers , H.V. Westerhoff & K.J. Hellingwerf E.C. Slater Institute for Biochemistry and Microbiology, University of Amsterdam, The Netherlands Flash induced donor-recombination transients were recorded in isolated, solubilized Rb. sphaeroides reaction centres, supplemented with ubiquinon-0 and cytochrome cz. The concentration and redox state of both electron mediators were varied and experiments were carried out under background illumination to detect steady state kinetics. All transients revealed multiphasic exponential behaviour which could only be analysed using a kinetic mathematical model which contained 12 different intermediate reaction centre states. The main advantage of this strategy was shown in the possibility to register rate constants at the acceptor (QA-Qs) site while measuring at the donor site. The analysis revealed steady state rate constants for re-reduction of the donor by cytochrome c (l.5Xl0~.M-1.sec-1), by direct recombination from QB (2.0 sec "I) and the rate constant for QJQBHz-exchange (8.5Xl06.M-1.sec'1). No recombination from QA occurred under steady state illumination. Control analysis on these data showed a high response coefficient of the Q-pool reduction state on the overall flux through the reaction centre.

Poster P-3-4-078

P-3-4-081

SIDE CHAIN CONFORMATIONALDISTRIBUTIONS. DYNAMICS AND ENERGETICS IN MODELAND PHOTOSYSTEMII TYROSYLRADICALS

RATES OF ELECTRON TRANSFER FROM QA TO QB IN RIIODOBACIOR SHAEROIDES R-26 RCS

K. Wamcke.X.-S.Tang1, C. Tommos2, G. T. Babcock.B. A. DinerI & J. McCracken Dept. of Chemistry.MichiganState Univ., E. Lansing,M[ 48864, 1Cen~alResearch& Development Dept., Expt. Station, E. I. DuPont de Nemours & Co., Wilmington. DE 19880-0173,2Dept. of Biochemistry,ArrheniusLab. for NaturalSciences.StockholmUniv.,S-10691Stockholm,Sweden Systematic characterizationof static distributionsin hydrogen hype~me interactionsin randomly orientedradicalsin the solidstateby using2H electronspin echoenvelopemodulationspectroscopyhas been described,a The hyperfineinteractionbetweenthe two ~-methylene2H nucleibondedto C[}and couplingn-spin density at ring carbon Cl in the tyrosineradicaltrapped in low temperatureaqueous glass was addressedspecifically.The model radicalexhibitsa non-uniformangulardistributionabout CI-CI5 (full extent, 36°). A theoretical treatment has been developed to extract the equilibrium thermodynamicparametersthat characterizemotionaboutCI-C~ priorto mediumvitrification,wherein the observed"frozen-in"relativerotaraecpopulationsare assumedto representthe quantummechanical probabilityamplitudes for harmonicCI-CI~oscillators,b The three potentialenergy curves obtained (arisingfromthe three CI~-CYconformations)and librationalfrequenciesidentifykey factorsthat control the reactivityof calalytictyrosineradicalsin enzymes. Applicationof the analysisto the YZ radical trapped by freezing isolated YD-less mutant Synechocystis PS It under illuminationreveals Ci-Cll dispersion of ---29°, whichcorrespondsto high frequencymotionin a singlepotentialwell. A model linking tyrosine sidechain dynamics about CI-Cjt and C[t-Cyand hydrogen transfer reactivity is developed, aK.W.& J.M., J. Chem. Phys. subm.;t~K.W.,G.T.B. & J.M., Z Phys. Chem., subm.

D.Gilrov. D.Tiede* and M. Gunner CCNY, Physics Dept. 138th & Conv e n t , N'~', N Y 10031,*Chem Div., A r g o n n e N a t L a b , A r g o n n e , IL 60439 Menaquinone3 (MQ) and UbiquinoneA (UQ) were substituted into the Q A and Q B sites o f bacterial RCs. A G for the M Q A - to U Q B electron transfer is slightly more favorable (20 eV) than for U Q A - to U Q B . M Q A - , and U Q A - or U Q B - have markedly different abserbance between 395-420 a m , as measured in R C s with trapped semiquinone. The electron transfer from Q A - to Q B c a n thus be measured directly from the optical transition between MQA- and UQB.. Previous measurements relied on Stark shifts in BCh or BPh in the near IR, or on a small shift in the UQ- spectrum at 397 run. Two rates for electron transfer have been characterized. At p H 8.1, 25" C: 65% is a t 5 5 0 0 / s (Ea--4.2 kcal), a n d 3 5 % a t 1 2 0 0 / s (Ea=7.8 kcal). Both rates are essentially p H independent b e l o w p H 9.5. A t all pHs, as temperature drops f r o m 25-0"C amplitude is transferred from the fast to the slow rate. However, the amplitudes of these two rates is only 50% of that expected from the trapped M Q A - and U Q B - spectra. Kinetics of the Stark shift in the near IR, with faster time resolution, do show a faster component (k>100,000/s). This could account for the missing amplitude, if it is also present in the 395.420 nm transition. N.I.H.RO1-GM48726-02

P-3 -4-082

P-3-4-079 I N V E S T I G A T I N G T H E R O L E OF W A T E R IN R E A C T I O N C E N T E R - Q U I N O N E F U N C T I O N BY O S M O T I C P P , ~ S S U R E J. W. Larson & C. A. Wraight Program in Biophysics, University of Illinois, Urbana, IL, 61801, USA The crystal structure of reaction centers (RCs) from Rhodobader sphacroides, at high resolution (Ermler, ct aL, 1995, 2, 925), reveals many buried water molecules, some of which are well positioned for a role in proton transfer to, and binding of, the secondary quinone, QB. We have attempted to explore the role of water in RC function by applying high oemotie pressures. RGs were titrated with miscible cosolvents to high concentrations, corresponding to oemotic pressures of up to 500 atm. Three effects were observed on the flash induced reaction kinetics as a function of rising cceolvent concentration: (1) the bark reaction of the electron transfer from Q~ to the primary donor slows down, (2) the second electron transfer from Q ] to O~ accelerates, and (3) the binding affinity for ubiquinone diminishes. The effects were independant of the chemical identity of the cceolvent, indicating a connection to solution osmoledity. These resul~ suggest that bound water may play a role in quinone binding as well as affecting the protein's ability to solvate the negatively charged semiquinone. Electrostatic effects may also cause the observed kinetic changes since the dielectric coustant of the aqueous medium changes as a function of cusolvent concentration. These two models are compared in discussion of the data.

P-3-4-080 CHARACTERIZATION OF A ~ C~ULA~S MUTANT WITH TI-IE QB SITE MADE MORE LIKE QA.

R C SYMMBTRIZED

DIMERIC AND MONOMERIC CORE COMPLEXES OF HIGHER PLANT PHOTOSYSTEM II- A FUNCTIONAL STUDY Giles Johnson and Engelbert Weis Institut for Botanik, Westf~ilisches Wilhelms-Universit~it Munster, Germany

A core complex o f higher plant photosystem II, consisting o f D I , D2, cytochrome b559, CP 43 and CP 47, can be isolated in a dimeric or a monomeric form, depending on the precise isolation procedure. Cores are isolated using digestion in dodecyl-flD-mahoside followed by ion-exchange chromatography. Monomerisation is favoured by detergent digestion at low salt concentrations at pH 8. Whilst the preparations vary in the activity o f the oxygen evolving complex, they are in other respects similar, in terms o f the presence and stoichiometry o f different redox components. We are conducting measurements, using various spectroscopic techniques, to characterise these preparations and to establish whether dimerisation is important in the functioning o f photosystem H

P-3-4-083 S T R U C T U R A L AND MECHANISTIC STUDWoS OF PSII IN ABSENCE O F MAGNETIC A C C E P T O R SIDE NON-HEME IRON.

THE

Y. Deligiannakis, S. Un, A. Boussac a n d A. W. R u t h e r f o r d L Li, W.J. Coleman*, D.C. Youvan #, M.R.Gunner.Phys.Dept. C C N Y , 1 3 8 t h St. a n d C o n v e n t Ave., NY, N Y 10031. *Dupont C R & D , Expt. Station, P.O.Box 80173, Wilmington, DE,19880. #Palo Alto Inst. Mt. View, C A 94043 Rb. c a n s u l a ~ RCs from a photosynthetically competent revertant of the mutant with M220-M261 f r o m the Q A site substituted for LI93-L227 in the Q B site have been investigated. Despite the huge change in the QB pocket, secondary quinone function still can be reconstituted. The UQ4 binding constant for the QB site is the same for mutant and wild type RCs. The rate of charge recombination f r o m P+QB-, knP, is 0.02S -t vs 1S -1 for wild type RCs at p H 7.3. Thus, the equilibrium constant between QA-QB and QAQB- is 400 in the mutant and 10 in wild type, showing large stabilization of QB-. In the wild type, kBP increases above p H 9.0 and decreases below pH 6. In the mutant, ksp is p H independent f r o m p H 4 . 6 - 1 0 . 3 . T h e electron transfer rate f r o m QA" to QB in the mutant (15S -t) is 3 orders of magnitude slower than the wild type as determined by the c y t o c h r o m e e oxidation with different intervals between first and second flash. Only 5 0 % P+QB- is formed, as determined by the back reaction kinetics, when the QB site is saturated with quinone, again supporting a very slow electron transfer rate from QA to QB. Supported by NIH RO1-GM48726.

Service de Bio~nerg~tique, Y v e t t e Cedex, France.

DBCM,

CEA

Saclay,

91191

Gif

sur

The acceptor side Fe2 + (S = 2) of PSII reaction center was converted to its low-spin state Fe2 + (S = 0) by treatment with CN. The anion radicals of the primary quinone QA and the pheophytin were studied in Tris-treated PS II by c.w. X-band EPR, c.w. High Field (245GHz) EPR and Electron Spin Echo spectroscopy:(a) In the presence of the diamagnetic iron the triplet state 3P680 is EPR detectable only after double reduction of the plastoquinone. This shows that the magnetic state of the iron does not influence the lifetime of the 3P680.(b) The relaxation profile of the state Ph-QA'Fe2 ÷ (S = 0) is different from both the PhOA'Fe 2 + (S = 0l or Ph'QAFe2 + (S = 0) states, probably due to a weak magnetic coupling between the radicals. In the high spin state (S = 2) the iron enhances the spin-lattice relaxation of the pheophytin.(o) At high magnetic fields (8.7-r) the usual magnetic interaction between the plastoquinone radical and the high spin ferrous iron (g = 1.9 signal at X band) has an negligible effect on the EPR spectrum o! the QA" radical. The principal g-vatues of the plastoquinone anion radical were resolved in the high field EPR spectrum, gx = 2.00659, gy= 2.00515, gz = 2.00228.(d)From the ESEEM in 14N- and 15N- PSII it is shown that there is a superhyperfine coupling between the QA radical and a protein nitrogen nucleus with a quadrupole coupling constant e2qQ/h = 3.4MHz,an assymmetry parameter n =0.49 and A= 2.1MHz.

65

Poster P-3-4-084

P-3-4-087

CHARACTERIZATION OFTHE PURIFIED REACTION CENTER FROM THE PURPLE BACTERIA RHODOCYCLUS TENUIS

DYNAMICS OF ELECTRONIC AND VIBRATIONAL C O H E R E N C E S IN P H O T O S Y N T H E T I C R E A C T I O N C E N T E R S

I A,,alidis & F. Reiss-Husson Centre de G6n6tique Moleculaire, CNRS, B~t 24, 91198 Gif sur Yvette France.

M. Sugawara, M. Hayashi, S. Suzuki and S. H. Lin

Reaction centers (RCs) of Rc. tenuis have been prepared by dodecyldimetbylamine oxide treatment of chromatophores followed by chromatographic purification in presence of dodecyl maltoside. Initially a tetraheme cytc was bound to the RC, but it was removed at 90% during purification. RCs displayed photochemical activity as observed by the bleaching of 865nm absorption band of the donor P870 and the IR absorption band arising at 1260 nm attributed specifically to P870 +. Flash induced absorbunce changes showed that 85% of QB (secondary quinone) was still bound to the RC. P+QB'"-)PQB recombination rate was very slow (0.1s-1) relative to P+QA----)PQA rate (33s-1) where QA is the primary quinone. Extraction of quinones from the RC allowed us to identify QA with menaquinone 8 and QB with ubiquinone 8. The shape of flash induced QAabsorption band is different from those measured in other bacterial RCs where QA is a menaquinone, probably because of different interactions with other pigments and/or residues of QA pocket..

Institute of Atomic and Molecular Sciences, A c a d e m i a Sinica, Taipei, Taiwan, ROC Effects of electronic and vibrational coherent motions on the stimulated e m i s s i o n signals are theoretically investigated for the elucidation of the m e c h a n i s m o f the q u a n t u m beats appeared in the stimulated e m i s s i o n profiles of the reaction center of Rhodobacter sphaeroides. We demonstrate that rapid vibrational coherent motions result in significant phase c h a n g e s in q u a n t u m beats as a function of probing frequency. T h e s e phase changes, in addition to vibrational relaxation, lead to transient Stokes shifts in the stimulated emission profiles. Electronic coherence, on the other hand, prevents such phase c h a n g e s in q u a n t u m beats.

P-3-4-085 ELECTRON TRANSFER REACTIONS IN CRYOPHILIC ALGAE Atsuko Kanazawt;1, Antony R. Croftst'2 and David M. Kramer3 'Program in Biophysics and Computational Biology, "Dept. of Microbiology and "Dept. of Plant Biology. University of Illinois at Urbana-Champaign, 393 Morrill Hall, 505 S. Goodwin Ave., Urbana, IL 61801 Cryophilie algae are photosynthetic organisms normally found on the surface of permanent snow or ice. We have obtained ten strains of these isolated from the surface of Antarctic snow, and are making a comparative analysis of the energeties ofphotosystem II (PSI/) in them. Previously, in temperate plants, we found high activation barriers for the electron transfer reactions from Qa" to Qb, and recombination from S2Qa" and from S2Qb'. In the eryophilie algae, Chlorella antarctica, we found significant lower activation barriers for these reactions. We suggest these represent specific adaptations ofPSII bioenergeties for life at low temperatures.

P-3-4-088 The non heme iron of P h o t o s y s t e m xl investigated b y FTIR difference spectroscopy R. H i e n e r w a d e l , A. Boussac, a n d C. B e r t h o m i e u DBCM, CEA-Saclay, 91191 G i f - s u r - Y v e t t e - C e d e x , France The Fe 3+ to Fe 2+ t r a n s i t i o n of the n o n h e m e iron of Photosystem II w a s i n v e s t i g a t e d by light-induced FTIR difference spectroscopy with Tris-washed m e m b r a n e s in the p r e s e n c e of ferricyanide. The F T I R difference spectrum between 1800-1000 cm -I shows c h a n g e s of I R - m o d e s f r o m l i g a n d s a n d n e i g h b o u r i n g r e s i d u e s of the iron. T h e b i n d i n g of b i c a r b o n a t e to Photosystem II is shown b y exchange with 13Cbicarbonate. The Vas(C00-) a n d Vs(CO0-) modes of b i c a r b o n a t e are found at approx. 1560 cm "I and at 1338 c m -I, respectively, which suggest bidentate l i g a t i o n in the Fe2+-state. In order to g a i n m o r e detailed information on the interaction of bicarbonate with protein side chains we will compare FTIR difference spectra obtained in presence of various other small anions like glycolate, g l y c o x y l a t e etc.

P-3-4-089

P-3-4-086 ELECTRIC FIELD EFFECTS ON FLUORESCENCE EMISSION OF Rb. sphaeroides CHROMATOPHORES

T Y R O S I N E D R A D I C A L IN P H O T O S Y S T E M FTIR DIFFERENCE SPECTROSCOPY

Jana Steiger and Kenneth Sauer Chemical Biodymanics Division, Lawrence Berkeley Laboratory and Dept. of Chemistry, University of California, Berkeley, CA 94720 (U.S.A.)

R. Hienerwadel, Berthomieu

Room-temperature fluorescence emission of Rb. sphaeroides chromatophores shows a linear dependence on electric field strength for both positive and negative applied fields. The direction is opposite to that expected for an electric-field induced change of charge separation/recombination rates in the reaction center; also opposite to the fluorescence change previously observed in spinach thylakoids (Dau, H. and Sauer, K., 1991, Biochim. Biophys. Acta, 1098, 49-60). The maximum of the electric-field induced change (Fe) is at 890 nm corresponding to the maximum of (quorescence emission from LH1. Trap closure by high light intensity leads to an increase in the magnitude of Fe without altering its spectrum or linear dependence on applied field strength. Reduction of the primary quinone QA with sodium dithionite causes a reversal of the direction of the linear electric-field induced effect and a shift of the Fe emission maximum to 910 nm. These results are consistent with an electricfield effect on the flugrescence of antenna pigments in Rb. sphaeroides chromatophores. The effect of QA reduction is interpreted in terms of a diffusion-limited model for excitation transfer to the reaction center.

66

A.

CEA-Saclay (*URA Bio~nerg~tique, DBCM, France.

Boussac*,

CNRS 91 191

II

J.

INVESTIGATED

BY

Breton

C.

and

1290) Section de Gif-sur-Yvette, Cedex,

TyrD (D2 160) of p h o t o s y s t e m II (PS II), w h i c h has a still mostly unknown function, easily forms the s t a b l e r a d i c a l TyrD ° . The p h o t o o x i d a t i o n of TyrD has been studied by FTIR difference spectroscopy in c o n d i t i o n s w h e r e the e l e c t r o n a c c e p t o r side of PS II is r a p i d l y r e o x i d i s e d b y ferricyanide. The TyrD°/TyrD FTIR difference spectrum is compared to FTIR difference spectra of tyrosine and phenol radicals g e n e r a t e d b y U V i r r a d i a t i o n a t l o w temperature. TyrD signals can be identified at 1513 a n d 1252 can-~. These f r e q u e n c i e s p r o v i d e an e x p e r i m e n t a l evidence t h a t Tyro is p r o t o n a t e d in its r e d u c e d state. A b a n d a t 1504 c m -I is a s s i g n e d to the v7a (CO) m o d e o f Tyr~ ° . C o n t r i b u t i o n s f r o m o t h e r a m i n o a c i d s to the TyrD°/TyrD s p e c t r u m will b e a n a l y s e d to p r e c i s e the s p e c i f i c e n v i r o n m e n t w h i c h s t a b i l i z e s Tyr~ ° .

Poster

P-3-4-090 FEMTOSECOND INFRARED SPECTROSCOPY OF BACTERIAL REACTION CENTERS FROM RB. SPHAEROIDES P.Hamm. M.Zurek, W.Zinth Institut ffir Medizinische Optik, Universit~tt Mnnchen, Barbarastr. 16, 80797 MOnchen, Germany With the impovcment in laser technology, time resolved femtosecond infrared spectroscopy is able to give new insights into structural changes of molecules during photochemical reactions. In this presentation we will demonstrate recent experiments on the initial electron transfer reaction of bacterial reaction centers ofRb. sphaeroides testing the I g absorbsnce changes of the sample between 1000 and 1800 cm-1 with a time resolution of 200-300 fs. The discussion of the difference spectra is focussed on into two spectral regions: (i) the high frequency region (>1600 cm-l) where a detailed assignment of the spectrum to distinct vibrational modes in the special pair can be given, and (ii) the low frequency range (<1600 cm-1) where only s qualitative assignment is possible. Nevertheless, especially in the low frequency range, a pronounced general absorption increase is observed, which does not turn up instantaneously but within an ultrafast initial reaction occurring on a time scale of 200 fs. This reaction is related to conformational changes of the special pair P altering its electronic structure.considerably

P-3-4-091

P-3-4-093 RE-EVALUATION OF THE PIGMENT STOICHIOMETRY OF ISOLATED PSII REACTION CENTRES D.Zhel~va, B. Hankamer, A. Telfer & J. Barber Biochemistry Department, Imperial College, Imperial College Rd, London SW7 The number of pigment molecules associated with isolated PSII reaction centres (RC) prepared according to Chapman D., et al., (1989) Photosyn 23:411 and Vacha F., et al., (1995) PNAS in press, were determined by reverse phase HPLC. The HPLC system was calibrated using standards chlorophyll a (chlA) and 13- carotene (13-~ar) (purchased from Sigma) and extinction coefficients E mM for chlA- 76.79 (at 663.6 in 80% acetone, reported by Porra R.J., et al., (1989) BBA 975:384) and E mM for 13-car 139 (at 452 nm in 100% hexane; Zechmeister L., (1943) J.Am.Chem.Soc 65:1522). Pheophytin a (pbeoA) standard was prepared by acidification of chlA with HCI (2mM). The pigment stoichiometries of higher plant reaction centres were found to vary slightly depending on seasonal growth conditions of the pea plants. The best RC preparations isolated by the method of Chapman et al., (1989) were associated with 6.4 chlA : 213-car : 2 pheoA while the Vacha type RCs contained 5.2 chlA : 0.6 13-car : 2 pheoA. As RC chlA]pheoA stoichiometries were also determined by calibrating the HPLC system using an method, independent of extinction coefficients we estimate our values to be accurate to within 3%.

P-3-4-094

TRAPPING AND ENERGY TRANSFER IN THE DzD2-cyt-b-5$9 REACTION CENTER COMPLEX AT 1.5 IC

THE CONFORMATION OF QA" SEMIQUINONE HEADISOPRENYL TAIL DIFFERS BY 90 g IN PSII VS BACTERIA

T.J. Aar~ma. P. Scbellenberg and R.LW. Louwe. Dept. of Biophysics, Huygcns Laboratory, University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands.

M. Zheng a,v. Pctrouleas b and G. C. Dismukes a* aprinceton University, Dept. Chemistry, Princeton, NJ 08544 USA blnstit. Material Science, NCSR "Democritos',15310 Aghia Paraskevi Attikis, Greece

The excited state dynamics in the DtDz-cyt-b-559 reaction center complex at 1.5 K has been investigated by means of accumulated photon echo measurements. The photon echo decay is multiexponential, and depends on the wavelength of excitation in the 680 nm region. The various decay components are attributed to primary charge separation, energy transfer, and trapping by a long-wavelength absorbing pigment, respectively. We will also relxxt on the temperature dependence of these processes in the temperature range of 1.5 to 30 K, and on experiments to identify the nature of the pigments involved. The experimental results may be compared with those of hole-burning measurements.

Quinones found in bacteria (type I: ubquinone, menaquinone, Vit-K1) cannot effectively replace plastoqninone (PQ) in the QA site in PSII. Using 1H ENDOR spectroscopy we find that the head-tail conformation at Ct(isoprenyl) differs by 90~for Qa- in bacteria (both MQ and UQ) vs PSII, with the latter case having a coplanar quinone ring-Cl~C~isoprenyl) conformation. These two distinct conformations are found to be the lowest energy conformations for the isolated anion radicals free in solution. This difference is due to non-bonded repulsions between the ring C6methyl and isoprenyl tail in solution which axe present in type I semiquinones (9(P ring-isoprenyl) but which are absent in PQ-9 owing to the lack of the C6methyl. QA- in PSII has two 2H20 exchangeable hyperfme couplings, suggesting two H-bonds analogous to bacteria. The hyperfine couplings are 15-20% smaller in PSII indicating either slightly longer H-bonds or lower spin density at carbonyl-O atoms. We propose that the aforementioned binding affinity difference at the QA site derives in part from the implicit structural difference between PSII vs bacterial RC proteins that must exist to accomodate the different isoprenyl conformations. NIH-GM39932.

P-3-4-092 P680 + REDUCTION IN PS II CORE PARTICLES AND THE EFFECT OF UV-B ON THE DONOR SIDE OF PS II

P.B.Lukins 1 , Adele Post2, P.J.Walker 1 and A.W.D.Larkum 2 1 School of Physics; 2 School of Biological Sciences, University of Sydney, NSW 2006, Australia The kinetics of P680 + reduction in spinach photosystem II (PS II) core particles were studied using both repetitive and single-flash 830 nm transient absorption. Single-flash measurements indicate decay times of 7 ns, 40 ns, 95 ns, 4 Its, 35 Its and 90 Its. The 40 ns and 95 ns components are assigned to the normal S-state controlled Yz ---> P680 + electron transfer. The 4 Its component appears to be associated with the water-splitting catalytic site and the 90 Its component is assigned to P680+ - QA- recombination. The data gives results that are consistent with the current model of the oxygen-evolving complex but the overall kinetics are slightly faster than for BBY preparations. Treatment of these core particles with UV-B radiation leads to (a) conversion of the nanosecond kinetics to microsecond kinetics in a fashion similar to that for hydroxylamine and (b) a gradual decrease in the initial amplitude. These observations, together with the results of single-flash measurement.s, show that the primary UV-B damage is to the oxygen-evolving complex and primary charge separation.

P-3-4-095 THE EFFECT OF PS II-NON QB REDUCING ON THE MEASUREMENT OF THE FLUORESCENCE QUENCHING PARAMETERS qp AND q. Philip D. Laible, Jeffrey L. Mullert, Phil Ginsburg, and Thomas G. Owens, Section of Plant Biology, Comell University, Ithaca, NY 14853-5908 USA Depending on growth conditions, between 10 and 30% of PS II reaction centers are incapable of electron transport beyond Q^ (PS II non-Q B reducing). These non-Q B centers are thought to be an intermediate in the repair of damaged PS II reaction centers. As a consequence of the slow (seconds) oxidation of QA- in non-Q B centers (compared to ps or ms kinetics in functional PS II), the non-Qs centers are closed at very low actinic intensity. Thus, at physiological light intensities, all of the non-Q 8 centers are closed while a large fraction of functional PS II centers remain open. Calculation of photochemical (qp) and nonphotochemicel (qN) quenching assumes a homogeneous population of PS II units - an assumption that is clearly not met. Using a simple model, we demonstrate the effects of non-Q a centers on fluorescence quenching parameters qp and %. In the case that nonphotochemical quenching affects normal and non-Q a PS II similarly and that the ratio Fv/F o is identical in the two types of centers, the presence of non-Q a centers reduces the actual value of qp by a factor of (l-B) where B is the fraction of total PS II that are non-Q a centers. The values of qN are not affected. We describe a method to estimate B using standard modulated fluorescence techniques.

67

Poster

P-3-4-096 ISOLATION OF REACTON CENTRES FROM PLANT PHOTOSYSTEM II AND PHOTOSYNTHETIC PURPLE BACTERIA BY RAPID PERFUSION CHROMATOGRAPHY Margrit Roobol-Boza 1, Susana Shochat2,3 , Peter Gast3 and Bertil Andersson1,2 1 Dept. of Biochemistry, Stockholm Univ, Sweden, 2 Dept. of Biological Chemistry, Hebrew Univ. of Jerusalem, Israel, 3 Dept. of Biophysics, Leiden Univ. The Netherlands. The new method of ion-exchange perfusion chromatography has dramatically reduced the time for isolation of soluble proteins while retaining separation efficiency. We here report that this chromatographic technique can also be successfully applied to hydrophobic membrane proteins allowing rapid isolation of photosynthetic reaction centres from various biological sources. Solubilized PS II reaction centres from spinach could be isolated after a chromatographic step requiring only 6 minutes compared to previous procedures requiring several hours or over night separations for completion. The isolated PSII reaction centres showed at least the same purity and functional intactness as the previous preparations. Through a modification of established procedures combined with perfusion chromatography, reaction centres from wild type and mutant Rhodobacter sphaeroides cells could be isolated completely free from light-harvesting complex and other contaminations. The separation time was significantly reduced compared to previous isolation procedures.

P-3-4-099

SOLUBILIZATION AND PURIFICATION OF THE PHOTOSYNTHETIC UNIT FROM RHODOSPIRILLUM RUBRUM. Janine Kessi and Reinhard Bachofen. Institut fiir Pflanzenbiologie der Universitiit Ziirich, Zollikerstrasse 107, CH-8008 Ziirich. The chromatophore membrane from R. rubrum is solubilized using the short chain lecithin Diheptanoyl-sn-phosphatidylcholine (DHPC). This detergent leads to the solubilization of photosynthetic units (LH-RCcomplex) which are purified to homogeneity using ionic exchange chromatography and ultracentrifngation in a sucrose gradient. Absorption and near-IR CD spectra as well as RC-activity of the purified LH-RC-complex are well preserved. SDS-12%-PAGE analysis reveals the presence, together with the expected subunits of the LH- and RC-complexes, of a diffuse band migrating slightly above the H-subunit of the RC-complex. On 2D-gel electrophoresis this band focuses exactly at the same pI's as the H-subunit, suggesting a great homology of structure and function between the two polypeptides and, therefore, the existence of two different states of the H-subunit.

P-3-4-097 THE B I N D I N G E N V I R O N M E N T OF THE R E D U C E D P R I M A R Y QUINONE ELECTRON ACCEPTOR, Q A ' , OF PSII.

P-3-4-100 T H E T R I P L E T S T A T E 3p6s0 IN P H O T O S Y S T E M II STUDIED W I T H T I M E - R E S O L V E D EPR

X.-S. Tan81, B. Diner 1, J. Peloquin 2, G. Lorigan 2, L. Gilchrist 2, D. Randall 2, R. D. Britt2 and V. Petrouleas 3 - 1) CR&D, E. I. Du Pont de Nemours & Co.; 2) U.C., Davis; 3)NRC Demokritos

A. Kamlowski, A. van der Est and D. Stehlik Physics Department, Free University, Arnimallee 14, 14195 Berlin, Germany

The binding environment of QA- has been investigated using CN--treated PSII core complexes isolated from the cyanobacterinm Synechocystis 6803. ESEEM results obtained from PSI] isotopieally labeled with 14N and 15N show that nitrogen is coupled to QA'. Possible candidates for such nitrogen coupling are D2-His214 and D2-AIa260, the PSII homologues of the bacterial reaction center M-His217 and M-Ala258, respectively, that hydrogen bond to QA. W e have incorporated 14N-histidine into globally 15N-labeled PSII core complex, and confirmed that one of the ESEEM nitrogen couplings indeed arises from histidine, proving that histidine hydrogen bonds to QA" in PSII. In order to investigate the interaction between QA and D2-Ala260, we have made site-directed mutants at this site. In addition, site-directed mutants have also been constructed at the site of D2-Trp253, the homologue of the bacterial reaction center M-Trp250 that closely interacts with QA. Biochemical and spectroscopic characterization of those mutants has been carded out.

In Photosystem H (PSII) the triplet state of the primary donor, 3p680, is formed via recombination of the primary radical pair, P680÷Pheo-, if the light-induced electron transfer to the secondary acceptor, QA, is blocked. At low temperature, the characteristic spin-polarized EPR spectrum of 3p680indicates that P680 is a chlorophyll-a monomer the plane of which is tilted 30 ° out of the membrane [1]. This peculiar orientation argues against a strict structural analogy of PSU and bacterial reaction centers (bRC). Temperature dependent studies feasible with time-resolved EPR are presented which give further information. Indeed, a different EPR spectrum could be observed above - 150 K, which is narrowed as a result of triplet delocalization. Possible structural arrangements which account for the spectra are presented and discussed in relation to the results in bRC and Photosystem I. In addition, at low temperature the EPR spectrum of 3p680 is detected independent of the state of QA (extracted, singly or doubly reduced) which shows that P680+Pheo- is created in PSII with singly reduced QA. [ 1] F. van Mieghem et al., Biophys. Biochim. Acta 1058 (1991) 379-385.

P-3-4-098

M U T A G E N E S I S OF THE cd- AND D-HELICES OF THE D1P O L Y P E P T I D E IN S Y N E C H O C Y S T I S PCC 6803: EFFECTS OF T H E PROTEIN ENVIRONMENT ON THE PROPERTIES OF P680

W-J. Coleman, P.J. Nixon & B.A. Diner E.I. du Pont de Nemours & Co., Central Research and Development, Experimental Station, P.O. Box 80173, Wilmington, DE 19880-0173 USA Site-directed mutagenesis was used to change a number of amino acid residues in the putative cd- and D-helices of the Dl-polypeptide (psb A3 gene) of Synechocystis PCC 6803. Photosynthetic growth tests and spectroscopic measurements were used to determine whether any of these residues affects the functional properties of P680, the primary electron donor of PS II. Mutagenized residues include D1-His198 in the D-helix and several in the cd-helix. Results of optical and EPR measurements on the primary donor will be presented.

68

THE CONSTRUCTION

P-3-4-101 O F C Y T O C H R O M E B559 M U T A N T S O F

CHLAMYDOMONAS REINHARDTII F.Mor'~is, J.Barber & P.Nixon Biochemistry Department, Imperial College of Science Technology and Medicine, London SW7 2AY, U K The role of cytochrome b559 in Photosystem II function is unknown although a role in protection from photoinhibition is suspected. In order to test this hypothesis we describe the construction and charactedsation of cytochrome b559 mutants in Chlamydomonas reinhardliL

Poster P-3-4-102 ELECTRON REACTION

TRANSFER MONITORED CENTERS FROM PURPLE

BY CHLOROPHYLL BACTERIA

FLUORESCENCE

IN

Sz. O s v ~ t h I, G. L a c z k 6 I, P. S e b b a n 2 and P. Mar6ti ~ IDept. of Biophysics, J6zsef A t t i l a University, H-6722 Szeged, E g y e t e m u.2., Hungary; 2CNRS CGM, F-91198 Gif sur Yvette, France In spite of the r e l a t i v e l y low quantum y i e l d (4x10 -4) and n e a r infra red (920 nm) detection, fluorescence of the c h l o r o p h y l l d i m e r was u s e d to m o n i t o r changes in redox states in the reaction center of photosynthetic purple bacteria Rb. sphaeroides and Rb. capsulatus. The yield of f l u o r e s c e n c e of the dimer was followed either u n d e r r e c t a n g u l a r shape of illumination ("fluorescence induction") or under flash e x c i t a t i o n of laser diode fireing at 810 nm. F l u o r e s c e n c e y i e l d s of the dimer in different redox states of the r e a c t i o n centers were t a b u l a t e d for the two bacterial species. Finer d i s t i n c t i o n than o n l y "open" or "closed" reaction c e n t e r from the point of v i e w of p h o t o c h e m i s t r y can be established. E s s e n t i a l e l e c t r o n transfer parameters as sharing rate and e q u i l i b r i u m constant of electron d i s t r i b u t i o n b e t w e e n the two q u i n o n e s could be d i r e c t l y determined. The i n v e s t i g a t i o n s c o n t r i b u t e d to the c l a r i f i c a t i o n of the o r i g i n of the o b s e r v e d f l u o r e s c e n c e and d e m o n s t r a t e d its use as a s u p p l e m e n t a r y m e t h o d to track the p r i m a r y steps of electron transfer.

P-3-4-105 PARALLEL ELECTRON PARAMAGNETIC RESONANCE AND LoW-TEMPERATURE FLUORESCENCE MEASUREMENTS OF Mn-DEPLETED PHOTOSYSTEM II MEMBRANES. Robert H. Schweitzer and Gary W. Brudvig. Department Chemistry, Yale University, New Haven, CT 06511, USA.

Parallel electron paramagnetic resonance (EPR) and lowtemperature fluorescence measurements were made on the same Mn-depleted photosystem II m e m b r a n e s . The redox state was controlled by chemically preoxidizing Cytochrome b559 followed by illumination at different temperatures (200-270 K ) in t h e p r e s e n c e o f 3 - ( 3 , 4 - d i c h l o r o p h e n y l ) - l , 1 dimethylurea. P 6 8 0 + f o r m e d b y t h e i l l u m i n a t i o n is r e d u c e d b y e i t h e r c h l o r o p h y l l Z ( C h l z ) or t y r o s i n e D d e p e n d i n g o n the illumination temperature. In t h i s w a y , samples with varying amounts of oxidized Chiz (measured by EPR) were prepared. A correlation between the amount of Chlz + present and the fluorescence intensity at 85 K was found i n d i c a t i n g t h a t C h l z + a c t s as a f l u o r e s c e n c e q u e n c h e r .

P-3-4-106

P-3-4-103 ACID-BASE

TITRATION

OF BACTERIAL

REACTION

CENTRE

L. K~im~nl, P. S e b b a n 2 and P. Mar6ti I IDept. of Biophysics, J6zsef A t t i l a University, H-6722 Szeged, E g y e t e m u. 2., H u n g a r y 2Centre de G e n e t i q u e Moleculaire, CNRS, Gif/Yvette, France

The a m i n o acids and the cofactors of the reaction centre (RC) p r o t e i n from p u r p l e b a c t e r i a have been l o c a l i z e d w i t h atomic resolution. The light induced p r o t o n u p t a k e b y the RC has b e e n d e t e r m i n e d b y m a n y authors and less than one p r o t o n per RC was found. O n l y few p r o t o n a t a b l e residues can be i n v o l v e d in the light induced p r o t o n uptake. The p r o t o n a t i o n of the p r o t e i n as a w h o l e (in d i f f e r e n t redox states) has not b e e n the subject of attention. The problem, however, is of great importance as it can be r e l a t e d to the a v a i l a b i l i t y of p r o t o n a t a b l e amino acids to the w a t e r phase. U s i n g a c i d - b a s e titration, the p U - d e p e n d e n c e of p r o t o n u p t a k e b y the whole RC p r o t e i n was d e t e r m i n e d from the b u f f e r i n g c a p a c i t y of the system and was d e c o m p o s e d into p r o t o n a t a b l e groups w i t h pKs close to their sulutional values. The d e t e r g e n t s d e c r e a s e d the a c t i v i t y of protons about o n e - t h i r d of that in aqueous solution. It was found that all of the p r o t o n a t a b l e groups were a c c e s s i b l e (on an e x t e n d e d time scale) to the w a t e r phase. The light induced differential p r o t o n u p t a k e was also d e t e r m i n e d from b u f f e r i n g capacities u s i n g the acid m i x i n g technique and g o o d agreement was found w i t h the earlier data.

of

ACTION OF TRYPSIN ON ILLUMINATED AND DARK-ADAPTED BACTERIAL PHOTOSYNTHETIC REACTION CENTERS: AN INDICATION OF LIGHT-

INDUCED CONFORMATIONAL CHANGES l.A.Smimova I, A.Binmberg2, L.-E.Andr~assonI & P.BrzezinskiI IDept. of Biochemistry & Biophysics, and 2Dept. of General & Marine Microbiology, University of G6teborg, Medicinaregatun 9C, S-41390 G6teborg, Sweden Reaction centers (RCs) from the photosynthetic bacterium Rhodobacter sphaeroides contain three polypeptide subunits (L, M and H) and photoehemically active cofaetors associated with the L and M subtmits. Isolated RCs were partly digested by trypsin under illumination and in the dark. Trypsinolysis resulted in different polypeptide patterns detected by gel electropboresis and reverse-phase high-performance liquid chromatography. The RC degradation was found to be more prominent in the illuminated samples. A partial amino acid sequence analysis of some of the resulting polypeptides was carried out to identify positions of the trypsin attack. The must hydrophilic H-subunit was attacked preferably. This is consistent with the previous data that trypsinolysis of the illuminated RCs brings about a 100-fold retardation of the electron wansfer from the primary to the secondary quinone acceptor (P.Brzezinski & L.-E.Andr6asson, in press) which is known to depend on the H-subunit integrity. It is suggested that the light-induced charge separation in RCs is accompanied by a rearrangement of the protein making it more accessible to the trypsin degradation.

P-3-4-107

P-3-4-104 INVESTIGATION ON HIGH ENERGY QUENCHING: EFFECTS OF pH AND LIGHT ON FLASH-INDUCED ABSORPTION CHANGES AT 820 run IN THYLAKOID MEMBRANES AND PS lI PARTICLES

THE TEMPERATURE EFFECT ON DIRECT CHARGE RECOMBINATION FROM D÷QAQI~ TO DQAQ B IN BACTERIAL REACTION CENTERS FROM RHODOBACTER SPHAEROIDES

Anja Krieger SBE, DBCM, B~tt. 532, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France on leave from J.v.Sachs Inst., Mittl. Dallenbergweg 64, D-97082 Wfirzburg

A. L a b a h n t ' 2 , J . M. Brueea, M.Y. Okamura 2 and G, Feher 2 1Univ. Freiburg, Inst. f'dr Phys. Chemie, D-79114 Freiburg; 2Univ. of Calif., San Diego, Phys. Dept., L a Jolla, CA92093, USA; 3Univ. of Manchester, Chem. Dept., Manchester M139PL, UK

Effects of high proton concentrations on the activity of PS II were analyzed in thylakoid membranes and PSII enriched membrane fragments. Flashinduced absorption changes were measured at 820 am with a maximal time resolution of 20 ns reflecting the transient P680 + formation and electron donation. Signals obtained with PS II particles, pretreated at low pH, and thylakoids, in which a proton gradient was built by hydrolysis of ATP, were compared. The effect of additional, illumination during the measurement and the light saturation behaviour were studied in presence and absence of electron acceptors. These results are interpreted in regard to chlorophyll fluorescence quenching and pH-depcndent regulation in the reaction centre of PS II.

The charge recombination from D+QAQ]~ to DQAQ B in native reaction centers (RCs) proceeds via the inlermediale state D÷QAQB(Kleinfeld et al. (1984) Biochim. Biophys. Aeta 766, 126). To observe the direct charge recombination rate, kBD, we increased the energy difference between D÷Q2QB and D÷QAQ~ by raising the energy of the D÷QAQB state t h r o ~ h substitution of Qlo by low potential quinones. Based on a total of 32 independent measurements we found an increase in the observed recombination rates from Q~ from 0.115+-0.002 s -1 at 277 K to 0.176 ~ 0.006 s -1 at 313 K. After correcting for the thermal expansion of the RCs (5X) and the temperature dependent contribution of klndireot the direct rates kBD were found 1o be 0.100±0.004 s -1 and 0.13"t:0.01 s -1, respectively. We fitted the data with the Marcus theory using an enthalpy of AHOD=0.39 eV and an entropy of A S ~ D = - 0 . 5 0 meV/K. The reorganization energy of ~BD=0.9t0.1 eV is in agreement with our previous result (Labahn et el. (1995) Chem. Phys. (submitted)).

69

Poster

P-3-4-108 INVESTIGATIONS OF PHOTOINDUCED ELECTRON TRANSFER IN PYROPHEOPHYTIN-ANTHRAQUINONE MODEL SYSTEMS

P-3-4-111 ISOLATION AND CHARACTERIZATION OF A D1/D2/ CYTOCHROME b-559 COMPLEX FROM PHORMIDIUM

A. Y. Tauber j, J. P. Helaia1, N. V. Tkachenko 2, H. Lemmetyinen2 and P. H. Hynninen I [Department of Chemistry, University of Helsinki, P.O. Box 55, FIN-00014 Helsinki, Finland; 2institute of Materials Chemistry, Tampere University of Technology, P.O. Box 589, FIN-33101 Tampere, Finland

LAM1NOSUM.

The second electron transfer (ET) step in the bacterial photoreaction centre

(Rhodopseudomonas

viridis) involves photoinduced El" from bacteriopheo-

phytin (BPheo) to menaquinone (MQ). The time for the ET (=200 ps) is determined by the environment, mutual orientation and distance between the BPheo and MQ molecules. We have studied the influence of these factors on the El" process in the novel donor-acceptor compounds, P4(R)- and P4(S)-oxy-[9,10anthraquinone-2-carbonyl]- pyropheophytin a, by NMR, time-resolved fluorescence spectroscopy and molecular modelling. The flexibility of the phytyl chain

M. Aured and R. Picorel. Department of Plant Nutrition, Estaci6n Experimental de Aula Dei-CSIC, Apdo. 202, 50080 Zaragoza, Spain. We have developed a method to isolate the Dl/D2/Cytochrome b-559 from the cyanobacterium Phormia~um laminosum from a highly purified oxygen evolving core complex (OECC) preparation. Thylakoid membranes were diluted with buffer containing the detergent LDAO and separated by a sucrose gradient centrifugation. The lower pigmented fraction was loaded onto a DEAE-Toyopearl column and a fraction with a red maximum at 675 nm which corresponded to the OECC was obtained. This particle was solubilized with Triton X-100 and the chaotrophic agent tanrine. The resultant extract was rechromatographiedon a Q-Sepharose column. A D 1/D2/Cytochromeb-559 complex was obtained.

in our model compounds allows the presence of two main types of conformers, the folded one and the opened one, with different photochemical properties. The time for the ET in the folded conformer was found to be less than 10 ps.

P-3-4-109 EARLY STEPS IN PHOTOSYNTHETIC REACTION CENTERS P R O B E D BY ACCUMULATED P H O T O N ECHO E X P E R I M E N T S P. Schellenbex$ , R.J.W. Louwe, T.J. Aartsma Department of Biophysics, University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Nederlands Accumulated photon echo experiments are performed to probe early steps in photosynthetic reaction centers. The systems investigated include the reaction centers of Rhodobacter (Rb.) 8phaeroides R26, the mutant Rb. sphacroides M(Y)210W and the PS II of green plants. The mutant M(Y)210W is distinguished from Rb. sphacroides IL26 by its slow electron transfer rate in the first step of charge sepaxation, wkich is also reflected in the photon echo experiments. In all of the reaction centers investigated, the echo signal show a non--exponential decay pattern, indicating a dispersive kinetics. The assumption of a ~parking state' being responsible for the multioxponential decay pattern can be excluded from the results. The results axe compexed with hole burning, time resolved fluorescence and transient absorption measurements.

P-3-4-112 INTERACTIONS THAT INFLUENCE THE REDOX POTENTIAL OF THE BACTERIOCHLOROPHYLL DIMER IN REACTION CENTERS FROM RItODOBACTER SPHAEROIDES J. C. Williams, K. Artz, X. Lin, X. Nguyen, and J. P. Allen Dept. of Chemistry and Biochemistry, and Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, AZ 85287-1604, USA Mutagenesis studies of reaction centers from Rhodobacter sphaeroides have shown that additions of hydrogen bonds from histidine residues to carbonyl groups of the bacteriochlorophyll dimer are correlated with increases in the redox potential of the dimer (Lin et al., 1994, Proc. Nail. Acad. Sci. USA 91, 10265-10269). In wild-type reaction centers, His L168 forms a strong hydrogen bond with the ring I acetyl group of the A-side bacteriochlorophyll of the dimer, and the mutation His to Pbe at L168 results in a 95 mV decrease in the dimer redox potential. A variety of mutants are being designed to develop models of the particular bacteriochlorophyll-protein interactions that are responsible for the changes in redox potential. For example, Asn L166 probably forms a hydrogen bond with His L168 and thus should influence the strength of the hydrogen bond between His L168 and the dimer. To test this hypothesis, the mutations Ash to Asp, Ash to His, and Asn to Leu are being made at L166, and the effect of these substitutions on the properties of the mutant reaction centers, particularly the midpoint potential of the dimer, will be characterized.

P-3-4-U0 NEW EPR SIGNALS ASSOCIATED WITH CYANIDE BINDING ON THE IRON-QUINONE COMPLEX OF PSII

P-3-4-113 PHOTOACOUSTIC DIAGNOSTICS OF LASER INDUCED PROCESSES IN REACTION CENTERS OF Rhodobacter Sphaeroides

Y. Sanakis and V. Petrouleas Inst. Mat Sci., N C S R Democritos, 15310 Aghia Paraskevi, Athens, Greece.

O.V. Puchenkov ,Z.Kopf& S.Malkin The Weizmann Institute of Science, Biochemistry Department, Rehovot, Israel

C N - binds at/or in the vicinity o f the non-heme iron of PSII in a stepwise fashion. At relatively low concentrations CKd 0.1-0.2 mM) it modifies the QA-Fe2+ state inducing an EPR signal at g=1.98 [Koulougliotis, D. et al. (1993) B.B.A. 1141, 275-282]. At high concentrations (K d ca 1.2 m M in PSII membrane fragments) it converts the iron to a low spin (S=0) state [Sanakis, Y. et ai (1994) Biochem. 33, 9922-9928]. We s h o w here that (i) the g=1.98 signal is accompanied by a ground state signal at g=2.0038 (ii) the split pheophytin signal is significantly modified in the presence of cyanide and the spectra can be explained on the basis o f a theoretical model (iii) new EPR signals result from the Fe 3+ state; at high concentrations of cyanide these signals are typical of a low spin (S = 1/2) configuration.

70

A theoretical model of photoacoustic signal generation in a suspension of bacterial reaction centers (RCs) is suggested, and a technique of dynamic separation of ditferent contributionsin photoinduced volume changes is proposed. In practical implementation of the technique it was found, that electron transfer in the RC isolated from Rhodobacter sDhaeroides is associated with a fast (<100 ns) contraction 32q-1 As which has the same value for wild type and R-26 preparations. The enthalpy of the P÷QA"state deduced from our measurements is 0.56~.04 eV. The quantum yield of photochemistry dpwas measured in a broad range of background light intensities. Its value for dark adapted RCs, ~b--l.0, decreased continuosly to qb=0.3 at background light intensity = 1 photon/(RC "s). Long background light illumination induces reversible structural changes which relax in about 15-20 rain after charge recombination. In photochemically "closed" reaction centers a deactivation process, following excitation, involves a long lived intermediate state characterized by an exponential decay with a lifetime 620:~80 ns and an amplitude 0.14±0.04.

Poster

P-3-4-114 NOVEL EFFECTS OF NITRIC OXIDE BINDING ON PHOTOS Y S T E M II. Charilaos Goussias, Yiannis Sanakis and Vasili Petrouleas.

Institute o f Materials Science, N C S R ParaskevL Athens, Greece.

"Demokritos", 15310 Ag.

Nitric oxide (NO) interacts with i m p o r t a n t P h o t o s y s t e m II (PSII) components. N o t a b l e is the interaction o f N O with the n o n h e m e iron(lI) o f the acceptor side, a n d the reversible binding o f N O to the tyrosyl radical YD + o f the d o n o r side (Petrouleas V. a n d Diner B.A., Biochim. Biophys. Acta, 1015, 131-140). Two new E P R signals are induced by N O treatment, coupled to the photosynthetic electron transfer. (a) D u r i n g incubation o f PSII preparations with N O a precursor complex is f o r m e d by redox interaction o f N O with a donorside component, p r o b a b l y the M n - W . O . C . This u p o n illumination f o r m s a stable nitroxide radical signal centred at g=2.0053 a n d charaeterised by AlI=44G a n d A 1=22G. (b) Following charge separation at -30oC, N O interacts slowly with a PSII c o m p o n e n t a n d forms a transient - 4 0 G wide E P R signal with an inflection p o i n t at g~2.013. T h e origin o f these signals is currently u n d e r investigation.

P-3-4-117 PHOTOACTIVITY MEMBRANE MODELS

OF

REACTION

CENTER

IN

L. Catucci, A. A g o s t i a n o , G. C o l a f e m m i n a a n d M. Della Monica Dip. Chimica, Universit~ di Bari, V. Orabona 4, 70126 Bari, (Italy) Photosynthetic reaction centers (RC) from Rhodobacter sphaeroides have been reconstituted in p h o s p h o l i p i d vesicles, in reverse micelles and in organogels. The kinetics of charge recombination between the primary p h o t o - o x i d i z e d electron donor (P) of the r e a c t i o n center and both the primary (QA) and secondary (QB) q u i n o n e acceptors have been i n v e s t i g a t e d in e a c h s y s t e m by time-resolved absorption spectroscopy. In all the systems examined the recombination kinetics of the charge separated state P+QB- never fit to a single exponential decay. N M R s t u d i e s of the d i f f e r e n t RC h o s t i n g s y s t e m s are in a d d i t i o n reported, in a first a t t e m p t of a s t r u c t u r a l characterization of the phospholipid microenvironment in w h i c h the RC is embedded.

P-3-4-115 PROTEIN ENGINEERING O F T H E PltOTOSYSTEM 11 REACTION CENTRE IN THE GREEN ALGA CHLAMYDOMONAS REINHARDTII C. Andronis & R Nixon Department of Bioebemistry, Imperial College of Science, Technology and Medicine, London SW7 2A~ UK

We are probing strueture2function relationships within the photosystem H comple~t by ennstrueting mutants of the I)2 polypeptide in Ch/amydomonas re/nhaMt//. This is done by oreating site-directed mutations in the psbD gene, which encodes I)2 and then introducing the mutated gene back to the chloroplast genome using biolistic technology. We have constructed sitedirected mutants of D2 at positions D2-Le~05 and D2-Thr2. D2-Len205 is thought to be analogous to residue M-Tyr210 of the reaction centre from the purple photosynthetic bacterium Rb. sphaeroides. Thus, D2-Let~05 has been replaced by Tyr in order to study the role of this residue in primary electron transfer. Initial chameteri.~tion of the D2-Leu205"Pyr mutant has shown reduced rates of oxygen evolution as well as alterations in the eplical properties of 1'680. Mutations at D2-Thr2 have been constructed in order to study the role of D2 phosphorylalion.

P-3-4-116 REACTION CENTER-PHOSPHOLIPID KINETIC OF CHARGE RECOMBINATION

REVERSE

MICELLES:

A. M a l l a r d i I, R. A n g e l i c o I, M. D e l l a M o n i c a 2, M. Giustini I and G. Palazzo 2 I C S C F I L M (CNR), V. O r a b o n a 4, 70126 Bari; 2Dip. Chimica, Universit~ di Bari, V. Orabona 4, 70126 Bari (Italy) Reverse micelles formed by phospholipids in n-hexane h a v e b e e n used as h o s t - s y s t e m for r e a c t i o n c e n t e r (RC) from Rb. sphaeroides. The protein in this system shows to r e t a i n its p h o t o c h e m i c a l activity. T h e behaviour of the charge recombination kinetics of the RC in reverse micelles is discussed. The o b s e r v e d m u l t i p h a s i c character of the p h o t o x i d i s e d p r i m a r y donor (p+) decay could be explained by c o n s i d e r i n g the exchange of the quinone between the p r o t e i n and o r g a n i c bulk. The rate of charge r e c o m b i n a t i o n in d e p e n d e n c e on the t e m p e r a t u r e and p h o s p h o l i p i d composition is also investigated.

DIFFERENT

P-3-4-118 P* VIBRATIONAL COHERENCE IN MUTANT RCs

~ t , M.R. Jones 2, C.N. Hunter2, J. Breton3 & J.-L. Martin 1 Laboratoire d'Optique Appliqu~e, INSERM U275, CNRS URA 1406, Ecole Polytechnique-ENSTA, 91120 Palaiseau, France; 2Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S10 2UH, United Kingdom; 'SBE/DBCM, CEA de Saclay, 91191 Gif-sur-Yvette Cedex, France Oscillations in the femtosecend kinetics of stimulated emission in bacterial reaction centers have previously been shown to reflect coherent nuclear motion in the P" excited state, the precursor of physiological electron transfer. A series of single residu-mutants of R. sphaeroides RC's, at positions M210 and L181 was studied, where the time scale of primary ET varies over about two orders of magnitude without major structural changes in the vicinity of P. These mutants displayed a direct correlation between the overall decay of P* and the apparent dephasing of the oscillations. This result conftrms the assignment of the oscillations to excited state vibrational motion. Moreover, in mutants with a long-lived P*, a lower limit of the vibrational dephasing time is obtained that is longer than the overall ET time t,f wild type reaction centers. In contrast to the above mutants, mutants designed to modify the H-bonding between P and the protein also affect the vibrational pattern of the nuclear motions activated by the P-->P* transition.

P-3-4-119 EFFECTS OF CAVITI/~S IN THE BACTERIAL REACTION CENTER M. Schiffer. Y.-L. Deng, A. Marrufo, & D. K. Hanson. Center for Mechanistic Biology & Biotechnology, Argonne National Lab., Argonne, IL USA We have previously found that cavities near the macrocycles in the RC of

Rhodobacter capsulatus that are generated by replacement of the symmetryrelated residues M208Tyr and L181Phe by threonines ('IT mutant) or M207Leu and L180Phe by alanlnes (AA mutant) yielded nonpbotosyotbetic (PS') swains. In contrast, replacement of the L187Phe-M214Leu symmetry-relatedpair by Ala did not affect the growth phenotype. The latter residues lie at the interface of the L and M chains along the twofold axis of the complex, but are not as deeply buried as the T'r or AA sites, which are closer to the special pair. We have also replaced the conserved L156Trp-M183Trp residues, which lie between the periplasmic surface of the RC and the special pair, with either Phe or Ala. The L 156Phe-M 183Phe double mutation impacts photosynthetic growth, and the L156AIa-M183Ala double mutant is IS-. The suppressor mutation M271Ala Pro carried by a spontaneous photocompetent (PS +) revertant of the TT mutant swain also yielded a PS + strain when coupled with the AA mutations but did not restore the PS + phenotype to the L156AIa-M183Ala double mutant. Thus, not only the cavity volume but its nature and location are important in determining the growth phenotype. Supported by U.S. Dept. of Energy, OHER, under Contract No. W-31-109-ENG-3g and by U.S. PHS Grant GM36598.

71

Poster P-3-4-120 COMPENSATION FOR L212GLU IN BACTERIAL REACTION CENTERS D. K. Hanson 1, Y.-L. Deng 1, P. Sebban 2, and M. Schiffer 1. ICMB, Argonne National Lab., Argonne, IL USA; 2CNRS, Gif/Yvette, France

P-3-4-123 CHARACTERISATION OF REVERTANTS FROM DIRECTED MUTANTS IN THE HYDROPHILIC LOOP OF THE PHOTOSYSTEM II SUBUNIT CP 43 FROM SYNECHOCYSTIS sp. PCC 6803 M. Kuhn, U. Fink & C. Steinkopff

In wild-type reaction centers (RC), residue L212Glu in the QB site is involved in delivery of the second proton to QB2-. In Rhodobacter capsulatus RCs, the L212GIu ~ Ala mutant is photocompetent (PS+), while the L212Glu -->Gin mutant is not. To detemaine the function that is lost in the L212Gln mutant but restored by Ala at that site, we selected four PS+ revertants from the L212Gln strain. None of the revertants carried the single base transversion that would restore the wild-type Gin codon; each carried a different second-site substitution in addition to the original L212GIn mutation. These compensatory mutations are, in order of relative growth phenotype: L228Gly ~ Asp < L231Arg ~ Cys < M231Arg --->Cys < L227Leu ~ Phe. Some of these residues lie at the interface of the L and M chains, thus the mutations might cause chain rearrangement as was observed for the L222Tyr ~ Phe mutant ofR. viridis. However, some of these substitutions also change the electrostatic environment which could restore the proton transfer function which is lost in the L212GIn mutant. Proton mmsfer pathways in these revertants must be different than that of the wild type, or that of the L212Ala strain where a water molecule could substitute for the function of L212Glu. Supported by U. S. Dept. of Energy, OHER Contract No. W-31-109-ENG-38 and by U. S. PHS Grant GM36598.

Max-Volmer-lnstitut for Biophysikalische und Physikalische Chemie, Technische Universit~t Berlin, Stral3e des 17. Juni 135, 10623 Bedin, Germany. In order to study the function of the large hydrophilic loop of CP43 different deletions in this loop were introduced by site directed mutagenesis of the psbC gene. All deletion mutantsresulted in a obligate photoheterotrophic phenotype. These mutants were screened for revertants capable of photoautotrophic growth. Only one of the eight different deletion mutants yielded photoautotrophic revertants. Two different revertants were found. These two revertants showed different insertions at the site of the former deletion. The insertions were derived from the psbC sequence upstream of this deletion. Both revertants were characterized regarding the function and the biochemical and biophysical parameters of Photosystem II. Charactedsations include sequence analysis, oxygen evolution, electron transfer rates and the protein composition of the Photosystem II complex.

P-3-4-121 TRAPPING IN MUTANT PHOTOSYNTItlcrlC BACTERIA A. Freiber~.. J. P. Allen J. C. W'dliamsand N. W. Woodbury Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, AZ 85287, USA The trapping of light energy in mutant membranes of Rhodobacter sphaeroides was studied via picosecond emission decay kinetics. The primary charge separation time, Xrc, in seven different reaction centre (RC) single, double and triple hydrogen bond mutants varied from 3.4 ps to 52 ps. The mutants have only a core antenna. Three decay times were found in emission kinetics. The shortest one increased linearly with "Crc, like x(in ps)~55+2Xrc, while the other two, the 250 ps and 680 ps times, were nearly the same in all mutants. There was a substantial non-linear rise of the aptitude of the 250 ps component with respect to one of the fastest component with Xrc. An analysis of the observations concluded that the fastest antenna emission component reflected the interplay between the antenna-RC energy transfer rate and the Xrc. The 250 ps component was a recombination lumine-~,,ence due to back reaction from the charge-saparated state. The 6g0 ps emission was mainly caused by the antenna complexes uncoupled to PC.

P-3-4-122 SITE-SPECIFIC MUTAGENESIS AROUND THE SPECIAL PAIR OF THE REACTION CENTRE IN RHODOPSEUDOMONAS VIRIDIS

P-3-4-124 PROTEIN INTERACTIONS MODULATING THE PHYSICOCHEMICAL PROPERTIES OF THE PRIMARY DONOR IN BACTERIAL REACTION CENTERS: A COMPARATIVE STUDY Tonv A. Mattioli Sect]on de Biophysique des Prot~ines et des Membranes, DBCM, CEA and URA CNRS 1290, C.E. de Saclay, 91191 Gif-sur-Yvette cedex, FRANCE. The binding site and pigment-protein interactions of the bacteriochloroph),ll pair constituting the primary electron donor (P) in various bactefiai reaction centers (RCs) have been studied using FT Raman resonance Raman spectroscopy. For RCs whose structures are unknown but whose primary sequence data are available, amino acid residues donating H-bonds to the conjugated carbonyl groups of P may be proposed, assuming structural homologies with respect to Rb. sphaeroides. For RCs whose primary sequence data are not available, the presence of similar H-bonding amino acids may be inferred. Physico-chemical properties, such as redox midpoint potentials, of P in RCs of several species correlate with specific H-bonds to the conjugated carbonyl groups donated by the proposed residues, and are consistent with results recently reported for H-bonding mutants in Rb.

sphaeroides.

P-3-4-125 OF QB PHOTOREDUCTION IN PROTON TRANSFER MUTANTS OF RB. SPHAEROIDES

FTIR DIt't't.alF.,NCE S P ~ O S C O P Y

1, j. Breton1, R. Hlenerwadell, C. Fogel2, W. Mltntele2, M. M. Okamura3

M. Bibikova 1, Th. Arlt 2, W. Zinth2 and D. Oesterhelt 1, 1Max-Planck-lnstitut ftir Biochemie, D-82152 Martinsried and 2lnstitut far Medizinische Optik, Ludwig -Maximilians-Universit~t, D-80797 Mttnchen.

ISBE/DBCM, CEA-Saclay, 91191 Gif-sur-Yvette, France; 2Universi~t Erlangen, RFA; 31Tc'U.CSan . Diego, USA

Three groups of amino acids located close to the special pair of the reaction centre have been mutagenized. The histidines M200 and L173 liganding the special pair from both sides have been exchanged for phenylalanine, isoleucine and threonine, but in no ease photosynthetic growth or expression of the reaction centres could be observed. A second group comprises histidine L168 and tyrosine M195 which form hydrogen bridges to the special pair. Loss of these H-bonds causes blue shifts of the absorption maximum between 15 and 50 nm and changes in the redox potential of the special pair which influence the initial electron transfer steps. Specifically the L168 His-:>Phe mutation shows acceleration of the initial electron transfer from 3.5 to 1.1 psec and allows the unambiguous demonstration of the accessory bacteriochlorophyll b as the first intermediate. A third group of mutations concerns tyrosine M208 and the corresponding symmetric position L181 Pha which have been investigated in detail in Rhodobactersphaeroides for their influence on the electron transfer kinetics. In Rps. viridis more pronounced changes in kinetics were found and all effects observed will be discussed in terms of the Marcus electron transfer theory.

In order to investigate the possibility that protonation changes of carboxylic residues occar upon photoreduction of QB in Rb. sphaeroides 2.4.1 RC, we have performed steady-state FHR studies at pH 7 of RC fa'om wild-type (Wt) and several mutants where carboxylic amino acid residues lying in the binding pocket of QB, i.e., Glu L212, Asp L213 and Asp L210 are substituted by Gin or Asn. The assignment of an IR band to the protonation/deprotonation of a particular carboxylic side chain is analyzed by combining the effects of sitedirected mutagenesis and 1H/2H isotope exchange. The protonatinn of Gin L212 in Wt, DN L210 and DN L213 RC is revealed as a 13ositive band at 1728 cm-1 in the QB-/QB spectra that shifts to 1717 + i cm-'l in 2H20, and that is absent in the mutants EQ L212 and EQ 1.212 + DN L213. Proton uptake by Gin L212 is comparable in Wt and DN L210 (m 0.3 H+/QB-) and is increased in DN L213 (== 0.5 H+/QB-), in agreement with the decrease of pica of Giu L212 in this last mutant. In contrast, our data do not indicate changes of protonation of Asp L213 upon photoreduction of QB. Furthermore, a signal at 1685 cm-1 in Wt RC is tentatively assigned to a change of the amide I mode of Glu L212. This signal is lost in EO L212 and the double mutant.

72

Poster II II

P-3-4-126 LIGHT-INDUCED FTIR DIFFERENCE SPECTROSCOPY OF THE PRIMARY DONOR CHLOROPHYLLS IN THE D1/D2 REACTION CENTER OF PHOTOSYSTEM II E. Nabedryk, S. Andrianambinintsoa, D. Dejonghe, C. Berthomieu, and IL IIienerwadel SBE/DBCM, CEA-Saclay, 91191 Gif-sur-Yvette Cedex, France The conversion of the primary donor P680 into its triplet state 3p680 has been investigated between 170 K and 10 K by FTIR difference spectroscopy. Comparison with the triplet state of P860 in bacteria and of chorophyll a in solutmn [J. B. & E. N., Chem. Phys. Letters 213, 571, 1993] suggests possible assignments for the 9-keto and the 10a ester carbonyl groups in the ground and triplet states of P680. The 3p680/P680 difference spectra change significantly with temperature suggesting the involvement of two chlorophylls with an increased localization on one of them at the lowest temperatures. Although the spectra in the 170 K to 80 K range agree very well with those previously reported by Nognchi et al. [Biochemistry 32, 7186, 1993], the spectrum at 10 K is difficult to reconcile with the assignments proposed by these authors. We rather assign the differential bands at 1713/1721 and 1701/1707 cm-1 to the 10a ester carbonyls and those at 1625/1668 and 1655-1660/1676 cm-1 to the 9-keto carbonyl groups of P680. In the presence of silicomolybdate and ferricyanide, a reversible photooxidation of chlorophyll(s) is observed at both 220 K and 20 K. The carbonyl bands of the neutral chlorophyll species do not match those observed in the ~IP680/P680 spectra.

P-3-4-127 SYMMETRY OF THE CARBONYL INTERACTIONS AND CLOSE EQUIVALENCE OF THE QB VIRATIONS IN RB. SPHAEROIDES AND PP. VIRIDIS AS PROBED BY LIGHT-INDUCED FFIR DIFFERENCE SPECTROSCOPY USING ISOTOPICALLY LABELED QUINONES C. Boullais, G. Berger, C. Mioskowski, and E. Nabedryk SBE/DBCM, CEA-Saclay, 91191 Gif-sur-Yvette C.edex, France The photoreduction.of the secon.da.d.a~.....quinoneacce.ptol-.QB in ~ . . tion.centers (RC) of Rb. sphaeroides and Pp. v/r/d/s has been lnv.esti..gntedby li.ght-ma.u.c..e.o. l'-l'lg difference spectroscopy of RCs reconstltuteo warn several isotoplc,mW labeled ubiqninones. The labels used were le;O on both carbonyls as well as 13C either uniformly or selectively at the 1- or at the 4-pnsition, i.e., on either one of the two carbonyls. The C=O frequency of each of the carbonyls of the unlabeled quinone is revealed at 1641 cm-1 for both species. This demonstrates a moderate and symmetrical hydrogen bonding of the two C=O groups to the protein at the QB site. In contrast, the C=C vibrations are not equivalent at C1 and C4, although they are found at almost identical energies in the RC of both species. These FTIR observations provide compelling evidence for identical interactions of the ubiquinone in the OB binding site of Rb. sphaeroides and Rp. viridis in contrast to the X-ray structures which yield different descriptions of the QB site. In the semiquinone state the bonding interactions of the C._..O groups are also symmetrical and the C._-.C are inequivalent at C1 and C4. However, the interactions are the same in both RCs.

P-3-4-128 DEGENERATE FOUR-WAVE MIXING ON PIIOTOSYNTIIETIC REACTION CENTERS T. Arlt, R. Heinecke, H. Penzkofer & W. Zinth Institut for Medizinisehe Optik, University of Munich, Barharastr. 16 80797 M(inchen, Germany Investigations of the ultrafast processes in photosynthetic reaction centers are one of the most interesting subjects in biophysics today. Optical spectroscopy of the electron transfer reactions of photosynthetic reaction centers does not always deliver complete information. As a consequence additional techniques are required. Degenerate four-wave mixing is an interesting coherent technique to characterize the nonlinear response of a medium. In the experiments intense ultrashort laser pulses induce a transient grating and the decay of the grating is monitored by properly delayed pulses. Using femtosecond pulses (150 fs FWHM, 700 - 900 nm range) the dephasing times of electronic transitions of dye molecules and photosynthetic reaction centers Rb. sphaeroides were determined. The consequences to the principles of the primary reaction are discussed.

73

Poster Ill

P-5-003 E X C I T O N I C R E L A X A T I O N AND E N E R G Y T R A N S F E R IN T H E R E A C T I O N C E N T E R C O M P L E X F R O M C..HLOROBIUM TEPIDUM Y.-Z. Ma, M. Miller & T. Gillbro 1Department of Physical Chemistry, Umeh University, S-901 87 Ume~t, Sweden, 2Institute of Biochemistry, Odense University, Campusvej 55, DK-5230 Odense M, Denmark

Poster session 5

Reaction centers: green sulfur bacteria and PSI P-5-001

- P-5-054

The isotropic and anisotropic kinetics in the FMO containing reaction centers from Cb. teoidum were examined by pump-probe technique using 50-80 fs pulses at several wavelengths between 300K and 77K. An obvious wavelengthand temperature dependence were observed especially for the isotropic decays. The polarization measurements in the spectral range of FMO absorption show a bi-exponential anisotropic decays, the fast one has a time constant about 100200 fs and the slow one is between 2-4 ps. Moreover, an ultrafast anisotropic decay of 100 fs was resolved at 834 nm at 77K, followed by 1 ps component with an initial anisotropy is considerably larger than 0.4. Moreover, clearly fast isotropic decays were observed at 77K in comparison to that obtained in the isolated FMO complex fl'om same species. Based on these observations, the process of excitonic relaxation and energy transfer was discussed.

P-5-004

P-5-001 SITE DIRECTED AND RANDOM MUTAGENESIS OF THE C H L O R O P L A S T G E N E PSAC O F C.REINHARDTII. N. F i s c h e r I, P. S ~ t i f 2 a n d J.-D. R o c h a i x l, I Dep. of Mol. B i o l . , U n i v e r s i t y of G e n e v a . 2 C.E.A Saclay, Paris.

The 8-9 KD p r o t e i n e n c o d e d by t h e c h l o r o p l a s t p s a C g e n e is t h e a p o p r o t e i n of t h e t w o 4 F e - 4 S c l u s t e r s F A a n d F B . T o t r y to u n d e r s t a n d t h e e l e c t r o n f l o w in t h i s t e r m i n a l p a r t of PSI, a set of m u t a t i o n s h a v e b e e n created via biolistic transformation: D9N, W 3 1 Y / Y 8 1 H , E46K, E 4 6 Q a n d Y68F. E P R m e a s u r e m e n t s w e r e m a d e on p u r i f i e d t h y l a k o i d s f r o m the t r a n s f o r m a n t s . The EPR s p e c t r a of E 4 6 K a n d W 3 1 Y / Y 8 1 H s h o w t h a t FB is m o r e r e d u c e d in t h e s e t r a n s f o r m a n t s indicating that these m u t a t i o n s a l t e r t h e e l e c t r o n flow. In p a r a l l e l , a r a n d o m m u t a g e n e s i s s y s t e m b y P C R h a s b e e n d e v e l o p e d for t h e psaC g e n e in w h i c h t h e transformants are s c r e e n e d for t h e l o s s of PSI a c t i v i t y a n d for s t a b l e a s s e m b l y of t h e PSI c o m p l e x .

ELECTRON SPIN ECHO STUDIES OF ~Mg COUPLING IN PHOTOSYSTEM I: IMPLICATIONS FOR PToo+ STRUCTURE Michelle Mac l,Xiao-songTang2,BruceDiner2,JohnMcCrackenIand GeraldT. Babcockl Department of Chemistly, Michigan State University, E. Lansing, MI 48824 USA and 2 Central Research and Development Department, Experimental Station, P.O. Box 80173, E.I. DuPont de Nemours and Company, Wilmington, DE 19880 USA. Oxygenic photosynthesis in higher plants and cyanohaeteria requires the interplay of two pigment protein reactions, Pbotosystem I and Photosystem II (PSI and PSII). In each of these reaction centers the primaxy charge separation event proceeds via the light induced generation of a chlorophyll a cation radical species. In PSI, this species is known as P7oo+ and is the primmy electron donor in an electron transfer chain that ultimately effects the reduction of NADP+. Although both monomeric and dimerie structures of chlorophyll a have been suggested for P~co, neither have been proven conclusively and the ambiguity surrounding its nnclcarity remains. The electronic structure of the cation radical is controversial as well. The pulsed-EPR technique of electron spin echo envelope modulation (ESEEM) was used to identify an interaction between the uni~red x electron in PToo+and the "~'Mgnucleus (Iffi5/2; natural abundanco, 10%) in ISN labeled PSI reaction centers from the cyanobaeterium, S.vnechocystlsPCC 6803. The magnitude of this coupling (a~=2.94 MI-Iz) and the resulting unpaired spin density on the 25Mg indicates a monomeric electronic strncturcforthe radical.

P-5-005

P-5-002 SYMMETRIC STRUCTURAL FEATURES OF THE PRIMARY ELECTRON DONOR, P840, FROM CHLOROBIUM REACTION CENTRE

TIME-RESOLVED FLUORESCENCE STUDIES ON PHOTOSYSTEM I ANTENNA COMPLEXES

~Jte Feller, Delphine Albouy, Bruno Robert and Tony A. Mattioli DBCM/SBPM, CEA & URA 1290 CNRS, CE de Saclay, F-91191 Gif-sur-Yvetre

N. Schneebauer and G. Hauska Lehrstuhl flit Zellbiologie und pflanzenphysiologie, Universitit Regensburg, Germany

Near-infrared Fourier transform Raman (FTR) spectroscopy has been performed on the isolated reaction centre (RC) of Chlorobium. to determine the protein binding interactions of P840, and the electronic symmetry of the radical cation P840÷°. The FTR spectrum of P840 indicates that it is constttuted of a single population of baeteriochlorophyll (BChl) a molecules. The BChl a molecules of P840 are pentacoordinated and the x-conjugated C2-acetyl and Cg-keto carbonyls are free of hydrogen bonding interactions. The P840 +° FTR spectrum exhibits a 1707 cm-I band attributable to a BChl a Cg-keto carbonyl frequency that has upshifted 16 cm-1 upon oxidation of P840. This upshifl indicates that the resulting +1 charge is equally shared between two BChl a molecules. This situation is markedly different from that &the oxidised electron donor in BChl a-containing purple bacterial RCs: i) an asymmetric distribution &the resulting +1 charge over two BChl a molecules was detemlined, and ii) the H-bonding pattern with respect to the 7~-conjugated carbonyl groups is asymmetric. According to our data, P840 of Chlorobium is a highly synunetnc dimer of BChl a molecules; with respect to the protein interactions of P840 and the +1 charge distribution in P840+°. Our results are consistent with the proposal of a homedimeric structure of the Chlorobmm RC.

74

We have investigated the time-resolved fluorescence spectra o f P S I particles from Anabaena variabilis in the time-region from sub-ns to 1 ms. In the observed wavelength region (i.e. 670 nm to 800 nm) no delayed fluorescence can be detected. The analysis o f the measurements with oxidized and reduced samples is consistent with a model o f excitation transfer, in which the decaytime o f about 1 ns reflects antenna systems which are connected to a functional reaction-center. Furthermore we show that the reaction center complex in the oxidized form quenches fluorescence better than in the reduced form.

Poster

P-5-006 SEARCH

FOR

THE

PHYLLOQUINONE

PRESENCE

IN THE

AND

CORE

Thomas Schwartz and Klaus Section de Bio~nerg~tigue,

OF

ROLE

OF

P-5-009 MUTATIONAL ANALYSIS OF [4FE4S] CLUSTERS F A AND F e OF PHOTOSYSTEM I IN SYNECHOCYSTI$ SP. PCC 6803

A

SECOND PHOTOSYSTEM I

Brettel CEA-Saclay

photosystem I (PS I) c o r e f r o m Synechocystis 6803 w a s t r e a t e d w i t h h e x a n e o r hexane/methanol according to a n e s t a b l i s h e d m e t h o d ( B i g g i n s , J. a n d M a t h i s , P. (1988) B i o c h e m i s t r y 27, 1 4 9 4 - 1 5 0 0 ) in o r d e r to e x t r a c t o n e or b o t h p h y l l o q u i n o n e (PhQ) molecules, respectively. The electron transfer k i n e t i c s a r o u n d t h e s e c o n d a r y a c c e p t o r A I (a PhQ) in the different s a m p l e s w e r e r e s o l v e d by f l a s h absorption spectroscopy a n d r e l a t e d to t h e P h Q c o n t e n t as d e t e r m i n e d by HPLC. It is c o n c l u d e d that only one PhQ molecule (non-extractable by h e x a n e a l o n e ) is i n v o l v e d in e l e c t r o n t r a n s f e r . T h e P h Q w h i c h is e x t r a c t a b l e by h e x a n e is p r e s u m a b l y n o t an integral constituent of t h e P S I core, b u t r a t h e r l o c a t e d c l o s e to t h e p e r i p h e r y at a n o n - s t o i c h i o metric amount. The

J. Yu 1, Y.-S. Jung2, J. Golbeek 2 & L Mclntosh1 Iplant Research Lab, Michigan State Univ, E Lansing, MI 48824-1312, USA; 2Dept Biochem, Univ Nebraska, Lincoln, NE 685834)718, USA Identities of the ligands for terminal electron acceptors F^ and F e on PsaC were determined by/n vitro reconstitution of mutant PsaC proteins onto PT00-Fx cores (Zhao et al., Biochem, 1992, 31: 5093). We created a set of/n v/vo site-directed mutations on PsaC in Synechocystis sp. PCC 6803, in which a cysteine ligand to F^(C51) and Fn(C14 ) was substituted by aspartate(D), serine(S), or alanine(A). Mutant CS1D cells were deficient in photoautotrophic growth but were capable of whole-chain oxygen evolution. Compared to wildtype, C51D thylakoids have lower levels of PsaC, PsaD, and PsaE; and a lower rate of NADP + photoreductinn. EPR studies showed that in CS1D the characteristics of the F B cluster were unchanged, and a fastrelaxing spin system was observed in lieu of F^.

P-5-010

P-5-007 The role of long-waveleglh absod:)Ing chlorophylls In PS I particles Isolated from cyanobacterium Syrmchococcuselongotus. 1 . D. Sofrovd l,J,Hlad~ ~,B.A.Gulyayev 2,V.L.Tetenkin2 ~Dept, of Biochem., Faculty of Natural Sci.Charles Unlv.~Ibertov 2030, 128 40 Praha 2, The Czech Republic; 2Fac. of Biology,M G U , LeninskieGory, Moscow, 119899, GSP, The Russian Federation B.SODkO

Isolated trimeric particles were used to investigate spectroscoplcal properties of PS I, We have found changes in fluorescence emission spectra when we subdued these particles to higher concentration of dithionite. The main peak of the difference spectra (reduced minus oxldLsed) was located at 703 nm which we contributed to to fluorescence of "closed' PT00.Considering that the main emission of antenna chlorophylls occurs at 720 nm we have estimated the activation energy neccessary for excffation energytransfer to PT~,but we have found that this activation energy is sufficient to transfer energy only to chlorophylls with absorption maximum at 710 nm. We have proved the existence of these non-tiuorescent chlorophylls absorbing at previously estimated wavelegth. We have also determined these chlorophylls to be in dimeric form oriented paraleUy to PT~.

CORRELATION OF LIGHT-INDUCED FLUORESCENCE QUENCHING AT 760 nm WITH P700 PHOTOOXIDATION IN MEMBRANES AND PHOTOSYSTEM I TRIMERIC COMPLEXES OF SPIRULIN.4 N.V, Karanetvan, R.J. Strasser*, V.V. Shubin & I.N.Bezsmertnaya Biochemistry Institute, Moscow, Russia; University of Geneva, Switzerland* PSI trimeric complexes of Spirulina show an emission band at 760 n m (F760) originating from Ch1735 which is located close to P7O0. Photooxidation of P700 quenches F760 as a result of energy migration from Ch1735 to P700 ÷ (77K). The rate of F760 decrease is higher than that of simultaneously measured P7O0 ÷ formation. Action spectra of P700 photooxidation indicate the efficient energy transfer from the long-wavelength Chls to P700 at 293-77K. The linear dependence of the rate o f P700 photooxidation on the fraction of P700 ÷ in PSI m o n o m e r s and trimers at 293K, the equal rate of P700 photooxidation in m e m b r a n e s and PSI trimers and m o n o m e r s at 77K, and the quenching of F760 by P700 ÷ indicate no energy exchange between subunits of PSI trimers.

P-5-OI 1

P-5-008

bound to photosystem

h kinetic and

ON THE SPATIAL DISTRIBUTION OF CHLOROPHYLL SPECTRAL TYPES IN PHOTOSYSTEM I

Ferredoxin chemically structural properties

G.Trinkunas~ and A.R.Holzwarth2 ~Institute of Physics, Vilnius, 2600, Lithuania; 2Max-Planck-Institut far Strahlenchemie, D-45470, Miflheim a.d. Ruhr, Germany

C. Lelong, P. S6tif, B. Lagoutte and H. Bottin Section de Bio6nerg6tique, DBCM, CEA Saclay, 91191 Gif sur Yvette Cedex France

The mathematical procedure (genetic algorithm) and the results of the search for spatial distributions of spectral types of chlorophylls (Chls) in the cyanobacterial photosystem (PS) I core antenna/reaction center (RC) particle (100 Chls/P700) are presented. The structure of the PS I core particle was modelled by a threedimensional regular pigment lattice. The spectral content was determined from the deconvolution of the absorption spectrum in terms of Chl absorption spectra. The least deviation of the model excitation decay kinetics from the picosecond fluorescence kinetics at 280K temperature served as criterion for the selection of the optimal spatial distributions of the spectral types (Trinkunas & Holzwarth, 1994, Biophys.J. 66, 415-429). The obtained three-dimensional models predict the "red" pigment pool to be spread on the surface of the antenna system in the vicinity of the RC pigment but without direct contact to it. These models are intermediate between the migration- and trapping-limit~l excitation transfer ones with a trend to the former. Our simulations suggest that the charge separation rate from P700* to A0 schould be up to an order of magnitude higher than that in purple bacteria.

Electron transfer kinetics from photosystem I (PS I) to ferredoxin (Fd) studied in the cross-linked complex show fast reactions (t½< llJS and t½ = 10 IJS) comparable to the reactions observed in the non covalent complex (Setif & Bottin, 1994, Biochemistry, 33, 8495-8504). This similarity extends also to the visible spectrum of each phase. Following the orientation of the cross-linked complex (PS I--Fd) on Mylar sheets, the measurements of the g-tensor of Fd by EPR shows that Fd is present in a single orientation with respect to PS I. The results are compared to structural data obtained by electronic microscopy.

75

Poster P-5-012 PURIFICATION AND CHARACTERISATION OF AN NADHDEHYDROGENASE IN HELIOBACILLUS MOBFLIS A. Kolpaskyl, U. Mithlenhoff 1, A. Atteia 2 & W. Nitschke 1

P-5-015 HIGH ENRICHMENT OF P700 BY ETHER-ACETALDEHYDE EXTRACTION OF ANTENNA PIGMENTS IN PS-I PARTICLES lsarnu lkegami I, Shigeru ltoh 2 & Masayo lwaki 2 IFaculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan; 2National Institute for Basic Biology, Okazaki 444, Japan

Biologic II, Biochemistry, Universi_ty of Freiburg/FRG; Dept. of Plant Biology, G6teborg Oniversity/Sw&len An NADH:MK--oxidoreduetase activity was detected in membranefragments from H.eliobacillus mobilis. The enzyme giving rise to this actt-vity was somailisen using a .r4nge of detergents. T h e solubilisation pr(3cegure was sansequenuy optimise?d with resL)ect to yield and stability and me sombilised sample was furtl~er purified on sucrose density. gradients, lncreasen concentrations ot oetc.rgent resulted in separation ot a ~oeompiex retaining me major part of me enzyme activity. 1he pai'tialty purifi&l enzyme was found to contain flavin and EPRdetectable FeS clusters. The isolate was further characterised with resEeet"to protein eompo,sition by S.DS-PAGE and Western-blotting using antabodies against t:omplex t trom Iv. crassa. The characteristics of the enzyme from H. mobilis is compared to those of NADH-dehydrogenases from other organisms and a possible role in the photosynthetic electron transport chain of H. mobilis is discussed.

Lyophilized spinach photosystem I particles were treated with diethyl ether that contains various organic solvents with different dielectric constants. The more pigments were extracted when the dielectric constants of the solvents added to ether became higher. The reaction-center chlorophyll-dimer, P700, was more resistant to the extraction than the other chlorophyll. The particle which contained only 6 chlorophylls in addition to WOO and the primary electron acceptor (A0) in one reaction-center unit, was prepared by extraction with ether-acetaldehyde mixture. A distinct shoulder at 695 nm due to P700 or at 686 nm due to WOO÷ was shown in the absorption spectra of the reduced or oxidized panicles, respectively, even at room temperature. No secondary aceeptor phylloquinone remained in the particle. A stable charge separation was recovered on addition of 2-amino-anthraquinone, even though the particle has the lowest chlorophyll/P700 molar ratio ever isolated.

P-5-013 EFFECT OF NAD(P)H DEHYDROGENASE INHIBITORS ON FERREDOXIN-PLASTOQUINONE OXIDOREDUCTASE ACTIVITY IN THE CYANOBACTERIUM Synechocystis PCC 6803. Mereedes Roncel and Manuel Losada. lnstituto de Bioqufmica Vegetal y Fotosfntesis, Universidad de Sevilla y C.S.I.C, Apto ] 113. 41080-Sevilla, Spain.

P-5-016 PHOTOSYSTEM I / FERREDOXIN INTERACTION : SITE DIRECTED MUTAGENESIS OF PSI-E Lagoutte. B.,Barth, P. and Sttif P., CEA-Service de Bio~nergttique/CNRS-URA 1290, DBCM, C.E. de Saclay 91191 Gif sur Yvette Cedex, France

The participation of NAD(P)H-dehydrogenase complex (NDH) in the cyclic electron flow around PSI of cyanobacteria is under strong controversy. Manasse and Bendall (Biochim. Biophys. Acta (1993) 1183, 361-368) have developed an assay to study cyclic electron transport in chloroplasts and cyanobacteria by monitoring the specific ferredoxinplastoquinone oxidoreductase (FQR) activity. Such an assay has been used in this work by following the absorbance changes of ferredoxin at 463 mn in Synechocystis thylakoid membranes in order to determine the action mode of a number of NDH complex inhibitors. The FQR activity was sensitive to rotenone, Hg2+ ions and 2-thenoyltrifuoroacetone(TTFA). The results agree with other previous conclusions (Mi et al., Plant Cell Physiol. (1992) 33(8), 1233-1237) and support the hypothesis that NDH is involved in cyclic electron flow around PSI in the cyanobacteriumSynechocystis. This work was supported by DGICYT (Grant PB 91-0085).

The Photosystem I (PSI) is a muhisubunit complex of the photosynthetic membrane whose main function is to catalyze the reduction of soluble ferredoxin. The reaction occurs through a precise, transient interaction of PSI with this small, very acidic, electron carrier. Two peripheral proteins of the reducing side of PSI are thought to provide the optimized site for this interaction: PSI-D and PSI-E. Extensive site directed mutagenesis of the later subunit allowed to sort some of the amino acid residues which are important -i) for the structuration of this protein, -ii) for its integration in PSI and -iii) for the fast photmeduction of ferredoxin. Some mutations in this last series are described with more details in this work, stepping forward in the understanding of the ferredoxin binding site on PSI.

P-5-014 MECHANISM OF ELECTRON TRANSFER FROM PLASTOCYANIN AND CYTOCHROME C6 TO PSI IN A NUMBER OF EVOLUTIONARILY DIFFERENTIATED ORGANISMS

P-5-017 KINETICS OF PSaC REDUCTION IN PHOTOSYSTEM I REACTION CENTERS

M, Herv~is1, J.A. Navarrot, A. Dfaz 1, H. Bottin2 and M.A. De la Rosa 1 lInstituto de Bioqufmiea Vegetal y Fotosflatesis, Universidad de Sevilla y CSIC, Facultad de Biologfa, Sevilla, Spain; 2I~partement de Biologic Cellulaire et Mol&ulaire, CEA Saclay, Gif-sur-Yvette, France The reaction mechanism of electron transfer from the interchangeable metalloproteins plastocyanin (Pc) and cytochrome e6 (Cyt) to photooxidized PT00 in photosystem I (PSI) has been studied by laser-flash absorption spectroscopy in a number of evolutionarily differentiated organisms such as eyanobacteria, green algae and high~ plants. The experimental data within each organism can be well fitted to one of the following kinetic models: i) an oriented eollisional reaction mechanism; ii) a minimal two-step mechanism involving complex formation followed by intracomplex electron transfer; and iii) rearrangement of the reaction partners whitin the complex before electron transfer takes place - this is the most evolved mechanism exhibiting a bi-phasic reaction course. A comparative thermodynamic analysis of the three kinetic models reveals interesting data on the activation parameters of the different reaction steps. Our findings suggest that PSI was able to first optimize its interaction with positively charged Cyt and that the evolutionarily replacement of Cyt by Pc would involve structural modifications in both the donor protein and PSI.

76

Josenh T. Warden Department of Chemistry, Rensselaer Polytechnic Institute, Troy, NY, USA 12180-3590 Recent advances in purification methodology for membrane proteins have enabled the isolation of highly purified photosystem I reaction centers either conmining or deficient in the Ix, FA and FB clusters. The identity of which of these clusters serves as the terminal aceeptor under physiological electron transport has not been established, although FA is preferentially reduced at cryogenic temperatures. We have proposed that in both Synechococcus sp. PCC 6301 and spinach photosystem I preparations the iron-sulfur cluster Fx is reduced within the 5 ns time constant of our flash spectrometer. However, the kinetics of the reoxidation of this cluster, corresponding to the reduction of the non-heine iron acceptor (either FAor F a) bound to the PsaC peptide, has not been determined. We present a careful comparison of the difference spectra for Fx and PsaC and demonstrate that spectral differences between these species provide the capability for monitoring the kinetics of electron transfer to Fx and from Fx to the PsaC-associated iron-sulfur clusters. (Supported by Grant GM26133from the National Institutes of Health.)

Poster

P-5-018

P-5-021

XLEC"~ROGlg3~ZCLIGI~]? I~gACTZONS IN PHOTOSYflTID( Z : RISOLUTION OF ELZCTRON-TRANSFER RATES BE~qEKIq THE ZRON-SULIrITR CENTERS

EPR Studies Of Site-Directed Mutant PsaC Protein Of Photosystem I In The Filamentous Cyanobacterium Anabaena Variabilis

K. siafridsson, ~. Hansson and P. Brzezinski, Dept. of B~ochem. and Biophys., G6teborg Univ. and Chalmers Univ. of Techn., Lundberg Lab., Medicinareg. 9C, S-413 90 G~teborg, Sweden

W. -Z. He 1, R. M. M a n n a n 2, H. B. Pakrasi 2 and R. Malkinl tDept of Plant Biology, University of California, Berkeley, CA 94720. 2Dept of Biology, Washington University, St. Louis,/dO 63130

F l a s h - i n d u c e d electrogenic events in Photosystem (PS) I particles from spinach, oriented in a phospholipid layer, have been studied at room temperature on a time scale ranging from 1 ~s to several seconds. A phospholipid layer containing P S I particles was adsorbed to a Teflon film separating two aqueous compartments. Voltage changes were measured across electrodes immersed in the compartments. In the absence of added electron donors and acceptors a multiphasic voltage increase, associated with charge separation, was followed by a decrease, associated with charge recombination. Several kinetic phases were resolved: a rapid (
P-5-019 PHOTOACTIVE REACTION CENTER COMPLEX OF

Several site-directed mutants of photosystem I PsaC protein, k n o w n to bind iron-sulfur d u s t e r s A and B, are generated in the filamentous cyanobacterium Anabaena var/ab/l/s ATCC 29413 and studied with EPR spectroscopy and redox potentiometry. In the C13D mutant, conversion of Cys13 to an aspartate residue leads to loss of the iron-sulfur d u s t e r FB. There is no [3Fe-4S] cluster found in the mutant. In contrast, the [4Fe-4S] cluster of FA is not affected as evidenced by normal EPR g-values. The redox potential of FA in C13D is -510 mV, comparable to w h a t found in the wild type. Despite the absence of FB, FA in C13D can still be photoreduced at cryogenic temperature. Furthermore, thylakoid m e m b r a n e s isolated from C13D are capable of photoreducing NADP + using ascorbate/DCIP as electron donor and in the presence of spinach ferredoxin and ferredo~Sx~ N A D P oxidoreductase. The roles of these t w o iron-sulfur d u s t e r s in the photosynthetic electron transfer reaction are discussed.

P-5-022 E L E C T R O C H E M I C A L AND SPECTROSCOPICAL CHARACTERIZATION OF THE PRIMARY DONOR OF PHOTOSYSTEM I, P700, IN THE M O N O M E R I C AND T R I M E R I C F O R M

CHLOROBIUM Hirozo Oh-oka 1, Saki Kakutani 1, Shoichiro Kamei t, Hiroshi Matsubara 1, Masayo Iwaki 2, & Shigetu Itoh 2, Dept. of Biol., Fac. of Sci., Osaka Univ. 1, Osaka 560, & National, Inst. Basic Biology 2, Okazaki 444, Japan The photoactive reaction center (RC) complex, comprising only two types of subunit, was isolated from Chlorobium limicola. The complex showed two subunit bands at 68 and 21 kDa on SDS-PAGE analysis. The former was assigned to the reaction center core subunit and the latter, with a heme moiety, to the cytochrome c551 subunit, respectively. The complex contained 1.6 - 1.7 c-type hemes per P840, as determined from the chemically oxidized-minusreduced difference spectrum. At room temperature, the complex did not show stable charge separation under continuous illumination. At 77 K, laser flash excitation induced an absorption change around 836 nm which is ascribable in + part to oxidation of P840. The charge separated state, P840 BChl-671-, were detected after 10-ns laser flash excitation. Recombination between 1'840 + and BChl-671- occurred within 100 ns and P840 T was generated. We will also present the photoactive RC complex isolated from C. tepidum under anaerobic conditions, whose complex contained five subunits as revealed on SDS-PAGE analysis. Some detailed spectroscopic studies are now in progress.

P-5-020 STRUCTURAL ORGANIZATION OF THE PHOTOSYSTEM I POLYPEPTIDES

E.Hamacher*, J.Ktuip #, M.ROgner#, and W.Mantele* *Institut £ Physik. Chemie, Egerlandstr. 3, 91058 Erlangen, Germany #1nstitut f. Botanik, ScldoBgarten 3, 48149 Mfinster, Germany The primary donor of Photosystem I, P700, from Synechocystis PCC 6803 has been characterized by electrochemistry in combination with FTIR difference spectroscopy and vis spectroscopy. Between 1800 to 1200 cm-1, highly structured difference spectra were obtained, reflecting the molecular changes at P700 and its protein site upon cation formation without any contributions from other cofactors. The IR difference bands titrate reversible and in unison, revealing the midpoint potential of P700 (Em= +260 mV vs. Ag/AgCI), corresponding to the values determind by monitoring the electrochemically-induced oxidation/ reduction of P700 at 700 nm and by measuring the amplitude of the flash induced absorbance change at 707 nm as a function of the applied potential. These values agree between monomeric and trimefic PS I, demon strating that proteinprotein contact has little impact on on the redox properties of P700. The lack of the 2600 cm -1 band (found in bacterial RC and interpreted as a low lying electronic transition of the dimeric structure of P+) indicates that the positive charge on P700 is rather localized on one of the chlorophylls.

P-5-023 THE OXIDIZING SITE OF PHOTOSYSTEM I MODULATES THE ELECTRON TRANSFER FROM PLASTOCYANIN TO PT00+

Q. Xu. V. P. Chitnil, A. Ke, P. R. Chitnis

Division of Biology, Kansas State University, Manhattan,KS 66506, USA We are studying the architecture of cyanobacterial photos/stem I (PSI) complex using polydonal antibodies, chemical cross-linking, surfacelabeling, chaotropic extraction, protease treatments, N-terminal amino acid sequencing, and subunit-deficient mutants in the cyanobacterium Synechocystis sp. PCC 6803. These studies have revealed many aspects of topography and subunit organization in the PSI comple~ (1) Identification of domains in PraB that interact with peripheral subtmits: PsaC, PsaD and PsaE are the peripheral subunits on the reducing side of PSI. Topographical studies in the mutants lacking specific subunits (e.g. PsaD, PsaE, PsaF, PsaL, PsaJ, or psaI) of PSI have shown that two putative extramembrane loops in the C-terminal domain of PsaB may interact with PsaD. (2) Subu-it contacts: Chemical cross-linking using the wild-type and subunit-deficient PSI complexes has revealed that the following subunits interact with each other. PsaC-PsaD, PsaE-PsaD, PsaC-PsaE, and PsaDPsaL. We are isolating and purifying these species to identify the interacting domains among these cross-linked PSI subunits.

M. Hippler, E Drepper and W. Haehnel Lehrstuhl ffir Biochemin d. Pflanzen, Institut fiJr Biologie II, Albert-Ludwigs-Universitfit, ScMnzlestr. 1, D-79104 Freiburg, Germany. The fast electron transfer within the complex between plastocyanin (Pc) and photosystern I (PSl) as well as after cross-linking Pc to PSI has been investigated, by measuring flash-induced absorption changes of P700 +. The midpoint potential of soluble Pc is shifted from 360 mV to about 410 mV when Pc is bound to PSI. The pH-dependence of the electron transfer from bound Pc to P700 revealed that inactive pmtonated reduced Pc, which was predicted at low pH (Gusset al., 1986, J. Mol. Biol. 192, 361387), is rapidly converted to its active form upon binding to PSI, suggesting a shift of the pK value of N 1 of His87 of Pc. An altered surrounding of the copper ligands is also indicated by a less pronounced temperature dependence of the midpoint potential of bound Pc as compared to that of soluble Pc. Furthermore the activation energy of the electron transfer of bound Pc to P700 was determined to be 9 kJ/mol. We have used the activation energy and the temperature dependence of the midpoint potential of bound Pc to estimate the reorganisation energy ~.. Its low value indicates that the complex provides conditions where a change in the oxidation state is coupled to a small contr~ution of solvent molecules. This supports the fast inner-protein electron transfer in spite of the relatively low driving force.

77

Poster i

P-5-024 HIGHLY RAPID SUBFRACTIONATION OF SPINACH P H O T O S Y S T E M I I N T O ITS C O R E C O M P L E X AND L H C I S U B F R A C T I O N S USING P E R F U S I O N C H R O M A T O G R A P H Y and BertiI Andersson. Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S- 106 91 Stockholm, SWEDEN Rapid subfractionafion of native spinach photosystem I into its core complex and different LHC I antenna sub-components was performed by the new method of anion exchange perfusion chromatography. LHC I subcomplexes were obtained in as short as only 10-25 minutes as compared to 50 hours using traditional density centrifugation. The rapid perfusion chromatography yielded LHC I subfractions highly enriched in Lhca2+3 (LHC 1-680) and Lhcal+4 (LHC 1-730) proteins. Also pure Lhca2, Lhca3, and Lhcal+2+4 subfractions were obtained. Notably, the two latter fractions were for the first time obtained in a soluble form and without the use of harmful SDS. Absorption and 77 K fluorescence-spectroscopy and SDS-PAGE of the various LHC I subfractions demonstrated their identifies, functional intactness and purity. Furthermore, the analyses specifically located C716, responsible for 730 nm fluorescence, to the Lhca4 polypeptide and chlorophyll b to preferentially deliver excitation energy to C716. Furthermore, the photosystem I core complex was by rapid perfusion chromatography isolated with high functional integrity. Work is also in progress to rapidly isolate native photosystem I directly from thylakoids.

P-5-027 QUINONE E X C H A N G E AT THE A 1 SITE IN P H O T O S Y S T E M I [PSI] A. Barkoff 1, N. Brunkan 1, S. W. Snyder I , A. Ostafin 1, M. Werst 1, J. Biggins2, M. C. Thurnauer 1. 1Argonne National Laboratory, Argonne, IL, 60439, USA; 2Brown University, Providence, It[, 02912, USA In the PSI reaction center, phylloquinone [K1] is believed to be the secondary electron acceptor A,. Photoinduced electron transfer [et] in PSI is initiated by reductton of the acceptor [A0] by the excited pnmary donor PT00" A1 acts as a transient intermediate between A 0 and iron-sulfur centers ~eS]. Quantitative extraction of K 1 from PSI requires chemical treatment. Extraction blocks room temperature reduction of FeS, and chemical reconstitution of K1 restores et. The characteristic electron spin polarized [asp] electron paramagnetic resonance [epr] signal of the interacting radical pair P~00A] is a probe of occupation and structure of the AI binding site. Using the esp epr signal we demonstrated that deuterated K 1 [~K1] can be exchanged with native K 1 without prior extraction of K 1. Exchange required extended incubation of the PSI particle with 2K 1 at 4°C, or shorter incubation at elevated temperatures. Quinones lacking the phytyl group did not exchange. Effects of incubation time, temperature, and light on K 1 exchange, and possible functions of exchange will be presented

P-5-028

P-5-025 Temperature Dependence of the Reoxidation Kinetics of At" in PS I

E. Schlodder 1, K. Brettel z, K. Falkenber~l~land M. Gergeleit' 1Max-Volmer-Institut ffir Biophvs. Chemic, TU-Berlin, Str. des 17. Juni 135, 10623 Berlin. ZSection de Bio~nerg~tique and CNRS, URA 1290, D6partement de Biologic Cellulaire et Moldculaire, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France The rcoxidation kinetics of the reduced secondary acceptor A,- have been examined as a function of temperature in PS 1 complexes from Synechococcus elongatus using absorbance difference spectroscopy. 60% (v/v) glycerol has been used as a cryoprotectant. Between 310 K and approx. 180 K, Al" is oxidized by forward electron transfer to the ironsulfur-cluster Fx. The measured half-life of this reaction decreases from 150 ns at 308 K to 12 Its at 200 K. An activation energy of 220 ± 20 meV has been determined from an Arrhenius plot. In the temperature range between 200 and 160 K, the electron transfer to Fx becomes blocked in about half of the P S I centers. In this fraction AI" decays with a half-life of about 200 /as by charge recombination with P700 +. In the other half, the forward electron transfer competes with the charge recombination of A~'P700 +. Also at lower temperatures (160 - 5 K), three fractions of P S I centers can be distinguished according to their capability to perform charge separation up to FA/B, Fx or only up to A1. The influence of the solvent on the heterogeneity of low temperature photochemistry in P S I has been investigated.

MUTATIONAL ANALYSIS OF THE BIOGENESIS, STRUCTURE FUNCTION OF THE PHOTOBYSTEM I REACTION CENTER IN CYANOBACTERIUM BYNECHOCYSTIS SP. P C C 6 8 0 3

AND THE

R. Schulz I, L.B. Smar~ I, J. Yu I, Y.-S. Jung 2, J.H. ~olbeck z & L. McIntosh ¢ DOE-Plant Research Laboratory, Michigan State University, East Lansing, MI 48824-1312, USA; Department of Biochemistry, University of Nebraska, Lincoln, NE 68583-0718, USA The photosystem I (PSI) reaction center is comprised of a heterodimer of homologous polypeptides, PsaA and PsaB. The corresponding genes were inactivated by interposon mutagenesis and selection under lightactivated heterotrophic growth (LAHG) conditions in a glucose-tolerant strain of Svnechocvstis 6803. We are employing site-directed amino acid substitutions and deletions to investigate the ligands of F.A and amino acid residues which may interact with A_ and A~. Thls technique is also being used to study t~e assembly of the PSI reaction center; specifically, the proposed leucine "zipper" between PsaA and PsaB and residues which may be required for interaction between the PsaA/PsaB core and PsaC.

P-5-026 INTERACTION OF PsaC WITH THE PSI CORE HETERODIMER

P-5-029 SITE DIRECTED MUTATIONS OF PsaB FOR THE ASSIGNMENT OF STRUCTURAL ELEMENTS OF THE PHOTOSYSTEM I

S u z a n n e M. R o d d a y a n d | o h n Bi i[~oL~ D e p a r t m e n t of M o l e c u l a r Biology, Cell B i o l o g y a n d B i o c h e m i s t r y , B r o w n U n i v e r s i t y , P r o v i d e n c e , RI 02912, USA.

L. Krabben., M. Kuhn, A. Webber+ & W. Lubitz.

W e h a v e p r o p o s e d a m o l e c u l a r s t r u c t u r e for the b i n d i n g site of the P s a C s u b u n i t o n the P S I r e a c t i o n center core h e t e r o d i m e r a n d s u g g e s t e d a m e c h a n i s m for the i n t e r a c t i o n b e t w e e n the s u b u n i t s [ R o d d a y et al. 1993, Photosyn. Res. 36: 1-9]. The p r o p o s a l has b e e n s u p p o r t e d b y c h e m i c a l m o d i f i c a t i o n a n d p r o t e o l y t i c c l e a v a g e of surface r e s i d u e s , a n d the s i t e - d i r e c t e d c h a n g e of the c o n s e r v e d a r g i n i n e r e s i d u e i n the i n t e r h e l i c a l l o o p of the d o m a i n of the FX c l u s t e r i n PsaB s u b u n i t . W e h a v e n o w c h a n g e d the n e g a t i v e l y c h a r g e d r e s i d u e s o n the surface of PsaC s u b u n i t b y site-directed m u t a g e n e s i s to create the m u t a n t s D9R, D24R, E46R-D47R a n d E72R. W e w i l l r e p o r t o n the f u n c t i o n a l i n t e r a c t i o n b e t w e e n these m u t a n t P s a C s u b u n i t s w i t h b o t h w i l d t y p e a n d R561EPsaB m u t a n t FX cores a n d p r e s e n t a m o d e l of the PsaC s u b u n i t i n the core b i n d i n g site.

78

Max-Vo/mer-lnstitut for Biophysikalische und Phy~ika/ische Chernie, Technische Universit~t Berlin, Stre/~e des 17. Juni 135, D-10623 Berlin, Germany. * Department of Botany, Arizona State University, Tempe, AZ 85787-1601, U.S.A. The Photosystem I core consists of the two main subunits PsaA and PsaB. In order to study the function and structure of the Photosystem I site directed mutations were introduced into the psaB gene of Ch/amydomonas reinhardtii. Mutations were designed in order to find amino-acid residues with a specific role for the function of the primary donor P7oo. Photosystem I from Chlamydomonas reinhardO'iwere purified and used to charactense the effect of the different mutations on the properties of PTooand on the reduction of P~oo by plastocyanin. Results from the biochemical and biophysical characterisations will be discussed. A model for the arrangement of Photosystem I cofactors will be presented which fits the stnJctural data known so far.

Poster

P-5-030

P-5-033

P R O P E R T I E S OF M I X E D - L I G A N D [4Fe.4S] CLUSTERS IN PS IR E C O N S T I T U T E D CSID AND CI4D MUTANTS OF PsaC

HEAT-INDUCED PIGMENT ALTERATION REACTION CENTERS

Yu z, Yean-Sung Jung 1, Donald A. Bryant 2, and John H. Golbeckl L Dof Biochemistry, e University i aof Nebraska, n Lincoln, NE 68583p arJmant 0718; ZDepartment of Biochemistry and Molecular Biology, The Pennsylvania

M. Kobayashi ~ , M. Iwaki 2, T. Tomo "-3, H. Kise ~, K. Satoh a.3, S. Itch 2 ~Inst. Materials. Sci., Univ. Tsukuba, Tsukuba, 2Natl. Inst. Basic Biol., Okazaki, 3Fac. Sci., Okayama Univ., Okayama, JAPAN

State University, University Park, PA 16802

Reconstituted CI4D-PS I and C51D-PS I complexes show light-induced EPR signals with g-values, linewidths, and photoreduction behavior characteristic of F^and FB, respectively. In C14D-PS I, a new center, termed FB' is EPR-visible after illumination at 8 Kand shows gay of -1.97. In C51D-PS I, a new center, termed FA', is EPR-visible after illumination at 6 K in the prestmce of prereduced F/~and shows g-values of 2.105, 1.922 and 1.847. Both centers represent S = [4Fe-4S] clusters in the mixed-ilgand (3Cys.lAsp) sites. The E m values are 600 m V for F s' in C I 4 D - P S I and -640 m V for F 4' in C 5 1 D - P S I. N A D P + photoreduction rates are equivalent to the wild-type m C 5 1 D - P S I and 7 0 % of wild-type in C 1 4 D - P S I. Free P s a C - C 5 1 D and P s a C - C I 4 D proteins s h o w resonances near g = 5.5 which represent a population of S=3/2 [4Fe-4S] clusters

in the mixed-ligand sites. It is proposed that the P700-Fx cores selectively rebind those mutant proteins that contain two [4Fe-4S] clusters, and that the spin state of the mutant [4Fe-4S] cluster crosses over from S = 3/2 to 1/2 on binding. These studies indicate that primary photochemistry and electron transfer occur at high efficiency in the presence of mixed-ligand iron-sulfur clusters in FA and FB.

P-5-031

IN THE PBOTOSYSTEM

I AND II

Primary donor of photosystem (PS) I, PYO0, can be concentrated by Ikegami's ether extraction method. In this sample, ca. 10 molecules of chlorophyll (Chl) a, one nolecule of Chl a' and one molecule of Chl b per PY00 are present. Recently we found a heat-induced drop in P700 photoactivity in ether-treated PSI particles. Photoactivity of P700 decreased with increasing incubation temperature for ten min: relative activity is 100 at 0 , 75 at 3 0 ~ , 50 at 3 5 ~ , 25 at 4 0 ~ , 7 at 4 5 ~ and 5 at 50~C. Certain Chls a were oxidized to yield CI32-OH-Chl a during heating: one molecule of Chl a at 3 5 ~ , two at 40~C and three at 4 5 ~ were oxidized. No alteration of Chl a' and Chl D, however, has not been observed. Under unaerobic condition, CI32-OH-Chl a was not produced, although same reduction of Chl a was observed. By contrast, no alteration of Chl a, Pheo a and B -Car (6:2:2) in Dl/D2/cyt b559 reaction center complex occurred by heart incubation even at 7 0 ~ . These results strongly suggest that certain molecules of Chl a in the core of RC I are very sensitive to heating, but pigments in the core of RC II are very tolerant to high temperature.

P-5-034

IN

Q U I N O N E B I N D I N G I N Rhodobacter Sphaeroides A N D P H O T O SYSTEM I STUDIED BY T R A N S I E N T EPR.

I. R. Vassilievl, Y.-S. Jungl, R. Schulz 2, L. McIntosh 2 and J. H. Golbeck ~ ~Deparm~nt of Biochemistry, University of Nebraska, Lincoln, NE 685880664, USA; 2 D e p o t of Energy Plant Research Laboratory, Michigan State University. East Lansing, MI 48824-1312, USA

A. van der Est 1, I. Sieckmann*, W. Lubitz 2 and D. Stehlik 1 ZFachbereich Physik, Freie Universitgt Berlin, Arnimallee 14, 14195 Berlin (Germany). 2Max Vollmer Institut, Technische Universitgt Berlin, Strafle des 17. Juni 135, 10623 Berlin, (Germany)

The [4Fe-4S] cluster Fx is bound in the reaction center beterodimer of P S I by two cysteines from PsaA and two cysteines from PsaB. PsaB mutants of Synechocysas sp. 6803 with substitutions in the Fx biding niche were generated to study the significance of cysteine and aspartate residues in elecuon transfer through F x. The isolated mutant P S I complexes were characterized by optical kinetic and EPR spectroscopy. Substitutions of the cysteinas (C556--->S and C565~S) lead to an increase in the contribution of Fx- and At- to the overall back reaction with PToo÷ at the expense of the back ~ ' t i u n from F^- and Fe-. Substitutions of the aspartates (D557--~A or K and D566--~A or K) induced minor or almost no changes in the P~o0÷decay kinetics. The results in C556S and C565S infer a lower quantum efficiency of F^ and Fe photoreduedon, and explain the inability of these mutants to grow photoautotrophically.

The binding of the primary quinone acceptors in bacterial reaction centres (bRC's) and photosystem I (PS I) is investigated using transient EPR. The spin polarized EPR spectra of P~sQA" and P+~oAi-" in bRC and P S I respectively reveal drastic differences in the two systems. Experiments in which the native quinones have been replaced by other quinones show that this is a result of differences in the binding of the quinone to the protein matrix. In bRC's the magnetic end structural parameters obtained from the EPR spectra are consistent with the fact that QA is hydrogen bonded to the protein. In contrast, the results in P S I suggest that A1 is bound via n - 7r* interactions with neighbouring aromatic amino acid residues.

ELECTRON TRANSFER VIA THE F x CLUSTER OF P S I PsaB M U T A N T S OF S Y N E C H O C Y S T I S SP. P C C 6803

P-5-032 IDENTIFICATION OF THREE PHOTOACTIVE [4FE-4S] CLUSTERS IN THE PHOTOSYNTHETIC REACTION CENTER OF THE GREEN SULFUR BACTERIA CHLOROBIUM VIBRIOFORME ~

t , Bedil Kj~r2, Henrik Vibe Scheller2and John H. Golbeck l University of Nebraska, Lincoln, USA; 2 Royal Veterinary and Agricultural University, Copenhagen, Denmark

Green sulfur-reducing bacteria are thought to contain type I photosynthetic reaction centers (RC), which are characterized by three [4Fe-4S] clusters. We

have analyzed a RC from Chlorobium vibr[oforme by EPR spectroscopy and have identified three photoactive [4Fe-4S] clusters which give EPR spectra similar to those previously interpreted to be derived from two clusters. Our RC shows an EPR spectrum at g--2.08, 1.90 and 1.85 when illuminated at 10K. When chemically reduced and photoaccumulated, a spectrum develops with a trough at g=1.88 and an inflection at g=1.94. We have de-convoluted these resonances by taking advantage of differences in their spin relaxation properties and have found that this signal represents two [4Fe-4S] clusters whose spectra are overlaid. Further, we were able to reduce one of these two clusters singly with dithionite. The redox behavior of the overlaid spectra suggest that they are derived from clusters analogous to FAand Fe in photosystem I of plants and cyanobacteria. We propose that the g=2.08, 1.90 and 1.85 signal represents Fx. A unique feature of Fx in Chlorobiura is that it does not appear to donate electrons forward to F^ or Fe or back to Pu0* at 10K.

P-5-035 AG DEPENDENCE OF THE RATE CONSTANT OF THE P+Ao---~P+Q REACTION IN THE QUINONE-RECONSTITUTEDPSI REACTION CENTER.

Nasayo Iwaki 1, Shlgeichi Kumazaki 2, Keitaro Yoshihara2, Tarsus Erabi 3 and Shi~eru I t o h 1 1Natl. I n s t . Basic B i o l . , ~ I n s t . Nol. S c i . , Okazaki 444 and 3Tottori Univ., T o t t o r i 680 (Japan) The rate of r e a c t i o n between A0 (chlorophyll a-690) and phylloquinone or a r t i f i c i a l quinone in P S I r e a c t i o n center (RC) was measured by p s - n s p h o t o l y s i s in spinach P S I p a r t i c l e s at 280 K. The r a t e depended on the energy gap (-AG) of the r e a c t i o n that was varied between -0.6 and +0.1 eV by the quinone r e c o n s t i t u t i o n . Total and medium reorganization energies were estimated to be 0.30 and 0.12 eV, r e s p e c t i v e l y , and the e l e c t r o n i c coupling to be 14 ca -1 by the quantum mechanical Marcus theory. The reaction i s found to be optimized with phylloquinone t h a t gives -AG of -0.34 eV. The edge-to-edge distance between A0 and quinone i s estimated to be about 8 A. The distance and medium reorganization energy are smaller than those in the reaction between pheophytin and quinone in the purple b a c t e r i a l BCs.

79

Poster

P-5-039

P-5-036 GENETIC MANIPULATION OF PHOTOSYSTEM I FROM BARLEY Slaren Ki~erulff, Vibeke Skovgaard Nielsen, Birger Lindberg M¢ller and Henrik Vibe Scheller. Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agrieultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg, Denmark In order to obtain a detailed knowledge of the function and structure of photosystem I (PSI), a transformation system for barley has been established using the particle bombardment method. Since plants can not yet be transformed by homologous recombination with high efficiency, it is not possible to make gene deletions or gene inactivation and instead the antisonse approach is used. Antisense constructs have been made with full length cDNAs encoding PSI-E, which is thought to be involved in cyclic electron transport and PSI-N, which is localized on the thylakoid lumen side of PSI. The antisense cDNAs are initiated by the maize ubil promoter followed by an intron and terminated by nos 3'. The constructs contain the bar gene as seleetable marker. Analysis of regenerated green transformants with PCR shows that the antisenso psaE construct has been integrated in the genome of barley. Further characterization of the antisense transformants will be performed.

ELECTRON DOUBLE NUCLEAR RESONANCE (ENDOR) STUDIES OF A QUINONE ELECTRON ACCEPTOR IN THE PHOTOSYNTHETIC REACTION CENTRES OF GREEN SULPHUR PHOTOSYNTHETIC BACTERIA. I.P. Muhiuddin~, S.E.J.Rigbyz, M.C.W. Evans2 and P. HeathcoteI. 1. School of Biological Sciences, Queen Mary and Wesffield College, London E1 4NS. 2. Department of Biology, University College London. Although the reaction centre of green sulphur photosynthetic bacteria resembles photosystem I (PSI), evidence for a quinone electron acceptor like the phylloquinone A t is indirect and disputed. We report here the observation of an electron paramagnetic resonance (e.p.r.) signal arising from a semiquinone electron acceptor in the reaction centre of Chlorobium limicola ) thiosulphatophilum. This quinone was reduced by photoaccumulation at 205K, in membranes isolated from cells grown in the presence of acetylene to inhibit bacteriochlorophyll c synthesis. This quinone and its environment has been studied in detail using ENDOR spectroscopy, and the results compared with those obtained from similar studies of the phylloquinone electron acceptor in the reaction centre of PSI.

P-5-037

P-5-040

RECONSTITUTION OF BARLEY PHOTOSYSTEM I WITH MODIFIED PSI-C, EDENrllqlgS THE REGIONS OF PSI-C WHICH ARE IMPORTANT FOR INTERACTION WITH THE PSI CORE AND PSI-D.

ELECTRON NUCLEAR DOUBLE RESONANCE (ENDOR) STUDIES OF THE PHYLLOQUINONE ELECTRON ACCEPTOR (At) IN THE PHOTOSYSTEM I REACTION CENTRE

Helle Naver, M. Paul Scott, Hanne Linde Nielsen, Birger Lindberg Meller, John H. Golbeck and Henrik Vibe Scheller Department of Plant biology, Royal Veterinary and Agricultural University, DK-1871 Frederiksbcrg C. Copenhagen, Denmark The PSI-C subunit of photosystem I (PSI) shows similarity to soluble 2 [4Fe4S] from Peptococcus aerogenes. Alignment analysis clearly shows that PSI-C contains an 8 residue internal loop and a 15 residue c-terminal extension not present in the ferredoxin. Two PSI-C deletion mutants, lacking either the 8 residue internal loop or 10 residues of the e-terminal, were made and the oteins were expressed in E. coll. The modified proteins and the wild type I-C were used to reconstitute a barley PSI core devoid of PSI-C, -D and -E. The modified proteins carded 2 [4Fe-4S] dusters similar to those of native PSIC as shown by EPR analysis of the reconstituted complexes. Flash photolysis of the reconstituted complexes showed that in the absence of PSI-D, the PSI-C protein without the loop was very inefficient in binding to the core. The eterminally tnmeated PSI-C was unable to interact with PSI-D. These results suggest that the int.e.e/'nalloop of PSI-C must face the membrane. B)r analysis of the published 6 A structure of PSI (Krauss et al. 1993) in relation to me reconstitution experiments we reach the conclusion that FA is the duster nearest tOFx.

p. Heathcote ~, S.E.J. Rigby2 and M.C.W. Evans 2. 1. School of Biological Sciences, Queen Mary and Westfield College, Mile End Road, London E1 4NS, U.K. 2. Department of Biology, University College London. Comparison of the ENDOR spectra from the A f electron paramagnetic resonance (EPR) signal in photosystem I preparations, and of the phylloquinone semiquinone/n v/fro, demonstrates that the A f EPR signal arises from the phyUoquinone secondary electron acceptor. The hyperfine couplings to the methyl protons and hydrogen bonds to the quinone from the solvent and/or protein have been identified. The ENDOR spectra of A f show that the electron spin density distribution on the phylloquinone in vivo is perturbed. This is attributed to the binding pocket for this quinone in the photosystem I, and may in part account for the low redox potential of this electron acceptor.

P-5-041

P-5-038 ARABIDOPSIS T ~ A

PHOTOSYSTEM I GENES.

V. S. Nielsen, S. Kjmrulff, B. L. MeHer and H. V. Scheller. Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University,Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark. P h o t e s y s t e m I f r o m A r a b / d o p s / s thaliana contain all n u c l e a r encoded p h o t o s y s t e m I s u b u n i t s previously identified in h i g h e r plants, n a m e l y PSI-D, PSI-E, PSI-F, PSI-G, PSI-H, PSI-K, PSI-L a n d P S L N . T h i s is s h o w n by w e s t e r n blotting u s i n g barley antibodies. Sequences of p h o t o s y s t e m I gene eDNA clones obtained from t h e Arabidopsis Resource C e n t e r indicate t h a t t h e r e axe two genes encoding PSI-D, PSI-E a n d PSI-H, w h e r e a s there is only one gene encoding t h e r e m a i n i n g p h o t o s y s t e m I subunits. The identity is 83% b e t w e e n t h e two PSI-D genes, 69% b e t w e e n t h e two PSI-E genes a n d 84% b e t w e e n t h e two PSI-H genes showing highly homologous genes. T h e barley PSI-L antibody recognizes two protein b a n d s in Arabidopsis thylakoids, while t h e r e is identified only one PSI-L gene. T h i s s u g g e s t s two different processing sites in t h e PSI-L precursor.

80

EARLY ELECTRON ACCEPTORS IN HELIOBACILLUS M O B I U S ~

,

Hung-Cheng Chiou and Su Lin

Department of Chemistry and Biochemistry, Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, AZ 85287-1604 Picosecond transient absorption difference spectroscopic experiments were performed on membranes of the antenna/reaction center complex of Heliobacillus mobilis to study the electron transfer processes. Particular emphasis was placed on the blue spectral region, where the difference spectra of iron sulfur centers and quinones are significantly different. The absorption difference spectra of the primary and the secondary electron acceptor were constructed from different charge-separated states. The A0- - A0 spectrum resembles the spectrum of the same state from Photosystem I, which also contains a Chl a molecule as the primary electron accepter. The secondary electron acceptor X, has a X- - X difference spectrum similar to Px in Photosystem I from higher plants. The spectra do not give any evidence in favor of a quinone acceptor in heliobacteria, although they do not rule out the possibility that such an acceptor is present. The P+X- recombination is monophasic at 300 K with "c=15 ms, and also monophasic at 50 K with x=2 ms, but biphasic at intermediate temperatures, with temperature dependent amplitude of the two kinetic phases.

Poster P-5-045

P-5 -042 MUTATIONAL ANALYSIS OF PUTATIVE LIGANDS TO THE P700 REACTION CENTER OF PHOTOSYSTEM I

DEGRADATION OF PHOTOSYSTEM I REACTION CENTER PROTEINS

Andrew N. Webber. Hui Xu and Scott E. Bingham, Hanno Kii[~*, Matthias Kuhn* and Wolfi, an~, Lubitz*. Department of Botany and Center for the Study of Early Events in Photosynthesis, ASU, Tempe, AZ 85287-1601, and *MaxVolmer-Institut, TU Berlin, Str. d. 17. Juni 135, D-1000 Berlin 12, Germany.

K. Baba 1, S. Itoh2 and S. Hoshinal: Dept. Biol., Fac. Sci., Kanazawa University l, Kakuma, Kanazawa 920-11 and National Institute for Basic Biology2., Myodaiji, Okazaki 444, Japan

The PsaA and PsaB proteins of the PSI reaction center bind the P700, A0, A1 and FX electron transfer cofactors. Specific mutagenesis of PsaB is being used to identify regions of the polypeptide involved in coordinating these cofactors. Two putative membrane spanning regions of PsaB contain conserved histidines that possibly coordinate P700 -- these are His 523 of membrane span VI]/and His 656 of membrane span X. Site-directed conversion of H523 to Leu results in impaired assembly of PSI but does not alter spectroscopic characteristics of P700 + (Cui et al. 1995 Biochemistry 34, 1549-1558). H656 has been altered to Leu, Ash, Ser, Arg, Cys and Gly. H656L does not stably accumulate PSI. However, mutant H656N accumulates high levels of PSI, which is consistent with the hypothesis that this residue coordinates a chlorophyll. The 1HENDOR spectra of P700 + from H656N is significantly altered from wild-type, suggesting that the P700 + environment has been altered by this mutation. These results provide preliminary experimental evidence that H656 is a ligand to P700.

DURING PHOTOINHIBITION IN VITRO

Degradation of photosystem I (PSI) reaction center proteins under photoinhibitory illumination was studied. PSI core complex with Chl/P700 ratio of 100 (PSI-100) was prepared from spinach thylakoid membranes by the treatment with Triton X-100, followed by sucrose density gradient ultmcentrifugation. Strong light irradiation (1.1 x 104,uE m -2 s -1) of PSI-100 resulted in a 50% decrease of the electron transfer activity from the reduced dichlorophenol indophenol to methyl viologen with bleaching of the bulk chlorophyll. However, the primary donor, P700 assayed by chemical oxidation-reduction was not destroyed. Measurements of the flash-induced absorbance change indicated that about 50% of the secondary electron acceptor, A1 (phylloquinone) was inactivated by the irradiation. Photoinhibitory illumination of PSI-100 caused the degradation of the reaction center proteins. Histidine, a singlet oxygen scavenger, protected significantly the proteins against degradation. These results suggest that toxic oxgen molecule generated in the PSI reaction center complex during illumination is responsible for the in vitro degradation of the proteins.

P-5-043

P-5-046

LIGHT-INDUCED CHARGE SEPARATION IN CHLOROBIUM P840REACTION CENTER PREPARATIONS - STABILIZATION BY THE PRESENCE OF THE 42 kDa BChl a-PROTEIN AND A 17 kDaPOLYPEPTIDE Christine HAGER-BRAUN 1, Dian-Lin XIE2, Rainer ZIMMERMANN 1, Nathan NELSON 2, Astrid RIEDEL 3 and G/inter ItAUSKA I 1 Lehrstuhl fiir Zellbiologie and Pflanzenphysiologie, Universit~t Regensbnrg, Germany, 2Roche Institute of Molecular Biology, Nutley, New Jersey, USA, 3Lehrstuhl f/ir Biophysik, Universit~t Regeasburg, Germany. Simple procedures for the anaerobic preparation of photoactive and stable P840RCs from Chlorobium tepidum and Chlorobium limlcola in good yield are presented. The preparation from C. limicola consists of 4 subunits: The P840reaction center protein PscA, the BChl a-antenna protein FMO, the FeS-protein PscB with centers A and B, and a positively charged 17-kDa protein denoted PscD. The RC from Chlorobium tepidurn additionally contains the cytochrome c551 PscC. Removal of PscD and FMO destabilizes the preparations. The ratio of the BChl a-absorption peak at 810 nm to the shoulder at 835 nm is 4 which is only slightly below the ratio in chlorosome free membranes and corresponds to about 80 BChl a-molecules per reaction center. Lif~ht-induced bleaching at 610 nm demonstrates that up to 80% of the reaction centers are photoactive. Three FeS-centers can be photoreduced in the preparations - an FeS-center reminiscent of FeS-B of photosystem I at 4K, and two more resembling FeS-A and FeS-X during photoaccumulation at 200K. At least 90% of the photoactive P840reaction centers transferred electrons beyond the primary electron accepter As), as shown by triplet measurements.

T W O D I M E N S I O N A L F O U R I E R T R A N S F O R M E L E C T R O N SPIN E C H O E N V E L O P E M O D U L A T I O N S P E C T R O S C O P Y OF THE P700 ~ 14N R E A C T I O N C E N T R E C H L O R O P H Y L L IN PHOTOSYSTEM 1 Peter J. Bratt, Pierre Motnne-Loccoz, Peter Heathcote and Michael C. W. Evar~ University College London and Queen Mary and Westfield College, London, UK The two-dimensional ESEEM pulsed EPR experiment, HYSCORE, has been used to examine the electron distribution ratio's in the reaction center chlorophyll dimers of Rhodobacter sphaeroides, (P870+), and of green plant photosystem I, (P700+). By using the HYSCORE spectroscopy technique it has been possible to characterise the 14N quadrupolar and isotropic coupling constants of the high electron density halves of these chlorophyll dimers. In both the bacterial and the photosystem I reaction centres the spectrum of the low electron density half of the dimer can be identified, although the spectral resolution is not sufficient to identify the spectral parameters of the individual nitrogen atoms. Detection of the spectrum of this low electron density chlorophyll in photosystem I shows that the reaction centre chlorophyll cation P700 + is a dimeric structure. The results for P870 ÷, and P700 ÷, indicate electron distribution ratio's of 2 : l and 3:1 respectively.

P-5-047

P-5-044 Isolation of trimeric Cyanobacterial Photosystem I Complex by recombinant Antibody Fragments

G. Tsioti~ 1,2 W. Haase I , A. Engel2, S. Miiller2 and Hartmut Michel 1 1Max-Planck-Institut, Heinrich Hoffmannstr. 7, D-60528 Frankfurt am Main; 2M.E. Miiller Institut for Microscopical Structural Biology, Biozentrum Universit~it Basel, Klingelberg str. 70, CH-4056 Basel A monoclonal antibody (mAb) was derived from mice immunized with the native trimeric photosystem I (PSI) complex from the cyanobacterium Synechococcus PCC 7002. The mAb reacts with a conformational epitope of the PSI complex. The ability of the mAb to recognize native PSI was quantified by analysis of the PSI-mAb interaction using the biosensor technology. As seen by immunoelectron microscopy the mAb bound to the stroma side of the thylakoid membranes. The DNA sequence encoding variable regions of the mAb was cloned in recombinant plamids, sequenced and expressed in E.coli. Further, an isolation procedure was developed for an one step purification of the PSI using the expressed Fv fragment. Analysis by gel electrophoresis and N-terminal sequencing revealed the presence of the psaA, psaB, psaC, psaD, psaE, psaF and psaL gene products. Flash induced absorption change measurements of the complex showed that it exhibited electron transfer from the primary donor P700 to the Fe-S center FA/FB. Quantitative mass analysis of the PSI/Fv complex by scanning transmission electron microscopy yielded a mass peak at 983+81 kDa at a dose of 352_'.-L27 e/nm 2 confirming that the eluted complex is a PSI trimer.

A NASTY mutagenesis strategy for studying the reaction centre of photesystem I in Chlamydomonas. V. Patel, B. Hallahan, S. Purton & M. Evans Dept of Biology, University College London, Gower Street, London WC1E 6BT. The reaction centre core of photosystem I is a heterodimer of two homologous proteins, PsaA and PsaB, which bind the PT00 reaction centre and the early electron accepters Ao, A1 and the 4Fe-4S iron-sulphur centre Px. This centre is believed to be coordinated by four cysteine ligands: two provided by PsaA and two by PsaB. In order to investigate the binding niche of Fx and its role in electron transfer we are using the strategy of NASTY (nature ~hould ~all ]tou) mutagenesis. We have created a PSI- mutant of Chlamydomonas in which the PsaA gene is disrupted with a spectinomycin resistance cassette (1). We have also introduced silent changes into the cloned gene to create two unique restriction sites upstream of the Fx binding site. We are currently creating a plasmid library comprisIng 2.6 x 105 random changes to the coding sequence between these sites and testing these mutant psaA genes for their ability to complement the PSImutant. Sequence analysis of complementing clones should provide valuable information on the structure and function of this region of PsaA. (1) Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4090

81

Poster I

P-5-051

P-5-048 PRIMARY ELECTRON TRANSFER REACTIONS IN HELIOBACTERIA AND PHOTOSYSTEM I B. Toupance, & J. Breton CEA Saelay, Section de Bic~nergrtique, 91191 Gif-sur-Yvette, FRANCE Heliobacteria contain a photosynthetic reaction center (RC) which belongs to the same family of RCs as PSI from higher plants or cyanobacteria. These RCs are characterized by the presence of iron-sulfur clusters as secondary, low-potential electron aeceptors. In both systems flash-induced electrogenie reactions in the picosecond and nanosecond time scale have been studied by a photeeleetric technique. In PS I membranes from b],nechocystis, the following kinetics and dielectrically weighted transmembrane distances between the cofactors have been determined (time constant/relative distance): <25 ps/1.0 (F'/00~Ao); 50 ps/0.25 (Ao--,AI);220 ns/0.35 (A1--,Fx); <50 ns/0.48 (Fx--,FA,B). In Heliobacteria also several phases are observed but the kinetics and relative amplitudes are quite different. The structural and kinetic information obtained by photoelectric measurements on Heliobacteria and PSI will be compared and discussed with respect to the similarities and differences of both systems.

P-5-049 ANALYSIS OF THE Fe-Sx REGION OF PHOTOSYSTEM 1 BY SITE DIRECTED MUTAGENESIS IN C H L A M Y D O M O N A S REINHARDTII. D. Wright, B. Hailahan, S. Purton & M.C.W. Evans Dept. of Biology, University College London, Gower St, London WC1E 6BT, UK. The photosystem 1 reaction centre is a heterodimer of two homologous proteins PsaA and PsaB. This dimer is believed to bind the reaction centre redox components P700, A0, A~ and Fe-S x. Fe-Sx is a 4Fe-4S centre which is predicted to be coordinated by four cysteine ligands, two from PsaA and two from PsaB. Each polypeptide has two conserved cysteine residues arranged in an appropriate structure for the formation of an iron sulphur centre• The role of the cysteine residues of PsaA has been investigated by the preparation of site directed mutants of C578, C578H and C578D, and of the adjacent aspartate residue, D579L. The cysteine mutants are extensively disrupted, confirming the importance of the conserved cysteine in photo system 1, the D579L mutant synthesises essentially normal photosystem 1 reaction centers. Detailed structural and spectroscopic analysis of the mutants will be presented.

P-5-050 SUBUNIT ORGANIZATION OF THE HIGHER PLANT PHOTOSYSTEM I (PS I) HOLOCOMPLEX Stefan Jansson1,2, Birgitte Andersen 2 and Henrik Vibe Scheller2 1Dept of Plant Physiology, University of Ume~, S-901 87 Ume~t, Sweden 2plant Biochemistry Laboratory, Plant Biology, Royal Veterinary and Agricultural University, Thorvaldsenvej 40, 1871 Frederiksberg C, Denmark PSI preparations from barley and spinach was subjected to chemical crosslinking using Dithiobis(succinimidylpropionate) (DTSP) and its hydrophilic analog DTSSP. The pattern of cross-linking products were analysed with non-reducing/reducing 2-D electrophoresis and westem blotting using monospecific antibodies, and a large number of cross-linking products were identified. The snbunits PsaL cross-links to PsaH, PsaI and PsaD, PsaC cross-links to PsaD and PsaE. PsaF forms cross-linking products PsaE and PsaJ. The two largest LHC I subunits (Lhca2 and Lhca3) seem to be organized as homodimers, located close to PsaG and K, respectively, presumably at opposite sides of the complex. The LHC 1-730 subunits Lhcal and Lhca4 are present either as homo- or heterodimers, and Lhcal cross-links to Lhca2. Put into the framework of the structure of the cyanobacterial PS I, we have used our data to suggest a model for the higher plant PSI holocomplex.

82

OF A psaF MUTANT OF CHLAMYDOMONAS REINHARDTII: EFFICIENT INTERACTION OF PLASTOCYANIN WITH THE PHOTOSYSTEM I REACTION CENTER IS MEDIATED BY THE PsaF SUBUNIT. ISOLATION

Joseph Farah ! , Fabrice Rappaport 2 , Yves Choquet 2, Pierre Joliot2 & Jean-David gochaix t ~Departments of Molecular and Plant Biology, University of Geneva, 1211, Geneva, Swazerland and Instttut de Biologic Physlco-Chimique,Pans, France. •

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The PsaF polypeptide of photosystem I (PSI) is located on the lumen side of the thylakoid membrane and its precise role is not yet fully understood. We have generated a psaF deficient mutant of the green alga Chlamydomonas reinhctrdtii by cotransforming the nuclear genome of the cwl5-arg7A strain with two plasmids: one harboring a mutated version of the psaF gene and the other containing the argininosuceinate lyase gene conferring arginine prototrophy. This psaF mutant still assembles an active PSI complex and is capable of photoautotrophic growth. However electron transfer from plastocyanin to P700 +, the oxidized reaction center chlorophyll dimer, is dramatically reduced in the mutant, indicating that the PsaF subunit plays an important role in docking plastocyanin to the PSI complex.

P-5-052 MORPHOLOGY AND SPECTROSCOPY OF CHLOROSOMES FROM CHLOROBIUM TEPID UM BY ALCOHOL TREATMENTS Z.'Y. Wan~,. G. Marx & T. Nozawa Dept of Biochem & Eng, Faculty of Eng, Tohoku Univ, Sendai 980-77 Japan Chlorosomes from Chlorobium tepidum have been treated with alcohol saturated buffers, followed by dilution to the buffers with half the saturated concentrations. Morphologic changes during this process have been statistically investigated by dynamic light scattering technique combined with electron microscopy to obtain the complete information on shape, size and distribution, while spectral properties have been studied by absorption, CD and magnetic circular dichroism. Three alcohois ( 1-hexanol, 1-butanol and phenol ) have been found to produce nearly reversible conversion of absorption spectra despite more than 15 times differences in the alcohol concentration. It is shown that the degree of saturation is the key factor for the complete conversion of bacteriochlorophyll c in chlorosomes from aggregated state to monomeric form. We have observed substantial changes in the shape, size and distribution at each step of 1-hexanol treatment, indicating that the whole process is morphologically irreversible. Comparison of the morphotogic changes with the corresponding spectroscopic behaviour suggests that the size rather than the shape and distribution may be a more important factor affecting the spectral properties.

P-5-053 Fluorescence polarization of the long wavelength emission observed in isolated photosystem I particles of Synechococcus sp. L.-O. p~ilssont, E. Schledder ~, R. van Grondelle I and J. P. Dekker. 1 1. Department of Physics and Astronomy, Vrije Universteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands. 2. Max-Volmer lnstute ftir Biophysikalische and Physikalische Chemie, Technische Universitat Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany. Polarized steady state fluorescence spectra have been measured at 4 K as a function of excitation wavelength for isolated photosystem I (PS I) particles of Synechococcus sp. and the results are compared with those previously obtained with monomerie and trimeric P S I complexes from Synechocystis PCC 6803 (Gobets et al (1994) BBA 1188, 78-85). Absorption spectra at 4 K revealed a much higher absorption around 710 nm in Synechococcus particles than in Synechocystis particles, suggesting a considerably higher content of long wavelength absorbing chlorophylls in Synechococcu.s'. The 4 K emission maxima were at 718 nm in Synechocystis and at 730 nm in Synechococcus. In all investigated particles a very low anisotropy of N 0.05 of the emission maximum was observed upon excitation around 690 nm. Upon more red excitation the anisotropy increases significantly and for 716 nm excitation it equals ~ 0.20 in Synechococcus. The possibilty of energy transfer between the several long wavelength pigments in Synechococcus is considered.

Poster

P-5-054 Trapping kinetics in the LHI only mutants of Rhodobacter Sphaeroides Vesa Helenius 1, Lucas Beckman 1, John Olsen2, Neil Hunter 2 and Rienk van Grondelle 1 1. Department of Physics and Astronomy, Free University of Amsterdam, 1081 De Boelelaan, 1081 HV Amsterdam, The Netherlands, 2. Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S 10 2UH, United Kingdom Kinetics of excitation energy trapping by a photosynthetic purple bacterium Rhodobacter Sphaeroides are reported. A mutant strains containing only the light harvesting complex I (LHI) and the reaction center (RC) are used. Sitedirected mutations at Trp+ll in tx-polypeptide of the ct[~-polypeptide pair of LHI antenna induce a blue-shift the energy level of the antenna relative to the RC. Picosecond pump-probe spectroscopy is used to record the excited state kinetics of the antenna pigments and the formation of the oxidized special pair. At room temperature the decay of the excited state absorption of the antenna pigments is very weakly dependent on the blue-shift of the antenna. At 77K a more pronounced effect on the trapping time of the excitations by the RC is observed. Complementary steady state fluorescence and absorption spectroscopy results on the three mutants will also be presented.

83

Poster

P-6-003 THE LATER STAGES OF CAROTENOID BIOSYNTHESIS IN HIGHER PLANTS Poster

session

6

Julia Savill 1, George Brittoa 2 & Andrew J. Young 1 1 School of Biological & Earth Sciences, John Moores University, Liverpool L3 3AF UK. 2 Dept. of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX U.K.

Carotenoids P-6-O01

- P-6-031

P-6-001 CALCULATION OF THE ELECTROCHROMIC SHIFT OF THE ABSORPTION OF ANTENNA CAROTENOIDS INDUCED BY THE CHARGE SEPARATION IN BACTERIAL REACTION CENTERS Stefan Grzybek, Werner M~ntele Institut f/it Physikalische und Theoretische Chemie, Universitgt Erlangen, Egerlandstr. 3, 91058 Erlangen, Germany Antenna carotenoids in the photosynthetic membranes of several purple bacteria respond with electrochromic absorption shifts to the charge separation in the photosynthetic reaction center (RC) between the hemes of the RC associated cytochrome subunit and the primary or secondary quinone [e.g. Matsuura and Shimada (1993), BBA, 1140, 293-296]. An a t t e m p t was made to calculate this shift with continuum electrostatic methods employing an all atom model of the RC based on the crystal structure of the Rhodopseudomonas viridis RC [Deisenhofer, Epp, Miki, Huber and Michel (1985), Nature, 318, 618-624] and an atomic model for antenna carotenoids around the RC in different relative orientations. It was found t h a t calculated absorption shifts depend mainly on the orientation of the carotenoids and the distance from the semiquinone, but very little on which of the four hemes is oxidized.

P-6-002 T R I P L E T STATES IN L I G H T - H A R V E S T I N G C O M P L E X II T R I M E R S , M O N O M E R S AND A G G R E G A T E S STUDIED AS A F U N C T I O N OF TEMPERATURE

The later stages of the biosynthesis of carotenoids have been studied in seedlings of Hordeum vulgare and Raphanus sativus m a i n t a i n e d under white light and during the greening of etiolated tissues. The incorporation of radiolabelled substrates (CO2, acetate and MVA) into B-carotene and the i n d i v i d u a l x a n t h o p h y l l s (together with squalene) has been determined. Dual-labelling experiments with 14 C O 2/1 4C-acetate and 3 H - M V A have shown differential incorporation of these substrates into the individual carotenoids. The behaviour of 14C_acetat e as a precursor in B-carotene synthesis is quite different from its role in the synthesis of the xanthophylls.

P-6-004 CAROTENOID S1 ENERGY LEVEL AND QUENCHING IN LHCIIb. Denise Phillip 1, Sasha Ruban 2, Peter Horton 2, A1 Asato 3 & Andrew J. Young 1 1Biol. & Earth Sci., John Moores University, Liverpool L3 3AF UK. 2Dept. Mol. Biol. & Biotech., Sheffield University, Sheffield SIO 2UH, UK. 3 Dept. Chemistry, Hawaii University, Honolulu, Hawaii 96822 USA The role of carotenoids in n o n - p h o t o c h e m i c a l q u e n c h i n g ( q N ) o f c h l o r o p h y l l fluorescence in the m a j o r light-harvesting complex of Photosystem II has been studied. The extent of q u e n c h i n g observed in isolated LHCIIb has been i n v e s t i g a t e d on the basis of the S1 (2lAg) energies of a series of 19 carotenoids with different structures. A clear correlation was f o u n d to exist between t h e i r S1 e n e r g y a n d the ability to q u e n c h fluorescence: i.e. carotenoids whose S1 e n e r g y levels are lower t h a n chl a (14,700 cm -1) (e.g. z e a x a n t h i n & c a r o t e n e ) were able to q u e n c h fluorescence whereas those with h i g h e r e n e r g y levels t h a n h a d no observed effect (e.g. v i o l a x a n t h i n & fucoxanthin). In contrast, w h e n qN was i n d u c e d b y a lowering of pH, i n h i b i t i o n of q u e n c h i n g was observed for all carotenoids with h i g h S1 e n e r g i e s . However, of the carotenoids tested only v i o l a x a n t h i n i n h i b i t e d q u e n c h i n g i n d u c e d by dibucalne.

~L~I~SCENCESTUDIES

P-6-005 ON THE~CHANXS~OFPHOTONPROTECTION

Erwin J.G. Peterman, Florentine Calkoen, Fred M. Dukker, Claudiu Gradinare, Rienk van Grondelle and Herbert van Amerongen Dept. of Physics and Astronomy, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands

C. Hagen I, A. A. Pascal 2, P. Horton 2 & Y. Inoue 3 IInstitute of Botany, Friedrich Schiller U n i v e r s i t y Jena, A m P l a n e t a r i u m I, D-07743 Jena, Germany; 2Robert Hill Institute, Dept of Mol Biol & Biotechnol, U n i v e r s i t y of Sheffield, P.O. Box 594, Sheffield SI0 2UH, UK; 3Solar Energy Research Group, RIKEN Institute, Hirosawa 2-1, Wake, Saitama 351-01, Japan

With laser-flash induced Wansient absorbance we measured carotenoid (Car) and chlorophyll (Chl) triplet states in different aggregation states of lightharvesting complex H. We measured triplet contributions of different Car's. Under aerobic conditions at room temperature we could resolve at least two different Car triplets because of their different decay times caused by a difference in the rate of quenching by oxygen. In case of monomeric LHCII one of these components was absent. The Car triplets induce absorption changes in the Chl region. These changes depend on temperature and reveal interesting information on the most red absorbing states of LHCII. The Chl to Car triplet U'ansfer is complete at room temperature; at temperatures below 77 K however some Chl triplets survive. At 4 K in trimeric LHCH the Chl triplet show a narrow bleach at 680 nm. This is in agreement with fluorescence and holeburnig data, both suggesting thast a narrow absorption band at the same wavelength is the lowest absorbing state in LHCII.

T h e r m o l u m i n e s c e n c e peaking at ii0 K (Z-band) is sensitive to changes in the envirorument of the emitting chlorophyll species. Factors increasing non-photochemical chlorophyll fluorescence quenching enhanced the Z-band detrapping efficiency of red light. In LHC II microaggregates, acidification induced s i m u l t a n e o u s l y formation of larger aggregates, fluorescence quenching and e n h a n c e m e n t of Z-band emission. A similar pHdependent and antimycin A-sensitive Z-band increase was d e t e c t e d in thylakoid membranes. In both microaggregates and thylakoids, d e - e p o x i d a t i o n of v i o l a x a n t h i n resulted in stronger signs for aggregation of the PS II antenna. Our resuls are consistent with the hypothesis that p h o t o n protection of PS II is b a s e d on antenna aggregation, and that v i o l a x a n t h i n cycle carotenoids m o d u l a t e the aggregation capability of LHC II.

84

Poster P-6-006 THE REVERSIBLE D I S S O C I A T I O N OF THE B 8 8 0 COMPLEX CONTAINING CAROTENOIDS OF R S . RUBRUM INTO THE B 8 2 0 SUBCOMPLEXES DURING PAGE WITH O C T Y L - ~ - D - O L U C O P I R A N O S I D E A.MOSKALENKO, O.

TOROPYGINA & N.

KUZNETSOVA

INSTITUTE OF S O I L SCIENCE & PHOTOSYNTHESIS, PUSHCHINO, 142292, RUSSIA The procedure of the dissociation of the B880 complex containing carotenoids (Car) of Rs. rubrum into the B820 subcomplexes and the back process of the partially dissociated B880 complex with different Car content during t h e PAGE w i t h OG w e r e d e v e l o p e d . The former is accompanied by release of Car's from the complex. A correlation is observed between the Car content and the presence of the B880 complex. A small amount of the subcomplexes is tightly b o u n d w i t h t h e RC a n d r e tain the native state ( b a n d a t 8 8 0 nm) o f p i g m e n t s . Two C a r b i n d i n g sites were shown to be present in the B880 complex: one was modified during the dissociation-reassociation process whereas the second is changed only in the case of the complete dissociation of the B880 complex to the subcomplexes. The organization of the B880 complex, the principles of their dissociation into subcomplexes and the role of Car in the stabilization of the B880 complex structure are discussed.

P-6-009 XANTHOPHYLL CYCLE PIGMENTS IN INTERMITTENTLIGHT GROWN PEA PLANTS A. Father & P. Jalms, Institut for Bioehemie der Pflanzen, Heinrieh-HeineUniversitltt, Universitgtsstr. 1, D-40225 Dfisseldorf, Germany Intermittent fight grown pen plants (IML-plants; 2" light, 118" dark) are particularly characterized by the absence of chlorophyll a/b-binding (CAB) proteins and grana stacks. The amount of the xantbophyil cycle pigments violaxanthin, antheraxanthin and zeaxanthin (V+A+Z) per photosynthetic reaction centre (RC), on the other hand, was found to be about twofold increased. Under high light illumination, nearly all V was converted to Z in IML-plants, in contrast to the situation in control plants, where a portion of V is unavailable to de-epoxidation. The synthesis of CAB-proteins and the formation of grana in IML-plants can be induced by continuous illumination. We investigated the changes of the pigment composition and the xanthophyll cycle activity under these conditions. With increasing mounts of CAB-proteins, the V+A+Z contem per RC decreased, and some of the V became unavailable for de-epoxidation. This was paralled by an acceleration of the epoxidation kinetics of Z. The results are discussed in terms of the protective role of the xanthophyll cycle with emphasis on the function of CAB-proteins and grana stacking in these processes.

P-6-010

P-6-007 STUDY OF 'I'HE CAROTENOID BINDING POCKET OF THE PHOTOSYNTHEYIC REACTION CENTRE FROM RHODOBACTER SPHAEROID ES. P. Fyfe1, R.J. Cogdeil1, M. Jones2 1 Institute of Biomedical and Life Sciences, University of Gtasgow, Glasgow G12 8QQ, UK. 2 Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2UI-I, UK. The carotenoid binding pocket o f the reaction centre (RC) o f the purple bacterium Rodobacter sphaeroides is being investigated. Several amino acid residues which lie in close proximity to the carotenoid, such as W M I 5 7 , in the reaction centre have been changed by site-directed mutagenesis. The effect o f these nmtations on the structure and function o f the carotenoid have been studied by a range o f spectroscopic techniques and X-ray crystallography. The mutant reaction centres were expressed in a background where the antennae are not expressed. This has made it possible to carry out some o f the spectroscopy o f the RC's whilst they remain within the membrane. Details o f the mutagenesis, spectroscopy and the crystallography will be presented in the poster.

P-6-008 REGULATION OF VIOLAXANTHIN DE-EPOXIDASE A C T I V I T Y BY pH A N D A S C O R B A T E C O N C E N T R A T I O N Charlotte E v a Bratt, Per-Ola Arvidsson, Marie Carlsson & HansErik ~kerlund. Dept o f Plant Biochemistry, Lund University, Box 117, S-221 00 Lund, S w e d e n The activity o f violaxanthin de-epoxidase (VDE) has been studied both in isolated thylakoids and after partial purification, as a function o f p H and ascorbate concentration. W e demonstrated that V D E has a K m for ascorbate that is strongly dependent on pH, with values o f 10, 2.5, 1.0, and 0.3 m M at pH 6.0, 5.5, 5.0 and 4.5, respectively. These values can be expressed as a single Kin = 0.1+ 0.02 m M for the acid form o f ascorbate. Release o f the protein from the thylakoids by sonication was also found to be strongly p H dependent with a cooperativity o f 4 with respect to protons and with an inflexion point at pH 6.7. These results can explain some o f the discrepancies reported in the literature and provide a more consistent view o f zeaxanthin formation in vivo.

INHIBITION OF ASTAXANTHIN H A E M A T O C O C C U S PLUVIALIS

SYNTHESIS

IN

Sandra Orset, Mark Harker, Alex Tsavalos and Andrew J. Young. School of Biological & Earth Sciences, John Moores University, Liverpool L3 3AF UK. The inhibition of s e c o n d a r y carotenoid synthesis in the green microalga Haematococcus p l u v i a l i s is described. The inhibitors d i p h e n y l a m i n e (DPA) and 1 - a m i n o b e n z o t r i a z o l e both e f f e c t i v e l y block astaxanthin synthesis resulting in the a c c u m u l a t i o n of Bcarotene in treated cells. In contrast to previous reports DPA does not inhibit the earlier d e s t a u r a s e r e a c t i o n s involved in c a r o t e n o i d biosynthesis in this alga. Cyclisation reactions were inhibited by CPTA, resulting in l y c o p e n e a c c u m u l a t i o n , and d i f f e r e n t i a l inhibition of the £- and B-cyclases was evident. The utilisation of the lycopene and B-carotene (accumulated as a result of inhibition by CPTA and DPA, respectively) in the biosynthesis of astaxanthin was effectively d e m o n s t r a t e d by w a s h i n g these inhibitors f r o m the cultures and adding norflurazon to inhibit subsequent de novo synthesis of carotenoids.

P-6-011 THE EFFECT OF NORFLURAZON ON THE PHOTOSYNTHETIC APPARATUS OF THE RED ALGA CYANIDIUM CALDARIUM J. Marquardt Fachbereich Biologie/Botanik, Karl-von-Frisch-Str., D-35032 Marburg, Germany The effect of Norflurazon, an inhibitor of the carotenoid biosynthesis, on (~yanidium caldadum was analysed using an algal strain which is capable to grow heterotrephically and which retains its full pigment complement in the dark. In the presence of Norflurazon cells could not grow autotrophically. When sucrose was added to the nutrient medium, they were able to grow in the light, but were totally bleached. In the dark, the algae showed an identical growth rate in the presence and absence of Norflurazon. Under these conditions the inhibitor caused a nearly total loss of caretenoids and an enrichment in e chlorophyllide. The ratio of phycocyanin to chlorophyll was nearly identical, although there was a significant reduction of PSII core proteins. PSII activity could not be observed. The PSI core complex was present and photochemically active. An LHCI po[ypeptide could be detected by immunoblotting, but it showed a significantly lower molecular weight than the LHCI polypeptide from control cells.

85

Poster

P-6-012

P-6-015

FUNCTION OF CHLOROPHYLL AdB BINDING PROTEINS IN THE XANTHOPHTLL CYCLE AND PHOTOINACTIVATION OF PS H

XANTHOPHYLL CYCLE COMPONENTS AND ENERGY DISSIPATION IN SUN AND SHADE LEAVES OF C 3 AND C 4 PLANTS

P. Jahns Institut ffir Biochemie der Pfl.nzen, Universitit D~sseldort~ Universitiitsstr. 1)-40225 Diisseldor~ Germany

E. Brumaoli. A. Scartazza, M. Lauteri & M.C. De Tullio CNR, Istituto per l'Agroselvicoltura, via Marconi 2, Porano (TR), 05010, Italy

The xanthophyll cycle was investigated in pea plants (Pisum sativum L.) that were grown under intermittent light (IML-plants) and thus devoid of nearly all Chlorophyll a/b binding (CAB) proteins. The kinetics of the de-epoxidation reactions were found to be unchanged in comparison to control plants. The limited convertibility ofviolaxanthin (Vio) to zcaxanthin (Zen) (only 60-70% of the total Vio pool can be converted to Zea in controls), however, was abolished in IML-plants. The epoxidation kinetics, on the other hand, were drastically slowed down in IML-plants (factor 5-10). In parallel, IML-plants were found to be nmch more susceptible to PSII inactivation at high photon flux densities ( > 1 mE m-2 s'~). Moreover, the kinetics of recovery fiom photoinactivation were closely related to the kinetics ofZea epoxidation in IML-plants. It is concluded from these results that CAB-proteins have a crucial function in both the deepoxidatiun and epoxidation reactions of the xanthophyU cycle. The consequences of these functions for the inactivation processes of PSH and the possible functions of Zea in thermal energy dissipation are discussed.

The content in xanthophyll cycle components (XCC) was compared with the availability of violaxanthin (V) for deepoxidation and with non-photochemical fluorescence quenching CNPQ) measured in sun and shade leaves ofC 3 and C4 species. The capacity for NPQ was compared with the contents of zeaxanthin (Z) and antherexanthin (A) formed under conditions of excessive energy. The results indicated that sun leaves not only contained a larger pool of XCC, hut also a considerably higher capacity for the light-dependent conversion of V to A and to Z, in all the species analyzed. The proportion of photoconvertible V was linearly related with the ehl a/b ratio and inversely with the neoxanthin content, in both C3 and C4 plants. These results are in agreement with previous findings on Ligustrum ovalifolium (Brugnoli et al., 1994, Photosynth. Res., 41: 451-463) and suggest that the availability of V for deapoxidation can be related to the degree of thylakoid membrane appression and to the organization of pigment-protein complexes. This evidence indicates that C3 and C4 species share common features in the acclimation to different light environments.

P-6-013 AN O2-BARRIER IN THE ANTENNA COMPLEX LHC-IIB PROTECTS CHLOROPHYLLS AND CAROTENOIDS FROM PHOTOOXIDATION

• . 1& A. Angerhofer2 i Universitnt Karlsmhe, Botanisebes Institut I, D-76128 Karlsrube, FRG 2Universitltt Stuttgart, 3. Physikalisches Institut, D-70500 Stuttgart, FRG In the antenna complex LHC-IIB chlorophylls (Chl) and xanthophylls (Car) are protected from photooxidation (S.-H., 1990, J. Photochem. Photobiol. B: Biology 4, 283-295). Comparison of photostable LHC with LHC destabilized by the addition of linolic acid (+LA) or Triton-X-100 (+TR) showed: (1) Efficient energy transfer from ICar* to 1Chl-a took place in both, LHC and LHC+LA but not in LHC+TR. (2) When exposed in air-saturated solution to white light at 3000 taF-Ym2s, Chl and Car of LHC were slowly oxidised (5-10% in 30 min), in LHC+LA oxidation of Chl and Car was enhanced (40-60% in 30 min) and in LHC+TR Chl were rapidly destroyed (80% in 30 rain) while Car were little affected. (3) Laser flash excitation of Chl-a yielded 3Car* (Z-max at 512 nm) in LHC and LHC+LA but SChl* in LHC+TR. (4) Lifetime x of 3Car* was 6.7_+0.1 las for LHC and 4.2_+0.1 ~ts for LHC+LA in air-saturated solution but 8.8_+0.1 Its for both preparations without O2 .- It is concluded that Car in close proximity with Chl (as in case of LHC+LA) are not sufficient to protect Chl from photooxidation. In addition an 02 barrier reducing the 02 pressure at pigment sites is present in photostable LHCII.

P-6-014

P-6-016 NATURAL SELECTION OF CAROTENOID CONFIGURATIONS BY REACTION CENTERS AND LIGHT-HARVESTINO COMPLEXES: CONFIGURATIONAL DEPENDENCE OF EXCITED-STATE PROPERTIES

Y. Koyama F a c u l t y of Science, Kwansei N i s h i n o m i y a 662, Japan

Gakuin

University,

Uegahara,

In purple p h o t o s y n t h e t i c bacteria, 15-cis carotenoids are b o u n d to the reaction centers (RCs) for the p h o t o - p r o t e c t i v e function, while all-trans carotenoids are b o u n d to the lighth a r v e s t i n g complexes (LHCs) for the l i g h t - h a r v e s t i n g function. Further, 1 5 - c i s - B - c a r o t e n e was found in the RC of s p i n a c h photosystem II. 15-cis-Carotenoids are characterized by e x t r e m e l y rapid, o n e - w a y isomerization from 15-cis to all-trans in the T 1 state, and therefore, the natural s e l e c t i o n of the 15-cis c o n f i g u r a t i o n b y the RCs can be a s c r i b e d to this T 1 s t a t e p r o p e r t y ; a h y p o t h e t i c a l m e c h a n i s m of t r i p l e t - e n e r g y d i s s i p a t i o n is p r o p o s e d b a s e d on Raman s p e c t r o s c o p y of the RCb o u n d spheroidene. A l l - t r a n s - s p h e r o i d e n e fluoresces f r o m b o t h the B u and 2Ag states, w h i l e 1 3 ' - c i s - s p h e r o i d e n e f l u o r e s c e s f r o m the B u state alone. Therefore, a v a i l a b i l i t y of the two channels for s i n g l e t - e n e r g y transfer to b a c t e r i o c h l o r o h y l l for the a l l - t r a n s c a r o t e n o i d is r e g a r d e d as the r e a s o n for the natural selection of the all-trans configuration by the LHC.

P-6-017

ARABIDOPSIS THALIANA MUTANTS DEFINE PLASTOQUINONES AS ESSENTIAL COMt~NENTS FOR CAROTENOID DESATURATIONS

XANTHOPHYLL CYCLES AND THEIR POSSIBLE ROLE IN PLANT EVOLUTION

and Susan R. Norris Depts. of Biochemistry and Plant Sciences, Univ. of Arizona, Tucson, AZ

H. Y. Yamamoto H1TAHR, University of Hawaii, 3050 Maile Way, Honolulu, Hawaii, 96822, USA

In order to further understanding of the mechanisms and components involved in carotenoid synthesis, we have isolated mutants in Arabidopsis thaliana that disrupt the pathway. Here, we describe two loci, pdM andpds2(pds = p..hytoene de,~mration defective), whose disruption results in accumulation of phytoene. Surprisingly, neither mutation maps to the phytoene desaturase enzyme locus (PDS3), indicating at least three gene products are required for phytoene desaturation in plants. As phytoene desaturase contains an FAD eofactor and catalyzes an oxidation reaction, it is presumed that a chloroplast redox component and/or electron transport chain is involved in desaturation. Analysis ofpdM and pds2 indicates both affect specific steps of quinone (e.g. ¢x-tocopherol and plastoqulnone) biosynthesis. The pdsl mutation affects the enzyme p-hydroxyphenylpyruvate dioxygenase as it can be rescued by growth on the product but not the substrate of this enzyme. The pals2 mutation is not rescued by either compound, lacks plastoquinone and a-tocopberol and most likely affects the common prenyl/phytyl transferase enzyme in the quinone biosynthetic pathway. Together, these data provide the first genetic evidence that plastoquinones are essential components for carotenoid dasaturafious in plants.

86

Xanthophyll cycles occur only in the more recently evolved oxygenic photosynthetic organisms. Two types of cycles are known. The one in higher plants and green algae involves the interconversions of violaxanthin (diepoxide), antheraxanthin (monoepoxide), and zeaxanthin (non-epoxide).The other in diatoms involves similar conversions between the diadinoxanthin (mono-epoxide) and diatoxanthin (nonepoxide). In higher plants, the xanthophyll cycle, in conjunction with thylakoid acidification, dynamicallymodulatesthe transfer of energy from the PSII antennae to the reaction center over a wide range of light conditions, from sun-flecksto the longterm growth environment. This dynamic range is mainlydue to the kinetic and growth responses of the xanthophyll cycle. It is hypothesized that the evolution of xanthophyll cycles and associated modulation of energy transfer was an advantage necessary for photosyntheticorganisms,particularlyhigherplants, for adaptation to the various terrestrial light environments. The dynamic and regulatory nature of the xanthophyll cycle distinguishesit as a third functionfor carotenoids in photosynthesis, the other two being the essentially static functions of light harvesting and protecting against photooxidation. The distribution of violaxanthin de-epoxidase is explored by Western blots with antibodies to higher-plant violaxanthinde-epoxidase.

Poster

P-6-018 DIFFERENT LOCALIZATION OF ZEAXANTHIN DEPENDENT AND INDEPENDENT QUENCHING MECHANISMS RESOLVED BY A FLUORESCENCE DECAY STUDY ON ISOLATED PEA THYLAKOIDS AT PICOSECOND RESOLUTION

~.

M. Richter ~, B. Wagner2, andA. R. Holzwarth 2 Inst. of General Botany, Joh. Gutenberg-University, 55099 Mainz; 2MaxPlanck-Institut fiir Strahlenchemie, 45470 Mfilbeim, Germany

P-6-021 SPECIFICITY OF CAROTENOIDS IN CHLOROSOMES OF THE GREEN FILAMENTOUS BACTERIUM, CHLOROFLEXUS A URANTIACUS Kimihiro Tsuji, *Shinichi Takaichi, Katsumi Matsuura and Keizo Shimada Dept. Biol., Tokyo Metropolitan Univ., Minamiohsawa, Hachioji, Tokyo 192-03 • Biol. Lab., Nippon Medical School, Kosugi, Nakabara, Kawasaki 211, JAPAN

The influence of the transthylakoid proton gradient on the kinetics of picosecond fluorescence decay was examined using isolated pea thylakoids having high or low zeaxanthin contents. Fluorescence measurements were done with open (Fo) and closed (Fm) PSII reaction centres. Energization of isolated thylakoids decreased the average fluorescence lifetime at Fm in zeaxanthin free thylakoids while a more pronounced decrease in the average fluorescence lifetime at Fm and in addition a significant decrease at Fo was observed with zeaxanthin containing thylakoids. By the help of the chosen measurement conditions and subsequent kinetic analysis of the fluorescence data it was possible to separately locate and quantify the effects of zenxanthin and the proton gradient in terms of rate constants of individual processes within PSII. It is shown that the enhanced non-photochemical fluorescence quenching in the presence of zeaxanthin mainly originates in the antenna while without zeaxanthin smaller changes in the Fm-state are due to processes located at the reaction centre.

The characteristic light-harvesting apparatus of the green photosynthetic bacteria, chlorosomes, contain significant amount ofcarotenoids as well as bacteriochlorophylls(BChl) a and c. Carotenoids found in Chloroflexus aura~tiacus chlorosomes are 13- and y-carotenes, OH-v-carotene, OH-v-carotene glucoside and newly found OH-y-carotene glucoside fatty acid esters (Takaichi et al., Plant Cell Physiol., in press). Upon treatment with 0.08% SDS, the OH"/-carotene derivatives were removed as well as Bchl a, and only 13- and 3'carotenes remained with BChl c The molar ratio of 13- plus y-carotenes to BChl c was about 1/5. The efficiency of energy transfer from these carotenes to BChl c was estimated to be more than 80%. Carotenoid composition and excitation spectra for BChl c fluorescence in the SDS-treated chlorosomes obtained from cells grown under different growth conditions indicated specific interaction of 13- and 3'-carotenes with BChl c.

P-6-019 VARIATIONS OF THE XANTHOPHYLL CYCLE IN TOBACCO PLANTS IN OXIDATIVE STRESS CONDITIONS.

P-6-022 P A R T I A L P U R I F I C A T I O N O F T H E V I O L A X A N T H I N DEEPOXIDASE

S. Pasqualini, P. Batini and M. Antonielh Dept of Vegetal Biology, University of Pemgia, Borgo XX Giugno, 74Perugia-Italy Three specific xanthophylls, violaxanthin, antheraxanthin and zeaxanthin, typically found in the light-harvesting apparatus of higher plants and algae, are interconverted into one another in the chloroplast membrane in a process referred to as the xanthophyll cycle. The exposure of the plants to fight exceeding those that can be used for photosynthesis induces the enzymatic de-epoxidation of violaxanthin to anteraxanthin and zeaxanthin. Given a positive correlation between the xanthophyU cycle and dissipation of this excess light energy, it has been suggested that the de-epoxidation of violaxanthin plays an important role in the photoprotection. In the present work the effects of UV radiation, the ozone and toxic species derivated by O3-decomposition (H202,O~OH) xanthophyll cycle were studied. The experiments were conducted on tobacco plants (Nicotiana tabacum L. cv. Havana 425) by examining the content of three pigments separated in a high performance liquid chromatography system (HPLC). Moreover, seeing that the ascorbate plays a fundamental role in the systems protecting against free radical species in the chloroplasts and it is required also as cofactor for the reaction of de-epoxidation of violaxanthin, the activities of the enzymes of ascorbate-glutathione cycle were investigated.

Hans-Erik ,2~kerlund, P.-O. Arvidsson, C. Bratt & Marie Carlsson Dept o f Plant Biochemistry, Lund University, Box 117, S-221 00 Lund Zeaxanthin, f o r m e d f r o m violaxanthin in the xanthophyll cycle, under high light conditions, has been ascribed a protective function.The e n z y m e violaxanthin de-epoxidase, responsible for zeaxanthin formation has earlier been partly purified. H o w e v e r , this preparation gives several protein bands on S D S - P A G E , dominated by molecular weights o f 33, 37, 54 and 62. In this w o r k w e have further purified the violaxanthin de-epoxidase f r o m spinach thylakoids. By improved gel filtration on a 2 m Sephacryl S-300 c o l u m n and a further purification on a poros ET c o l u m n a total purification of more than 1000 times was obtained. This resulted in an enrichment o f a 40 kDa protein and a strong reduction in the level of the 33, 37, 54 and 62 kDa proteins.

P-6-020 DISTRIBUTION OF XANTOPHYLL CYCLE PIGMENTS IN W/LD TYPE ARABIDOPSlS AND IN A MUTANT BLOCKED IN ZEAXANTHIN EPOXIDATION.

P-6-023 THE EPOXIDATION STATE OF THE VIOLAXANTHIN CYCLE AND THE QUANTUM YIELD OF PHOTOSYSTEM 11 ARE LINEARLY CORRELATED UNDER FIELD CONDITIONS

P. Pesaresi,F. Morales(l)& R. Bassi Univetsit~di Verona- Facolthdi ScienzeMM FF NN - IstitutoPolicattedra. Strada Le Grazie - 37134 Verona- Italy. (1) L.U.R.E. ORSAYCEDEX.

Wolfgang Biluer and Maria Lesch Julius-von Sachs-Institut ftir Biowissenschafien, Mittlerer Dallenbergweg 64, D-97082 Wtirzburg, Germany

Xantophyll-cycle pigments (Violaxanthin, Antheraxanthin and Zeaxanthin) are involvedin the processof Non PhotochemicalQuenchingof chlorophyllfluorescence which is the mostimportantmechanismof photoproteetion.The distributionof these pigments within the chlorophyll proteins of the thylakoid membrane is under discussion and might be influencedby the epoxydation state. In this study we have isolated pigraent-pmtein complexesfrom the w.t. and aha-3 mutant of Arabidopsis which respectively contain violaxanthin and zeaxanthin. The results .~howthat zeaxanthin is mainlylocated in the minorchlorophylla / b proteinsCP 29, CP 26 and CP 24 whileLHCIIcomplexonly containsmmallamountsof this pigment according with previousresultswith maize. The mutantplants showhigh level of NPQ.

The epoxidation state of the violaxanthin cycle (EPS) and the quantum yield of photosystem II (PS II) as assessed from chlorophyll fluorescence (AF/Fm') were compared in leaves of higher plants in the field and during light response curves in the laboratory. A wide variety of plants including fast growing crop plants and trees was used. In all cases a good correlation between EPS and AF/Fm' was observed with significantly different slopes for sun and shade leaves. It is concluded that EPS may serve as an indicator for actual photosynthetic efficiency under the sampling conditions. Assuming that the steady state fluorescence yield stayed constant over the observed range of light intensifies the data indicate also a good correlation of EPS and non-photochemical fluorescence quenching.

87

Poster P-6-027

P-6-024 ROLE

OF

ApH

IN

THE

MECHANISM

OF

ZEAXANTHIN

DEPENDENT AMPLIFICATION OF NPQ

INFLUENCE OF OZONE TREATMENT ON COMPOSITION IN SUNFLOWERPLANTS.

PIGMENT CONTENT

AND

A. Wild, R. Goss, and M. Richter Institute of General Botany, Johannes Gutenberg-University 55099 Mainz, Germany

A. Castagna, A. Ranieri & G.F. Soldatini I s t i t u t o di Chimica Agraria, UniversitA degli studi di Pisa, Via S. Michele degli Scalzi 2, Pisa, 56124, I t a l y

The influence of zeaxanthin on nonphotochemical quenching 0XlPQ) and the pH dependence of the maximum chlorophyll fluorescence yield (Fm) was examined in spinach thylakoids. High contents of zeaxanthin were achieved using different pretreatments. A pronounced, zeaxanthin dependent amplification of NPQ was found in thylakoids containing zeaxanthi~ synthesized in the dark via the buildup of an artificial ApH. These thylakoids also showed a significant quenching of chlorophyll fiuoreseence in the range pH 5.5-6.3. Regarding these two aspects they behaved like thylakoids with high zeaxanthin contents achieved by a natural ApH, i. e. during light pretreatment. On the contrary, thylakoids containing high amounts of zeaxanthin from a dark pretreatment at pH 5.5 (in the absence of ApH) resembled zeaxanthin free thylakoids, that showed no pronounced amplification of NPQ and were insensitive to an incubation at lower pH values. Therefore, we propose that it is the ApH during ongoing zeaxanthin synthesis that activates the mechanism of zeaxanthin dependent amplification of NPQ.

In sunflower plants fumigated in the light with ozone f o r 4 days (150 ppb for 4 hs), the separation of total pigments by HPLC revealed no damage at level of both photosynthetic pigments chlorophyll a and b. On the contrary the oxidative stress, induced by ozone, caused a change in the carotenoid p r o f i l e , resulting in a significant reduction in the content of t o t a l carotene (-50%) and zeaxanthin, component of the violaxanthin cycle (-43%). Regarding to the other components of violaxanthin cycle we observed a slight increase (+15%) in the amount of antheraxanthin in the treated plants in comparison with the untreated ones, but the sum of carotenoids involved in this cycle remained constant in both the plants. The data obtained suggests a preferential action of ozone against carotenoid pigments rather than chlorophylls. Inside the xantophyll cycle this influence is revealed by a d i f f e r e n t d i s t r i b u t i o n of the components in the pool size.

P-6-025

P-6-028

CAROTENOID BIOSYNTHESIS IN GREEN ALGAE PROCEEDS VIA A NOVEL BIOSYNTHETIC PATHWAY

A NOVEL CAROTENOID GLUCOSIDE ESTER IN GREEN FILAMENTOUS BACTERIA

H. K. Lichtenthaler 1, J. Schwender 1, M. Seemarm2, M. Rohmer2 1 Botanical Institute II, University of Karlsruhe, D-76128 Karlsruhe, Germany; 2 Ecole Nationale Suprrieure de Chimie de Mulhouse, 3, rue Alfred Werner, F-68093 Mulhouse Cedex, France;

S. Takaichi, K. Tsuji*, S. Hanada*, K. Matsuura* & K. Shimada* Biological Lab., Nippon Medical School, Kawasaki 211, JAPAN * Dept. Biology, Tokyo Metropolitan Univ., Hachioji 192-03, JAPAN

Carotenoid biosynthesis in green algae could not be inhibited by the specific HMG-CoA-reductase inhibitor mevinolin. This pointed to a pathway for carotenoid biosynthesis which is different from the classical acetatemevalonate pathway. By applying 13C-labelled glucose to Scenedesmus obliquus cultures we evaluated the origin of carbon atoms in isoprene units of B-carotene and lutein. 13C-NMR spectroscopy of the isolated carotenoids revealed that the label of the C-atoms in the two carotenoids did not come from acetate via the acetate-mevalonate pathway. In contrast, a Ca-unit is condensed with a C2-unit to form a Cs-precursor, which undergoes a transposition step leading to the c5-isoprene carbon skeleton. This new pathway is the same as recently found in eubacteria for the formation of the isoprenoid hopanoids (Rohrner et al., 1994, Biochem. J., 295,517-524). This novel carotenoid biosynthesis pathway seems also to occur in higher plants, where mevinolin also cannot block carotenoid formation.

P-6-026 XANTHOPHYLL CYCLE OPERATION AND PHOTOPROTECTION IN BROWN ALGAE- EFFECTS OF HIGH LIGHT AND DESSICATION Y. Lemoine ', M. Harker 3, N-E. Rmikl l, B. Rousseau 2, C. Berkaloff2, J.C. Dural 2, A.J. Young 3 and G. Britton 4. 1- Laboratoire de Cytophysiologie Vdgdtale et Phycologie, U.S.T. Lille FRANCE, 2- Ecole Normale Supdrieure, Paris FRANCE, 3- John Moores University, Liverpool UK, 4- University of Liverpool, Liverpool U K The characteristics of high light response were studied in two brown algae, Pelvetia canaliculata and Laminaria saccharina both for fully hydrated thalli and when thalli were subjected to varying degrees of dessieation in air. All along the high fight treatment in sea water, a good correlation exists, in both species, between increases of zeaxanthin amounts and in the levels of non-photochemical quenching of fluorescence (NPQ). Owing to the higher violaxanthin pool size in Pelvctia ,a better photoprotection can be obtained as expressed by NPQ levels and by times needed for photoinhibition at equal photon flux. When both algae are submitted to progressive dessication, an earlier decrease of rate of the xanthophyll cycle conversions is noticed in Laminaria which quickly becomes poorly efficient in terms of photoprotection.

88

Carotenoids of Chloroflexus aurantiacus have been reported to be /3-carotene, T-carotene, OH-T-carotene, OH-T-carotene glucoside. On reidentification without saponification, we found a large amount of a novel carotenoid glucoside ester, carotenoid B - G - F A ( 1 '- [ ( 6 - ~ a c y I - B -Dglucopyranosyl)oxy]-I ',2'-dihydro-B,$-carotene) with C16:1 and C16:0 dominating. The f a t t y acid composition of the cellular lipids was different. The carotenoid composition in strain J-10-fl cells was 40% carotenoid B G- F A, 3% carotenoid B - G, 4% carotenoid B, 25% ~,-carotene and 28% B-carotene. Another strain, OK-70-fl, had the same composition. A newly found species, Chloroflexus aggregans MD-66, also had the same carotenoids with similar composition. Thus, it is likely that carotenoid glucoside esters are common major carotenoid component in the green filamentous bacteria. Subcenular distdbutions of carotenoids and BChls are also discussed. S. Takaichi et al. (1995) Plant Cell Physiol. Vol. 36, in press.

P-6-029 MEMBRANE POTENTIAL INDUCES A BATHOCHROMIC B A N D S H I F T OF T H E C A R O T E N O I D L I N K E D T O LH I IN RHODOBACTER SPHAEROIDES. C o l e t t e l u n e a s * . Mike Jones**, A n d r 6 Verm6glio* a n d C. Neil Hunter** *CEA-DPVE/LBC- CE Cadarache, 13108 St. Paul fez Durance, France **Krebs Institute and Robert Hill Institute of Photosynthesis, University of Sheffield, Western Bank, Sheffield SI02UH (UK) Light-induced absorbance changes have been analysed in whole cells of a mutant of Rhodobacter sphaeroides deleted in LH II antennae. The kinetics of the photoinduced reactions of the different carriers involved in the cyclic electron transfer are not affected by these deletion. A blue shift of the carotenoid band of the LH I antennae complexes is associated to charge separation across the membrane. A linear relationship is observed between this blue shift and the number of charge separation. This result will be discussed in terms of sign and strength of permanent potential due to fixed charges in the LH I complexes. Moreover, we observed that LH I associated carotenoids are more susceptible to oxidation in air if compare to those of LH If.

Poster P-6-030 ABSENCE OF THE 13, 13-EPOXYCAROTENOIDS IN DEVELOPMENTAL PHENOTYPES OF SCENEDESMUS. WITH OR WITHOUT THE 13, g-CAROTENOIDS, DOES NOT ALTER PHOTOSYNTHETIC CAPACITY Norman I. Bishot~t and Horst Senger 2 1Dept. Botany, Oregon State Univ., Corvallis, OR 97331, USA; 2Fachbereich Biologie/Botanik,Philipps-Univ., D-35032 Marburg, Germany Three n e w carotenoid-deficient strains o f the C - 2 A ' mutant o f Scenedesmus were isolated to study the d e v e l o p m e n t o f photosynthesis. These strains do not form the 13, e-carotenoids, (C-2A'-34), the 13,13, epoxycarotenoids ( C - 2 A ' - 6 7 ) and the combination o f these two strains (C-2A'-67,3). All strains develop photosynthesis w h e n illuminated; h o w e v e r only C - 2 A ' - 67 forms normal levels o f chl _a, chl b and LHC-II. Formation o f LHC-II is restricted in the 13, 5- carotenoid deficient strains. A b s e n c e o f the 13, 13-epoxycarotenoids does not cause any increased photolability in C - 2 A ' - 6 7 or C-2A-67,3; this suggests either that the increased level o f z e a x a n t h i n in these stlains provides adequate protection against photooxidative d a m a g e or that the zeaxanthin cycle is not an essential protective m e c h a n i s m in Scenedesmus.

P-6-031 MATRIX AND TEMPERATURE EFFECTS ON ABSORPTION SPECTRA OF [~CAROTENE AND PHEOPHYTIN a IN SOLUTION AND IN GREEN PLANT PS H I. Ren~e, 1 R. van Grendelle 2 & J. P. Dekker2 llnstitute of Physics, Estonian Academy of Sciences, EE2400 Tartu, Estonia; 2Dept. of Physics and Astronomy, Institute of Molecular Biological Sciences, Vrije Univarsiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands Absorption spectra of I~-carotene(It-Car) and pheophytin (Pbeo) a have been recorded in a number of solvents and polymers and the properties are compared with those observed in the isolated reaction center complex of green plant Photosystem 1I and in its core antenna complex CP47. The peak maxima shift to the red upon increasing the refractive index of the solvent. The temperature dependence was studied in poly(vinyl butyral) and polystyrene matrices between 80 and 295K. The purely thermal shift of the absorption maxima was calculated from the difference between the observed shift and the estimated dispersive shift. The absorption bands of ~Car in CP47 are remarkably narrow, suggesting a non-polar and highly uniform environment. The temperature-induced bandshift is much larger in CP47 than in the polymer matrices, which probably is related to a phase transition of the protein matrix at about 200 K. At room temperature, the effective refractive index of the CP47 host was estimated to be 1.53. The absorption bands of ~Car in the isolated Photosystem H reaction center complex are distorted because of excitonic interactions. The Pheo a Qx absorption band is considerably red-shifted compared to that in solvents and polymers, which is ascribed to specific interactions with the reaction center proteins.

89

Poster I

P-7-003 SELECTIVELY 13C-LABELLED TYROSINE RADICALS STUDIED BY ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY R.J. Hulsebosch1, J.S. van d e n B r i n k 1, S.A.M. Nieuwenhuis2, P. Gast 1, J. Raap2, J. Lugtenburg2 & A.J. Hoff1. ~Deparmaent of Biophysics, P.O. Box 9504, 2300 RA; 21nstitute of Chemistry, P.O. Box 9502, 2300 RA, Leiden University, The Netherlands.

Poster session 7

Oxygen evolution P-7-O01

Tyrosine radicals play an important role as electron carriers in several biological systems. Electron Paramagnetic Resonance (EPR) powder spectra of tyrosine radicals exhibit only partially resolved features. The EPR lineshape mainly depends on the spin density distribution over the phenoxyl ring and geometrical structure of the radical. Using X- (9.2 GHz) and Q-band (34.8 GHz) EPR spectroscopy, we have obtained new insights in the basic structure and major hyperfine interactions of selectively 13C- and 2H-labelled tyrosine radicals in alkaline frozen NaOH solution. Since a 13C-label not only probes local free-electron spin density but also provides information about spin density at "bliod" positions of the aromatic ring, i.e. at molecular positions which do not carry hydrogen atoms, we were able to determine the complete spin density distribution for the in vitro neutral tyrosine radical.

- P-7-063

P-7-001

P-7-004 II

RECONSTITUTION OF THE WATER OXIDIZING COMPLEX IN MANGANESE DEPLETED PHOTOSYSTEM II COMPLEXES BY USING SYNTHETIC BINUCLEAR MANGANESE COMPLEXES

STRUCTURAL ORGANISATION EXTRINSIC POLYPEPTIDES

I 2 2 2 S.I~Allakhverdiev , M.S.Karacan , G.Somer , N.Karacan , E.M.~,S~Y.Rane 3, S.Padhye 3, V.V.Klimov I, G.Renger 4 IInst.of Soil Sci.and Photosynth.RAS, Pushchino, I42292, Russia; 2Dept.of Chemistry, University of Gazi, Turkey; 3Dept. of Chemistry, University of Poona, India; 4Inst. for Biophysical and Physical Chemistry TU, Germany

A. Seidler a n d A.W. Rutherford S6ction de Bio4nerg4tique, D 6 p a r t e m e n t de Biologie Cellulaire et Mol4culaire, CEA Saclay, 91191 Gif-sur-Yvette, France

The efficiency of synthetic binuclear Mn complexes in reconstituting the PS II electron flow and the oxygen evolution capacity was analyzed in PS II enriched preparations deprived of their Mn and of the extrinsic regulatory subunits. It was found: i) as in the case of PS II electron transport, the synthetic binuclear Mn complexes are more efficient than MnCl2; ii) with respect to the Mn concentration the maximum effect is achieved with a u-oxo bridged binuclear Mn (III) complex at concentrations corresponding with four Mn per PS II, and iii) at all concentrations of this binuclear Mn complex a significantly higher restoration of the 02 evolution rate is achieved if the reconstitution assay contains, in addition, the extrinsic regulatory 33 kDa protein.

OF

PHOTOSYSTEM

Three extrinsic polypeptides of P h o t o s y s t e m II (PSII) with apparent molecular m a s s e s of 33, 23 and 16 kd are k n o w n to be involved in the regulation of o x y g e n evolution. Despite intensive biochemical studies, their precise function a n d binding sites are still u n k n o w n . A powerful technique to s t u d y direct protein-protein interaction is the covalent connection u s i n g the zero-length crosslinker EDC. U s i n g this m e t h o d the 33 kd protein has been s h o w n to be b o u n d to the chlorophyll a-binding protein CP47 (Bricker et al., 1988, FEBS-Lett. 231, 111-117). W e used a modified protocol for EDC crosslinking and obtained the following results: 1. the 33 kd protein is b o u n d to CP47 a n d a small intrinsic subunit, 2. the 23 kd protein is b o u n d to cyt. b559 a n d to at least one other intrinsic subunit, 3. the 16 kd protein is b o u n d to at least one intrinsic PSII subunit, 4. no evidence for binding of the extrinsic proteins to each other was found.

P-7-002 ADVANCED EXAFS STUDIES OF THE S1-STATE MANGANESE CLUSTER IN PLANT P H O T O S Y S T E M II

P-7-005 RAPID PROTON TRANSFER UNDER FLASHING LIGHT AT BOTH FUNCTIONAL SIDES OF PHOTOSYSTEM II CORE PARTICLES

M. Kusunoki1, T. Takano 1, T. Ono 2, T. Noguchi2, Y. Yamaguchi3, H. Oyanagi3 & Y. Inoue2: IDep. Phys., Meiji Univ., Kawasaki 214, Japan;

O. Btgershausen and W. Junge Universi~t Osnabnack, Abt. Biophysik, D-49069 Osnabr0ck, Germany

2Solar Energy Research Group, RIKEN, Wako 351-01, Japan; 3Electrotechnical Lab., Tsukuba 305, Japan.

Proton release during the four steps of water oxidation of dark adapted photosystem II core particles was investigated under flashing light. The signalto-noise ratio was improved by averaging under repetitive dark adaptation. The previously observed kinetical damping of pH-transients was prevented by detergent. The complicating superimposition of protolytic events at the donor side (water oxidation) and at the acceptor side (quinone oxido-reduction) was unravelled by characterizing the rate constants of electron and proton transfer at the acceptor side. Contrasting with the pronounced period of four stepping of the oxygen evolving center the extent of proton release was practically constant. The apparent half-rise time of the acidification was shortened upon lowering the pH (250 Its at pH 7.5, 70 ~ts at pH 6.0 and 12 Its at pH 5.2). This kinetic behaviour was independent of the nature and the concentration of the added pH-indicator. We conclude that it reflects the protolysis of several electrostatically interacting acids at the surface of the protein in response to a new positive charge on TyrZ. The extent of deprotonation persisted upon electron transfer from the manganese cluster to Tyrz+.

Manganese K-edge X-ray absorption spectra have been measta'ed for condensed spinach PSII membranes in the S1 state at 30 K at the Tsukuba Photon Factory BL-4C station with use of a single-element Si0Li) solid state detector placed 1.2 cm apart from the sample. A new method o f data analysis of the EXAFS, which can generate a non-wavy smooth background spectrum and a properly normalized EXAFS spectrum via successive least-square proc~nres and can estimate standard deviations of highly correlated EXAFS parameters in terms of a direct fitting of a raw data to EXAFS formula, has been developed to abstract more reliable information on the structure of the Mn cluster than conventional ones. We found that in 67 % probability a Mn cluster must involve 2.7-5.4 Mn-O interactions at 1.81-1.85 ~ (lst shell), 18-36 Mn-N(O) interactions at 2.36-2.41 A (2nd shell), 2.3-5.5 Mn-Mn interactions at 2.73-2.79 A (3rd shell), and at least four kinds o f Mn-Mn or Mn-Ca interactions at 3.38-3.48 A, 3.66-3.85 A, 4.32-4.51 A, and 4.61-4.77 /~ (4-th through 7-th shells). Some possible model Mn clusters, which are required to be consistent with observed EPR signals, will be presented.

90

Poster P-7-006 ADRY AGENT-INDUCED CYCLIC AND NON-CYCLIC ELECTRON TRANSFER AROUND PHOTOSYSTEM II Vitalv D. Samuilov & Eugene L. Barsky Dept. of Cell Physiology and Immunology, School of Biology, Moscow State University, Moscow 119899, Russian Federation Deactivation of the photosynthetic water-oxidizing complex in the S2 and S3 states is accelerated by ADRY agents. The protonophoric uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), pentachlorophenol (PCP) and 4,5,6,7-tetrachloro-2-fluoromethylbenzimidazole (TI'FB) are powerful ADRY agents. Similar to 2-(3chloro-4-fluoromethyl)anilino-3,5-dinitrothiophene (ANT2p), these compounds at low concentrations are catalysts of the cyclic electron transfer around photosystem (PS) II. They are oxidized on the PS II donor side and reduced by the membrane pool of plastoquinone. The tested uncouplers serve as proton donors, and not merely as electron donors on the PS II oxidizing side. At high concentrations (0.05-0.1 mM), CCCP, PCP, TI'FB and ANT2p maintain the PS II-dependent non-cyclic electron transfer to silicomolybdate. Their redox potentials are approx. +1.0 V (Samuilov et al., 1995, FEBS Lett. 357, 55-57).

P-7-007 X-RAY ABSORPTION SPECTROSCOPY

ON THE MANGANESE

C O M P L E X O F O R I E N T E D P H O T O S Y S T E M !I P A R T I C L E S STRUCTURAL CONSEQUENCES OF AMMONIA BINDING

H. Dau 1, J.C. Andrews 2, T.A. Reelers 2, M.J. Latimer 2, W. Liang2, V.K. Yachandra 2, K. Saner2 and NiP. Klein2 1FB Biologie/Botamk, Univ. Marburg, Lahnberge, D-35032 Marburg, Germany; 2Structural Biololgy Division, Lawrence Berkeley Laboratory, UC Berkeley, 1 Cyclotron Rd., CA 94720, U.S.A. After partial orientation of ammonia-modified PS [I membrane particles, electron paramagnctic resonance spectra of the Cytb559 and manganese X-ray absorption spectra were measured for several detection angles. Based on the results, the following aspects are discussed: (1) the orientation procedure (quantification of mosaic spread), (2) the data evaluation approach used for analysis of the Extended X-ray Absorption Fine Structure (EXAFS) spectra, (3) the ligation site ofNH 3 derived Mn ligands, (4) structure and orientation of the PS II manganese complex in its S1 and $2 state.

P-7-008 A T O P O L O G I C A L A N A L Y S I S OF T H E P S B B AND PSB 0 GENE PRODUCTS OF P H O T O S Y S T J ~ II U S I N G E P I T O P E - D I R E C T E D ANTIBODIES K.-D. I r r g a n g M a x - V o l m e r - I n s t i t u t e for B i o p h y s . & Phys. Chem., TU Berlin, 10623 B e r l i n Strasse des 17. Juni 135, F R G

P e p t i d e - d i r e c t e d antibodies have b e e n induced against the psb B gene- (CP47) and psb 0 gene products (extrinsic 33 kDa protein) of spinach P h o t o s y s t e m II (PS II). Purified IgGs were used to s t u d y the t o p o l o g y of these proteins in various PS II complexes in v i t r o and situ. Western b l o t t i n g studies showed that the IgGs d i r e c t e d against ~ sequence d o m a i n n e a r b y Cys 28 of the e x t r i n s i c 33 kDa subunit o n l y recognises its epitope in situ after reducing the intramolecular d i s u l f i d e b r i d g e (Cys28-Cys51) found in the isolated protein. This result d i r e c t l y d e m o n s t r a t e s the presence of the cystine in situ. P e p t i d e - s p e c i f i c IgGs elicited against a peptide fragment of the lumenal loop E of CP 47 revealed the a c c e s s i b i l i t y of this epitope in PS If, T R I S - w a s h e d PS II and O2-evolving PS II core complexes. Measuring the oxygen e v o l u t i o n / f l a s h as a function of the IgG c o n c e n t r a t i o n an activation of the enzyme was found. The results o b t a i n e d are d i s c u s s e d in the light of possible structural and functional roles of these p r o t e i n domains w i t h i n the water oxidase.

P-7-009 DEUTERIUM ISOTOPE EFFECTS ON THE KINETICS OF Yz" REDUCTION IN OXYGEN EVOLVING PHOTOSYSTEM H MEMBRANES.

N. Lvdakis Simantiris 1'2.C. W. Ho~mson2, D. F.Ghanotakis I and G. T. Babcock2 Departments of Clgmistty, tUniversity of Cre~e ~ , and 2Michigan State University, East Lansing, MI 48824, USA. The role of the tyrasine (Yz) at the Dl-161 position on the oxidizing side of Photosystom II (PSII) is being reexamined in the light of new electron magnetic resonance spectroscepie data on its radical form, (Diner, B. et al., in preparation, Tommos, C. et al., in preparation, Warncke, K.et al., in preparation, Babcock, G.T. et al., ESF workshop, Paris, November 1994), pulsed ENDOR data from Ca2+-deplfted PSII membranes, (BritL D. et al., ESF workshop, Paris, November 1994), and proton tranffer data, (Lavergne, J, Jun~, W., Photos. Res. 38, 279-296, 1993). A new model is tested in which the Yz residue acts as a hydrogen atom extraction agent in a (Mn4)/Yz" active site, rather than a simple electron transfer mediator. This model predicts that significant isotope effects will occur on each Sstate transition. Our initial results, from ~ t s in which the decay of Yz" as measured by time-resolved EPR with 35its lime resolution is monitored under steady state conditions in BBY membranes where either 1-120or 2I-I20is used in the buff~ medium, indicate that such isotope effects can be observed. These experiments are now being refined so that the isotope effects on the kinetics of Yz" reduction in dark adapted material can be assessed which will allow as to specify lq4ko ratios for each individual S-state transition.

P-7-010 HETEROGENEITY IN SUBSTRATE WATER BINDING TO PHOTOSYSTEM H

J. Messinger, M. Badger and T. Wydrzynski Research School of Biological Sciences, The Australian National University, Canberra, A C T 0200, AUSTRALIA The following two questions concerning the process of photosynthetic water oxidation in photosystem II (PS II) are addressed in this study by mass spectrometric measurements of 0 2 and rapid H2160/H21sO exchange: a) in which of the five different redox states, S i (i = 0, 1,.., 4), do the two substrate water molecules enter the catalytic site of water oxidation and b) does a partial oxidation of the substrate water, e.g. to a peroxidic intermediate, occur at an intermediate Si state? Due to the improvement of time resolution by a factor of more than 1000 compared to the original experiments of Radmer and Ollinger (1986, FEBS Letter 195, 285 -289) we have observed one slow (tl/2 = 500 ms, 10°C) and one fast (tit2 < 25 ms) exchanging substrate water molecule in the S 3 state of PS II. The slow exchange is coupled with an activation eoergy of about 75 kJ/mol. Measurements of the isotope exchange reactions in the $2 state are in progress. Considering the surprising heterogeneity in the binding of the two substrate water molecules current models for the mechanism of water oxidation are critically discussed.

P-7-011 PLASTOQUINONE REDOX REGULATION AND FEEDBACK ON THE DONOR SIDE OF PHOTOSYSTEM II IN VIVO

0ndi'ej Pr~il 1'2, Zbigniew Kolber 1, Joseph A. Berry3 & Paul G. Falkowski1'* 1Oceanographic and Atmospheric Sciences Division, Depar~ent of Applied Science, Brookhaven National Laboratory, Upton, NY, 11973-5000. Pemmnent address: Institute of Micro~,iology, Academy of Sciences of Czech Republic, Tfebo/i, 379 81, Czech Republic. Carnegie Institution of Washingtun, Department of Plant Biology, Stanford, CA, 94305. Simultaneous measuremmts of the changes in variable fluorescence (Fv) and oxygen flash yields 0 ( 0 2 ) were obtained in intact Chlorella cells on a bare platinum oxygen rate deotrode using a newly developed fast repetition rate (FRR) fluoromoter. The changes in quantum yields of fluorescence and oxygen were measured as a function of background irradiance following a transition from light to darkner,s. Following a light/dark transition the relationship between primary charge separation in PSH and the fraction of PSII reaction centers capable of evolving 0 2 became temporarily uncoupled. Maximum variable fluorescence yields recover to the preilluminafion levels rapidly (<5 s), while the 02 flash yields require up to 60 s to recover under aerobic conditions, and is longer under anaenbic onndifions. The hysteresis between the water oxidation and the reduction on the aceeptor side of PSlI reveals a large potential capacity for denetivetion of S 3 states either by oharg¢ recxxnbinafion from PQH2 or by cyclic eieetron flow around PSII. The results establish that the quantum yield of linear eleotron transport in a fluotuating light environment is not solely determined by the concentration of open PSII reaction centers and their ¢t~ficieneyof excitation energy capture, but also by the redox state of the plastoquinone pool.

91

Poster P-7-012 PHOTOACTIVATION AND PHOTOINHIBITION OF PHOTOSYSTEM II IN R E I N H A R D T I I LACKING THE 23 A MUTANT OF C H L A M Y D O M O N A S kDa EXTRINSIC SUBUNIT.

Mafia Roval*, Per-Olof Fredriksson* and Stenbj~m Styring I. tDept, of Biochemistry, Stockholm University, S-106 91 Stockholm, Sweden. *Dept. of Chemistry, University of Karlstad, S-651 88 Karlstad, Sweden. Photoactivation was performed in vivo at different light intensities in dark grown wild-type and FUD 39 cells ( mutant lacking the 23 kDa protein). In wild-type, photoactivation is very efficient at all light intensities and is completed after a few minutes. In the mutant it takes much longer time to complete. Furthermore, the final level of functional manganese clusters is dependent on the light intensity of the activating light with a maximum at intermediate light intensities. At higher intensities the fraction of centres capable of water splitting decreases although PS II mediated electron transport from the exogenous donor DPC remained constant in all the experiments. We also found that centres photoactivated with high light for short periods of time can be further photoactivated to the maximum level in intermediate light, in contrast, centres illuminated with high light for longer time can not be further photoactivated to a higher level. Our interpretations of these experiments are 1) the photoactivation capability of PS II is an early target for photoinhibition 2) the quantum yield of photoactivation is lower in the mutant compared to wildtype. This possibly reflects slower or inefficient chloride binding to the Mncluster in the absence of the 23 kDa subunit.

P-7-013 THE P R O T O N B I N D I N G FROM SPINACH

PROPERTIES PHOTOSYSTD~ II.

OF T H E

EXTRINSIC

33 K D A

PROTEIN

P-7-015 PROPERTIES OF CI'-DEPLETED PSII STUDIED BY EPR " van Vliet I & A.W. Rutherford BE, DBCM, URA 1290, CEA Saclay, 91191 Gif-sur-Yvette, France. CI- depletion in PSII was done by CI" free washes of PSII membranes followed by treatment at pH=10. The CI" free washes alone, prior to the pH=10 treatment, only slightly affected the oxygen evolving activity of PSII. Nevertheless, the S~ EPR properties of the functional Mn cluster were reversibly altered, exhibiting an enhanced g=4 signal at the expense of the multiline signal. In contrast to CI" free washes, CI" depletion by treatment at pH=10 resulted in inhibition of oxygen evolution and resulted in the ability to generate an EPR signal from S 3. In addition, the S2-state was m~.ified, exhibiting a more dark-stable g=4 signal. Tffe anions SO 4 " and F- added to the already Cl'-depleted PSII after pH=10 treatment had additional effects, resulting in EPR properties of S9 similar to those when these anions were present during CI" depletion of PSII. The distinct CI" depletion effects observed after CI- free washes at pH=6.5 and pH=10 treatment lead us to consider the presence of two Cl'-sites in PSII.

P-7-016 Effects of mild trypsinization on the properties of a Photosystem II core complex

Shutova. T.*, Irrgang, K.-D. °, O p a n a s e n k o V.*, Klimov, V.V.*, Renger, G. ° *Inst.of Soil S c i e n c e & Photosynthesis, Russian A c a d e m y of Science, R u s s i a ; ° M a x - V o l m e r Institut f~r Biophys. & Phys. Chem., TU Berlin, FRG

Elena Kouimtzoglou, Ranjit K. Mishra and Demetrios F. Ghanotakis Department of Chemistry, University of Crete, Iraklion, Crete, GREECE

The 33 kDa p o l y p e p t i d e of PS II is closely a s s o c i a t e d to the m a n g a n e s e cluster of the water oxidase and supposed to act as a r e g u l a t o r y subunit. B u f f e r capacity measurements were u s e d for studying the p r o t o n - a c c e p t o r properties of this protein. The following results were obtained: t i t r a t i o n of the 33 kDa p r o t e i n with acid or base revealed hysteresis curves, which were r e p r o d u c a b l e in repetitive cycles. This p h e n o m e n o n was neither r e l a t e d to kinetic effects nor to d e n a t u r a t i o n of the p o l y p e p t i d e as was shown by measurements of the t r y p t o p h a n e fluorescence. From these data and CD-measurements at various pH it can be c o n c l u d e d that -COO--groups of A s p / G l u are involved in p r o t o n - i n d u c e d c o n f o r m a t i o n a l changes of the 33 kDa protein. Based on these results and a computer-assisted secondary structure analysis a model of H + - i n d u c e d conformational changes and its p o s s i b l e role in water o x i d a t i o n are presented.

Solubilization of Photosystem II (PS II) membranes with the non-ionic detergent 6-O-(N-heptylcarbamoyl)-methyl-ct-Dglucopyranoside (HECAMEG) under various conditions, allowed the isolation of an oxygen evolving PS II complex which retained the 23 and 17 kDa extrinsic polypeptides. Mild trypsinization of the PSII complex resulted in proteolysis of the D1 protein while all three extrinsic proteins (17 23 and 33 kDa) and the manganese complex remained intact. The structural and catalytic properties of the trypsinized complex were examined by a series of biochemical and biophysical techniques.

P-7-014

P-7-017

S T R U C T U R A L S T U D I E S OF T H E O X Y G E N E V O L V I N G C O M P L E X

EVIDENCE OF SINGI,ET OXYGEN EVOLUTION BY WI1OLE LIVING CEI.LS OF CHLAMYDOMONAS REINHARDTII.

Sandra Turconi, Jonathan H.A. Nugent and Michael C.W. Evans Department of Biology, UCL, Gower st., London WC1E 6BT, U.K.

S.N.Roudyk, A. Moxhet, R.F.Matagne*, J.Aghion, Biochimie and

*G~n~tique, D~partement de Botanique (B22), Universit~ de Liege, B-4000 Liege, Belgium The structure and mechanism of function of the oxygen evolving complex of Photosystem II (PS II) is not known to the present. Its catalytic site is thought to consists of four Mn atoms. One of the most promising techniques for elucidating the structure of this Mn-cluster is X-ray absorbtion, which provides information on the oxidation state (from the position of the rising absorption edge) and on the immediate surrounding of the Mn (from EXAFS, Extended X-ray Absorption Fine Structure). Apart from the investigating directly the Mn-cluster, valuable information can be obtained by comparison with suitable model-compounds. We have performed X-ray absorption measurements on PS II Mn-cluster in different conformations and oxidation states. In addition, a series of model compounds of identical coordination with different metal ions included in a crown-ether moiety was studied and the results compared to our existing data on PS II and discussed in terms of the role of cofactors such as Ca2+ in the regulation of structure and function of the Mn-cluster in PS II.

92

The oxygen evolved by Chlamydomonas reinhardtii in the light is measured simultaneously with a Clark electrode and with the nitrosodimethylaniline-imidazole method, specific of singlet oxygen. Experiments with Fud7 mutant cells (unable to synthesize the D1 protein of photosystem IlL with dichorophenyldilnethylurea (which blocks electron and proton transfer between the 2 photosystems), with dibromothymoquinone (artificial electron acceptor between the photosystems) or with hydroxylamine (electron and proton donor to photosystem II but it does not yield oxygen) all point to a link between the singlet oxygen detected out of the cells and photolysis of water by their photosystem 11.

Poster

P-7-021

P-7-018 SIMULATION OF THE $2 STATE MULTILINE EPR SIGNAL OF PS-H & R.J. Pace, Department of Chemistry, Australian National University; CRC for Plants Science, Canberra, Australia Simulations have been performed at Q-band, X-band and S-band frequencies, based on a model assuming that the signal arises from a magnetically isolated MnIII-MnIV dimer, with a ground state electronic spin S= 1/2. The spectra are generated from exact numerical solution of a general spin Hamiltonian with anisotropic hypeffine and quadrupolar interactions at both Mn nuclei. The features that distinguish the muifiline from spectra of model Mn dimer complexes can be explained assuming an unusual ligand geometry, giving rise to normally forbidden transitions from quadrupole interactions. The fitted parameters indicate that hyperfine and quadrupole interactions arise from Mn ions in low symmetry environments. For a quadrupole interaction of the magnitude indicated here, the Mn ions must be 5 coordinate or have a 6th, weakly bound ligand. The hyperfme parameters indicate quasi-axial anisotropy at Mn~I and corresponds to the unpaired spin being in the ligand deficient, z-direction of the molecular reference axis. The parameters for MnlV show a high degree of anisotropy. This anisotropy can be qualitatively accounted for by a histidine ligand providing n backbonding into the metal dxy orbital, together with a weakly bound or absent ligand in the x-direction. Experiments using one-dimensionally ordered PSI] samples are currently underway. The experimental data will be used for simulations, using the current minimised parameter set, to obtain an orientation of the Mn-Mn vector in the membrane plane.

Ma2+ and dipheayi carbaalde 0DPC) electron donors to photosystem I1 studied in Chtamydomonas rdnhavdtti mutants.

~tlLd~, R.T. SayerI and S. Styrtng*. Dellagflrw,nt of Natural Science, Uraversity o f ~ Sweden. I D e p ~ of Plant Biology and Biochemistry, Ohio State University, ColumbuLUSA. *Deparlmentof Biocaemistry, Arrhe~us Laboratories, Stoek~,Im University. Phot3systemII can pbetcoxidize the exogeno~3eleetren donon nmaSanese (Mnt~) :anddiphenyl carbazide (DPC). Kinetic analysis reveal that both M_nz+ and DPC domtt~ele~ons ia a bifimotionalmanner in wildty~ Chlamydc, mo~ms r¢tnhardtfi. For both lvln~*arid DPC there exists one high atYmJty-and one low affinity-site, respectivelyTwo mutants in C. relnh~rdtTt have. been studied with the same methods. Mutant FuD 39, lacking the 23 kDa extrinsic subunit in the oxygeniccomplex, showedaltered donation kinetic;. For d¢¢tron donation from Mnz" onl5, one site was fore,d, while DPC sail had txvosites. The other mutant is a sito-dirccted mutation, where ~stidinz 190 on the D 1 protein has been changed into ~ , In this mutant the kisses fiom Mn:" and DPC wexealso altered. Again we found two affiuit3-sites fer DPC while we only found on+:affinity-sitefor/Vln2+. DI-His190 is ~oug~t to be situated Lathe ,Acinityof Yyrz. It has been shown to be involved in eleetren transfer from Tyrz to P680. Our slradiesindicate that this residue: migl~ also be involved in the intnraetionbetweenPSII and Mm~÷,

P-7-022

P-7-019 SYNTHESIS OF STEREO-SPECIFICALLY I~-METHYLENEDEUTERATED L-TYROSINES.

Binding of the 33kDa Extrinsic Protein to Manganese-Sufficient and Manganese-Depletod Photosystem lI Preparations

S.A.M. Nieuwenhuis,C. Mul, J. Raap, J. Lugtenburg. Leiden Institute of Chemistry, PO. Box 9502, 2300 RA Leiden, The Netherlands.

Carola Leuselmer and Terry M. Bricker Department of Plant Biology, Louisiana State University Baton Rouge LA 70803

L-Tyrosines, stereospecificallydeutemted at the 13-methylenepositions((2S,3S)[2,3,3',5'-D4]-tyrnslae 1 and (2S,3R)-[3,Y,5'-D3]-tyrosine2) were synthesized. Chiral reduction of (4-methoxy)-tx-benzamido-cianamicacid with D2, and of (4-methoxy)-ctbenzamido-13-deutero-cinnamicacid with H2, affordedthe protected labelled tyrosines. Depmtection by DBr led to deuteration of the 3',5' positionsof the aromatic ring yielding tyrosines 1 and 2. Incorporationof these two tyrosinesin photosystemII and EPR-measurememtsof the thus obtained isotopicaliy c n n ~ reaction centers will lead to the unambiguousdeterminationof the orientationof the aromatic ring of Y~'. D D OH 1

D

OH 2

1: R=CD0qH2)CO2H 2: R=CHfNH2)CO2H

D

The 33 kDa extrinsic protein acts to enhance oxygen evolution within PS II and stabilizes the manganese cluster at low chloride concentrations. Due to controversies concerning the stoichiometry of this protein (Miyao M. and Murata N (1989), Biochim. Biophys Acta, 977, 315 - 321 versus Xu Q.and Bricker T.M. (1992) J Biol Chem 267, 25816 - 25821) we have analyzed the rebinding of the 33 kDa extrinsic protein to PSII membrane preparations which contain 0, 2 or 4 manganese per reaction center. Immunoquantification of the 33 kDa component demonstrated that each of these preparations binds two moles of the 33 kDa protein per mole of reaction center. This binding appeared to be cooperative. Additionally, our results indicated that the binding of the 33 kDa protein to manganese-depleted membranes was weaker than the binding observed for 4 manganese-containing preparations.

P-7-023

P-7-020 A T W O D I M E R M O D E L O F M n ORGANISATION IN PS-H

P.J. Smith* & R.J. Pace. Department of Chemistry, Australian National University; * CRC for Plant Science, Canberra, Australia The $2 state of Photosystem II (PS-II) exhibits two EPR signals arising from the Mn of the oxygen evolving complex (OEC). These are a hyperfine structured signal at g=2, (muitiline) and a broad feature around g=4 (4.1 signal). The temperature drpendenco of the muitiline signal conf'LrmSit to be a spin 1/2 ground state. There are at least two conflicting interpretations of the 4.1 signal however; a) that it arises from the first excited (spin 3/2) state of the system with multiline ground state or b) that it is a trapped intermediate electron transfer or structural state (spin 3/2 or 5•2) between the EPR silent S1 and multiline $2 states of the OEC. We have now established that two different froms of the 4.1 signal may be generated. One exhibits excited state behaviour, is co-generated with a multline signal and is quantitatively consistent with a) above. The other exhibits ground state behaviour and is generated alone by 130K illumination of PS-II in ethylene glycol cryoproteeted material. Although similar at X band, these signals are distinguishable at Q band, with a resolvable splitting of the g_l_ components. Both are most likely near axial spin 3/2 states. Our data suggest that these signals arise from two separate, exchange isolated, bridged Mn pairs. One functions as a) above, the other as b). The latter involves a three spin system (spin 3/2 ground state) comprising two Mn in equal oxidation states (III or IV) with a bridging oxidised (spin 1/2) ligand. We have observed an excited state spin I/2 signal from this system with both CW and spin echo EPR.

HIGH AFFINITY BINDING OF THE Ca 2+ ESSENTIAL FOR 0 2E V O L U T I O N IS DEPENDENT ON THE EXISTENCE OF THE MNCLUSTER AND INDEPENDENT OF THE EXTRINSIC PSII PROTEINS. Changguo Chen and G. Cheniae U. Kentucky, Lexington, KY Analyses of the Ca2+ abundance of PSlI membranes [Vo2 >__800 0 2 • (Chl • h)"1 and 2 Ca, 4Mn/RC] showed that VO2 is linearly correlated with bound Ca2+ between the limits of 2 to 1 Ca/RC O2-evolving Core Complex particles lack the 1 Ca/RC not required for VO2, are partially depleted of the 1 Ca/RC required for Void, and require Ca2+ reconstitution for maximum VO2 [ _> 1400 02 • (Chl • h)" ]. NI-I2OR Tris treatments which dissociate the Mn-cluster also dissociate the essential 1 Ca/KC Photoactivation of NH2OH-PSII results in parallel assembly of the Mn-chister and the essential 1 Ca/RC. Over a wide range of conditions, the ratio of Mn assembled per Ca2+ bound was 4, thus indicating that the binding site for the essential Ca2+ is "created" during photoassembly of the Mn-cluster. Analyses of the Ca2+ concentration dependence for VO2 by Ca2+ depleted PSII membranes containing all or none oftbe PSII extrinsic proteins gave Ca2+ Km values (pH 7.2) of< 131xM in the absence of Ca2+ antagonistic cations. (Supported by DOE)

93

Poster ii iii

i

P-7-027

P-7-024 CHLORIDE DEPLETION AND AMINO ACID COFACTORS OF PHOTOSYNTHETIC WATER OXIDATION M. Haumann,W. Drevenstedt& W. Junge Universit~itOsnabrtick, Binphysik, Barbarastr, 11, 49076 Osnabriick, Germany The catalytic center of PSII accumulates four oxidizing equivalents in the water oxidation cycle. Whetherthese charges are all stored on manganeseor in part on amino acids is under debate. The oxidation of histidine has been proposedto occur in chloride and calcium depleted material and in controls. Without chloride the sequence of redox steps is unclear (S1Yz=--:*S2*Yz=:,S2*Yz+ or S1YzHis~S1YzHis+:-~,S2*YzHis+). We compared electrou transfer and proton release in chloride depleted and control 0 PSII core particles in the first two transitions. P680+ reduction was fast, 35 (15) and 100 (40/300) us, indicating that the electron was provided by YZ' YZ+ was reduced with half-rise times of 300 tts (60 Its) and about 300 ms (300 its). 1 proton was released on the I st transition under both conditions. This transition produced a UV difference spectrum (-CI) similar to the one of $2~S 3 in controls. We conclude: In chloride depleted material YZ+ oxidizes (in 300 its) the same componenton the first

MODULATION BY THE S-STATES OF THE EQUILIBRIUM C O N S T A N T B E T W E E N THE P R I M A R Y D O N O R S OF P H O T O S Y S T E M II, Fabrice Rappaport and Jertme Lavergne. lnstitut de Biologie Physico-

Chimique. 13 rue Pierre et Marie Curie 75005 Paris, France. The absorption changes caused by a series of single turnover flashes given to dark-adapted algae were measured in the blue region of the spectrum. A component that oscillates with the number of flashes was detected in the 10 ms range. Its spectrum appears consistent with a shift of the equilibrium constant [Yz+P-680]/[YzP-680+] towards a lower value (< 6) in the presence of states $2 and $3 compared with So and $1. This is interpreted as due to the electrostatic potential gradient between Yz and Pss0 caused by the net positive charge residing on the catalytic center in states $2 and $3. The mechanism and rate of decay of the additional P-680 + fraction, initially. present in states S2Yz+ and S3Yz+, are discussed.

transition as in normal S3 formation.Ist difference spectrumresemblesthe one of His+ in vitro. YZ+ stays oxidized on the second transition. The redox sequence: S 1Yz(His)~ S 1YZ(His+)~S l Yz+(His+)•

P-7-025 EVIDENCE ON T H E S P E C I F I C I N T E R A C T I O N B E T W E E N MANGANESE AND CYT B-S$9 ON T H E PS II O X I D I Z I N G SIDE. Noriaki Tamura t, [kuko lwasaki 2, Sayo Shibano t, Izumi Oka t and Shigeki Okayama 2. Dept. Environ. Sci., Fukuoka women's Univ., Fukuoka 8131 and Dept. Biol., Kyushu Univ., Fukuoka 810"-, Japan. Cyt b-559 is an essential component for structural integrity of PS II in oxygenic photosynthesis, and exhibits several types of redox forms such as high potential-(HP-), intermediate potential- (IP-) and low potential(LP-) forms. However, functional role of the forms of Cyt b-559 in PS I1 remains to be solved. Depletion of functional Mn from the PS I1 membranes by hydroxylamine treatment induced little decrease in the HP content. Further treatment of EDTA caused approx. 40% decrease in the HP content, resulting in a concomitant increase in the ]P content. The IP form was partially converted to the HP by dark-incubation of Mn 2+ with EDTA-treated membranes. Other divalent cations (Mg 2+, Ca 2+ and Zn 2÷ ) did not effect such conversion. Furthermore, we observed a drastic increase in the HP content after photoactivation of the apo-water-oxidizing complex. It is discussed whether the binding/ ligation of Mn 2+ at the Mn binding sites is correlated with the transition of redox forms of Cyt b-559.

P-7-026 STRUCTURAL STUDIES OF THE S2-STATE MANGANESE CLUSTER IN PLANT PHOTOSYSTEM II. THEORETICAL ANALYSIS OF "MULTILINE" EPR SIGNALS. Koji Hasegawa and Masami Kusunoki: Dep. of Phys., School of Sci. &

Techng., Meiji Univ., 1-1-1 Higashimita, Tama-ku, Kawasaki 214, Japan The so-calledmultilineE P R signalof the $2 statewas quantummechanically interpretedas arisingfrom tetrarnericM n spins exchangecoupled to each other (Kusunoki, 1992). What values of six supcrcxchange interactionsbetween M n spins can give riseto an S2-stateMn-teramer being in the S=I/2 ground statewith such four cffccive hyperfme coupling constants as determined by specwal simulation and having the firstexcited stateof 30-40 cm -l above it? W e have examined ca. 700 million sets of six exchange interactionsand found thatthese features mentioned above can be equally reproduced by a number of either Mn(3,4,3,3) or Mn(3,4,4,4) tetramers excluding a "dimer-of-dimcrs" type of complexes with respect to strong antiferromagnetic couplings involved, but notably including a "Uimer-plus-monomer" type in addition to a "dimcr-plus-two monomers" type proposed before. Next, an anisotropic g-tensor and anisotropic hyperfmc coupling tcnsors have been dctermincd by a least-square spectral simulation of orientation-dependent signals,suggesting that the oxidation stateof the M n ten'amer in $2 would be more likelyMn(3,4,4,4).

94

P-7-028 The characterization o f chloride-depleted Photosystem H by UV-VIS spectree~py. H. Wincenciusz. I-I.J. van Gorkom Dept.of Biophysics, State University of Leiden, P.O.Box 9504, 2300 RA Leiden, The Netherlands Chloride is an obligatory cofactor for water oxida~on by Phomsystem I I i n plants. There is some controversy whether inhibition of the Oxygen Evolving Complex in the absence of chloride occurs after one or two electron transfer events. The character of the oxidized species is also uncertain: is it manganese or an amino acid residue? The UV-VIS absorption difference spectra obtained for cldofide-depleted PSI] prompt us to suggest manganese oxidation upon first flash. However in the region of 300-350nm th~ change is followed by a small absorbents decrease in a few miliseconds. We propose that this absorbance decrease reflects a structural change or reduction of the S2 state by another donor. On the second and subsequent flashes the absorbance changes appeared to be due to Qa" T y r m i ~ + formation and recombination.

P-7-029 AMINO ACID RESIDUESOFTHE EXTRINSIC33 kOa PROTEIN INVOLVED IN ELECTROSTATIC INTERACTION WITH INTRINSIC PROTEINS OF PS II T. Miura 1,2, S. Takahashi1' M. Kamo1, E. Nakamura1, H. Ohta1, Y. Incus2 & I. Enaml1 1Dept of Biol., Fac of Sci., Science University of Tokyo, Tokyo 162, Japan; 2Dupt of Appl.Biol.Sci., Fac of ScLTesh., Science Univemity of Tokyo, Node 278, Japan Amino acid residues of the 33 kDa protein (Mn-stabitizlng protein} involved in electrostatic interaction were examined by chemical moditioatlonof the protein. Rebindingof the protein to PSII core complex and reactivationof oxygen evolution by reconctifution with the protein were inhibited by chemical modification of amino groups of the 33 kDa protein with N-succinlrnidy[ propionete, but not by chemical moditlcation of carboxyl groupswith glycine methyl ester in the presence of EDC. This indicates that positive charges but not negativecharges of the 33 kD= protein are essential for binding of the 33 kDa protein to intrinsic proteins of PS tl. In order to determine lyslne residues of the 33 kDa protein involved in electrostaticinteraction, the Isolated33 kDa protein and the bound 33 kDa protein which attaches to intrinsic proteins of PS II were treated with tdnitrobenzene-sulfonic acid (TNBS)which modifies amino groups. Mess-spectroscopic analysis and amino acid sequence of modified peptides from both prolstns after dtgestionwith V-S protesse revealed that no I/sine residues were moditlad with TNBS in the bound protein, but a number of b/sine residues, at least Lye-4, -101, -105, -233 and -236, were modified in the lactated protein, indicating that these b/sine residues contact with intrinsic proteins of PS II.

Poster i P-7-030 ISOLATION AND CHARACTERIZATION OF A HIGHLY PURIFIED OXYGEN-EVOLVING PHOTOSYSTEM II CORE COMPLEX FROM AN ACIDOPHILIC AND THERMOPHILIC RED ALGA, Cyanldlum cnldarlum I. Enaml. H. Murnyama, M. Kamo, H. Ohta & K. Nakazato Dept of Biol., Fac of Sci., Science Univ. of Tokyo, Tokyo 162, Japan A highly purified and active photosystem II (PS II) core complex was isolated from thylakoid membranes of an acidophillo and thermophilic red alga, Cyanidium caldarlum, by nhaptyithioglucoaidn and dodacyl-15-D-maltosldo solubilizatlon followed by single-step anion exchange chromatography. This PS II core complex consists of the D1 and D2 reaction center proteins, the 47- and 43-kDa chlorophyll-carrying proteins and the three extdnslo proteins of 33 kDa, cyt c550 and 12 kDa which were found in PS II core complex from a thermophilic cyanobactedum, Synochococcus vulcanus. The core complex was completely free from phycobillproteins and linker polypeptidss, and did not contain the extdnslo 23 kDa and 17 kDa proteins involved in O2-nvoiving PS II complex from higher plants. The preparation showed a high activity'at a rate of 2,000-2,500 ttmol OJmg chYh even in the absence of CaCI2. The oxygen-evolving activity was considerably inhibited with EDTA and the lost activity recovered by addition of not only Ca but also Mg. These results suggest that oxygen-evoiving system of rod algae is similar to that of cyanobacteria but not of higher plants.

P-7-031 EVIDENCE FOR ELECTROSTATIC INTERACTION OF THE LOOP A ON CP 47 WITH THE EXTRINSIC 33 kDa PROTEIN H. O h t a 1, N. Y o s h i d s 1, M. S a n e 1, M. Hirano 2, K. Nakazato I and I. Enami 1

1Dpt of Biol., Fac of Science, Science University of Tokyo, Kagurazaka, Shinjuku-ku, Tokyo 162, Japan and 2Toray Reg. Center, Kamakura 248, Japan The extrinsic 33 kDa protein (Mn-stabflizing protein) and the cl-dorophyll-binding 47 kDa protein (CP 47) were covalently linked by using a water-soluble carbodiimide to yield an adduct of the two proteins without any significant effects on the oxygen-evolving activity [Bocltim. BiOl~ys. Acts 1060 (1991) 224-232]. Paplide mapping and sequencing studies revealed two domains of linkage between the two proteins. One domain of linkage is deduced to be formed between carboxyl group(s) of Asp-87 and/or Gin-94 in the extrinsic loop A on CP 47 and e-amino group(s) of Lys(s) on the extrinsic 33 kDa protein. The other domain involved the extrinsic loop E on CP 47 and the 33 kDa protein. Thus, the 33 kDa protein was found to electrostaticaily interacts with the extrinsic loop domain A (Pro49-Gly95) of CP 47, in addition to the large extrinsic loop domain E (Tyr258-Try450) of CP 47.

P-7-032

P-7-033 E P R / E N D O R STUDIES OF T H E W A T E R OXIDIZING C O M P L E X IN P H O T O S Y S T E M II R. Fiege ~, W.Zweygart1, K.-D. Irrgang 1, J. Messinger 1, N. Adir 2, G. Renger l and W. Lubitz 1 l Max-Volmer-lnstitut fi~" Biophysikalischeund Physikalisd~eChemie, TechnischeUniversit~t Berlin, Str. d. 17. Juni 135, D-10623Berlin, Germany, 2 Department of Physics, 0319, University of California, San Diego, La Jolla, CA. 92093-0319,USA The catalytic site of the water oxidizing complex (WOC) of Photosystem lI (PSII) contains a bridged tetranuelear manganese cluster ligated to subunits of PSI1. The sequence of univalent oxidation steps within the WOC is energetically driven by electron donation to P+s0 that is formed by the light induced primary charge seperation. Thereby the WOC cycles through 5 different redox states (So to $4). We have studied the S2-state in three different typesof PSII preparations: (i) membrane fragments with all extrinsic proteins present, (it) core particles containing only the 33kDa protein and (iii) membrane fragments without any extrinsic proteins. The multiline signal (MLS) can be detected in preparation (i) to (iii). In addition we observed the unresolved g=4.I signal of the $2 state in (i) and (it). The MLS's of (i) and (it) are almost identical but some changes are discernable in (iii). The EPR spectra of the So state and of the super-reduced states S-1 and S-2 indicate light induced changes correlated with redox transitions. Proton ENDOR shows several hyperfine couplings upon generation of the $2 state disappear almost completely upon D20 exchange. The detected couplings are most likely associated with exchangeable protons in the ligand sphere of the WOC or to closeby water molecules.

P-7-034 X-RAY ABSORPTION SPECTRA OF HYDROXYLAMINE I N D U C E D S. l AND So S T A T E S O F T H E O X Y G E N E V O L V I N G MANGANESE COMPLEX P. DeMarois t, P. Riggs-Gelaseo2, J. Penner-Hahn2, C. Yocum 2,3 1Biophysins Research Division, 2Department of Chemistry, 3Department of Biology, University of Michigan, Ann Arbor, MI 48109, U S A Changes in Mn oxidation states in PS II can be monitored by x-ray absorption spectroscopy. Previous workers have reported advances in xray absorption near-edge spectra (XANES) edge energies for the St--~S 2 and $ 2 ~ S 3 transitions and a shift to lower energy has been observed for S3--~$4--,S 0 transition. We have examined the XANES edges in dark samples exposed to NH2OH; low concentrations of the reductant produce a two electron reduction of the M n complex, which would correspond to an S. t state. Continuous illumination at 0 ° C returns the edge energy to the control, S 1 value. However, continuous illumination at 200 K, which limits P S I / t o a single turnover, advances the XANES edge to an energy between the S. t value and the control. This intermediate edge energy value indicates a one electron oxidation, which would correspond to an S 0like state.

P-7-035

PROPERTIES OF TIlE Mn-CLUSTER O F P H O T O S Y N T H E T I C OXYGEN EVOLVING CENTER IN CI" AND/OR Ca2+-DEPLETED PHOTOSYSTEM H

SYNTHETIC MODELS FOR THE REACTIVITY OF MANGANESE IN P H O T O S Y S T E M II

T. One I , T. Noguchi 1, M. Kusunoki2, Y. Inoue 1, H. Yamaguchi3 & H Oyanagi3 So]'~'~arEnergy Research Group, The Institute of Physical and Chemical Research (RIKEN), Wake, Saitama 351-01, Japan; 2School of Science and Technology, Meiji University, Kawasaki, Kanagawa 214, Japan; 3Electromechanical Laboratory, Tsukuba, Ibaraki 305, Japan.

Vincent L. Pecoraro, Michael Baldwin, Mathew T. Candle, Andrew Gelasco and Nell Law, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055

Changes in the properties of the Mn-cluster of the photosynthetic oxygen evolving center (OEC) induced by depletion of Cl- and/or Ca 2+ were studied by thermoluminescence (TL) and X-ray absorption spectroscopy. Upon a single flash illumination in the presence of DCMU, the control photosystem(PS) II membranes showed a TL band peaking at around 15 °C arising from S 2 and QA-. In C1-depleted PSI1, the peak temperature markedly upshifted to around 42 ° C indicating the formation of the modified S2-state when Ca2+ was retained in OEC. The peak temperature, however, downshifted to around 32 °C when Ca 2+ was removed. In the presence of CI-, the presence of Ca2+ did not affect the TL peak temperature. These may suggest that Ca2+ and C1- were mutually effects the properties of the M-n-cluster. The structure and/or electronic states of the Mncluster detected by X-ray absorption near edge structure (XANES) in the Ca 2+depleted OEC were, however, substantially different from those in the O--depleted OFA2.

Manganese is an essential participant in the enzyme responsible for oxygen evolution in photosynthesis. W e have been investigating the structure and reactivity o f Mn(IV)202 aggregates in order to gain greater insight into biological water oxidation chemistry. Specific structural and reactivity models for this enzymatic system will be presented. Particular emphasis will be placed on strucutral and magnetic changes associated with the protonation and methylation of Mn(IV)202 cores. We observe that [Mn(IV)(bis-1,3(salicylldeneamino)propane)(tl20)]2, [Mn(IV)(Salpn)(P.2-O)] 2, can be converted to Mn(llI)(Salpn)(I-I20)2 by adding protons. Additionally, the reaction of that [Mn(III)Cois-l,3-(salicylideneamino)(2-hydroxypropane)]2, [Mn(m)(2-OH-Salpn)] 2, with alkyl peroxides yields singlet oxygen. The relationship of this chemistry to the possible mechanism of the oxygen evolving complex will be presented.

95

Poster

P-7-036

P-7-039

CHARACTERIZATION OF K COLD-SENSITIVE ASSEMBLY M U T A N T O F MRIqGJdCESE-STABILIZIMG P R O ~ M

A C C E L E R A T E D P H O T O I N H I B I T O N O F P H O T O S Y S T E M II BY T H E AZIDE RADICAL PHOTOPRODUCED FROM AZIDE ANION

Scott D. B e ~ s , Jeannine R. Ross, Eran Pichersky, and Charles F. Yocum, Departments of Biology and Chemistry, The U n i v e r s i t y of Michigan, Ann Arbor, MI 48109 U S A

Kunio Ka/~ a m o t o , Junichi Mano and Kozi Asada The Research Institute for Food Science, K y o t o University, Uji, K y o t o 611, JAPAN

The O 2 - e v o l v i n g complex (OEC) of PSII includes three extrinsic polypeptidea that regulate 02 evolution through direct or allosteric interactions with inorganic cofactoru. The largest and most tightly bound extrinsic eubunit in the OEC is the 33-kDa Manganese-stabilizing Protein (MSP}. R e c o n e t i t u t i o n studies show that wild-type MSP assembles into PSII with equal efficiency on ice as at room temperature. The m u t a t i o n V235A inhibits functional assembly of MSP into PSII only on ice; however, the mutant protein assembles efficiently at room temperature. Differences in the electrophoretic mobility of wild-type and V235A-MSP make it possible to perform competition experiments using mixtures of the two proteins. Samples of reconstituted PSII can also be probed for unoccupied MSP binding sites by using either the mutant or wild-type protein in consecutive reconstitutions. Results from these studies are consistent with a stoichiometry of two MSP per PSII and suggest a model in which the two copies serve non-overlapping functions.

Azide ion inhibited 02 evolution in PS II membranes from spinach in a timeprogressive manner under light to null activity. The illumination in the presence of azidc (azide-phototreatment) irreversibly inhibited (1) both the w a t e r oxidation and the electron transport between the redox-active tyrosine 161 in DI protein (Yz) and the secondary quinone electron acceptor (Q~) site to the same extent, (2) the donation of electrons to the primary quinone electron acceptor (Q.d as measured by maximum variable fluorescence of Chl, and (3) the photoproduction of the Yz radical (Y z'). The Y z ~ QB activity in Tris-treated PS I1 m c m b r a n e s was inhibited by the azide-phototreatment more rapidly than that in the oxygenic PS II membranes was. Thus the primary inhibition site lay between Yz and QA. Azidyl radical was photogenerated as detected by spin-~apping ESR in Tris-treated PS I1 membranes with azide. The spin-trapping reagents D M P O and PBN suppressed the inhibition c a u s e d by the azidephototreatment. Therefore azidyl radical was produced via a univalent oxidation of azide by Y z" and irreversibly inhibited the electron transport from Y z to

P-7-037 SPINKCH~J~IDARABXDOPSIS 3 3 - K I ) A pRO'JL~IN8 I N SPINACH P S I Z FUNCTIONAL

IDEMTITY

P-7-040 R E G U L A T I O N O F T H E O 2 - E V O L V I N G M E C H A N I S M D U R I N G N2FIXATION IN THE DIAZOTROPHIC CYANOBACTERIUM CYANOTHECE SP. A T C C 51142

SlrJL~4EEN

Scott n. Betts, Jeannine R. ROSS, Eran Pichersky, and Charles F. Yocum, Departments of Biology and Chemistry, The U n i v e r s i t y of Michigan, Ann Arbor, MI 48209 U S A The 33-kDa M n - u t a b i l l z i n g Protein (MSP) of PSII stabilizes the O 2 - e v o l v i n g M n cluster and increases its catalytic rate. We previously reported a method for recovering MSP from bacterial inclusion bodles (8ette at al., Plant Mol Biol 26 (1994) 117-130). This method yields a mixture of assemblycompetent and aesea~01y-inoompetent MSP, including dimers and oligomers cross-linked through incorrect intermoleculnr disulfide bonds. A modification to this method yields pure monomeric protein. ~Lrabidopaia MSP and spinach MSP refolded and purified using the improved m e t h o d are functionally indistinguishable from each other and from the native protein. The two homologs, which differ at 43 of their 247 amino acid residues, assemble stoichiometrically into MSPdepleted spinach PSII /n vitro and restore 02 evolution activity to the same level.

P-7-038 ABERRANT ELECTRON TRANSFER IN D1-Y161 F MUTANT FROM CHLAMYDOMONAS REINHARDTII.

Jun MinagawaI , David M. Kramer2, Atsuko Kanazawa] and Antony R. Crofts ~ 1Program in Biophysics, 2Dept. of Plant Biology, and 3Dept. of Microbiol University of IUinois, 607 S, Mathews Ave., Urbana, IL 61801, USA The electron donor to oxidized P680, tyrosine Z (Y 161) in the D1 polypeptide of photosystem II in Chlamydomonas reinhardtii, was replaced by phenylalanine via site-directed mutagenesis, to produce a pho- strain. We have examined the functional consequences by measuring flash-induced changes in fluorescence yield (~f) and absorbance. In the presence of DCMU, #f was low after a single flash, indicating that no rapid donor was present; a rise in #f on later flashes showed a slow electron donor to accumulated P680 ÷. To identify this donor species, we looked at optical absorbance changes on moderate light illumination. Both chlorophyll(s) and carotanoid(s) were bleached at a rate which depended strongly on experimental conditions. In the absence of O2, or upon addition of DCMU or hydroquinone, carotenoid bleaching was inhibited. This suggests that mutation of the donor Y 16~ allowed photoaccumulation of oxidized P680, which could be rereduced by exogenous electron donors, such as hydroquinone, or endogenous pigments as alternative donors. We are now studying the mechanism of oxygen dependency of carotenoid bleaching. Additional DI mutants affecting donor side reactions are NI91 to L, D or A, and E189 to L, D, or Q. We have characterized the equilibria on the donor-side in these mutants using studies of the back-reaction, and thermoluminescence.

96

Q.v

Pascal C. Meunier, Milagros S. C o l o n - L o p e z a n d L o u i s A. Sherman, Department of Biological Sciences, Purdue University, Lilly Hall, West Lafayette, In 47907 USA W h e n the unicellular, d i a z o t r o p h i c c y a n o b a c t e r i u m , Cyanothece sp. A T C C 5 1 1 4 2 is g r o w n under N2-fixing conditions, the culture fixes N2 or evolves 0 2 with peaks every 24 h and the two processes are 12 h out of phase. During these cycles, the redox state of the plastoquinone (PQ) pool varies depending on respiration and the balance between PSII and PSI electron transport activity• The reduction of the PQ pool results in high probabilities of b a c k w a r d transitions in the S-state mechanism, super-reduced S-states (S-I and S-2) and up to a 90% loss of active M n complexes• The loss is especially rapid when light-grown ceils are incubated in the dark. However, the M n centers can then be reactivated readily by three min of 10 Hz xenon flashes• The fast deactivation and reactivation of M n are similar to what we have observed for the psbO deletion mutant of Synechocystis sp. PCC6803. B y contrast, the photoactivation treatment is less effective for cells grown in the dark. W e suggest that the regulation of PSII involves a feedback mechanism by which the redox state of the PQ pool exerts an influence on the Mn center; the magnitude of this influence depends on the state of the extrinsic lumenal proteins such as the Mn-stabilizing protein, MSP.

P-7-041 PAlaTIAL CARACTERIZATION OF T W O DIFFERENT MANGANESE BINDING SITES IN PSII: BINDING Alyt'IIqI'I'I'I'ESAND pH DEPENDENCIES ~ l l L M a g a u ~ and Lars-Erik Andreassen Department of Biocbemistry and Biophysics, GOteborg University and Chalmers University of Technology, Medieinaregatan 9C, S-413 90 Gsteborg, Sweden . . • Extraneous Mn 2 + acts as electron donor to PSII part,ties depleted of extnnstc . . . . . proteins and native manganese. Dissociation constants for Mn 2 * have earher been reported to range from less than 1 p.M to about 200 p.M. Sensitivity towards chemical modification by l-ethyl-3-((3-dimethylamino)propyl)-carbodiimide(EDC) and diethylpyroearbonate (DEPC) at these binding sites has also been reported. In . . . . the present study, the bmdmg afflntuy of Mn 2 + has been momtored as a function of pH, by following the steady-state reduction of 2,6-dichlorophanolindophenolby PSII under continuous illumination. The KMvalue for Mn2÷is shown to be pH dependent, with a pK value of about 6.5. DEPC and EDC modification changed this pH dependence, indicating the involvment of histidyl and carbxylic acid residues. • . . . . . The blndmg of Mn 2 + to a second, low-aff'mlty site, earher found by observmg the • + . electrostutie effect of added manganese on the reductmn of P680 after a seturatmg flash, was diminished after modification by DEPC. This indicates that a histidine elose to TyrZ acts as a ligand for Mn 2+.

Poster

P-7-045

P-7-042 I S O L A T I O N A N D C H R R A C T E R I Z A T I O N O F P H O T O S Y S T E M IX COMPLEXES WHICH HAVE INTACT OB SITES BUT HAVE NO QB PLASTOQUINONE H. E g a s h i r e , Y. K a s h i n o , H. K o i k e & K. S a t o h D e p t . o f L i f e S c i . , Fac. S c i . , H i m e j i I n s t i t u t e of Technology, Harima Sci. Garden City, Hyogo 678-12, Japan Oxygen-evolving photosystem (PS) II c o m p l e x e s with the intact QB site but without the QB plastoquinone molecule were isolated from a thermophilic Synechococcus vulcanus. The thylecyanobacterium, keid membranes were treated with heptyl-thioglucoside in the presence of an inhibitor of the QB site, 3-(3,4-dichorophenyl)-l,l-dimethylurea, and the solubilized PS II p a r t i c l e s were collected by centrifugation. T h e P S II p a r t i c l e s evolved oxygen when 2,5dichlorobenzoquinone was used as an electron accepter, b u t s h o w e d n o o x y g e n e v o l u t i o n in the presence of tetramethylbenzoquinone (duroqulnone).

P-7-043 THE ROLE OF CHLORIDE IN PHOTOSYSTEM II K. Lindberg & L.-E. Andr6asson Dept. of Biochemistry and Biophysics, G6teborg University and Chalmers University of Technology, Medicinaregatan 9C, S-413 90 G6teborg, Sweden We have previously shown that there is a site for C1- in PS II in slow exchange with CI" in the medium (t½=l h, Kd=20 ~M). Complete removal of all CI-, including the slowly exchanging CI-, by dialysis against Cl'-free medium (< 1 pM CI') for 24 h decreases the oxygen-evolving activity to 40% of the control, suggesting that CI" is not an absolute requirement for oxygen evolution but might be necessary for full activity. The O2-evolution activity lost by incubation in a Cl--free medium could be rapidly (in 15 s) recovered by addition of fairly high concentrations of CI- (Kap p = 0 •5 raM) . Complete removal of C1- led to a slow, gradual increase of the g---4 EPR signal at the expense oftbe multiline signal. The original relation between the signal amplitudes was promptly restored alter addition of CI-. This indicates the presence of a Cl--binding site with properties (affinity, kinetics) different from those of the high-affinity, slowly exchanging site. The significance of these observations in relation to possible models for the binding and function of C1- will be discussed.

P-7-044

SIG,E C T I V X E F I ~ C 2

OP ~ 1 " ~ [ ,

AND H Z S T I D I N X

N O D Z P I m U i ON BINDING

AND SINGZZ Tt/RNOVI~t PHOTOOZXD~TION OF Mea* DT p H O ' ~ N ~ J T ~

IX

M. L. Ghirardi. Thomas W. Lutton, & Michael Selbert National Renewable Energy Laboratory, Golden, CO 80401, USA Diphenylcarbazide (DPC} n n d M ~ 2÷, in the absence of the other, are donors to Yz+ in Mn-depleted PSII membranes, as monitored by the photoreduction of DCIP, and by the inhibition of flash-lnduced fluorescence decay in DCMU-traated samples, Addition of 10 gM MnCl 2 to a reaction mixture containing 200 gM DPC results in due to noninhibition of steady-state yz+ photoreduction, competitive interaction between the two donors. However, under the same experimental conditions, we still observe a slnole of Yz, due to photoreduction of the llght-ganerated-Yz ÷ but not by DPC. The inhibition of steady-state DPC photoox£dation by MnCl 2 can he deconvoluted into four coc~ponents, each associated with different amino acid residues (two carboxyls and two histidines) and each binding • Mn 2+ with high affinity. Chemical modification of carboxyl residues in P$II eliminates only two of the manganese binding c ~ p o n e n t s but cofapletely inhibits the single photooxidation of high affinity Mn 2+ both in the presence an6 absence of DPC. Modification of histidlnes eliminates the other two Components, but does not slgnificantly alter the single photooxidation of Mn 2+, again either in the presence or absence of DPC. Our results suggest that only one (or two) of the high affinity components bind a photooxldizable 2÷ Mn , and that the residue(s} must be a cnrboxyl.

~

P-7-046 S3-STATE OF THE O2-EVOLVING COMPLEX: STRUCTURE OF THE MN C O M P L E X OF PS II D E T E R M I N E D BY X-RAY ABSORPTION SPECTROSCOPY. Wenchuan Liang, Thee Rcelofs, Matthew J. Latimer, Holger Dau 1, RceM Cinco, Annette Rompel, Joy Andrews, Gary Olsen, 3fiI~LK,...,Y.~f~o~t, Kenneth Saner & Melvin P. Klein. Structural Biology Division, and Dept. of Chemistry, University of California, Berkeley, CA 94720, USA, and IFB Biologie/Botanik, Phillips-Universi~t Marburg, D-35032, Germany. We have prepared $3 state samples generated under physiological conditions by flash illumination. The S-state composition was determined by following the period four oscillation of the amplitude of the multiline EPR signal which is characteristic of the $2 state. We have obtained the first EXAFS (extended X-ray absorption fine, structore) spectra from such dilute samples that can be optically saturated using flash illumination. The Fourier transform of the EXAFS spectra show that there is a significant perturbation in the structure of the Mn complex in the $3 state, compared to that in the S1 or .$2 states. The Fourier peaks corresponding to Mn-O/N interactions at 1.8-2.1 A and the MnMn interaction at -2.7 A are considerably reduced in amplitude compared to those in the S1 or $2 states. The structural changes in the Mn complex that could be responsible for such changes in the X-ray absorption spectnnn will be discussed.

V I B R A T I O N A L STUDIES OF P H O T O S Y S T E M II AND RELATED M A N G A N E S E M O D E L COMPLEXES

P-7-047 A STRUCTURAL MODEL FOR THE PHOTOSYNTHETIC OXYGENEVOLVING MANGANESE CLUSTER.

Matthew T. Gardner t. Ganrt Deinum 1, Michael J. Baldwin2, Vincent L. Pecoraro 2, & Gerald T. Babcock I, tDepartment of Chemist~ and the LASER Laboratory, Michigan State University, East Lansing, MI, 48824, USA; 2 Willard H. Dew Laboratories, Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109-1055, USA

J. C. Andrews, H. Dau 1, M. J. Latimer, W. Liang, T. A. Rcelofs, A. Rompel, R. Cinco, IL Saner, V. K. Yachandra, & ~ Structural Biology Division, and Dept. of Chemistry, University of California, Berkeley, CA 94720, USA, and 1FB Biologie/Botanik, Phillips-Universit~L Marburg, D35032 Germany.

FTIR, light-induced FTIR difference spectroscopy, and resonance Raman specUnscopy, have been used to study oxygan-evniving preparations of Photosystem If, and related manganese containing model complexes. The model complexes, primarily [~(IM)(SAUPN)(~2-O)] 2 and derivatives that have one or both of the bridging oxo groups protonated, show significant shifts in the vibrational frequencies of the Mn202 core upon isotopic labeling with deuterium and/or oxygen-18. These w~orations, which have been satisfactorily assigned through the use of normal coordinate analysis, arc being used to assist in identifying low frequency vibrations exhibited in light-induced ZI'IR difference spectroscopy of thin films of Photosystem II reaction center particles. These vibrations may be indicative of intermediates in the oxygen evolving process, or may arise from fundamental stmcnires of the OEC such as di-~-oxo bridged diraers of manganese.

The photosynthetic 02 evolving complex of higher plants and cyanobacteria contains a cluster of four Mn atoms that act as the locus of charge accumulation, driven by the energy of four successive photons through a cycle of five so-called S-states. Ca 2÷ and C1- are essential cofactors that can be replaced by Sr2+ and Br-, respectively. XANES has been used to determine the oxidation states of Mn in each of the flash advanced S-states. From EXAFS a structural model has been derived. It suggests two distinct binuclear di-B-oxo bridged units with Mn-Mn separation of -2.7 A that are in turn bridged by a single B-oxo linkage with Mn-Mn separation of 3.3 A. ~r substituted samples provide evidence for one Mn-Sr(Ca) interaction at -3.5 A. Evidence for halide ligation to Mn is moot. EXAFS from oriented membranes of native and NH3 treated samples has permitted a determination of the relative orientation of these several vectors with respect to the membrane normal. Structural differences are observed between control samples and those treated by F-, or NH3 as well as those in the higher S-states.

97

Poster

P-7-048 ASPECTS OF THE COFACTOR FUNCTIONS OF Cab AND c r IN TIIE WATER OXIDIZING COMPLEX

P,H. Homann and J. Wang Dept. of Biol. Science, Horida State University,Tallahassee,FL 32306 USA

P-7-051 SPECTROSCOPICAL CHARACTERISATIONS AND 02 MEASUREMENTS OF WHOLE CELLS FROM WlLDTYPE AND MUTANTS OF SYNECHOCYSTIS sp. PCC 6803 H.Kretschmann

In chloroplasts of green plants, the interaction of the water oxidizing complex of PSI1 with its cofaetor CI" is modulated by the extrinsic OEE2&3 polypeptides. Removal of CI" typically results in a greater stability of oxidants due to a lowered redox potential. This effect becomes more pronounced at pH > 7. The resulting upshift of the temperature of thermolumineseence emission from the recombination of the oxidant S2(D+?)/Q^"charge pair is diminished when the OEE2 polypeptide is bound. Since a similar shift is caused by lowering the pH, the polypeptlde action may reflect an increasedprotonatiou of critical sites which, in turn, may be related to the OEE2-ieduced 10-foldincrease of the apparent binding constant for CI'. The constitutive absence of OEE2&3 from prokaryotie PSII apparently delegates control of CI" binding to the extrinsic OBEI polypeptide which often is more hydrophobie than the green plant counterpart (e.g. Mor et aL, 1993, BBA 1141,206). The existence of structural variants of OEEI even among higher plants may he responsible for the fact that CI" deficiency with an only marginal loss of functional Ca2+is more readily achieved with OEE2&3 depleted pSI] membranesfrom Phytolaccathanfrom Spinacia. All these polypeptide linked aspects of the cofactor relations possibly can provide information of biochemical and physiological significance. They certainly must be taken into account when comparing data collected after different CI" depleting procedures because they yield preparations differing with respect to their actual or potential interactions with OEE2&3.

P-7-049

Max-Volmer-lnstitut for Biophysikalische und Physikalische Chemie, Technische Univet~it~t Berlin, Stral3e des 17. Juni 135, 10623 Berlin, Germany. In order to characterize the different S-states in the oxygen-evolution complex different spectroscopical methods were applied on whole cells of Synechocystis 6803 wildtype and mutants. The methods include: UV and electrochremic absorption changes, fluorescence oscillation and 02evolution. For wildtype we found that whole cells are mainly in the So state after 20min dark adaption, Interestingly, in one mutant of Synechocystis 6803 (revertant of a CP43 deletion mutant; see abstract: Kuhn at al) whole cells were mainly in the $1 state after 20min dark adaption. Cells which are in the So state must be pretriggered by light after 24h dark adaption before the normal Oz-evolution pattern can be seen.For the mutant which is in the Sl-state no preincubation with light is necessary to see the normal 02evolution pattern.The data were discussed with regard to evolutionary aspects of the oxygen-evolution complex.

P-7-052

TERBIUM SUBSTIUTION OF THE CALCIUM-BINDING SITES IN THE OXYGEN EVOLVING COMPLEX OF PHOTOSYSTEM II.

A M I N O ACID RESIDUES THAT INFLUENCE THE BINDING OF M A N G A N E S E O R C A L C I U M T O P H O T O S Y S T E M I1

Christine E. Hatch, Russell LoBrutto, Ryan Bradley and Wayne D. Frasch Center for the Study of Early Events in Photosynthesis, Department of Botany, Arizona State University, Tempe, AZ 85281-1601.

Hsiu-An Chu, Anh P. Nguyen, and Richard J. Debus, Department of Biochemistry, University of California, Riverside CA, 92521-0129, U.S.A.

The calcium binding sites of the oxygen evolving complex were investigated by substituting calcium with terbium. Terbium inhibited water oxidation competitively versus calcium in spinach PS II preparations. Terbium substituted PS II preparations did not exhibit multiline or g= 4,1 signals after continuous illumination at 200K, but did produce the Fe-Q^- EPR signal. Subsequent illumination at 230K did not induce formation of the 150 G width EPR signal attributed to a hisidine radical. However, the change in the power saturation of YD radical upon illumination at 200 K indicates that this illumination did induce the SI to $2 transition. The results suggest that a Ca2+binding site is in close proximity to the Mn cluster of the OEC.

P-7-050

Site-directed mutations were constructed at all conserved carboxylate, histidine, anti tyrosine residues in the lumenal domains of the D1 polypeptide of the cyanobacterium Synechocystis sp. PCC 6803. Mutants were characterized in vivo by measuring changes in the yield of variable chlorophyll a fluorescence after a saturating flash or brief illumination given in the presence of DCMU. Mutants were also characterized after propagation in media having other cations substituted for Ca2+. We conclude that Asp-59 and Asp-61 may ligate Ca 2÷, that Asp-170 probably ligates Mn, that His-332, Glu-333, and His+337 :may ligate Mn, and that Asp-342 may ligate Mn, Ca2÷, or both. Several His332, Glu-333, His-337, and Asp-342 mutants are very light sensitive, possibly because toxic activated oxygen species are released from altered or partly assembled manganese clusters. We also conclude that that Asp-59, Asp-61, Glu-65, and His-92 influence the properties of the Mn cluster, that Glu-189 plays an important structural role in maintaining the catalytic efficiency of the Mn cluster, and that His-190 strongly influences the redox properties of Yz and either ligates Mn or serves as a crucial base or hydrogen bond donor.

P-7-053

PHOTOASSEMBLY OF THE TETRA-Mn/Ca CLUSTER OF PSII: EPR REVEALS LIGATION OF A Mn DIMER INTERMEDIATE

P H O T O A C T I V A T I O N AND S-STATE C Y C L I N G K I N E T I C S O F H20-OXIDATION M U T A N T S IN S Y N E C H O C Y S T I S 6803.

G. Ananvev. E. Bruntrager & G. C. Dismukes

Princeto-n University, Dept of Chemistry, Hoyt Lab. Princeton, NJ 08544, USA

Robert L. Burnap. Ming Qian, Sufian AI-Khladi and Coy Pierce, Dept. of Microbiology and Molecular Genetics, Oklahoma State University, StiUwater, OK 74078 USA

The apo-PSII protein complex, depleted of inorganic cofactors is capable of functional reassembly in the presence Mn 2+, Ca 2+, CI-, light and electron acceptors, a process called photoactivation. We have introduced three improvements to determine the yield and kinetics of recovery of oxygen evolution: 1) an ultrasensitive dioxygen cell (105 improved sensitivity); 2) integrated cell/LED illuminator for controlling the intensity, duration and dark interval of flashes; and 3) new chemical chelators for extraction of Mn, Ca and water-soluble extrinsic proteins. Kinetic studies reveal: 1) both an optimum flash duration 20-40 ms and dark interval 2-3 s; 2) kinetic intermediates have been trapped and charamerired; 3) each photoactivated center binds 4.0 Mn/P680 by a cooperative process; 4) the rate of photoactivation is slowed by increasing concentrations of Ca 2+, Mg 2+, Na +, H + and sucrose at non-specific sites; 5) complete inhibition of photoactivation occurs at _specific sites for the electron donors DPC and H 2 0 2 , Zn2+(20 I.tM), and La 3+ or Tb 3+ (4 IxM); 6) EPR spectra reveal that apo-PSII forms a Mn II cluster in the dark, possibly dimeric, evidenced by extensive Mn hyperfine lines with mean coupling 45 G. Formation of this signal is inhibited by Ca 2+, Mg 2+, Zn 2+ and by PSII turnover by illumination at ~ 200K. Support NIH-GM39932.

The kinetics of photoactivation and S-state cycling were examined in a variety of Synechocystis 6803 strains having defined mutations affecting the H20oxidation complex. Photoactivation of hydroxylamine-treated cells lacking the manganese-stabilizing protein (MSP), under flash or cont. illumination occurs at an intrinsically faster rate (approx. 5x) than the wild-type, although the process of photoactivation in both cell-types saturated at similar cont. light intensities. Experiments measuring the flash frequency dependence of photoactivation indicate a multiquantum process in the mutants resembling the wild-type, but with alterations causing an overall increase in yields at all flash frequencies examined. Results indicate the intrinsically faster rate of reactivation observed MSP-CeUs may be due to an increase in the yield of an intermediate prior to the rate-limiting dark rearrangement producing the first stable intermediate and point to a greater accessibility of Mn +2 to the active site in absence of MSP. Kinetics of S-state cycling examined using double flash techniques confirm a retardation of the $3-S0 transition to be on the order of 13-16 ms in the absence of MSP. Corresponding analyses of sitedirected and randomly produced PSH mutations will be presented.

98

Poster P-7-054 AN FTIR STUDY OF CALCIUMINTERACTIONSIN PHOTOSYSTEMII H. Zhang, G. Fischer and T. Wvdrzvnsld Research School of Biological Sciences, The Australian National University, Canberra, ACT 0200, AUSTRALIA The specific role whichcalcium playsin oxygenevolutionin PS II of higher #ants is still unknown even though calcium is generally considered to be a cofactor. In tiffs study, FTLR techniques that are highly sea~sitiveto changes in protein conformation are used to investigate the structural role of calcium. For samples that have been treated with 2M NaCI]lmM EGTA plus 20gM ionophore A23187 to create a calcium demand, PS II undergoes a conformational change with a loss of or-helical structure. Upon incubation of these samples with various amounts of CaC12 (2050raM), not only is part of the oxygen-evolvingactivity restored but also the cthelical structure, indicating calcium ~ ~ PS II. Results on the correlation between the calcium content of PS II and the extent of the FTIR-detectable conformationalchange will be reported.

P-7-055 FORMATE EFFECTS ON THE DONOR SIDE OF PHOTOSYSTEM II A. Stemler 1,2& J~r6me Lavergne I lInstitut de Biologie, Physico-Chimique, Rue Pierre et Marie Curie, 75005 Paris, France. 2Permanent address: Section of Plant Biology, University of California, Davis, CA, 95616, USA We observed flash-induced absorbance changes at 295nm in photosystem IIenriched membranes (BBY), as modulated by formate. These changes reflect primarily the S-state transitions in the oxygen-evolving mechanism. In the presence of formate, the period-of-four pattern is delayed one flash. That is, the change corresponding to oxygen evolution is maximal on the fourth rather than the third flash. This shift also occurs in spinach thylakoids, hut to a lesser extent. Apparently, formate causes an endogenous one-electron donor to reduce the S t state, forming a large (-80 percent) population in the SOstate. Alternatively, the donor could act after the flash train begins, reducing one higher S-state, or preventing its formation. These results provide additional evidence for direct effects of formate on the electron donor side of photosystem II.

P-7-056 NEW MAGNETICRESONANCESIGNALS FROMPHOTOSYSTEMI I

Jonathan NuQent, Sandra Turconi. Stephen Rigby & Amanda Cheater. Dept. of Biology, University College London, Gower St, London WCIE 6BT, U.K. We will report on EPR and ENDORexperiments on the oxygen evolving complex (OEC). P680+ and Qa- of photosystem II (PSII) This includes two new EPR signals which we have found in native PSII membranes. The f i r s t is an asymmetric 17mTsignal near g=2 which is reversibly formed on illumination at cryogenic temperatures. The signal is similar to. but distinct from, that formed by illumination of samples depleted of their Cab or CIcofactors at >250K. The origin of this new signal is probably an interaction between an S=I/2 Mn cluster and a S=I/2 organic radical (Mnm-MnrV--x+) in the OEC. The second signal, 25mTwide at g=5, is found in the dark in samples which can form the g=2 signal. Someof the g=5 signal is lost as the g=2 signal is formed. At present we assign the g=5 signal to a new trapped form of a S=>I/2 Mn state in the OEC of native PSII.

P-7-057 OXIDATION STATE CHANGES OF THE MANGANESE CLUSTER IN THE FLASH-INDUCED S-STATES OF PHOTOSYSTEM II: DETERMINATION BY X-RAY ABSORPTION SPECTROSCOPY T.A. Roelofs*, W. Liang, M.J. Latimer, J.C. Andrews, R. Cinco, A. Rompel, K. Sauer, V.K. Yaehandm and M.P. Klein - Structural Biology Div. of Lawrence Berkeley Laboratory and Dpt. of Chemistry, University of California at Berkeley, U.S.A. *New address: Dpt. of Physics, Free Univ. Berlin, Germany. We performed X-ray absorption edge spectroscopy on the flash-induced Sstates of the oxygen-evolving complex of Photosystem II. The S-state population distributions in samples given 0 through 5 single-turnover flashes was calculated by modeling the EPR multiline-signal amplitude oscillations. From the Mn K-edge spectra of the flash-treated samples, we calculated the pure edge spectra for the Mn-cluster in the different S-states. These spectra indicate that direct Mn oxidation occurs during the So --~ S r and the S l ~ Sgtransitions. However, going from $2 to S3 both the edge position and edge structure remain relatively unchanged, suggesting the absence of a direct Mn oxidation. Data on M_n-model compounds are presented to support this interpretation of both edge-position -structure in terms of oxidation state changes.

P-7-058 C O M P A R I S O N OF STRUCTURE AND F U N C T I O N OF T H E P H O T O S Y N T H E T I C O X Y G E N - E V O L V I N G C O M P L E X OF SCENEDESMUS SPINACH AND T H E GREEN ALGA

OBLIQUUS H. Schiller. S. Klingelhdfer, W. Ddrner, H. Senger and H. Dau 1713Biologie/Botanik, Philipps-Universit~it, 35032 Marburg, Germany Axe structure and function of the PS II oxygen evolving complex fully conserved in evolutionary diverse organisms? PS II-containing membrane particles were prepared fxom the higher plant spinach and the unicellular green alga Scenedesmus obliquus. A comparison of the enzymatic activity of PS II particles of both species demonstrates a comparable binding affinity for Hill acceptors and the same pH-optima for oxygen evolution. The analysis of ammonia inhibition indicates similar anunonia binding affinities in both organisms. X-band EPRspectra of these particles were collected with high spectral resolution.The presence of a g---4-signal in spectra of the green alga is discussed. The g=2 multifine-signals of both organisms are similiar, but exhibit clear difference in the fine structure of individual lines. We propose that these differences are due to differences in the protein environment of the PS l] manganese cluster.

P-7-059 IS PHOTOSYSTEM II KINETICALLY AND ENERGETICALLY HETEROGENEOUS IN SYNECHOCYSTIS 6803 ? Zs. Defik, L. Sass & I. Vass Institute of Plant Biology, Biological Research Center, P.O. Box 521, Temesvfiri krt. 62, H-6701, Szeged, Hungary A useful method to study the function of Photosystem II (PSII) in the naturally transformable cyanobacterium, Synechocystis 6803 is thermoluminescence (TL). Single-flash excitation of dark adapted Synechocystis 6803 cells results in three TL components with peak temperatures at 2025, 35-40 and 55-60 °C. This is in contrast to PSII containing preparations from higher plants, where only a single TL component is observed after one flash from the S2QB- recombination at 20-25 °C. Flash-induced TL oscillation in Synechocystis indicates that both of the 20-25 and the 35-40 °C components arises from the S2QB" recombination after the first flash, and from the $2(3)QB- after two or more flashes. The properties of the 55-60 °C component are very similar to those of the C band in higher plants, which is assigned to the Tyr-D + QA" recombination. Our results indicate that in Synechocystis 6803 cells the QA to QB electron transport is blocked in part of the PSII centers, which also lack functional S2 and S3 states. In addition, the PSII centers which have active electron transport appear to be heterogeneous in respect to the energetic stability of the $2(3)QB" charge stabilization state.

99

Poster

P-7-060

P-7-063

G-values as a probe of the local protein environment: High-ileld EPR of the tyrosine-D radical in Photosystem H

A DIFFERENCE INFRARED STUDY OF HYDROGEN B O N D I N G TO THE PSII Z TYROSYL RADICAL

Sun Un and A. William Rutherford Section de Bio6nerg6tique, URA 1290 CNRS, Dept. Biologic, Cellulaire et Moleculaire, CEA Saclay, F-91191 Gif-sur- Yvette, France.

M. Bernard 1, G. MacDonald l, A. Nguyen 2, R. Debns2 & B. B a i ~ 1 lDepartrnent of Biochemistry, University of Minnesota, St. Paul, Minnesota 55108; 2Department of Biochemistry, University of California, Riverside, California 92521

The stable tyrosyl radical Photosystem II (PSII) was studied by high-field 245 GHz 8.7T EPR. The g-values of several tyrosyl radicals were calculated using the unrestricted Hartree-Fock MNDO molecular orbital method with PM3 parameterization and the theory of g-values of radicals developed by Stone. The local environment of the radicals were incorporated into these calculations by inclusion of point charges and hydrogen bonds. The method describe here is independent of adjustable parameters except those inherent to the PM3-MNDO method. The calculated g-values were in good agreement with those measured experimentally. The differences in g-tensors of the tyrosyl radical in RNR, PSII and the model system are explained in term of stabilization of the non-bonding orbitals of the tyrosyl oxygen by electrostatic and hydrogen-bonding interactions. The g-value calculations also indicate that Tyr-D ° is less strongly hydrogen-bonded than in the model system. We have been able to localize the hydrogen-bond donor to Tyr-D ° relative the phenyl-ring: a hydrogen bond distance of ~l.7A, bond angle of -140 ° and dihedral of ~35 °. This approach which combines high-field EPR measurements of g-values with molecular orbital calculations is generally applicable and has been extended to quinones.

P-7-061 PRESENCE OF A MANGANESE REQUIRING L-ARGININE METABOLIZING ENZYME IN PHOTOSYSTEM II MEMBRANES OF SPINACH: ISOLATION AND PARTIAL CHARACTERIZATION OF THE PROTEIN Achim E. Gau and Elfi'iede K. Pistorius Universitat Bielefeld, Biologie VIII: Zellphysiologie, 33501 Bielefeld, FRG. In CaCI2 washed photusystem 11 (PSIl) membranes of spinach a Mn requiring Larginine metabolizing enzyme (L-AME) activity leading to omithine, urea, and minor additional products, could be detected. This activity was also present in PSII core and reaction center complexes - suggesting that the L-AME is tightly associated with the DI/D2 heterodimer. It was possible to isolate the detergent solubilized protein from spinach PSII membranes, Based on SDS PAGE the protein was homogenous and had an apparent molar mass of 7 kD. Sequence data indicate that it is a nuclear encoded and so far not identified PSII peptide. Based on a redox cycling assay there are indications that the protein contains a redox active group - most likely an amino acid derived quinonoid. The L-A/VIE required for activity besides Mn, addition of c r (anions, such as Bf or SO4~, can substitute), and a second cation, such as Ca2+, is also necessary for activity. Moreover, we could show that the protein has a low Mn dependent eatalase activity. On the basis of these and additional results the possibility of a functional role of this protein in photosynthetic water oxitdation will be discussed.

P-7-062 DIFFERENCE FTIR STUDY OF THE PHOTOSYNTHETIC WATER OXIDIZING COMPLEX J. Steenhuis & B. Barry Department of Biochemistry, University of Minnesota, St. Paul, MN 55108 Water oxidation occurs at the manganese cluster of Photosystem II. Illumination of the water oxidizing complex causes it to cycle through five oxidation states Sn, where n refers to the number of oxidizing equivalents stored. The St state is the dark stable state. The $4 state is unstable and converts rapidly to SO with release of oxygen. When illuminated at 200 K, the $2 state exhibits a multiline EPR signal centered around g=2 {Dismukes, G.C., & Siderer, Y. (1981)}. Illumination at 130 K generates a derivative shaped EPR signal at g=4.1 from the $2 state {dePaula, J.C., et al. (1985)}. We have obtained the difference FTIR spectra associated with the $1 to $2 transition in spinach photosystem II at 130 and 200 K. In the spectral region from 1900 - 1200 cm "1 differences in these data can he observed. These spectral differences could he due to structural changes in the protein environment of the manganese cluster. These data have been compared to the spectrum associated with chlorophyll oxidation and quinone reduction at 80 K. We have also obtained difference F'FIR spectra of the S1 to $2 transition in cyanobactedal photosystem II at 200 K. These spectra are similar to those obtained on spinach photosystem II at the same temperature. We are now able to use isotopic labeling and site directed mutants to identify the structural changes associated with the St to $2 transition.

100

Photosystem !1, the photosynthetic water oxidizing complex, contains two well characterized redox active tyrosines, D and Z. The vibrational difference spectra associated with the oxidation of tyrosines Z and D have been obtained through the use of infrared spectroscopy {MacDonald et al. (1993)}. Here, we examine the effect of deuterium exchange on these vibrational difference spectra. While the putative C-O vibration of stable tyrosine radical D downshifts in 2H20, the putative C-O vibration of tyrosine radical Z does not. This result is consistent with the existence of a hydrogen bond to the phenol oxygen of the D radical; we conclude that a hydrogen bond is not formed to the Z radical. In an effort to identify the amino acid residue that is the proton acceptor for Z, we have performed global 15N labeling. While significant ISN shifts are observed in the vibrational difference spectrum, substitution of a ghitamine for a histidine that is predicted to lie in the environment of tyrosine Z has little or no effect on the difference infrared spectrum or the EPR signal. Our results are inconsistent with the possibility that this residue, histidine 190 of the D1 polypeptide, acts as the sole proton acceptor for tyrosine Z.

Poster P-8-003 STABILIZATION OF THE HIGH POTENTIAL FORM OF CYTOCHROME b-559 BY CALCIUM OR LACK OF OXYGEN ~ 1 , K. Gounaris 2 and J. Whitmarsh1,3 IDept. of Plant Biology and 3Photosyntbesis Research Unit, USDA/ARS, University of Illinois, Urbana, IL 61801, USA; 2Dept. of Biochemistry, Imperial College, London, SW7 2AY, UK.

Poster session 8

Cytochrome

complexes

Cytochrome b-559 is an essential component of the reaction center of Photosystem H (PS II) although its role remains unre,~Ived. In the dark, the predominant high redox potential form (cyt b-559HP) is associawd exclusively with PSH complexes in granal domains of the thylakoid membrane whilst a low potential form (cyt b-559LP) is found only in stromal lamellae. Reversible interconversion between these two s m ~ s has been speculated to occur as part of a mechanism by which cyt b-559LP protects PS H against photodamage. Cyt b-559HP is frequently found to correlate with the ability of PS H to evolve oxygen although a causal relationship is considered unlikely since extrinsicpolypeptide-depleted PS H can evolve oxygen if re-constituted with calcium while cyt b-559 remains in its low potential form. Using PS H-enriched membranes containing almost exclusively cyt b-559HP, it is shown that the binding of calcium is necessary but not sufficient to stabilize this form of the cytochromc. In contrast, the 23 kDa extrinsic polypeptide of PS H is not required. Furthermore, it is shown that oxygen is needed for the conversion to cyt b-559LP. Further details of the relationship between components on the donor side of PS II, oxygen and the redox state of cyt b-559 will be presented.

P-8-O01 - P-8-032

P-8-001 THE CYTOCHRO~ B6F COMPLEX ~ND STATE TRANSITIONS: EVALUATION IN THE UNICELLULAR ALGA ~LARI"DOMONAS

Claire S. Tinq, Francia-Andr~ Wollman, Institut de Biologie Physico-Chimique,

A BIOCHEMICAL

REl~HARDTII

and Jean-Luc 75005 Paris,

Popot France

Transitions from State I to State II are induced by an increase in cellular ATF demand in C. r e i n h a r d t i i and are accompanied by lateral migration of cytochrome (cyt) b ~ complexes from the grana to the stromal lamellae. In order to determine if these state transitions result in biochemical alterations of the cyt ~ f complex, we have developed a procedure which allows us to purify dimeric cyt b ~ complexes from cells maintained in a predetermined state during the initial isolation of thylakoid-enriched membrane fractions. We have found that changes in polypeptide and cytochrome composition of cyt b ~ complexes do not accompany state transition-associated lateral migrations. Analysis of purified cyt b ~ complexes by SDS-PAGE revealed bands corresponding to cyt f, the Rieske FeS protein, cyt b6, and subunit IV in both State I and II cells. Low molecular weight subunits of 54 kDa were associated with the isolated complex in both states. Dissociation characteristics of the purified, dimeric cyt b6f complexes were similar between State I and II cells as well. Interactions between the cytochrome b ~ complex and other integral membrane proteins in States I and II are being further characterized using selective detergent solubilization techniques.

P-8-004 THE ROLE OF CYTOCHROHE /3559 IN PHOTOREACTIVATION OF OXYGEN-EVOLVING C E N T E R O F PHOTOSYSTEM-II M E M B R A N E S Naoki M i z u s a w a Mitsue Miyao I & T a k a s h i Yamashita, Inst. Biol. Sci., Univ. of T s u k u b a , Ibaraki 305, Japan, INatl. Inst. Agrobiol. Resources, Ibaraki 305, J a p a n In Tris-inactivated a n d M n - d e p l e t e d PS-II m e m b r a n e s about 1/3 of c y t o c h r o m e b559 (Cyt b559) w a s p r e s e n t as a high potential form a n d oxidized b y a d d i n g 1 M NaCI concomitant with a c h a n g e of the H P form to a low potential (LP) form. This oxidized C Y % /3559 w a s necessary as a n electron accepter for the photoreactivation or the reeonstitution of Mn--cluster. In the photereactivation of Tris-inactivated PS-II m e m b r a n e s 0.I m M M n 2+ a n d 50 m M C a 2+ w e r e a d d e d a n d reactivated with repetitive flashes. T h e L P form w a s firstly bl~3ught h a c k to the H P form only with t w o flashes, s u b s e q u e n t l y the C y t b559 H P form w a s p h o t o r e d u e e d a n d finally the O2-evolving activity w a s expressed. C a 2+ stabilized the flash-induced H P form of C M t b559. D C M U inhibited the r e c o v e r y a n d photereduction of H P form a n d the r e c o v e r y of O2-evolution in photereactivation suggesting that the C y t b559 H P form accepts a n electron via Q B or plastoquinone pool.

P-8-005

P-8-002 A MEMBRANE BOUND CYTOCHROME ANAEROBIC PHOTOSYNTHETIC HELIOBACTERIUM GESTII

C 5 5 2 IN T H E BACTERIUM

Inorid Albert and Hartmut Michel Max-Planck-lnstitut fur Biophysik, Heinrich-HoffmannstraSe 7, 60528 Frankfurt/Main, Germany Membrane preparations of Heliobacterium gestii (H. gesti0 show three major heme staining bands at 63, 30 and 19 kDa when analysed by SDS-PAGE. As in the other known heliobacteria no soluble cytochromes could be detected. The 19 kDa heme staining band corresponds to a c-type cytochrome with absorption maxima at 421, 522 and 552 nm in the reduced state. Equilibrium redox titrations carried out with enriched preparations of the cytochrome c 552 can be fitted with a single Nernst equation (n=l) with Em (pH7.5)=+215+15mV. We will present the sequence coding for the gene of the 19 kd cytochrome c 552 that is the most abundant cytochrome in this organism.

BIOCHEMICAL AND GENETIC ANALYSIS OF THE CYTOCHROME C y OF RHODOBACTER CAPSULATUS ~ , Francis Jenney, and Fevzi Daldal. ~ e n t University of Pennsylvania, Philadelphia, PA 19104, USA

of Biology,

Genetic and biochemical data indicate that in R. capsulatu.~ the photosynthetic reacfice center (RC) can be reduced either by the soluble cyt c2 or the membrane-bound cyt Cy, encoded by cycAand cycY,respectively. Cyt Cy can also replace cyt c2 during respiration by donating electors to the cyt c oxidase (cyt cb410) of R. capsulatus. Primary sequence and biochemical data indicate that cyt cy is an integral meanbr,me protein. Its C-terminal domaLnshows, despite a bacte-dalorigin, a high homology to soluble mitocheedrial c-type cytochromes. The N-terminal part of the protein is hydrophobic and possibly functions as a membrane anchor. Cyt Cyhas been purified using an epitope tagged version of this protein, and its rex]ucedminus oxized spectrum shows typical characteristics of a ctype c~tochrome. More detailed biochemical and biophysical characterizatic~ of cyt Cy ave underway. To study the specific run,ons of the different domains of a cyt Cy chimera between the cyts Cy and c2 are being constructed. Finally, the possibility that cyt c is closely associated with the photosynthetic apparatus will also be investigated,y

101

Poster

P-8-006

P-8-009

CYTOCHROME b559 PROTECTS PHOTOSYSTEM H AGAINST PHOTOINHtBITION BY ACCEPTING AN ELECTRON FROM PHEOPHYTIN.

C Y T O C H R O M E C O M P O S I T I O N A N D F U N C T I O N IN THE OBLIGATELY AEROBIC PHOTOSYNTHETIC BACTERIA FROM GENERA Erythromicrobium, Roseococcus AND Erythrobacter

Mary Poulson 1, and John Whitmarshl, 2 t Department of Plant Biology, University of Illinois, Urbana IL 61801 USA, 2 Photosynthesis Research Unit, U.S.D.A./A.R.S., Urbana, IL 61801 USA

Vladimir Yurkov and Andr6 Verm6glio CEA-DPVE/SBC- C.E. Cadarache, 13108 St. Paul lez Durance, France

The capacity ofphotosystem H (PSH) to evolve oxygen can be impaired when plants are exposed to excess fight. Photoinhibition is initiated by electron transport reactions that create damaging redox states within PSH such as the rarely-formed but long-lived state P680/Phen'/QA'. Experiments using thylakoid and PSH-enriched membranes poised at different ambient redox potentials demonstrate that light-induced damage to PSII is controlled by a redox component within the reaction center. We have provided evidence that the redox component is cytochrome b559 (Cyt b559), an intrinsic home protein within PSII. If the low potential form of the cytochrome (Cyt b559LP) is reduced prior to ilb~mlnation, the rate ofphotoinhibition is fast, whereas if it is oxidized the rate of photoinhibifion is slow. PreliminAry results suggest that the high potential form of Cyt b559 may also protect PSH from photoinldbition in PSH-enriched membranes. In continuous light under anaerobic conditions and without added redox mediators, Cyt b559LP aco~r~dntes in the reduced state prior to photoaccumulation of reduced pheophytin and loss of oxygen evolution capacity. These results support the proposal that electrons can flow from pheopbytin to Cyt b559 and thereby protect PSH against photoinhibition.

P-8-007

Obligate aerobic phototrophs is a g r o u p of bacteria which produce bacteriochlorophyll a, but can not g r o w photosynthetically under anaerobic conditions. The c y t o c h r o m e s from n e w l y described representatives of the genera Erythromicrobium ( E. ramosum, E.

hydrolyticum, E. ezovicum, E. sibiricum, E. ursincola ), Roseococcus (R. thiosulfatophilus ) and Erythrobacter ( E. litoralis ) were analyzed. Species E. sibiricum, E. ursincola and R. thiosulfatophilus, contain a heme cytochrome subunit bounded to the reaction center (RC) (like Roseobacter denitrificans). In E. ramosum, E. hydrolyticum, E. ezovicum and E. litoralis, the electron donor to the RC is a soluble cytochrome. Cytochrome b of bcl complex was also detected in these species. Light- and oxygen-induced electron transfer, kinetics of oxidation and re-reduction of the soluble cytochromes in whole cells and membrane fractions will be discussed.

P-8-010

THE CHLOROPLAST CCSA GBNE PRODUCT IS REQUIRED FOR CYTOCHROME C BIOSYNTHESIS IN CHLAMYDOMONAS

ELECTROGENIC PROTON RELEASE FROM THE CYTOCHROME bCl-COMPLEX OF RHODOBACTER CAPSULATUS,

Z. Xiea, R. Karasb, J. Girard-Baseoub, F.A. Wollmanb, and S. Merchanta a Dept. of Chem. & Blachem., UCLA, Los Angeles, CA 90095, USA. b Institut de Biologic Physico-Chlmtque, 13 rue Pieaxe et Marie Curie, 75005 Paris, France.

A. Mulkidianian and W. Junge Abt. Biophysik, Universit,'it Osnabriick, D-49069 Osnabriick, Germany

To study the function of the Chlamydomonas reinhardtii chloroplast ccsA (cytochrome c synthesis A) gune, agene knuek-out strategy was applied. The bacterial amino-glycoside adenine Waasferase (aadA) expression cassette was inserted into the coding region of the eosA gone, and the construct was introduced into the C. reinhardai chloroplast genome by particle gun transformation. Analysis of one aad+ strain, determitrM to be a heteroplastic knock-out strain, showed it to be non-photosynthetic and resistant to metronidazole. Western blot analysis of the abundance of two chloroplast ctype cytuehromes, cyt e6 and eyt f, showed that the photosynthetic deficiency may be attributed to the loss of these two proteins in the cesA knock-out mutant. The wild-type cosA gane was also found to rescue three previously chmactedmd cytochrome c-deficient mutant strains, B6, ct34, and ct59. Western blot analysis showed that eyt o5 and cyt f proteins appear at normal levels in rescued cells, indicating that the cosA gone product can complement mutations in tbeae strains. The possible function of the ccsA gene product and the nature of mutations in B6, ct34, and et59 strains will be discussed.

P-8-008 CRYSTAL S T R U C T U R E OF CYTOCHROME C6 FROM THE GREEN ALGA MONORAPHIDIUM B R A U N I L COMPARISON WITH THAT OF PLASTOCYANIN G. Sheldrick l, C. Frazao2, M.A. Carrondo 2, E. Pohl 1, Z. Danter3, K.S. Wilson3, M. Herv~is4, J.A. Navarro 4 and M.A. De la Rosa'* 11nstimt flir Anorganisehe Chemie, Universitiit GtRtingen, Germany 21nsfituto de Tocnologia Qufmica e Biol6gica~ Oeiras, Portugal 3European Molecular Biology Laboratory, Hamburg, Germany 4Institoto de Bioqufmica Vegetal y Fotosfntesis, Sevilla, Spain The crystal structure of cytochrome (Cyt) c6 from the green alga Monoraphidium brw~nii has been solved by ab initio methods from the single wavelength native data alone; the key to success was the collection of X-ray data to almost atomic resolution. Four a-helices and six reverse turns envelope the heine group in a hydrophobic pocket, accounting for ca. 70% of the 89 amino acids. The octaedral coordination of the iron atom is completed by the axial ligands Hisl9 and Met61. The only other histidine residue, His30, is at the molecular surface. The Cyt c6 structure compares well with that of plastocyanin, thereby allowing us to assign equivalent regions in Cyt c6: a hydrophobic "north pole"close to the exposed part of the heine group and an "east negative patch". An important difference, however, becomes evident when one fails to find an aromatic residue in Cyt c6 equivalent to Tyr83 in plastocyanins.

102

The reduction of the high-potential heme bh (at 561-570 nm) and of cytochromes c (at 552-570 nm), net charge translocation (by electrochromic transients at 523 nm) and proton release into the lumen (by neutral red, at 546 nm) were studied under flashing light in chromatophores of Rb. capsulatus in the presence of either antimycin A, myxothiazol, oligomycin or venturicidin. The kinetic behaviour of the transmembrane electron transfer to heme bh differed from the one of the reduction of cytochrome c, electrochromism and proton release. The three latter reactions were kinetieally coincident. This let us to the following conclusions: (I) the protons that are released at ubiquinol oxidation stay bound with the cytochrome bcl-complex and compensate the negative charges of the delivered electrons; (2) the transmembrane electron transfer between two b-heroes is accompanied by the proton rearrangement that makes it electrically silent; (3) the electrogenic proton expulsion into the chromatophore lumen follows the disappearance of negative charges due (i) to the oxidation of heme bh and (ii) to the electron delivery from the FeS protein to cytochromes c.

P-8-0 1 1 A MOSSBAUER STUDY OF THE CYTOCHROME B6/F COMPLEX J. Illerhaus1, V. Petrouleas2, Y. Sanakis2, and W. Haehnel1 1 Biochemie der Pflanzen, Univ. Freiburg, Sch~inzlestr. 1, 79104 Freiburg, Germany 2 Inst. Nat. Sci. NCSR "Demccritos", 15310 Ar. Paraskevi, Athens, GREECE The cytochrome(cy0 b6/f complex mediates electron transfer between Photosystem I and Photosystem II coupled to proton translocation across the chloroplast membrane. It contains four iron centers, two b-type cytochromes, one cyt f and a Rieske-type 2Fe-2S center. The importance of the complex and the central role of the iron has prompted us to examine the feasibility of the application of the M6ssbaner spectroscopy to the complete complex. To our knowledge the only related application has been the MOssbauerstudy of the isolated Rieske protein from the cyt b/cI complex of T. thermophilis (D. Kulian and J.A. Fee, 1986, J. Biol. Chem. 261, 2768). The cyt be/f complex was isolated by a modified method of Hanska (1986, Methods Enzym. 126, 271) from Spinacia oleracea grown with 57Fe in hydrocultare. The molar ratio of cyt b to cyt f was 2:1. One chlorophyll was present per cyt fi The oxidoreductase-activity was 17sd. Samples for M~ssbauer studies were 200 }aM in cyt fi The M6ssbaner spectra show good resolution among the various components. Because of the difference in the redox potential and the relaxation behaviour at low temperatures,the signalscan be attached to the different iron centers. We expect detailed information on the interaction between the iron centers and the protein.

Poster

P-8-012

P-8-015

THE MECHANISM OF CYT b PHOTOOXIDATION IN Chlamydomonasreinhardtll.

CYTOCHROME C6 FROM SYNECHOCOCCUS ELONGATUS

• , Deborah A. Berthoid and Richard Malldn Dept. Plant Biology. University of California. Berkeley, CA 94720-3102

M. Sutter 1, H. Sticht3, P. Wlasak] , R. Schmid2, P. R6sch3 & W. Haehnel ! IBiochemie der Pflanzen, Schiinzlestr. 1, 79104 Freiburg, Germany; 2Fachbereich Biologie/Chemie, Posffach 4469, 49076 Osnabriick, Germany; 3Lehrstuhl Biopolymere, Universi~tsstrafle 30, 95440 Bayreuth, Germany

At Eh < -200 mV a single flash photooxidizes cyt b(s) after a 2-3 ms lag (11/210-30ms). 1)This reaction is sensitive to low concentrations of stigmalellin or DBMIB. HQNO inhibits the oxida~onas well, sugge~ng ~at the reaction occurs at the Qc site. 2) A plastocyanin less (Pc-) mutant (containing normal levels of potentially ac~'e b6f compled is impaired in = b6f redox reactions, whereas ferredoxin photoreduotion is normally present 3) At Eh <-200 mV a slow phase of electrochromic signal (sensitiveto slJgmetellinand DBMIB) develops much faster (tl/2 ca. 3 ms). This signal is biphasic and can be deconvoluted in an HQNO sensitive (aftor a ~ of ca. 2 ms) and an HQNO insensitive phase. Probably, the former relates with the movement of an electron from the cyt b chain through the Qc site and the later with the movement of a semiquinone from the Qz to the Qc site. 4)The sensitivity to Qz site hhUtore and behavior of the Pc" mutant independen~ demonslrate that the photooxidation of o/t b b triggered by the appearance of an oxidant equivalent at the high potential chain and that ~ redudng side of PSI is not involved. 5) These results disprove the ~ i s m of " r e d u c ~ ~ " proposed by Chain, R (1982) FEBS Lett. 143, 273 where the oxidant is a semiquinone generated through photoreduced ferredoxin. Conversely, they are fully coherent with the possibility thai the semiquinone generated after a flash at the Qz site is the oxidant, at the Qc site, of ~ prereduced low potenSalchain, thus favoringa ~ m q , ~ cycle" as proposedby Joliot and Joliot (1994) PNAS 91,1034.

The soluble cytochrome (cyt) c 6 has been isolated from the thermophilic cyanobacterium Synechococcus elongatus. In this organism cyt ca is the only electron donor to Photosystem I (PSI), which has been crystallized by P.Fronune and H.T.Witt/Berlin. The cyt c6 has a molecular mass of 9795 Da including the covalently linked monoheme. The complete amino acid sequence has been determined and is consistent with our results from electrospray mass spectrometry, which allowed also to verify the two cysteines linking the monoheme. It has a high homology to the sequence of cyt ca from Synechococcus livldus. The visible spectrum, the amino acid sequence and first results from NMR measurements indicate that the heme iron has a methioninehistidine axial coordination. The redox midpoint potential is 342 mV at pH 7.0/25°C and shows slightly decreasing values at pH>7. The electron transfer from cyt c6 to photooxidized P700 has been studied by laser-flash absorption spectroscopy with PSI preparations from Synechococcus elongatus and with crystals of the trimeric PSI. The cyt c 6 has been crystallized and diffraction studies are in progress.

P-8-016

P-8-013 I S O L A T I O N OF G E N E S I N V O L V E D I N P L A S T I D I C C-TYPE CYTOCHROME BIOSYNTHESIS IN C H I A M Y D O M O N A S R E I N H A R D T I I

REDOX CONTROL OF THYLAKOID PROTEIN PHOSPHORYLATION BY A PLASTOQUINOL BINDING TO A REDUCED CYTOCHROME BFCOMPLEX

Beth Welty Dreyfuss and Sabeeha Merchant Dept. Chem. & Biochem., UCLA, 405 Hflgard Ave., Los Angeles, CA 90095, USA The cloning a n d characterization of Chlamydomonas reinhardtii nuclear genes that are required for attachment of heme during the assembly of the plastidic c-type cytochromes, cyt f and cyt c6, and the determination of the function of the gene products are underway. The work involves the generation and identification of tagged nuclear mutants that are defective in cyt c6 a n d cyt f accumulation. "ragged" mutants have been generated by random integration of the argininosuccinate lyase (ASL) gene into the nuclear genome by glass bead transformation of a recipient arg-minus strain. Several cyt b6f-minus mutants have been identified among the arg-plus transformants on the basis of their fluorescence induction kinetics. Among these mutants, those transformants failing to accumulate holocyt c6 a n d holocyt f were further identified by Western blot analysis, heme staining and pulse-chase analysis. The phenotypic characterization of the tagged mutants and initial analysis of the tagged locus will be presented.

A. V. Vener1, P. J. M. van Kan 1, A. Gal2, L Ohad2, and B. Ande~sson 1 1Dept. of Biochemistry, Stockholm University, Stockholm, Sweden, and 2Dept. of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel

P-8-014 M U T A T I O N A L A N A L Y S I S OF T H E R O L E OF E X T R I N S I C CYTOCHROME c-550 I N C Y A N O B A C T E R I A L PHOTOSYSTEM II J.-R. Shen 1, W. Vermaas 2, R.L. Burnal>3 & Y. Inoue 1 1Solar Energy Res. Group, The Inst. of'Phy. & Chem. Res. (R/KEN), Wako, Saitama 351-01, Japan; ZDept. of Bot., Arizona State Univ., Tempe, Arizona 85287-1601; 3Dept. of Micro. & Mole. Genet., Oklahoma State Univ. Stillwater, Oklahoma 74078. Inactivation of the gene coding for cytochrome (cyt) c-550 (named as psbV gene) in Synechocystis sp. PCC6803 resulted in a 60% reduction in P S I I 02evolving activity and to a less extent a destabilization of the PSII complex. The resultant mutant could grow photoautotrophically but with a slower growth rate. This indicates a role o f c y t c-550 in maintaining PSII 02-evolving activity and stability in vivo. When the 33 kDa extrinsic protein and cyt c-550 were simultaneously deleted, however, the resultant mutant cannot grow at all photoautotrophically. This double deletion mutant showed PSII O2-evolving activity below I0% of the wild type, and a PSII content of 20% of the wild type. Thus, the 33 kDa protein and cyt c-550 function independently in vivo; both of them are required for maintaimng optimal rate of 0 2 evolution and photoautotrophic growth. This implies that the 0 2 evolution and photoautotrophic growth in the 33 kDa protein-deletion mutant is maintained by cyt c-550 and vice versa. These results agree with our previous biochemical evidence indicating that cyt c550 is one of the extrinsic components of cyanobacterial PSII required for maintaining 02 evolution, which suggested a possible evolutionary linkage at the donor side of PSII-type reaction center from bacteria to higher plants.

Redox-controlled light dependent protein phosphorylation regulates the light harvesting process in thylakoid membranes in response to changes in light conditions. We found that phosphorylation can be induced in the dark without addition of reducing agents by a transient low pH treatment of thylakoids causing an increase in the plastoquinol/plastoquinone ratio from 0.05 to 0.25. Howeve~ plastoquinol is rapidly reoxidised (<1 min) in the phosphorylation medium at neutral pH in contrast to a slow deactivation of the kinase (tl/2, 4 min). This indicates that the phosphorylation is not directly controlled by the plastoquinol pool. Both, irrhibitors and mutant studies, demonstrate the involvement of the cytochrome bf complex in the low pH induced protein phosphorylation. EPR spectroscopy shows that the redox centers of cytochrome bf are reduced during the activation/deactivation cycle. However, the rate of deactivation correlates with the decrease in the EPR gz signal of the reduced Rieske Fe-S center. This signal is also affected by quinone analogs inhibiting the kinase activation. We conclude that the kinase activity is controlled by binding of plastoquinol to the reduced cytochrome complex in the vicinity of the Rieske protein. Thus, the plastoquinolcytochrome bf interaction acts as a receptor system leading to the kinase activation.

P-8-017 T H E D E L E T I O N OF p e t O I N ~ CYTOCBRO~BhfCGMPLEX

R

E

~

I

I

D.A. Berthold, C.L. S c h ~ d t , and R. M a l k i n Dept. of Plant Biology, University of California, 94720-3102, U S A

DISREPTS

Berkeley,

TRB¢

CA

T h e 4 kDa protein e n c o d e d by chloroplast petG copurifies with the cytochrome bf complex of spinach, and is found in a number of other photosynthetic organisms, including the eukaryotic alga Chlamydomonas reinhardtii. In order to determine w h e t h e r petG is involved in the function or asserably of the cytochrome bf complex, petG w a s r e p l a c e d with the aadA cassette in the C. reinhardtii chloroplast using biolistic transforn~tion. As a control, transformants were also isolated in which the aadA cassette was inserted into ORF56, located 23 bases d o w n s t r e a m from the stop codon for petG. The petG mutants are unable to grow photosynthetically on minimal media, whereas the ORF56 mutants retain photosynthetic function. Western iramunoblot analysis reveals greatly d e c r e a s e d l e v e l s of cytochrome b6, cytochrome f, Rieske Fe/S protein, and subunit IV in the petG deletion mutant, although wild-type levels of RNA transcripts for each of these subunits is present. T h e a b s e n c e of the petG protein product affects either the assembly or stability of the cytochrome bf complex in C. reinhardtii.

103

Poster

P-8-018 EXPRESSION AND I-STEP PURIFICATION OF A FULLY ACTIVE POLYHISTIDINE TAGGED BC-COM]PLEX IN RHODOBACTER SPHAEROIDES

G. Bechmann, R. Salcedo-Hernandez, M. Guergova, A.R. CroRs and R.B. Gennis Depts. of Biochemistry and Microbiology and Program in Biophysics, University of Illinois, Urbana, IL 61801 The fbcB gene of the fbc-operon, encoding cytoehrome b of the be-complex was extended with a segment to encode a poly-histidine tag (I-Iis0 linked to the C-terminal sequence of cytochrome b. The plasmid was constructed in a PUCderived background by creating suitable unique restriction sites and splicing the appropriate DNA-sequence using PCR, and amplified in E. coil The coding region was transferred to a PRK-derivative plasmid, and this was used to transform the foe- strain of Rb. sphaeroides, BC17. The transformants showed normal rates of growth. Chromatophores prepared from these cells showed kinetics of turn-over of the the be-complex which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement by redox potentiometry showed no difference from wild-type. Chromatophores were solubilized and applied to a Ni-doped agarose column. The be-complex was washed from the column with imidazole, giving a substantially pure preparation containing all the cytochromes of the complex. Gel electrophoresis showed four anbunits. This construction will be used in future mutagenesis work, to provide a facile method for preparation of mutant be-complex.

P-8-021 Cloning, Overexpression and Characterization of Subunit IV from a

cyanobaeterial Cytochrome b6f complex. U. Boronowsky, J. Kruip and M. ROgner Institute of Botany, University of Mtinster, Germany In the thylakoid membranes of cyanobacteria, the cytochrome btf complex is of central importance both for photosynthetic and respiratory electron transport. Due to the high instability of the isolated complex, up to now the yield of cyanobacterial preparations was very low. Overexpression and purification of the isolated subunits of this complex could help considerably for the understanding of structure/function relationship; it may also allow the reconstitution of a whole, functional complex. In this work, the sequences of the petB and petD genes from SynechocTstis sp. PCC 6803, encoding cyt b6 apoprotein and subunit IV, respectively, could be determined by nonradioactive methods. The genes could be amplified by PCR and cloned into expression vectors under control of the lacUV5 promoter. Overexpression of subunit IV in E. colt yielded up to 5% of the total cell protein as characterized by immunoblotting. The overexpressed subunit - which also would allow the introduction of site-directed mutations - is characterized by physico-chemical methods.

P-8-019 A CHAIN REACTION MECHANISMFOR OXIDATION OF THE FULLYREDUCED

CVTOCIIROMZ¥COMPI~X.

David M. Kramer Iand Antony R. Crofts2"3tDept, of Plant Biology, ZMierobiology, and ~Program in Biophysics and ComputationalBiology. 265 Morrill Hall 505 S. Goodwin Ave. Urbana, IL 61801 It has been suggested that the Q--cycle cannot turnover when the bfcomplex is fully reduced, because no aeeeptor for the electron on the Qo site is available when both ¢ytochromes b are reduced. However, many reports show that turnover of the complex does occur, though at a slowed rate. We propose that a chain reaction type mechanism can account for this behavior within the framework of a Q-cycle. This scheme relies on two processes: a slow reaction that produces a small fraction of"open" bf complexes (with cyt b oxidized), and subsequent more rapid reactions in which opened centers oxidize plastoquinol (PQH2) to plastoqumone (PQ) through the Q-cycle, which in turn opens more centers. A candidate for the slow nucleating process is the oxidation of the ferrocyt bL by the somiquinune at the Qo site followed by a second turnover at the site producing rice PQ. Complexes opened in this way can then turnover in the normal Q-cycle mode, producing PQ The entire process requires that plastocyenin (PC) be able to rapidly redistribute positive charges to allow a fraction of complexes to turn over multiple times, and we show that the movement of PC is much faster than the turnover of the bfcomplex. After a very weak flash of light, cyt bH and bL were oxidized in approx, equal amounts as predicted by our model, but in contradiction with the semiquinone cycle.

P-8-022 TOPOGRAPHICAL STUDIES ON THE CYTOCHROME BeF COMPLEX FROM SPINACH BY CROSSLINKING WITH EDC AND OPA A. Blokesch 1, K.-D. Irrgang 2, J. Salnikow I & J Vater I Technische Universitat Berlin: llnstitut ftir Biochemie und Molekutare Biologic, FranklinstraBe 29, 10587 Berlin. und 2MaxVollmer-Institut ftir Physikalisehe und Biophysikalische Chemic; Stral3e des I7. Juni 135, 10623 Berlin, Germany Purified cytoehrome btf complexes from Snina¢ia olerac¢~ were crosslinked with the zero-length crosslinker l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and ortho-phthalaldehyde (OPA). Conjugates were identified by heme and immunostaining using antibodies raised against the four main subunits of the complex and a peptide corresponding to residues 25 to 37 of the mature subunit V, the petE gene product. Main conjugates were formed between subunit IV and each of the two cytochromes. respectively. Upon EDC treatment two strong bands of 27 and 30 kDa were observed involving subunit IV. At high concentrations of OPA, the Rieske protein yielded a 23 kDa conjugate, indicating crosslinking of both the Rieske protein and subunit IV with small subunits of the c o m p l e x

P-8-023

P-8-020 CYTOCHROME

b$59 WITHIN PHOTOSYSTEM II: SPECTROSCOPIC STUDIES ON THE PHOTOREDUCTION AND PHOTOOXIDATION OF

THIS CYTOCHROME AND GENETIC CONSTRUCTION OF A psbF. LESS MUTANT IN CHLAMYDOMONAS REINHARTII.

CHARACTERISATION OF THE 32 kDa CYTOCHROME C553 FROM CHLOROBIUM LIMICOLA FORMA THIOSULFATOPHILUM D. Albouy 1, U. Feiler l, J.Sturgis 1, W. Nitschke 2 and B. Robert 1

Javier De Las Rivas 1, Francisco Morals 2, James Barber 2 & Peter Nixon 2 1 Departmentof Biochemistry& Molecular Biology.Faculty of Science. Universityof the BasqueCountry.P. O. Box 644 - BilbaoE48080 - SPAIN. 2 PhotosynthesisResearch Group. WolfsonLaboratories. Department of Biochemistry. ImperialCollegeof Science, Technology& Medicine - LondonSW7 2AY - UK.

lSection de Biophysique des Prot~mes et des Membranes, DBCM/CEA and ERA 1290/CNRS. Centre d'Etudes de Saclay, F-91191 Gif sur Yverte Cedex, France; 2Institnt flit Biologic 1], Albert-Ludwig Universit~it, Sch~inzlestr. 1, D-79104 Freiburg, Germany

A series of experiments on the photoreduction and photooxidation at different pHs of Cytochrome b559 (Cytb559) within isolated reaction centres and cores of photosystem II (PSII) from Pea have been conducted. At acid pH Cytb559 was reversibly photoreduced by red actinic light in the presence of Mn2*, while at basic pH a photooxidation was observed reversibly in the presence of hydroquinone. These pH dependent light induced changes were measured under anaerobic conditions and correlated with changes in the relative levels of high fliP) and low (LP) potential forms of the cytochrome as detected by dark titration with hydroquinone. Combined with these functional experiments the construction of a psbF deletion mutant in the unicellular green algae Chlamydomonas reinhardtii has been undertaken. The psbF gene encodes for the I~subunit of the Cytb559. The construction and further comparative study of the photosynthetic activity of deletion mutants in Cytb559 will be most useful to elucidate the role of this important haemoprotein in photosynthesis.

We have isolated and characterised a cytochrome from Chlorobium limicola £ thiosulfatophilum, exhibiting an cc band at 553 nm. Its apparent molecular weight is 32 kDa. Both absorption and EPR spectroscopy indicate that this cytochrome is tetrahaen~c. Time-resolved absorption spectroscopy performed on whole cells, as well as EPR spectroscopy on membranes show that cytochrome c553 is the most likely candidate for the electron donor to the reaction centre (PC) of Chlorobium. The halflime of the electron transfer between the RC and the cytochrome c553 is 18 ~ts in vivo. Other cytochromes absorbing at 555 and 551 nm which are involved in subsequent electron transfer reaction, were spectrally characterised.

104

Poster P-8-024 CYTOCHROME b J COMPLEX FROM C. REINHARDTII : PURIFICATION AND CHARACTERIZATION Y. Pierre a, C. Breytont, C. Tribet~'2, J. Olive3 & J.-L. Popot~ qBPC, 11 rue P. et M. Curie, F-75005 PARIS, France, 2ESPCI, 10 rue Vauquelin, F-75005, France, 3IJM, 2 p l a c e Jussieu, F-75005, France The cytochrome b j " complex from Chlamydomonas reinhardtii has been purified to homogeneity. It contains 2/7 hemes and one chlorophyll a per cytochromef and is highly active in transfering electrons from decylplastoquinol to plastocyanin. It comprises 6 integral subunits, whose identity has been established by N-terminal sequencing and peptide-specific immunolabeling, and one peripheral subunit, the Rieske protein. The solubilized complex has a MW of 300 _ 25 kDa, including bound detergent, within experimental error of that calculated for a dimer. Freeze-fractured lipid vesicles incorporating the b J d i m e r exhibit homogeneous particles 10-11 nm in diameter. At high detergent concentration, the dimer breaks down into inactive monomers that have lost the Rieske protein. The preparations appear suitable for crystallization (see poster by Mosser et aL).

P-8-027 EFFECTS OF INACTIVATION OF GENE CODING FOR THE REACTION CENTER-BOUND CYTOCHROME SUBUN1T ON GROWTH AND ELECTRON TRANSFER IN PURPLE PHOTOSYNTHETIC BACTERIUM,

R UBRIVIVAX GELATINOSUS Kenli V.P. Nagashim~ Keizo Shimada and Katsumi Matsuura, Dept. Biol., Tokyo Metropolitan Univ., Minamiohsawa 1-1, Hachioji, Tokyo 192-03, Japan Many purple bacteria have the reaction center-bound cytoehrome subunit which donates electrons to the photo-oxidized special pair of bacteriochlorophylls in the reaction center. Some species of purple bacteria do not have this subunit. To clarify the role of the cytoehrome subunit, we inactivated the gone coding for the eytochrome subanit in purple bacterium, RubrCwvaxgelattnosus by an insertion of the kanamycin resistant cartridge. The m u m ~ d m m m did not produce the cytochrome subunit but was able in grow phomsymhetic~ly. However, the growth m ~ of the mutated strain was approximately half of that of the wild type strain under the photosynthctJc conditions. Measurements of flash-induced absorption changes showed that the photo~xidized special pair in the whole cells of the mutated strain was re-reduced with comparative rate to that of the wild type. These results are consistent with our previous observation that in Rv. gelaanosus, the purified reaction center complex without the eytochrome subanit accept electrons from soluble cytochrome e as effeelively as in the complex with the suhanit.

P-8-025 4-kDa CHLOROPLAST POLYPEP~XDE OF C'~J~O~ffROI4E b 6 / f

COI~PT.w-X

~ C O D E D BY THB N U C L I L ~ p e t X OLDIE: NUCLEIC AND PROTEIN SEQUENCESt TARGETING SIG~IALSo A N D ~ TOPOLOGY.

CRYSTALLIZING THE CYTOCHROME b J FROM C~_AMYDOMONAS REINHARDTII.

P-8-028 COMPLEX

C. de V~trv, C. areyton, Y. Pierre & J.-L. Popot Laboratoire de Photosynth~se, Institut de Biologie PhysicoChimique, 13 rue Pierre et Marie Curie, 75005 Paris, FRANCE.

G. Mosser, J.-J. Lacap~re & J.-L. Rigaud Institut Curie, 11 rue P. et M. Curie, F-75005 PARIS, France

We have previously identified in Chlamydomonas reinhardtii by p r o t e i n sequencing and immunodetection a nuclear-encoded 4-kDa polypeptide preaent in purified b6f complexes and absent in b6fdeficient mutants and therefore considered as a b~f subunit (Pierre & Popot, C.R. Acad. Sci. 316:1404-1409, 1993). This aubunlt is also present in higher plants (Schmidt & Malkin, photoeynth. R e e . 38: 73-81, 1993). We have now sequenced the cDNA of petx encoding thls 4-kDa aubunit and the mature protein by Edman degradation in Chlamydomonas reinhardtii. The size of the transcript for pe~X gene is of about 0.5 kb. The transit peptide has a basic region typical for the transfer through the chloroplast envelope, but no hydrophobic segment at the C-terminal end as is found in most proteins transfered through the thylakoid membrane. The mature protein is 39 residues long and has a hydrophobic segment, which could form a transmembrane u-helix. The amphipathy of this u-helix is low. Upon extraction with chaotropic agents, the petX product behaves as an integral membrane protean. According to charge distribution and preliminary proteolyais experiments using synthetic peptide antiaera, the C-terminus of the petX product would be located on the stromal side of the membrane.

C. Breyton, D. Picot & J.-L. PoDot I.B.P.C., 11 rue P. et MI Curie, F-75005 PARIS, France The eytochrome baffrom C. reinhardtii can be purified to homogeneity using a novel procedure that yields mg amounts of pure complex, adequate for crystallogenesis experiments (see poster by Pierre et el.). Two routes to bJcrystals have been explored. First, 2D crystals have been grown following reconstitution of the bc,f dimer into.lipid vesicles. After negative staining, they diffract to at least 18 A resolution. Second, 3D crystallization has been attempted using the hanging drop method. Crystal-like objects have been obtained, up to 50 tim in their largest dimension. At the time of this writing, it is not known whether they diffract X-rays.

P-8-029

P-8-026 SITE DIRECTED MUTAGENESIS ON THE PETB GENE IN CHLAMIDOMONAS REINARD TII F. Zito 1, R. Kuras 1, H. K6ssel 2 and F.-A. Wollman 1. (1)Service de Photosynth~se, IBPC, Paris, France. (2) Albert-Ludwig University, Freiburg, Germany. W e used site directed mutagenesis o f the petB gene in Chlamydomonas reinhardtii to d r a w structure/function correlations at the level o f cytochrome b6. W e first addressed the rationale for editing the petB transcript from maize, which replaces Pro204 by Leu. In Cldamydomonas the codon for position 204 corresponds to Leu at both the D N A and R N A levels. W e therefore changed it to a Pro codon in two versions, one only being a substrate for the editing in maize. One construct allowed us to check whether editing could occur at that position in Chlamydomonas whereas the other allowed us to understand whether Pro204 prevents cytochrome b6 function. W e also investigated the function o f the Qi site by changing the polarity and the bulkiness o f several residues possibly involved in quinone binding. Preliminary results on the effect on photosynthetic growth and cytb6f assembly o f several transformants are presented.

INHIBITOR BINDING TO ISOLATED CYTOCHROME bfCOMPLEX A. B. Hope School of Biol. Set., Flinders Univ., GPO Box 2100, Adelaide, S.A. 5001, AUSTRALIA Stigmatellin and dibromothymoqninone (DBMIB) are well-known inhihitors of the chloroplast cyt bf complex, preventing electron transfer from quinol at p (Qo, qninol oxidation)-sites to the high potential components FeS and cyt ~ By using isolated bf complex their binding properties can be studied without competition from endogenous plastoquinol. The kinetics of electron transfer between bf complex and cyt f, and between cyt f and plastocyanin (PC) were measured as functions of inhibitor concentration. Both inlfibitors lowered the reduction potential of cyt f relative to plastocyanln by up to 20 mV, in agreement with redox titratinns (sttguratenin: Hanska, Herold, Huber, Nitscnl~e & Sofrova, 1989, Z. Naturf. 44c, 462467). The kinetic rate coefficient kFp (PC reduction by cyt /~) was increased by inhibitor while the reverse coefficient kl~ was unaltered. In agreement with earlier findings (Hope, Matthews & Valente, 1994, Photosynth. Res. 40, 199-206), in the absence of quinol, few electrons from the the FeS centre filled holes in cyt f or plastocyanin, and this limited transfer was inhibited by stigmatellin. Titration of the effect of inhibitor on the equilibrium between cyt f and PC enabled a sensible estimation of the binding coefficient of stigmatellin of the order of I07 M (K = 3.107 can be estimated from the effect of stigmatellin on electron throughput from di-tertbutyl hydroquinone to cyt c: Oettmeier, Godde, Kunze & H6fie, 1985, giochim. Biophys. Acta 807, 216-219). DBMIBappeared to fully inhibit at a ratio of one per bf dimes" (cf. Green & Ort, 1986, Arch. Biochem. Biophys. 248, 445-451). Stigmatellin binds 1:1 with monomeric bfbut this is statistically improbable for the DBMIB data. If two binding sites exist per dimer, DBMIB may oscillate rapidly between them, while this is not possible of stigmatellm.

105

Poster P-8-030 ARE CYTOCHROMEB/F COMPLEXESFROM GRANAAND STROHA MEMBRANESOF SPINACHCHLOROPLASTSDIFFERENT? ~. Romanowska1 and P-A. Albertsson 2 ~Dept of Plant PhysJol.II, University of Warsaw, Poland; Dept of Biochemistry, University of Lund, Sweden The cytochrome b/f complex was isolated from spinach grana and stroma lamellae vesicles. Electrophoretlc analyses indicate that the complexes are in dimeric form and composed of six polypeptldes. The 12 kDa protein was identified as plastocyanin (PC) by immunoblotting. There is significantly more PC and 4 kDa protein In the cytochrome b/f complex isolated from stroma lamellae than from grana. In additions there is a 15 kDa protein in the complex isolated from the stroma lamellae fraction. Immunoblot analysis after crossllnking indicated that the 4 kDa protein and the PC are associated in the cytochrome b/f complex. The cytochrome b/f complex isolated from both grana and stroma lamellae contain the 64 kDa protein claimed to be a klnase. The Rleske FeS protein from grana and stroma membraneswas separated into two polypeptlde spots having the Isoelectric points of 5.1 and 5.4, respectively.

P-8-031 ELECTROCHEMICAL AND SPECTROSCOPIC INVESTIGA-TION OF CYTOCHROME BC 1 COMPLEX FROM RHODOBACTER CAPSULA TUS F. Baymann~, D. Robertson2, W. Mantele3 qnstitut for Biophysik, Universitat Freiburg, Albertstr. 23, 79104 Freiburg, Germany; 2Director of Enzymology, 33 Evergreen Lane, Haddonfield, NJ 08033, USA; 3Institut for Physikalische Chemie, Universitat Erlangen, Egerlandstr. 3, 91058 Erlangen, Germany; The cytochrome bcrcomplex from Rhodobacter capsulatus was investigated by a combination of FTIR-spectroscopy and electrochemical titrations. IRdifference spectra, characterizing exclusively the redox reaction of one of the cofactors, i.e. cytochrome cl, cytochrome bH or cytochrome bL, the Rieske protein or the quinone bound to Qi-binding site were obtained. The spectra show signals from the hemes or the quinone as well as from the protein environment of all cofactors. Difference bands from the buffer systems in the infrared region could be evaluated, yielding information about the number of protons released or picked up by the complex concomittant with the redox reaction of each cofactor. The midpoint potentials of the cofactors at two different pH-values, their spectra and their extinction coefficients in the visible range have been obtained. The results provide further insight into the molecular mechanisms of the reactions of cytochrome bcl-complex.

P-8-032 A SHIFT IN A CHLOROPHYLL SPECTRUM ASSOCIATED WITH ELECTRON TRANSFER WITHIN THE CYTOCHROME b/f COMPLEX

A. Ioli0t & P. Joliot Inst. Biologie Physico-Chimique 13, rue P. & M. Curie, 75005 Pads, France Light-induoed spectral changes have been observed in the red region of the spectrum in green algae (Chlorella soroldniana or Chlamydomonas reinhardtii) in anaerobic conditions. This flash-induced spectral change is observed in the presence of dicyclohexanol-lB-crown-6 but not with other uncoupling agents. The spectrum of this signal corresponds to a red shift of a ehlorophyU peaking at 669 nm (negative peak at 661 nm and positive peak at 681 nm). The effect of specific inhibitors of the Qo site and the absence of this new spectral change in a mutant devoided of cyt b/f complex, shows that this signal is associated with electron transfer reactions occurring within the cyt b/f complex. The rise time of the signal is close to the reduction time of cyt f and its decay time (tin ~"2 s) is not correlated with any redox change of electron carders included in the cyt b/f complex. This spectral change is likely due to a storage of protons which induces a change in the electrostatic environment of a chlorophyll associated with the cyt b/f complex (see poster by Pierre et al.).

106

Poster

P-9-003 C H A R A C T E R I Z A T I O N O F RECCNBII4ANT W I L D - T Y P E AND M U T A N T FO~MS OH SPINACH F E R R E D O X I N I

Poster

session

L. Piubelli, F. Bellintani, A. Aliverti and G. Zanetti Dip. Fisiologia e Biochimica Generali, Universit~ degli Studi di Milano, Italy.

9

In photosynthesis, ferredoxin I (Fd I) acts as an electroncarrier protein for distribution of electrons coming from photosystem I to several metabolic routes. To study proteinprotein interactions between Fd I and its partners, the spinach protein was overexpressed in E. coli. The recombinant Fd I is correctly assembled with the [2Fe-2S] cluster, and is fully active in the NADP ÷ photoreduction using thylakoids deprived of endogenous Fd. Glu92, a conserved residue, has been proposed to be located at the molecular interface in the complex between Fd I and ferredoxin-NADP + reductase (FNR) (Zanetti, G., et al, 1988, Biochemistry 27, 3753). The corresponding residue in Anabaena Fd was shown to be required for electron transfer to FNR (Hurley, J.K., et al., 1993, Biochemistry 32, 9346) Three mutant spinach Fd I forms, i.e., E92Q, E92A, B92K, were obtained by site-directed mutagenesis, overexpressed in E. coli and purified. The Fd I mutant forms display absorption spectra superimposable to that of the wild-type protein. Further characterization of the mutants is in progress.

Electron transfer proteins P-9-O01

- P-9-037

P-9-001 PRIMARY STRUCTURE AND POST-TRANSLATIONAL MODIFICATION OF FERREDOXIN-NADP REDUCTASE FROM Chlamydomonas reinhardtii.

P-9-004 INVOLVEMENT OF THE FAD DOMAIN RESIDUE TYROSINE 308 ON STABILITY AND FUNCTION OF FERREDOXIN-NADP + REDUCTASE

p~ulette Decottignies 1, Pierre Le Mar6chal 1, Jean-Marie Schmitter 2, Val6rie Flesch 1 and Jean-Pierre Jacquot 1. llnstitut de Biotechnologie des Plantes, Universit6 Paris-Sud, 91405 Orsay Cedex, France., 2Laboratoire de Biochimie, Ecole Polytechnique, 91128 Palaiseau Cedex, France.

E. Ceccarelli, G. Pico, E. Orellano, J. Ottado & N. Calcaterra Fac. de Bioquimica. Universidad de Rosario. Suipacha 531. 2000 Rosario. Argentina

The flavoprotein ferredoxin-NADP-reductase (FNR) was isolated from the unicellular green alga, Chlamydomonas reinhardtii. FNR is a monomeric protein containing one FAD and exhibiting ferredoxin-dependent cytochrome c reduction activity. Its complete primary structure was investigated by sequencing overlapping peptides generated by cleavage with trypsin and SV8 protease, and confirmed by nucleotidic sequence. C. reinhardtii FNR contains 320 residues, corresponding to a calculated mass of 35,685 and 36,470 including FAD, in agreement with the values measured by laser desorption mass spectrometry. The combination of both amino acid and nucleotidic sequencing, in association with mass spectrometry of peptides allowed the identification of two Ne-trimethyllysines at positions 83 and 89 and one NEdimethyllysine at position 135. Comparison of the primary structure of C. reinhardtii FNR with the known sequences shows 41-46% identity.

The C-terminal tyrosine of plant ferredoxin-NADP + reduetases (FNR) displays extensive interaction with FAD. We investigated the role of this residue in FNR function using site-directed mutaganesis. Pea FNR was expressed in Escherichia coli and purified to homogeneity. Substitution of the terminal Tyr308 by Trp, Fen, Ser and Gly, or elimination of the Tyr, resulted in progressive reduction of the kcat for FNR reactions, without affecting the Km values. The results indicate that aromatieity is the most important factor to the function of Tyr308 in catalysis. Analysis of stability of wild-type and mutant FNRs by thermal and urea denaturation indicate direct participation of the Tyr30g phenol ring in FAD binding and maintenance of the FNR folded structure. These findings indicate that the C-terminal Tyr plays a key role in FNR assembly and function.

P-9-005

P-9-002 SPINACH FERREDOXIN -~h%DP+ REDUCTASE : S T R U C T U R E -F~NCTION R E L A T I O N S H I P AS STUDIED BY S I T E - D I R E C T E D MUTAG~NESIS.

MUTATIONAL 89 OF PEA

ANALYSIS OF THE CONSERVED FERREDOXIN-NADP + REDUCTASE

RESIDUE

A. Aliverti I, V.E. Pandini I, P.A. Karplus 2 & G. Zanetti 1 iDip. Fisiologia e Biochimica Generali, Universit~ deg~i Studi di Milano, via Celoria 26, 1-20133 Milano, Italy; Div. of Biological Science, Cornell University, Ithaca, NY 14853, USA

N. C a l c a t e r r a , E. O r e l l a n o & J. Fac. d e B i o q u f m i c a , Universidad 531. 2 0 0 0 R o s a r i o . A r g e n t i n a

Ferredoxin-NA/SP + reductase (FNR) catalyzes the last step in the photosynthetic electron transport chain transferring electrons from reduced ferredoxin (Fd) to NADP +. Residues located close to the FAD isoalloxazine ring, as revealed by the three-dimensional structure of FNR, i.e., Tyr95, Set96, Cys272, Glu312, have been changed by site-directed mutagenesis. Tyr95, which interacts with the si-face of the flavin, has been replaced with Phe with only minor effects on the enzyme features. Mutants at position 96 and 272, share similar properties: very low kca t values both in Fd-dependent and diaphorase activities, and impairment in the stacking between the re-face of the flavin and the nicotinamide ring of NADP(H), as observed by difference spectroscopy as well as by rapid-reaction studies. In addition, replacement of Set96 with Gly or Val apparently affected the redox properties of the bound FAD. The three-dimensional structure of FNR-S96V showed small conformational changes only in the isoalloxazine microenvironment. The FNR-E312L mutant has been obtained and its characterization is in progress.

The flavoprotein ferredoxin-NADP + reductase (FNR) is m a d e u p of t w o d o m a i n s , with FAD attached to the Nterminal region. The flavin is b o u n d to a ~-ba~el, interacting edge-to-face with a Tyr residue (Tyr in t h e p e a F N R ) . T h e r o l e of T y r 89 in F N R f u n c t i o n was, studied by site-directed mutagenesis on pea F~ expressed in Escherichia coll. Replacement of Tyr ~ by Fen rendered a stable protein with a 150-fold decrease of k c a t and an increase o f K m f o r t h e eacceptor. Substitution by Trp, Set, Gly strongly affected the stability of the protein and its ability for FAD binding. These mutant proteins were unstable and their Vmax and Km w~e strongly modified. Our results indicate that Tyr ~ plays a structural role in FAD binding, and that there is little size~ tolerance at t h i s p o s i t i o n . We conclude that FAD in FNRs is accommodated between a n edense aromatic system, with a defined volume and orientation .

Ottado de Rosario.

TYROSINE

Suipacha

107

Poster ii

PURIFICATION AND PROPERTIES EXPRESSED in E. coli

OF

PEA

P-9-006 THIOREDOXIN m

J. L6vea Jaramillo~ A, Chueca Sancho and I. L6pea Gorge.

Department of Plant Biochemistry, Estaeidn Experimental del Zaidfn (CSIC), Profesor Albareda I, 18008Granada (Spain). Thioredoxins (Td) are low molecular mass proteins (about 12 KDa) engaged in different processes. Chloroplast Tds activate of some organellar enzymes by a light mediated reduction mechanism. Td f modulates fruetnse-l,6-bisphosphatase (FBPase), whereas Td m aetivatea NADP-inalate dehydrogeanse (MDH). All the Tds so far sequenced show the active center -C-X-P-C-(Xffi Gly or Ala), wich fulfils the redox function throug the stablishment of a S-S bridge. To go deeply into the properties and specificity of Tds, a eDNA encoding pea thioredoxin m was cloned into pET12a. The recombinant protein, with 108 amino acids and a molecular mass of 12503 Da, shows 71% homology with Td m from spinach, but only 2527% with T d s f fsom pea and spinach. Like in some other algal and bacterial Tds, but not inftype ones, a FWA sequence appears before the active center. Td m was purified from E. coil rather easily, giving a protein (6 mg per liter of culture) homogeneus by SDS-PAGE and specific, as the native enzyme, againstTd m antibodies. This Td m activates the two chloroplastic enzymes, MDH and FBPase, using dithiothreitol asl reduetant. When the FPBase was incubated with Td ra and then filtered through a Superosel2 column, a FBPase-Td m complex was recovered, which shows FBPase activity without any exogen Td.

P-9-007 SPEC~ROSCOPZC AND KINETIC PLASTOCYANIN

CIIARACTERIZATZON

OF SITE-SPECIPZC

MUTANTS O F

~--~/IIi~, M. Ejdeb~ck, K. Sigfridsson, and O. Hansson, Dept. of Biochem. & Biophys., Lundberg Lab., G~teborg Univ. & chalmers Univ. of Techn., Medicinareg. 9C, S-413 90 06teborg, Sweden A system has been developed for the overexpression of the spinach plastocyanin (Pc) in E. coll and site-specific mutations have been introduced. When the Cu ligand His-87 is mutated to a Gln, the CII site is severely distorted to a nonblue (Type II) site. Tyr-83 in the acidic patch has been changed to a His. The protonation of His-83 (pKa 8.44 as determined by NMR) is accompanied by a 30 mv increase in the reduction potential. The electron transfer (ET) to photosystem (PS) I has been studied with flash-phctolysis techniques. The intracomplex ET rate for the Tyr83His mutant increases with pH, consistent with the pH-dependent driving force. Position 12 mutants display a much slower ET kinetics than the wild-type or the other mutants studied. The kinetics for the Leul2Glu mutant slows down with pH (pKa 6.6), presumably due to a deprotonation of Glu-12. The results suggest that Leu-12 in the hydrophobic patch is directly involved in the binding to P S I while Tyr-83 in the acidic patch is not.

P-9-009 LOCALIZATION OF FERREDOXIN-NADP REDUCTASE WITH AND WITHOUT N-TERHINAL BLOCK IN SPINACH THYLAKOID HEMBRANES N a s a t e r u S h i n ~, K o z o S a k a i 2, S h i g e h i r o O b a t a 3 & N a o k o ~ ~ Yamate College, Cbuo-ku, Kobe, 650; 2 S e k i s u i P l a s t i c s Co., T e n r i - s h i , N a r a 632; 3 K i s h i d a Chemicals, Co., ganda-shi, Hyogo 669-]3; ~Osaka Institute of Technology, Asahi-ku, Osaka 535, Japan K a r p l u s et al. r e p o r t e d t h a t N - t e r m i n a l a m i n o a c i d of s p i n a c h f e r r e d o x i n - N A D P r e d u c t a s e (FNR) is
P-9-010 NUCLEOTIDE BINDING TO P R O T O N - T R A N S L O C A T I N G TRANSHYDROGENASE FROM Rhodost?irillum rub~m T.Biz0u~rn t, C.Digglet , P.G.Quirk t , B.A.Levine t, C.M.Thomas 2 & I.B.Jackson t Schools of Biochemistry! & Biological Sciences2 , University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. Transhydrogenase couples the translocation of protons to the transfer of reducing equivalents between NAD(H) and NADP(H). I , vivo, it uses the protonmotive force (generated by photosynthctie or respiratory electron transport) to drive NAt)P* reduction for biosynthesis. Several groups have independently suggested that the energy-coupling steps occur at the level of the nucleotide-binding reactions. The enzyme from Rrubrura is unique in that its N A i l - b i n d i n g subunit Th, exists as a separate polypeptide. We have cloned the genes for this enzyme and over-expressed TI~ inE.coli; it can reconstitute coupled transhydrogenation activity to membranes depicted of Tl~. We are studying the nucleotide-blnding characteristics of Th, using three different techniques. (1) Directly, using radiolabelled nucleotides. (2) By recording the quenching offluoreacenee from a unique Trp. (3) By ~ the addition of NAD ÷ or NADH causes changes in the structure of a well-defined mobile loop which protrudes from the surface of the protein.

P-9-008 PURIFICATION AND CHARACTERIZATION OF THE SULFIDEQUINONE REDUCTASE FROM RItODOBACTER CAPSULATUS

P-9-Oll A N E W M E T H O D T O IDENTIFY F E R R E D O X I N - I N T E R A C T I N G P R O T E I N S AND C L O N E T H E GENES.

Sehfl~ M.*, Shahak, y.i, Padan, E&. and Hauska G.* */ Lehrstuhl f. Zellbiologie u. Pflanzenphysiolosie,Universitit Regensburg, 93053 Regensbur$ Germany;§/Instituteof Horticulture,the VoleaniCemer, AgriculturalResearchOrganization,P.O.Box 6, 50250Bet-Dagan,Israel;&/Divisionof Microbialand MolecularEcology,Instituteof Life Science, the HebrewUniversityof Jerusalem91904 Jerusalem;

M. I. Garcia-S~inchez, A. J. Vigara, C, Gotor, J. M. Vega. Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla y CSIC. Sevilla, Spain.

An enzyme catalizing sulfide qulnone oxtdoreduction (E.C.1.8.5..; SQR) has been purified in an active form from membranes of the non-sulfur purple bacterium Rhodobacter capsulatus. It is composed of a single polypeptide with an apparent MW of about 55kDa in SDS-PAGE. Gel filtration studies indicate, that it is active in a monomerlc form. The catalytic activity of the purified enzyme is similar to the membrane-bound form in its kinetic parameters. The UV and visible absorption and fiuoreacettee spectra are typical for a flavoprotein. We have sequenced 18 amino acid residues of the N-terminus and found, that this sequence contains the highly conserved fingerprint of the NAD/FAD-binding domain of many oxidoreductases (Wlerenga, R.IC et ai~ (1989), J.Mol.Biol.lg7; 101-107). All this suggests, that the SQR from Rhodobacter capsulatus is, like the SQR from the cyanobacterium Oscillatoria limnetica (Arieli, B. et al. (1994), J.Biol.Chem. 269(8); 5705-5711), a membrane-bound fiavoproteln and that the electron transfer between sulfide and qutnone Is mediated by FAD. The results of our efforts to clone and sequence the gene of the SQR will be reported.

108

Ferredoxin (Fd) is a central protein for transferring electrons to Fddependent enzymes in the chloroplasts of phothosynthetic organisms. Our final goal is to develope a method to clone cDNAs encoding proteins interacting with Fd. The first step has been to design a detection system of interacting proteins in crude extracts of Chlamydomonas reinhardtii with biotin-labeled Fd. For this purpose, we have used Fd-glutamate synthase as a positive control of Fd-interacting protein as has been previously described'. Different conditions as Fd concentration, incubation time and temperature, blocking agents, biotin-avidin interactions have been studied to get the best ratio signal/background. The procedure we will describe is efficient to detect Ferredoxin-interacting proteins, and it is actually being used to screen Chlamydomonas expression libraries. IVigara AJ, Garcfa-S~nchezMI, G6mez-Moreno C, Vega JM (1994) Biol. Plant. 36:S170.

Supported by DGICYT Grant PB93-0735.

Poster P-9-015

P-9-012 Site-Directed Mutagenesis of Acidic Patches of Plastocyanin

C H A R A C T E R I Z A T I O N OF THE NAD(P)H.PLASTOQUINONEOXIDOREDUCTASE FROM MAIZE THYLAKOID MEMBRANES

B.H.Lee*, T.Hibino**, Tet.Takabe* & Ter.Takabe** *BioScience Center, School of Agricultural Science, Nagoya Univ., Chikusa-ku, Nagoya 464-01, Japan and **Dept. of Chem., Fac. of Sci. & Teehnol., Meijo Univ., Tenpaku-ku, Nagoya 468, Japan

E. Funk & K. Steinmfiller Institut fiir Entwicklungs- und Molekularbiologie der Pflanzen, HeinrichHeine-Universitiit,Universittitsstral]e 1, 40225 Diisseldorf, FRG

To investigate the role of two highly conserved negative patches, residues #42-45 and #59-61, on the surface of plant plastocyanin, six mutants were constructed by site-directed mutagenesis. The mutants were designed to systematically incorporate positive charges into the negative patches, and net charge on negative patches was modified from -4 to + 1. The mutant proteins were expressed in E. coli and purified. Electron transfer reactions of mutant plastocyanin with cytoehrome f as well as P700 of PSI complexes from cucumber were examined. The electron transfer from plastoeyanin to P700 of PSI complexes from C h l a m y d o m o n a s , A n a b a e n a bariabilis, and Synechocystis sp. PCC 6803 were also examined.

P-9-013 I AND

ON THE INTERACTION OF PLASTOCYANIN WITH PHOTOSYSTEM CYTOCHROME B F -COMPLEX.

J.Reichert~ J.Illerhaus, L.Altschmled*, R.B.KI6sgen*, R.G.Herrman* and W.HaehneL Biochemie der Pflanzen, Unlversit&t Freiburg, FRG; *Botanisches Institut, Universit&t Mfinchen, FRG. The electron transfer fi'om the cytochrome bf-complex (cyt bf) to Photosystem I (PSI) is mediated by plastocyanin (Pc). We have studied both reactions using site-diracted mutated spinach Pc, over-expressed in E. cog The changes in amino acid sequence have been verified by electrospray mass spectrometry. W'~h respect to electron transfer from the copper center of Pc to the surface of the protein, three different routes have been discussed. One of them involves H87 at the hydrophobic northern end, another passes through Y83, and the third may be the string of aromatic amino acids in the interior of the Pc molecule. We could show that the path via H87 is the most likely for the electron transfer from Pc to PT00. We further demonstrated that the fiat hydrophobic surface is essential in the docking process. The two acidic patches near Y83 are the other important features, which we have converted to neutral residues to various degrees. The electron transfer from isolated cyt f t o the copper center of Pc via Y83 has been shown by He et al.. We have extended the previous studies using our mutant Pc for stopped flow experiments with intact cyt bf-complex of homologous origin.

P-9-014 DYNAMIC INTERACTIONS BETWEEN PLASTOCYANIN AND PHOTOSYSTEM I F. Drepper, M. Hippler, and W. Haehnel Lehrstuhl fiir Biocbemie der Pflanzen, Albert-Ludwigs-Universita't,Schttnzlestr. 1, D-79104 Freiburg, Germany We have studied in detail the interaction between plastocyanin (Pc) and Photosystem I (PSI) isolated from spinach. The laser-induced kinetic component of PT00+ reduction with a constant halftime of 11 ~s has been used to detect the complex between reduced Pc and PSI. The second-order binding of Pc to PSI with oxidized P700 was analysed from the reduction of the residual P700+. The competitive effect of oxidized Pc on the complex formation and the electron transfer rates with reduced Pc have been used to determine the binding equilibrium between each of the oxidized and reduced species. The dissociation constant of the complex between oxidized Pc and PSI is almost an order of magnitude larger than that between reduced Pc and PSI. Consistent with this finding is an increase of the midpoint redox potential of Pc from 360 mV in solution to about 410 mV when it is bound to PSI. Thus, the driving force of the electron transfer from Pc to P700+ is diminished at the expense of an optimized binding of reduced and release of oxidized Pc. Double-flash experiments show that the kinetic limitation of the turn-over of PSI is the release of oxidized Pc, enabeling the next Pc molecule to bind to the specific site at the PSI complex. All data are consistently described by a model with one single binding site. The structural orientation of the components in the complex is discussed.

The first complete sequences of plant plastid genomes revealed open re.ading flames homologous to genes for mitochondrial complex I (NADH-ubiqumoneoxidoreductase). These open reading frames were named ndh-genes and it was proposed that they encode subunits of a putative NAD(P)H-plastoquinoneoxidoreductase. Our objective is the isolation and characterization of this enzyme from higher plant plastids. We have used different ndh-genes from tobacoo and rice for the production of recombinant proteins via expression vectors. Antibodies raised against these proteins were able to detect the corresponding NDH-proteins in the chloroplasts of several species of higher plants. For the purification of the enzyme, the NDH-proteins were solubilized from maize thylakoids by limited, detergent-mediated lysis of the membranes. Two antibodies against NDH-A and NDH-H were separately coupled to proteinA-sepharose and used for immunoaffinity purification. With both antibodies we were able to isolate two identical protein fractions which consisted of about 20 different polypeptides. Western blot analysis of both fractions demonstrated the presence of NDH-H, -J and -K, indicating that they contain the oxidoreductase complex.

P-9-016 C H A R A C T E R I S A T I O N OF AN NAD(P)H D E H Y D R O G E N A S E F R O M PEA AND SPINACH THYLAKOIDS L . A . S a z a n o v & P.J.Nixon Dept of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AY, U.K.

Genes encoding possible subunits of an NAD(P)H dehydrogenase have been found in the plastid genome of several higher plants. However, a thylakoid-bound NAD(P)H dehydrogenase has yet to be purified and characterised. We have found that under suitable conditions thylakoid membranes from pea and spinach display significant NAD(P)H-dehydrogenase activity. The possible role of the n d h gene products in this activity will be discussed.

P-9-017 NON-PHOTOSYNTHETIC TYPE FERREDOXIN FROM MAIZE RETARDED PHOTOAUTOTROPHIC GROWTH OF THE CYANOBACTERIUM PLECTONEMA B O R Y A N U M T. Matsumura, Y. Fujita and T. Hase Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan

M~dze ( Zea mays L.) has two types of ferredoxin (Fd); FdI is a photosynthetic type and FdllI is a non-photosynthetic type. To examine the functional differentiation of the two Fds in photosynthetic cells, these Fd cDNAs were separately expressed under control of trc-promoter and lacl q in the cyanobacterium Plectonema boryanum. Both Fdl and Fdlll were induced by IPTG to a comparable to the cyanobacterial Fd, and both Fds were present in the holt forms in the cyanobacterial cells. When the FdllI-expressing cells were cultivated in photoautotrophic conditions, the growth was retarded. The extent of the retardation depended on the level of Fdlll expressed, and the transformants with the highest expression level became lethal. This growth retardation was partially recovered by supplying with glucose into the medium. In contrast, the expression of FdI did not show such effect. These results suggest that the maize non-photosynthetic Fd may disturb the photosynthetic electron flow in the cyanobacterial coils.

109

Poster

P-9-021

P-9-018 eDNA CLONING AND FUNCTIONAL EXPRESSION OF FERREDOXlN-DEPENDENT SULFITE REDUCTASE FROM MAIZE IN E . c o l l CELLS T. Ide~uchi, T. Akashi & T. Hase Inst. f o r Protein Research, O s a k a University, Suita, O s a k a 565, J a p a n In h i g h e r p l a n t , sulfite r e d u c t a s e (SIR) c a t a l y s e s p r o d u c t i o n o f S2f r o m SO3z u s i n g f e r r e d o x t n (Fd) as a n e l e c t r o n d o n o r . We h a v e c l o n e d a cDNA e n c o d i n g p r e c u r s o r o f m a i z e SiR c o m p o s e d o f 6 3 5 a m i n o acids. T h e d e d u c e d a m i n o a c i d s e q u e n c e exhibits s i m i l a r i t y to s u b u n i t o f E.coll NADPH-SiR a n d h a s a n N - t e r m i n a l e x t e n s i o n . T h e p r e c u r s o r s y n t h e s i z e d In vitro w a s t r a n s l o c a t e d i n t o c h l o r o p l a s t to be p r o c e s s e d to the m a t u r e f o r m w i t h a m o l e c u l a r m a s s o f 65kDa. A cDNA e n c o d i n g t h e m a t u r e p a r t of SiR w i t h a p u t a t i v e ATG c o d o n i n t r o d u c e d w a s e x p r e s s e d in E.coll, a l t h o u g h t h e e x a c t N - t e r m i n u s o f t h e m a t u r e p r o t e i n w a s n o t y e t d e t e r m i n e d . T h e t r a n s f o r m e d cells p r o d u c e d t h e 6 5 k D a p o l y p e p t i d e , a n d F d - d e p e n d e n t SiR a c t i v i t y was f o u n d . The level o f e n z y m a t i c a l l y active SiR w a s l i m i t e d in E.coli cells b y a v a i l a b i l i t y o f s i r o h e m e as is the case f o r E.coll SIR, a n d c o - e x p r e s s i o n o f s i r o h e m e s y n t h a s e r e s u l t e d in a n r e m a r k a b l e i n c r e a s e o f the activity. Km f o r Fd a n d specific a c t i v i t y o f the r e c o m b i n a n t SiR w e r e e q u i v a l e n t to t h o s e o f t h e a u t h e n t i c m a i z e SiR.

P-9-019 LOCALIZATION OF FERREDOXIN-NADP+-OXIDOREDUCTASE (FNR) IN THE THYLAKOID MEMBRANE Shl-Gui Yu & Per-~ke Albertsson DepertmentofBiochemistJy, University of Land, P.O. Rox 124, 8-221 00 Lund, Sweden Two groups of polypeptides of FNR identified by antibodies raised against purified FNR and microsequence analysis are present in the 2-D protein pattern. In order to estimate the distribution of the different types of FNR in the thylakoid membrane, the freshly prepared thylakoid membranes of spinach chloroplasts were washed 5 times in 5 mM NaC1/10m.M Tricine-NaOH pH 7.8, then fractionated with a combination of sonication and an aqueous two phase partitioning. It was observed: [I], FNR is distributed all over the thylakoid membrane. Its absolute amount is more in the stmma lamelino than in the grana stacks of the thylakoid membrane. The concentration of FNR pet PT00, in contrast, is higher in the grana than that in the stroma lamellao. [II], The amount of FNR removed from the grana-derived membrane fraction is much more significant than from the stroma lamellae-dedved membrane fraction. About a half was removed from the grana but only a few percentage was removed from the stroma lamellae, indicating that in the grana region of the thylakoids the loosely-membrane bound type of ~ is enriched while in the stmma la~eline region of the thyinkoids, the tightly-membrane bound type of FNR is enriched. These data would be in favour of a proposition that the tighdy binding type of the FNR located in the stroma lamellae region could carry out the cyclic electron transport around PSI~ while its loosely binding type preferentially located in the grana region of the thylakoids would mediate the linear electron transport from H20 to NADP* in this region.

P-9-020 M. HirasawaI , M. M. Dosex, Z. Salamonz, E. LewI , G. Tollin 2 and D. B. KnaffI ZDepartment of Chemistry & Biochemistry, Texas Tech University, Lubbock, Texas 79409, USA; Department of Biochemistry, University of Arizona, Tucson, Arizona 85721. Glutamate synthase (GOGAT) and n i t r i t e reductase are chloroplast-located enzymes that both use reduced ferredoxin as the electron donor for important reactions in the early stage of assimilatory nitrogen metabolism in algae and plants. We have recently re-examined the prosthetic group content of spinach leaf glutamate synthase and demonstrated that the only prosthetic group present in addition to a single [3Fe-4S] cluster is a single FMN. The redox properties of the enzyme have been characterized by cyclic voltammetry and deazariboflavin phototitrations. Flash photolysis was used to study the kinetics of GOGAT. N i t r i t e reductase and glutamate synthase appear to have similar, positively-charged binding sites for ferredoxin. Chemical modification experiments using phenylglycol and pyridoxal phosphate have established the presence of both arginine and lysine at the ferredoxin-binding sites of both n i t r i t e reductase and GOGAT. In the case of n i t r i t e reductase, specific amino acids at the ferredoxin-binding site have been identified.

I10

STRUCTURE OF THIOREDOXIN-2 FROM ANABAENA 7120 Markku $aarmen1, Florence K. Gleason2 and Hans Eklund1 1 Department of Molecular Biology, Uppsala, Sweden 2 Department of P!ant Biology, U of MN, St. Paul, USA The unusual thioredoxin-2 (TRX-2) from Anabaena 7120 IGtaason, 1992, J. BacterioL, 174, 2592-2598) was studied by protein crystallography. The structure was solv,)(J using the molecular replacement method, and refined to high resolution, in two crystal forms. This is the first high resolution structure of a thioredoxin from a photosynthetic organism, and gives an insight to how chloroplastic thioredoxins may interact with their target proteinio peptides (MDH, malate dehydm,'~erJase, FBPsse, fructose-bis-phosphatase). The main crystal contacts in the hvc crystai forms are made between amino- terminal five residues and residues in the protein interaction area of crystallographic.ally rebated molecules. Here parallel hydrogen bonds are found between the N-terminal peptide and residues right before the well conserved cis-pmline 76, close to the active site. Based on these observed contacts (as found in our Trx-2 crystals) and homology modeling of the Trx-f and Trx-m, we examined the interaction of thioredoxins with MDH and FBPase. The redox-regulated peptide of FBPase can form favorable charged intematinns with Trx-f and Trx-2, when bound in parallel mode to the protein interaction area of these thioredoxins. On the opposite, charge repulsion in parallel binding mode clearly can prevent both regulatory sites of MDH(NAPDH) from binding to Trx-f and Trx-2. The non-specifmity of Trx-m may be explained by the replacement of many charged residues with non-charged ones.

P-9-022 T H E D O C K I N G O F P L A S T O C Y A N I N W I T H C Y T O C H R O M E f. and D.C.Pearson, Jr. Dept. of Biochemistry, and Biophysics P r o g r a m , The Ohio State University, Columbus, OH, 43210, U S A In this study, an hypothesis is proposed to explain the docking between plastocyanin (PC) and cytochrome f (cyt f) prior to electron transfer. PC, a 10kDa copper protein, and cyt f, a 27 kDa home protein, are reaction parmers in the photosynthetic electron transport chain. Electrostatic calculations and manual docking procedures were used to analyze the docking o f the two proteins. Three potential docking positions were identified. The most significant electrostatic attraction between the two molecules (Dock 1) occurs when the negative patch o f P C (residues 42-44, 59-60) and positive patch o f cyt f (residues 58, 65, 66, 122, 187, 209) are oriented facing each other. However, in this dock, neither of the k n o w n electron transfer sites on P C (Y83 and H87) are within 10 A o f cyt f. Therefore this electrostatic attraction likely represents a pre-docking complex between PC and cyt f. The second dock (Dock 2) places D44 on PC next to K187 on cyt f a n d H 8 7 on PC adjacent to Y1 and the berne on cyt f. This is more likely to be a final electron transfer complex. D o c k 3, which shows more steric hindrance than D o c k 2 places Y83 on PC adjacent to Y1 and the home on cyt f.

P-9-023 THE ROLE OF THE PUFX PROTEIN IN THE PHOTOSYNTHETIC ELECTRON TRANSFER OF RHODOBACTER SPHAEROIDES W.P. Barz1, J.W. Farchaus 3, F. Francia 2, B,A. Melandri 2, D. Oestsrhelt 1 and G. Venturo!i 2 1Max-Planck Institut fur Biochemie, Martinsried, Germany; 2Dipartimento di Bioiogia, Bologna, Italy; 3Bacteriology Division, US Army Medical Research Institute for Infectious Diseases, Fort Detrick, Frederick. MD, USA Recent work indicated that the PufX protein is essential for photoheterotrophic growth of Rb. sphaeroides under unaerobic conditions end led to the proposal that the PufX protein facilitates the redox interaction between the reaction center (RC) and the cytochrome bc 1 complex. We tested this hypothesis by examining the kinetics of partial reactions of the chain in redox*poised chromatophores from a pufX', a pufX + and two suppressor mutants. The requirement of PufX exhibits an opposite redox dependence for the functionality of the RC and of the cyt bc 1 complex. Under oxidizing conditions the kinetics of single turnover cyt b561 reduction is dramatically inhibited in chromatophores from X- mutants showing that the availability of QH 2 molecules coming from the RC is drastically reduced in the absence of PufX. On the other side, multiple turnover of the RC primary donor is inhibited in pufX- chromatophores only under reducing conditions, indicating a requirement of PufX for the replacement of QBH2 by Q. The data point to a role of PufX in organizing a macromolecular antenna structure which allows efficient ubiquinol/ubiquinone exchange at the QB site of the RC.

Poster

P-9-024 THE HIPIP FROM RHODOFERAX FERMENTANS RAPIDLY REDUCES THE CYTOCHROME C-556 ASSOCIATED WITH THE PHOTOCHEMICAL REACTION CENTER A. Hochkoeppler~, S. Ciurlib, G. Venturolia, D. Zarmonia adept, of Biology, Bologna University, Irnerio 42, 40126 Bologna (1) bInst, of Agric. Chem., Bologna University, Berti-Pichat 10, 40127 Bologna (I) The functional role of the High Potential Iron-sulfur Protein (HiPIP) isolated from the phototroph Rhodoferax fermentans was studied through kinetic spectrophotometry. The HiPIP was found to deliver electrons to a c-type cytochrome ((x maximum at 556 nm, cyt c-556) associated with the photochemical reaction center (RC). Cyt c-556 reduction by the HtPIP gave almost identical results w h e n studied either in the c~ or in the Soret band. Moreover, direct detection of HLPIP oxidation was performed recording the light-induced absorbance changes at 589 nm, an isosbestic point for cytochrome(s) and RC difference spectral contributions. The kinetics of both cytochrome reduction (at 556 and 425 run) and HiPIP oxidation (at 589 nm) were found to be in good agreement, showing that complete cytochrome reduction by the HiPIP occurs in a ms time domain.

P-9-025 PHOTOSYNTHETIC IN PROTECTION

FERREDOXIN-NADP + REDUCTASE AGAINST OXIDATIVE DAMAGE

IS

INVOLVED

N. C a r r i l l o & A. K r a p p Dto. Cs. Biol6gicas. Fac. de Bioqu~mica. U n i v e r s i d a d de Rosario. S u i p a c h a 531. 2000 Rosario. A r g e n t i n a coli cells c a r r y i n g the m y r A m u t a t i o n are e x t r e m e l y s e n s i t i v e to o x i d a t i v e stress, and do not g r o w a e r o b i c a l l y in the p r e s e n c e of m e t h y l viologen, a radical propagator. The myrA gene was cloned (Bianchi et al. 1993, J. Bact. 175, 1590), and s h o w n to be r e l a t e d to p l a n t f e r r e d o x i n - N A D P + r e d u c t a s e s (FNR). A w i l d - t y p e p h e n o t y p e c o u l d be r e s t o r e d to m v r A c e l l s b y i n t r o d u c i n g a c l o n e d p e a FNR gene. C o m p l e m e n t a t i o n was d e p e n d e n t on expression of a f u n c t i o n a l FNR, since m u t a t e d i n a c t i v e e n z y m e s d i d not r e s c u e the w i l d - t y p e phenotype. F N R is a b e t t e r r a d i c a l s c a v e n g e r than s u p e r o x i d e d i s m u t a s e (SOD), as j u d g e d b y c o m p a r i n g the v i a b i l i t y of m y r A c e l l s a n d of a s o d A s o d B mutant. FN-R does not c o m p l e m e n t SODcells, s u g g e s t i n g that the two s c a v e n g e r s o p e r a t e t h r o u g h d i f f e r e n t pathways. We p r o p o s e that b e s i d e s its role in p h o t o s y n t h e s i s , F N R c o n t r i b u t e s to the p l a s t i d c o n c e r t e d d e f e n s e a g a i n s t o x i d a t i v e damage. Escherichia

P-9-026 CHARACTERIZATION OF THE ELECTRON TRANSFER COMPLEX BETWEEN HiPIP AND PHOTOSYNTHETIC REACTION CENTRE FROM R UBRIVIVAX GELA TINOSUS. B. Schoepp 1, W. Nitsehke 1 & A. Riedel2 BiOlogie II, Biochemie der pflanzen, University of Freiburg/FRG; Inst. ftir Biop,physik und Physikalische Biochemie, University gensourg/t~t.~J

of

Dried oriented membranes from Rubrivivax gelatinosus S-I were studied by EPR. The spectrum of membranes in the oxidized state showed a strong signal from oxidized HiPIP demonstrating that a significant fraction of this small electron transter protein remained associated to the membranes after ultracentrifugation. No observable orientation was detected. However, when membranes were prereduced by aseorbate and subsequently illuminated at 290 K followed by rapid freezing under illumination, the characteristic spectrum of the HiPIP appeared. The sig~l., corresponds to roughly 10 % of the full observed complement in oxidized membranes. This result shows that a ~action of the'HitPIP forms a complex with the reaction centre competent in electron transfer. This fraction was well-oriented showing that the complex exists in a well-defined conformation. The part of HiPIP involved in this complex was studied to more detail in samples where it was chemically erosshnked to the reaction centre. Furthermore, in order to examine the binding properties of the I-fiPIP to the tetraheme, we compared the redox potential oT the HiPIP in solution and in the complex.

P-9-027 AN NMR STUDY OF THE SOLUTION STRUCTURE OF PSAD Z Xia I , JH Golbeek 2, DA Bryant3, RW Broadhurst 1, ED Lane I & DS ~ndall I lDepartment of Biochemistry, University of Cambridge, England; 2Deparunent of Biochemistry, University of Nebraska, Lincoln, NE; 3Depastment of Cell & Molecular Biology, Pennsylvania State University, University Park, PA. PsaD is located at the stromal side of the PSI reaction centre and is believed to play an important role in transferring electrons from PsaC to ferredoxin. We are detenninin~ the solution structure of PsaD whh multi-dimensional NMR techniques. The PsaD gene ofNostoc sp. PCC 8009 was expressed in E.coli grown on a minimal medium containing ]5NH4CI, and the 15N-labelled protein was purified as previously described (Li et al, Biochemistry 30, 7863 (1991)). 15N-1H-HSQC spectra of PsaD indicate that a large ~ of the protein is not well structured and that there are flexible regions. From a 2D 15N-IH-HSQC-COSY spectrum we roughly estimate, using chemical shifts as an index (Wishart et al, FEBS Lett. 293, 72 (1991)), that the helical part accounts for 19% at mnst, [~strand around 13% and random coil at least 54%. At least three dynamic domains may exist in this protein, as indicated by 15N T2 relaxation measurements. 15N-1H-HMQCspectra at several different pH values from 5.7 to 8.5 suggest that there is a eonformational equilibrium in slow exchange with a pKa of about 6.3 and that the structured part of the acidic form is more open for proton exchange. Similar spectra in the presence of unlabelled PsaE show that thereis an interaction between the two proteins.

P-9-028 KINETIC AND MOLECULAR BIOLOGICAL STUDIES OF CYTOCHROME F FROM PHORMIDIUM L A M I N O S U M M.J. Wa~ner, J.C.L. Packer, C.J. Howe and D.S. Bendall Department of Biochemistry, Tennis Court Road, University of Cambridge UK Plastocyanin and cytochrome f are components of the electron transfer chain between Photosystems II and I. The reaction between the higher plant proteins has been studied previously and a model of the interaction has been proposed based on the the known structure of both proteins. A striking feature of most cyanobacterial plastocyanin sequences, however, is that these proteins lack socalled acidic patches, clusters of negatively charged residues, which are thought to play a key role in the interaction of higher plant plastocyanin with cytochrome f. Here we present the sequence of the gene for cytochrome fcloned from the moderately thermophilic cyanobacterium P. laminosum. This sequence and that of plastocyanin from the same organism show striking charge inversions compared with the higher plant counterparts. The effects of ionic strength on the rates of reduction of various redoxproteins by the cytochrome bf complex isolated from P. larninosum reveals a kinetically significant charge inversion. Based on the known structure of the higher plant proteins and the sequences of both P. laminosum proteins, a site of the interaction with plastocyanin is proposed for cytochrome f.

P-9-029 FERREDOXINFROM THE CYANOBACTERIUMSYNECHOCYS~IS PCC6803. Mireille Poncelet1, Corinne Chauvat2, St~phane VilloingI and Franck Chauvat1. 1Service de Biochimie et G6n6tique Mol~,culaire and 2URA D1290 CNRS, CEA Saclay, F-91191 Gif sur Yvette CEDEX,France. The (2Fe-2S) plant-type ferredoxins (Fd) are small acidic proteins that function as electron acceptor to photosystem (IS) I and are envolved in many metabolic pathways such as carbon and nitrogen assimilation. In cyanobacteria, iron-limitation triggers the synthesis of fiavodoxin (Fly) in place of Fd which is no longer produced under this condition. Our aim is to use saturation mutagenesis in order to analyse the structurefunction relationships of Fd and its interactions with PSI subunits. We have therefore isolated and sequenced the Fd encoding genes from the unicellular cyanobacterium Synechocystis PCC6803, which can grow in absence of photosynthetic activity. We will show that the Fd encoding gene is essential, even under heterotrophic growth conditions. The Fd gene-deleted strain was obtained using our temperaturecontrolled expression vector (Mermet-Bouvier P. and Chauvat F. (1994): Current Microbiology 28, 145-148.). The behaviour of this deleted strain in various environmental conditions (i.e. photnautotrophy, photoheterotrophy, heterotrophy and iron limitation) will be presented, as well as the phenotype of some strains harbouring mutations in the Fd gene. We will also report the analysis of the promoter region of the Fd gene.

111

Poster

P-9-030 CONSERVED NON LIGANDING RESIDUES OF R H O D O B A C T E R (R.) CAPSULATUS 'RIESKE' PROTEIN ARE ESSENTIAL FOR 2FE2S CLUSTER PROPERTIES AND QPOOL COMMUNICATION

CLONING AND EXPRESSION OF cDNAs CODING FOR THE SPINACH FERREDOXlN:THIOREDOXIN REDUCTASE

Ursula Liebl 1, Vladimir Sled2, Tomoko Ohnishi2, and Fevzi Daldal 1 1Dept. of Biology. and 2Dept of Biochemistry & Biophysics, Univ. of Pennsylvania, Philadelphia, PA 19104, USA

E. Gaymard and P. Sch0rmann, Laboratoire de Biochimie vd,g~tale, Universit~ de Neuch~tel, Rue Emile Argand 11, CH-2007 Neuchfitel, Switzerland

The amino acid residues T134 and L136 which are adjacent to the two designated cluster ligands C133 and H135 in the 'Riesk¢' 2Fe2S protein of R. eapsulatus were genetically altered to study the effect of these substitutions on the cluster properties, and to probe structure-function relationships. The Ti34R, H and G substitutions are photosynthetically competent (Ps +) and show EPR characteristics similar to wild type although their midpoint potentials are as low as +210 mV and the 2Fe2S clusters are sensitive to oxygen. Revertant analysis of T134D indicates that a negative charge is destructive at this position of the protein. All L136 substitutions (L136R, H, G. D) are Ps--Their EPR spectra show no response to the quinone pool in the membrane. An isolated second site suppressor revertant of L136H has a compensatory mutation of V4-4L. Hydrophobicity is the primary requirement for an amino acid residue at position 136 in order to be functional.

Ferredoxin:thioredoxin reductase (FTR) is the key enzyme of the ferredoxirVthioredoxin system, the light-dependent enzyme regulatory system in oxygenic photosynthesis. It is a nucleus encoded iron-sulfur protein, composed of two dissimilar subunits, of about equal size. Subunit A is of variable size when comparing different FTR and has no known catalytic function. Subunit B, the catalytic subunit, is rather well conserved between different organisms. It contains a redox-ective disulfide bridge functional in the reduction of thioredoxins and a [4Fe-4S] cluster. Based on the spinach protein sequence we have isolated and characterized fulllength cDNAs coding for both subunits. Using PCR we have made a dicistronic construction carrying, in phase and on the same DNA strand, the coding parts of the genes for both subunits. E.coli calls have been transformed with this construction and the recombinant protein has been expressed.

P-9-031 STRUCTURAL CONSTRAINTS FOR THE FORMATION OF [3Fe4S] AND [4Fe.4S] CLUSTERS IN FREE AND REBOUND PsaC

PHOTOSYSTEM I / FERREDOXIN INTERACTION : MUTAGENIC STUDY OF PSI-D

~

1 , Qiao Fengyu2, Donald A. Bryant3, and John H, Golbock 2 1.I.Depann~.nt of Chemislry, Addis Ababa University, Ethiopia; 2Departnlcnt of mucnemistry, University of Nebraska, Lincoln, NE 68583-0718; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802.

Cysteines 14, 21, 34, 51 or 58 were replaced with aspartate (C51D, C58D), serine (C14S, C34S, C51S), or alanine (C14A, C51A) in free PsaC of PS I. C34S contained two [4Fe-4S] clusters, consistent with the premise that C14, C21, (251, and C58, but not C34, provide figands to the cubane irons. C14D, C51D, C14S, and C51S contained S=1/2 [4Fe-4S] clusters, sub-stoichiometric [3Fe-4S] clusters, and S = 3/2 [4Fe-4S] clusters. The E m of the [4Fe-4S] clusters in the FA sites of C14S and C14A were -520 mV and -551 mV, and in the F n sites of C51S and CSIA were -495 mV and -575 mV. The C14D/C51D double mutant contained [3Fe-4S] and S=1/2 [4Fe-4S] clusters, showing that the 3Cys.lAsp motif acoomodates both types of centers. Electron transfer to FA/FB occured when C34S and C51S were rebound to P'/00-F x cores, but functional reconstitution has not yet been achieved for C14A, C14S, C51A and C14D/C51D. According to this study, PsaC requires two iron-sulfur clusters to refold, one of which must be a cubane. Two S=1/2 [4Fe-4S] clusters are invariably found in rebound complexes; hence, the presence of two cubanes appears to be a necessary precondition for in vitro binding to PT00-Fx cores.

P-9-032 BIOCHEMICAL CHARACTERIZATION AND THREEDIMENSIONAL STRUCTURE OF DIMERIC HIGH POTENTIAL IRON-SULFUR PROTEIN FROM CHROMATIUM PURPURATUM Cheryl A. Kerfe!0, Cheryl Chan and Todd O. Yeates Molecular Biology Institute, University of California, Los Angeles The High Potential Iron-Sulfur Protein (HiPIP) from the purple-sulfur bacterium Chromatium purpuratum has been purified and characterized and the crystal structure has been determined at 2.7/~ resolution. This is the first report of a HiPIP that is isolated as a dimer (based on size-exclusion FPLC, filtration retention and electrophoresis data), although EPR data and previous crystallographic studies of HiPIPs from other bacterial species suggested that the protein may form dimcrs. C. purpuratum HiPIP is further distinguished by its extremely high midpoint potential (~+400 mV). C. purpuratum HiPIP crystallizes in space group P21212 with three molecules (one and one-half dimers) in the asymmetric unit and the structure was solved by molecular replacement. The amino acid sequence of C. purpuratum HiPIP was determined by N-terminal sequencing of protein derived from crystals and from an examination of electron density maps. The biochemical characteristics and crystal structure of C. purpuratum HiPIP will be discussed in the context of its putative function as an electron donor to the reaction center.

112

P-9-033

P-9-034

Haniey. J.A.. Sdtif,P. and Lagoutte, B. CEA-Service de Bio~nergdtique / CNRS-URA 1290, DBCM,C.E. de Saclay 91191 Gif sur Yvette Cedex, France Photosystem-I (PS-I) is a multi-subunit pigment protein complex which functions as a plastocyanin-ferredoxin oxido-reductase. Crosslinking data and polypeptide analysis has lead to the proposal that binding and orientation of soluble ferredoxin is facilitated by two peripherally bound PS-I polypeptides, PSI-D and PSI-E. The mutagenic analysis of PSI-D from Synechocystis 6803 has indicated those regions of the polypeptide which are important for : a) the correct binding and orientation for first order reduction of soluble ferredoxin by photosystem-I, b: integration of PSI-D into the Photosystem-I complex, and c) the stabilisation and orientation of the peripheral bound iron sulphur protein, PSI-C. This work describes in detail the characterisation of both deletion and site directed mutants.

P-9-035 RECONSTITUTION OF THE 2Fe-2S CENTER INTO OVERPRODUCED CYANOBACTERIAL RIESKE PROTEIN AND MUTAGENESIS OF THE CYT B6 QUINOL BINDING DOMAIN X. Wu, A. I. Tsapin, Y.-S. Cho, L. Genzlinger, T.-X. Lee, B. Holton, T. Kall&s. University of Wisconsin, Oshkosh, WI 54901, U.S.A. We have overproduced in E. coli the Rieske protein from the cyanobacterium Nostoc PCC 7906. The 2Fe-2S center with its characteristic g = 1.90 electron paramagnetic resonance (EPR) signal have been restored in vitro and the protein purified as judged by SDS-polyacrylamide gel electrophoresis. To aid in purification and to investigate domains required for insertion of the 2Fe-2S center, we have genetically constructed Rieske proteins that carry a removable, N-terminal, "His-tag" for affinity purification and that bear N- or C-terminal deletions. One such deletion protein (RND2) lacks the N-terminal hydrophobic domain and is water soluble. In vitro reconstitution and chamcter~tion of Fe-S centers in these mutant Rleske proteins is in progress. To investigate inhibitor sensitivity and function of the quinol-oxidation domain, we have created the substitution mutations D141G, A147G, S 152A, A152T, and H202Q in the SynechococcusPCC7OO2petB geneforcytochromeb6. As determined by allele-specific p o l ~ chain reaction, each of the mutant alleles (with the exception of H202Q) has replaced the corresponding wild-type allele. Retention of wild-type gene copies in the H202Q mutant is consistent with an essential, heine-binding role for His202. Charaetedzation of these mutants is in progress.

Poster P-9-036 1H and 15N NMR sequential assignment and 3D structure model of [2Fe-2S] ferredoxin from Synechocystis sp. PCC 6803 C. Lelon~(a), P. S6tif(a), H. Bottin(a), F. Andr6(a) & J.-M. Neumann(b) (a) Service de Bio~nerg6tique / URA-CNRS 1290, (b) Service de Biologic des Prot6ines membranaires, D6partement de Biologic Cellulaire et Mol6culaire, C.E. de Saclay, 91191 Gif sur Yvette Cedex, France [2Fe-2S] ferredoxin 3D structure purified from Synechocystis sp. PCC 6803 h a s been studied by 1H a n d 15N N u c l e a r Magnetic Resonance. Sequence-specific 1H a n d 15N a s s i g n m e n t of a m i n o acid r e s i d u e s far from the p a r a m a g n e t i c cluster h a s b e e n p e r f o r m e d . I n t e r - r e s i d u e N O E constraints h a v e a l l o w e d the identification of the secondary structure. Following the analysis of inter-residue NOEs, the existence of a disulfide bridge between the c y s t e i n e r e s i d u e s 85 a n d 18 w a s s u g g e s t e d . S t r u c t u r e modeUisation w a s performed, u s i n g XPLOR calculations, with two h y p o t h e s e s : w i t h or w i t h o u t a d i s u l f i d e bridge. T w o s t r u c t u r e families were also o b t a i n e d w i t h a r m s d e v i a t i o n a r o u n d 2 A, respectively. A m o d e l of s e c o n d a r y s t r u c t u r e is proposed.

P-9-037 Interaction of Thioredoxin with CFoCF1 O. Schwarz. I. Ditmer-Knappmann and H. Strotmann Institut f'tir Biochemie der Pflanzen, Heinrich Heine Universit~it Dtisseldoff, Germany Investigation of thioredoxin specificity of the thylakoid ATPase for thiol modulation suggests that Td-f~ rather than Td-mr~ is the physiological reductand. Td-f can be replaced by the thioredoxin from E. colh. At light saturatiion the modulation process is a pseudo-first order reaction, at light limitation the kinetics are complex. The rate constants for the forward and for the backward reaction were determined. In reducing media the inactivation of the completely activated and thiol-modulated CFoCFI in the dark proceeds in three steps with half times of about Is, 30s and >5min with about equal amplitudes. ADP, phosphate, the previously employed ApH and the kind of the reductand significantly change the kinetics of inactivation.

113

Poster P-IO-O03 STRUCTURAL MODIFICATIONS OF PC2 P~INS INDUCE A HIGH V A R I A B I L I T Y OF H+-PteY.ea, s E S T O I C H I O ~ Y OF THE NATBR OXIDASE WITHOUT AFFECTING THEIR ENZYMATIC A C T I V I T Y

Poster session 10

Wacker. U. and Renger, G., M a x - V o l m e r - I n s t i t u t fur Biophysikalische und P h y s i k a l i a e h e Chemie, TU-Berlin, StraBe des 17. Juni 135, D-I0623 Berlin, G e r m a n y

ATPase, protons and energy transduction P-10-001 - P-10-050

PS2 membrane fragments from spinach w e r e subjected to conditions that hardly affect the o x y g e n evolution capacity of the water oxidase but severely c h a n g e the structure of proteins in the vicinity of the catalytic center. B+-relea@e stoichiometries w i t h i n the suspension m e d i u m (extrinsic H release stoichiometry) were investigated and were found to v a r y even under conditions that retain the oxygen evolution capacity as well as m a n g a n e s e binding of the water oxidizing complex. Based on these results it is inferred that the protons released into the suspending m e d i u m do not reflect the protolytic events of substrate water m o l e c u l e s associated with the catalytic center (intrinsic B + - r e l e a e e stoichiometry). It is further inferred that the extrinsic H+-release stoichiometry is influenced by membrane B o h r - t y p e effects and/or dynamic structural changes as suggested p r e v i o u s l y (Renger, G., 1978 in: P h o t o s y n t h e t i c O x y g e n E v o l u t i o n (Metzner, H. (ed.)), 229-248, Academic Press, New York).

P-10-004

P-10-001 INITIATION OF E L E C T R O N FLOW, P R O T O N T R A N S L O C A T I O N AND ATPG E N E R A T I O N BY C H R O M A T O P H O R E S F R O M PURPLE B A C T E R I A K. Knobloch Institute of B o t a n y and P h a r m a c e u t i c a l Biology, U n i v e r s i t y of Erlangen-Nuremberg, S t a u d t s t r a s s e 5, D-91058 Erlangen, G e r m a n y C h r o m a t o p h o r e s of Rhodopseudomonas palustris and Rhodospirillum rubrum were e x a m i n e d after being e n e r g i z e d with exogenous NADB in the n a t u r a l l y active, c o u p l e d state and after u l t r a s o n i c treatment u n d e r R F l - d e p l e t e d , u n c o u p l e d conditions. For comparative reasons, m e m b r a n e s from d a r k - g r o w n Paracoccus denitrificans were t r e a t e d in the same way. - E x p e r i m e n t a l conditions were adjusted to c o m p a r a b l e ranges on a photo-, pH- and luntlno-meter, i.e., 0.2 recorder units were equivalent to 8 nmol NADB, 7 nmol HCI and 7 nmol ATP per 1 nil reaction mixture. - With different amounts of th~ d i f f e r e n t m e m b r a n e s prepared, the p24amounts of injected N A D H were lowered steadily in order to find the resp. m i n i m a l c o n d i t i o n which w o u l d initiate (i.) electron flow, (2.) p r o t o n t r a n s l o c a t i o n and (3.) A D P - p h o s p h o r y l a t i o n . - It t u r n e d out that e a c h m e m b r a n e p r e p a r a t i o n could be c h a r a c t e r i z e d by a sim/lar capability, n a m e l y to initiate all the three events under the same ntlnimal c o n d i t i o n where one single smallest concent r a t i o n of m e m b r a n e p r o t e i n could initiate the three events by applying the same m i n i m a l c o n c e n t r a t i o n of NADH. - The relative amounts of p r o t e i n and e n e r g y n e e d e d a p p e a r e d to be p r o p o r t i o n a l to the coupling state of the membrane.

Cloning and expression of the F 1 - a Rhodoapirillum rubrum ATP synthase

A. M a l y a n & O. V i t s e v a I n s t i t u t e of Soil Science and Photosynthesis, Russian A c a d e m y of Sciences, Pushchino, M o s c o w Region, 142292 Russia I n c u b a t i o n of the chloroplast coupling factor (CF I) with Mg 2+ions results in reversible inactivation of CFI-ATPase. The rate and e x t e n t of inactivation increase with increasing concentration of M g 2+ and m e d i u m pH. We determined kinetic constants of i n a c t i v a t i o n and dissociation constants for groups i n v o l v e d in Mg 2+ b i n d i n g at pH 5-9. Incubation of M g - t r e a t e d CF 1 at pH 5.5 results in a reversial of A T P a s e activity. The a c t i v a t i o n rate i n c r e a s e s with ATP present. Sintilarly, activity of d a r k preinc u b a t e d t h y l a k o i d membrane H+-ATPase increases after its uncub a t i o n at pH5 in the presence of an uncoupler. The rate of i n a c t i v a t i o n increases with ATP present. The above suggests that i n a c t i v a t i o n of CFI-ATPase and H+-ATPase results from M g 2+ b i n d i n g to specific acid-base groups of CF 1 . P r o t o n a t i o n of these groups provokes dissociation of Mg 2+ and enzyme activation. The d e s c r i b e d m e c h a n i s m is probably the basis of energyd e p e n d e n t regulation of chloroplast H+-ATPase.

114

from

the

Ziyun Du and Zippora Gromet-Elhanan Department of Biochemistry, The Weizmann Institute of Science, Rehovot 76100, Israel F l - a and ~ subunits contain all nucleotide binding sites and form the F1catalytic core complex. A genetic approach will enable the application of site-directed mutagensis for identification of amino acid residues essential for assembly and function of F1(o¢~) complexes. The R.rubrum FI-~, subunit (RrF113) has been isolated in native form and also cloned and functionally expressed, whereas the RrFI~ subunit has never been isolated, nor expressed. We described here the cloning and expression of this subunit The RrFI¢ gene, containing its own ribosome binding site, was cloned by PCR directly from the R.rubrum genomic DNA. The cloned gene was inserted into a multifunctional phagemid and transformed into the unc-less E. coil DK8 strain. Expressed RrFla was identified by western immunoblotting, and unlike RrF 113, which was expressed in a functional soluble form, RrFla formed inclusion bodies under all tested conditions. The isolated inclusion bodies were purified and solublized with 6M Urea. Refolding and characterization of the expressed RrFla will be presented and discussed.

P-10-005

P-IO-O02 T H E R O L E O F S P E C I F I C A C I D - B A S E G R O U P S O F C F 1 IN M g 2+DEPENDENT INACTIVATION AND H+-DEPENDENT ACTIVATION OF C H L O R O P L A S T H+-ATPase

subunit

at Catalytic Sites of CFoFI in Thylakoids Sevbold, A. L. i and Gr~3er,P. 2 l Biol. Inst.,Universit~tStuttgart,pfaffenwaldxing57, 70550Stuttgart,Germany',2Inst. ~ Physikal. Chemie,Albert-Ludwigs-UniversitatFreiburg,Albertst~.23a, 79104Freiburg,Germany H+-ATPases from the FOFI type contain several nucleotide binding sites. Some binding sites have catalytic properties and it is assumed that they work cooperatively. The H+ATPasc from chloroplasts was brought into the reduced, active state and by washing procedure the content of endogenous nuclentides was minimized (1-2 ATPbo~ per CFoFI). After activation of the reduced H+-ATPase by light, alpha-[nP]-ATP was added in the concentration range betweon 1.5 nM and 6 laM ATP ([CFoF~] = 40 nM). Thirty seconds after starting hydrolysis the thylakoids were separated from the incubation medium by using the silicon oil centrifugation technique. After correction for the free nuclentides the amount of bound radioactive nuclentides was measured. Separation of the nueleotides by thin layer chromatography gives the ratio of bound ADP to bound ATP at the catalytic site. For ATP concentration up to 200 nM it resulted a ratio of 0.5 -+ 0.1 and for higher concentrations of ATP the ratio increased to values between 1 and 2. A similar experiment was earned out in the direction of ATP-synthesis. Alpha-[nP]-ADP was added in the range between l0 nM and 20 laM ADP. Ten seconds after starting synthesis by illumination the thylakoids were separated by the same technique as above. From this expenment we obtained a ratio between bound ADP and bound ATP of 0.6 + 0.2 in the investigated concentration range for ADP. Nucleotides

Poster

P-10-009

P-10-006 B i o c h e m i c a l C h a r a c t e r i z a t i o n o f T h y l a k o i d s and CFoCF~ I s o l a t e d f r o m Chlamydomonas reinhardtii c w l 5 and f r o m a t p A and atpB Deletion M u t a n t s •

I

2

H. Fmdler, S. Leu, N. Shavit 2 and H- Strotma nn~ 1Heindch Heine Universit~it Diisseldorf, Germany, 2Ben Gurion University of the Negev, Beer Sheva, Israel. Well coupled thylakoid vesicles were obtained by osmotic treatmet of intact chloroplasts isolated from the cell wall-deficient Chlamydomonas reinhardtii strain cw-15. They showed photophosphorylation rates of about 300 I.tmol ATP/mg chl per h at a ApH of 3. From the thylakoids CFt containing all five subunits and CFoCFI containing all nine subunits were isolated. The Nterminal amino acid sequences of the so far uncharacterized subunits were determined. In two mutants deleted in atpA or atpB, respectively, the CFoCFI complex seems to be completely absent. Thylakoid vesicles isolated from those vesicles allow the generation of much higher proton gradients than the wild type thylakoids illuminated at the same light intensity. These results suggest a significant contribution of CFoCFt to proton leakiness of the thylakoid membrane in the absence of ADP and Pi.

F-TYPE AND P-TYPE ATPases IN CYANOBACTERIA: IMPLICATIONSFOR ENERGY CONSERVATIONAND UTILIZATION S.Fromwald, A.Dworsky and G.A.Peschek, Biophysical Chemistry Group, Institute of P h y s i c ~ h ~ m ~ y , University of Wenna, W6hringerstrasse 42, A-I090 Wien, Austria, fax +43-I-3104597 Cyanobacteria are phototrophic prokaryotes uniquely capable of oxygenic photosynthesis and aerobic respiration. Chl-containing thylakoid membranes house a dual-functional photosynthetic/respiratory electron transport (PET/RET) system (I) and F-type ATPase (2). Chl-free plasma membranes (CM) catalyse RET only (3) and contain both F-type and P-type ATPases (4,5}. In stress conditions when photosynthesis does not work ATP can be made on the CM by light-insensitive RET. RET and P-type ATPase co-operate in maintaining a high trans-CM p.m.f, as the prerequisite for stress defence (4,6). Bioenergetic aspects of cyanobacterial electron transport and ATPase physiology will be discussed. I. G.A.Peschek et at. (1989) J. B. C. 264, 11827-11832 2. A.L.Cozens & J.E.Walker (1987) J.Mo1.Blo1. 194, 359-383 3. G.A.Peschek et at. (1989) Biochemistry 28, 3057-3063 4. A.Neisser et al. (1994) B.B.R.C. 200, 884-892 5. M.Geisler et al. (1993) J. Mol. Biol. 234, 1284-1289 6. A.Dworsky et ai..(1995) Bioelectrochem.Bioenerg. (in press)

P-10-010

P-10-007 APPROACH TO THE PROBLEM OF PHOTOPHOSPHORYLATION IN VIVO

FUNCTIONAL EXPRESSION OF WILD TYPE AND Glu195-~Gly MUTANT SUBUNITS OF THE RHODOSPIRILLUM RUBRUM FI-ATPase

Y-K Shen, J-Y Ye, H-M Ren, D-Y Li and J-M Wei Slaanghai Institute of Plant Physiology, The Chinese Academy of Science, 300 Fenglin Road, Shanghai 200032, PR China

I,, Nathanson and Z. Gromet-Elhanan Department of Biochemistry, The Weizmann institute of Science, Rehovot 76100, israel

It remains unclear whether all we know about the photophosphorylation (PSP) in vitro reflects the real process in wvo. We have compared the PSP by thylakoids in hypotonic and isotonic solutions, and found that lower concentration of Fd was required for PSP in isotonic medium. This results was supported by the experiment "that NADP + reduction by Fd in intact chloroplasts became less effective under mildly hypotonic condition. The osmotic pressure of the medium can also affect the millisecond delayed light emission (msDLE) of the thylakoids, an indicator of the proton motive force for PSP. Measurements of msDLE showed that proton motive force dissipated slower in vivo and in thylakoids in isotonic solution than in hypotonic solution. Therefore, the hypotonic medium conventionally used in m vitro experiments may change the dynamic structure of thylakoids and distort the results; and mildly hypotonic conditions in vivo during raining days may lower the efficiency of photosynthesis. This work was supported by NSFC (39230050).

The ~ subunit has been isolated from the R. rubrum chromatophore-bound FoF 1 ATP synthase (RrFI~), leaving a ~-Iess RrFoF1, that lacks also about 10% of its c~ subunit (RrFl(X), and can neither synthesize nor hydrolyse ATP. The purified RrFI~, could bind nucleotides, reattach to the J]-Iess R r F o F 1 , and restore its ATP synthesis and hydrolysis activities. It is therefore a very promising tool for identifying amino acid residues essential for these functions. We describe here the cloning and functional expression of wild type as well as a mutant E195G RrFI~. Both expressed subunits bind nucleotides. They also bind to the ~,-less RrFoF1 and restore its lost activities when supplemented with traces of RrFI~. However, the mutant E195G, unlike the native or expressed WT RrFI~, binds to ~,-less RrFoF 1 in an unstable manner that is removed by centrifugation. E195 in RrFll3 is identical to E192 in E. cofiFl~ and E199 in mitochondrial FI~, which bind the F1 inhibitor DCCD. A DCCD-modified native RrFI[], was earlier shown to be removed from reconstituted RrFoF1 by centrifugation. Our results indicate that this glutamic acid residue, which is conserved in all F 1~, subunits, plays an important role in their stable assembly into active FoF 1 complexes.

P-10-011

P-10-008 PNOTOPHOSPHORYL&TIOM

IN CHLOROPLASTS

AND HALOBACTERXA

Yasuo Mukohata. Fumiko Uchikawa, Naoka ¥amada, Koichi Oshita, ¥asuo Sugiyama and Kunio Ihara Department of Biology, School of Science, Nagoya University, NagOya 464-01 Japan In these twgnty years after photophosphorylation was first described for Halobacterium salinarium (halobium}, an extremely halophilic archaeon, the profile of halobacterial photopboephorylation has been greatly modified. Not only bacteriorhodopein and halorhodopsin in Hb. salinarium but their homologs in different genera have been known as light-energy transducing maohineries for proton-motive-force (pmf) generation. Driven by this pmf, FoF1 ATP eynthaee was readily assumed to form ATP in halohacteria. The synthaee, however, was found to be close to vacuolar ATPaee rather than CFOCFI ATP eynthase; as enzyme b y inhibitor study, as protein by imEuno-blotting and by genetic analysis. The halobacterial A(rchaeal)-type ATP synthase is a member of proton-tranelocating F-V-A ATPase family. A-type ATPace seems to have enzyme architecture similar to F-type ATP eynthaee; u3~37be for FI and ab2c12 for Fo, whereas V-type onel carries the c eubunit double in size. The "purple" photophosphorylation system in halobacteria is far from the "green" system of even procaryotic cyanobacteria.

THE SPINACH CHLOROPLAST CF1-~3~ 3 CORE COMPLEX: STABILITY AND CATALYTIC PROPERTIES

STRUCTURE,

M. Sokolov and Z. Gromet-Elhanan Department of Biochemistry, The Weizmann Institute of Science, Rehovot 76100, Israel The CF t(c¢~) core-ATPase earlier isolated from spinach thylakoids (Avital & Gromet-Elhanan (1991) J. Biol. Chem. 266, 7067-7072) was stimulated, but not inhibited, by the specific CF 1 effector, tentoxin. Pretreatment of this CFt(a~, ) with high, stimulatory tentoxin concentrations, led to stabilization of its hexameric structure and enabled the isolation of an active CFt-c(3~ 3 complex by size-exclusion chromatography. A detailed characterization of the MgATPase activity of this CFI-a3[~ 3 hexamer, as compared to a CFI(-~') complex, revealed similar apparent Km values and stimulation by 100 ~M tentoxin, but drastic differences in all other tested assays. Most pronounced were their different temperature profiles and different responses to all other inhibitors or stimulators of the F1-ATPase activity and to increasing concentrations of added Mg 2+ -ions. These results indicate that, although the ¥ subunit is not required for the CF 1-ATPase activity, it is absolutely necessary for obtaining the typical functional properties of the CF 1-ATPase, The stable CF1-~3[~ 3 hexamer and its parent C F I ( ~ , ) complex provide therefore excellent tools for elucidating the role of the CF1- ¥ subunit in the full spectrum of CF t activities, including its catalytic site cooperativity.

115

Poster

P-10-012

P-10-015

GENERATION OF AN INTERMOLECULAR TENSION BETWEEN CFo AND CFI AS CONSEQUENCE OF A TRANSMEMBRANE ELECTROCHEMICAL PROTON GRADIENT

IDENTIFICATION OF THE TRYPSIN CLEAVAGE PRODUCTS O F THE V SUBUNIT OF CHLOROPLAST COUPLING FACTOR 1

S.V.Ponomarenko I H.R.Fiedler2, H.Strotmann 2 tlPPGB NAS RK, Timirjasev St.45, Almaty-75, Kazakhstan 480075. 2lnstitut fiir Biochernie der Pflanzen, Heirtrich Heine Universit~it, Universit~itsstml3e 1, 13-40225 D/isseldorf, Germany

Kendra E. Hightower & Richard E. McCarty Dept. of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA

Treatment of isolated thylakoids with the chaotropic salt NaSCN results in release of CF1. At a eoneattration of 0.5 M NaSCN 20% CF1 is detached from dark kept thylakoids, but 70% CFI is released from illuminated thylakoids (Fiedler, Ponomarenko, yon C_Jetdenand Strotmann (1994) Biochim. Biophys. Aota 1188, 2934). The light-induced erdaaneement of CF1 solnhilization is abolished by uncoupling indicatingthat the transmembrane electreche~nical proton gradient is responsible. Light can be replaced by an acid/base transition. The ApH profile resembles the ApH dependency of phosphorylation. The enhancement of CF1 detachment by light is abolished by treatment of the thylakoids with DCCD indicating that proton translocation through CF0 is a necessary prerequisite. We conclude that protonnations at the interface between CF0 and CF1 decrease the electrostatic attraction between the subcomplexes. The light-nidueed enhancement of CF~ release is relieved by ADP and phosphate suggesting that the generation and decomposition of a tension between the subcomplexes are intermediate steps in energy transduetion in CFoCF1.

Trypsin cleavage of the 323 amino acid T subunit of CF1 lacking the e subunit results in the formation of three major fragments when the T disulfide is reduced. The trypsin cut sites have been identified. An N-terminal (amino acids 1-70, 7.9 kDa) and a C-terminal (amino acids 231-323, 10 kDa) T polypeptide are formed during proteolysis. A 14.8 kDa y fragment was purified and shown to be amino acids 71-204. A fourth small fragment, amino acids 205-215, was previously shown to be released from the enzyme (Schumann, Richter, & MeCarty, 1985). The reduced and trypsinized enzyme was very active as an ATPase and may be stored for weeks as an (NH4)2SO4 precipitate without loss of activity. The thermal stability of the trypsin-treated CF1, as revealed by differential scanning calorimetry, was not significantly different from that of CF1 and CFI(-e). This work was supported by the NSF.

P-10-013

P-10-016

IS H + T H E UNIQUE ION FOR P H O T O S Y N T H E S I S E N E R G Y TRANSDUCTION ?

COOPERATIVE INTERACTION BETWEEN THE ACTIVE SITES OF CHLOROPLAST CF1.

I Brown Cyanobact Biology Res. Lab., Odessa State University, Odessa, 270100, Ukraine

Y. Hochman & C. Carmeli. Dept. of Biochemistry. Tel-Aviv University. 69978 Tel-Aviv. Israel.

The cyanobacterial division proceeds very often under alkaline conditions as a result of CO2 exhausting from extracellular medium or its chemical composition, H + can not function as a coupling ion. It was postulated that the alternative coupling ion(s) for photosynthesis energy transduction should exist. It was found that Na ' and Ca 2+ , but not K ' , permit cyanobacteria to grow under alkaline conditions. Indications were obtained in our group that some halo- and alkalotolerant cyanobacteria possess primary electrogenic Na' - pumps in the cytoplasmic membrane using the light energy tbr generation of h l a N a ' Our further investigations showed that Ca 2", similar to Na", is able to permit Gloeobacter violaceus growth under alkaline conditions and to support light-dependent electrogenic import of extracellular H ~ against its concentration gradient by cells. So, H ÷ does not seem as a unique cation for photosynthetic energy transduction.

P-10-014 THE ATP SYNTHASE CFoCFI: PROTON FLOW UNDER SLIP AND ATP SYNTHESIS TRIGGERS SIMILIAR CONFORMATIONAL CHANGES O. Fritsche and W. Junge Universit~t Osnabrfick, Abt. Biophysik, 49069 Osnabr6ck, Germany The ATP synthase is probably a mechano enzyme. Crosslinking of a dark accessible cysteine residue with a light accessible one by o-phenylenebismaleimide (OPBM) is one indicator of the structural change during activation of CFoCF1. We studied the inhibition of photophosphorylation by the OPBM crosslink under conditions where the ATP synthase was under proton slip (no nucleotides present), blocked (with 20 p M ADP+P~) or synthesizing (more than 20 # M A D P + P i ) . The crosslink was formed both under slip and under ATP synthesis corroborating that the same type of conformational changes occurs during proton flow independent weather slipping or synthesizing. It could not be prevented by tentoxin or dequalinium chloride, which inhibited catalysis but not activation. The exposure of the light accessible cysteine residue is blocked by low concentrations of ADP or GDP, i.e. if a potentially catalytic site is occupied.

116

The kinetic parameters of chloroplast CF1-ATPase (CF1) were determined over a wide range of substrate concentrations. As substrate concentration was increased from 8nM (0.l of the CF1 in the assay) to lmM a three-phases change in the mechanism of CF1 activity was observed. Km's of 0.5, 3.5 & 72 vM and Vmax's of 4.3, 1.2xl02 & 5.5x103 nmoles/mgxmin were calculated for each phase respectively. The kinetic parameters for each phase were unique and could not be extrapolated to the other phases. The mode of interaction at the active site of CFI were studied with the transition state analog ADP-Vanadat (ADP-Vi). CF 1, at low substrate concentration, was not inhibited by ADP-Vi. At intermediate and high substrate concentration the ATPase was strongly inhibited by ADP-Vi. The result were interpreted to suggest that ATPase activity at a single, double or triple set of active sites of CF1 is responsible for the different parameters of activity observed and that ADP-Vi inhibited the cooperative interaction among the three active sites of CF1.

P-IO-OI7 L O C A L I Z A T I O N O F SUBUNIT ~ IN SPINACH CHLOROPLAST F1 A T P SYNTHASE H. Lill. W. Junge & S. Engelbrecht Abt. Biophysik, Universit~t Osnabriick, D--49069 Osnabrtick, Germany By coupling transmembraneous ion flows with ATP-synthesis or hydrolysis, F-type ATPases play a central role in the energy supply of cells. Despite growing knowledge about the enzyme's structure, the localization of several subunits is still unknown. We over-expressed five mutated spinach F1 subunits in Escherichia coli, with Cysteines replacing Serines in various positions. These residues were used for the introduction of reagents into 8 either prior to rebinding of the subunit into ~--free Fj-complexes or with the mutated subunits already bound. ~ was almost freely accessible to the bulky eosin-maleimide molecule when bound to F1. Cross-linking reagents coupled exclusively to either a or 13. We conclude a rather exposed position of b in the complex in close proximity to the large subunits. After introduction of fluorophores into ~ as well as into F1, the subunit was mapped by resonance energy transfer to a position intercalating between an oc/~-pair and on the level of the nucleotide binding sites.

Poster

P-10-018 PROTON-LINKED TRANSPORT ACTIVITY ACROSS CHLOROPLAST INNER ENVELOPE VESICLES

P-10-021 THE INFLUENCE OF DIFFERENT Mg2+ LIGANDS ON THE ACTIVITY OF ATPase IS IN FAVOR OF THE ROLE OF FREE ATP AS TRUE SUBSTRATE AND OF FREE Mg2+ AS ACTIVOR

Richard Shineles and Richard E. McCarty Department of Biology, The Johns Hopkins University 3400 N. Charles St. Baltimore, MD 21218 USA

G. BERGER, G. GIRAULT, S. PF_ZENNEC Section de Bioenerg@tique, D~partement de Biologie Cellulaire Mol~culaire, CEA Saclay, 91191 GIF-SUR-YVETTE CEDEX, France

Membrane Vesicles isolated from the inner envelope of chloroplasts are competent for transport studies, pH changes can be followed by loading the fluorescent indicator pyranine inside the vesicles either by a freeze/thaw technique or by using an extrusion apparatus. Vesicles prepared by freeze/thaw techniques are typically over 80% inside-out in orientation. Large proton concentration changes occur in the vesicle interior when ATP is added externally indicating that a proton-ATPase is active in these preparations (Shingles R. & McCarty R.E. 1994 Plant Physiology 106 731-737). Vesicles prepared by extrusion are usually about 80% right-side out in orientation and have small proton concentration changes in the vesicle interior when ATP is added. These results indicate that ATPase activity is localized on the inside (stromal side) of the chloroplast inner envelope. Vesicles prepared by freeze/thaw and extrusion techniques can be loaded with pyranine and phosphate to study 3-phosphoglycerate exchange via the phosphate translocator. In these preparations the extent of internal acidification upon the addition of 3-phosphoglycerate was similar indicating the bidirectional nature of the phosphate translocator. (This work supported by Department of Energy)

The hypothesis attributing the role of substrate to free ATP (instead of Mg ATP complex) and the role of activator to free Mg2+ ion (instead of inhibitor) in the course of ATP hydrolysis by chloroplast ATPnae has been vedfied by the use of Mg2+ ligands of different dissociation constants. In presence of excess of Mg2+, a low binding ligand as 8-hydroxyquinoline (PKMa = 2.8) has no effect on ATPase activity. Tropolone (PKMg = 21.1) or pyrophosphats (PKM~ = 4.6), the PKivlg of which are on the same order as that of ATP (PKMg = 4.4), but lower than that of CF1 (PKM~ > 5.1, have both an activating effect on the reaction rate. This result is i5 accord with the mentioned hypothesis, since ATP concentration is then increased at the expense of that of the Mg ATP complex, the Mg CF1 complex remaining unchanged. With a tight binding ligand of Mg2+ as EDTA (PKM. > 6.5) the activity first increases by dissociation of the MgATP complex, then decreases when the equivalence is reached and the MgCF 1 is dissociated.

P-10-019 FORC]~IPLUX 1UgLATIONSI'EIPSIN CIELOROPLASTS. 2. IgXPlgl[LIMBgNT&.L S T U D Y OF ATP SY1NTHIgSIS VS. ApH Ir~ THE THYLAK01DS F U N C T I O ~ ' G U~YD]~R THE CONDITIONS OF CYCLIC A N D NONCYCLIC ]gIAE;CTI$.ON T]I.&IMSPOIN.T

A.N.Tikhonov, S.G.Mngnitsky, M.Maanrova, A.Mueulin & L.A.Blumenfeld Deptnrtment of Biophysics, Faculty of Physics, M.V.Lomonosov Moscow State University, Moscow, 119899, Russia I1, this work we present experimental results of comparative study of foresflux relationships ( A T P synthesis vs. transmembrane proton gradient, ApH) in the thyhLkoids funetioi.[ng under the conditions of PSl-driven

cyclic anti n.',~ "=-I"- ~P°I ~ PS2) electron flow. The ApH values were determined by three independent methods (kinetic measurements, pH-indicating spin-label TEMPOamine, and the use of buffers penetrating into the thylakoid lumen). The data obtained demonstrate that photophosphorylation can efficiently proceed under the relatively small values of ApH in the region of stroma~exposed thylakoids &pH = 1.4-2.2 (depending on the composition of the suspending medium). The data obtained are consistent with the possibility of alternative mechanisms of energy coupling (localised and delocalized).

P-10-020 REGULATION OF THYLAKOID ELECTROGENESIS BY PROTONS

et

P-10-022 Q U A N T I T A T I V E A S S A Y OF A T P a s e S U B U N I T C F o II BY E L I S A USING OVEREXPRESSED POLYPEPTIDE FOR CALIBRATION J d r g e n Tiburzy, M a r t i n Zimmermann, R i c h a r d J. B e r z b o r n L e h r s t u h l f. B i o c h e m i e d. P f l a n z e n , B i o l o g y ND, R u h r Universit~t, D-44780 Bochum, FAX *49-234-7094322 P o l y p e p t i d e C F o II of p h o t o s y n t h e t i c A T P a s e seems to be an e s s e n t i a l subunit: It is c o - p u r i f i e d in the C F o CF I c o m p l e x u s i n g v a r i o u s m e t h o d s (1,2); in c y a n o b a c te@ia an ORF in the A T P a s e o p e r o n e n c o d e s its h o m o l o g b' (3). Its l o c a t i o n p a r a l l e l s the t o p o g r a p h y of C F o I (4). F r o m d e l e t i o n s t u d i e s it w a s c o n c l u d e d , h o w e v e r , t h a t u n l i k e C F o b or I s u b u n i t b' or II is n o t involv e d in b i n d i n g of CF 1 (5). We h a v e o v e r e x p r e s s e d s p i n a c h CFo II in E.coli. The p r o p e r t i e s of CFoII, p u r i f i e d s o l u b l e to h o m o g e n e i t y , are studied. N e w a n t i s e r a are r a i s e d to r e e x a m i n e the in situ l o c a t i o n of CFoII. W i t h the p r o t e i n a q u a n t i t a t i v e E L I S A is c a l i b r a t e d to d e t e r m i n e its c o n t e n t in t h y l a k o i d s a n d in CFoCFI, i.e. its s t o i c h i o m e t r y . I) 2) 3) 4) 5)

U.Pick, E . R a c k e r , J . B i o l . C h e m . 254(1979)2793-2799 R . J . B e r z b o r n et al. Z.Naturf. 4 5 c ( 1 9 9 0 ) 7 7 2 - 7 8 4 N.Nelson, Biochim.Biophys.Acta 1100(1992)109-124 J.Otto, R . J . B e r z b o r n , F E B S Lett. 2 5 0 ( 1 9 8 9 ) 6 2 5 - 6 2 8 H.Lill et al. B i o c h i m . B i o p h y s . A c t a 1184(94)284-290

P-10-023 CROSSLINKING T H E F I - P A R T O F C H L O R O P L A S T A T P n s e I N DII~'~'ERENT CONFORMATIONAL STATES

T, van Voorthuysen, J.H.A. Dassen, J.F.H. Snel & W.J. Vredenbergt tDept of Plant Physiology, Wageningen Agricultural University, Arboretumlaan 4, 6703 BD, Wageningen, The Netherlands

D. Schumann and S. Bickel-SandkOtter, Institnt fur Biochemie der Pfianzen, Heinrieh-Heine-Universit~it, Universit~tsstr.1, D-40225 DUsseldorf, FRG

Light capture by the antenna complexes of photosystem I and I1 and subsequent charge separation in the associated reaction centers initiate redox reactions involving binding and release of protons at the thylakoid membrane boundary layers. The light-driven accumulation of protons inside the thylakoid reversibly suppresses the flash-induced electrical potential as monitored with a new patchclamp method and with P515 spectroscopy. The reversible suppression is discussed to be caused either by an energy-dependent down-regulation of photosystem II activity or by changes in thylakoid membrane properties, notably the electrical capacitance. The patch-clamp technique is explored here for its use as a practical means for direct recording of the number of reaction centers turning over and of changes in membrane properties. A light-induced resistance increase of the thylakoid resistance network and its stimulation by nigericin are discussed in relation to chan~es in the low conduction barriers between the thylakoid membrane and its adjacent lumenal and stromal phases.

ATP-Synthases consist of a membrane-spanning F0 -part for proton-conduction and a Ft -part containing the sites of ATP formation. Six nocleotide binding sites have been determined in the Fl-part, three of them catalytic sites, which are most probably located on the interface between a and [~ subunits. EDC, a watersoluble earbodiimide, forms zerolength bonds between the sidechains of basic and acidic amino acids, if located in direct neighbourhood to each other. Covalent modification of CFI with EDC causes a loss of Ca-dependent ATPase activity. Coincubation with ITP as a substrate analogon protects the enzyme against inactivation and crosslinking. This indicates a covalent modification in the active site of the enzyme, because ITP has been reported to bind to catalytic sites only. EDC forms two different crosslinks in higher yields. SDS-PAGE and Westemblot analysis of the products resulted in an a - 8 (78 kD) and an a-13 (110 kD) product. Activation of the ATPase acitvity via incubation via ITFT at 60 ° C and subsequent gel filtration resulted in a complete loss of subunit 6. Consequently no ~x-8 erosslinkprodaet could be found. Our results confirm the role of subunit 6 in proton conductance.

117

Poster P-10-024 ENERGIZATION CHANGES THE REACTIVITIES OF LYSINE RESIDUES OF THE CHLOROPLAST ATP SYNTHASE ¥ SUBUNIT

Mizuho Komatsu-Takaki Department of Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, K a n a g a w a 199-01, Japan In chloroplasts, the formation of the transmembrane electrochemical potential of protons (AttH+) activates CFoCFi and drives the production of A T P from A D P and Pi. To understand the mechanism of ATP synthesis, it is necessary to know which regions of the CFoCFt respond to A~tH+. 1 have searched for such regions by measuring the reactivities of lysine residues of the c o m p o s i n g subunits. If Attrl+ changes the conformation around a lysine residue, the reactivity of this lysine residue with pyridoxal-5' phosphate is expected to change. I have previously shown that L y s l 0 9 of the e subunit and Lys399 of the I~ subunit change their reactivities depending on AttH+ (Komatsu-Takaki, 1992, J. Biol. Chem. 267, 2360-2363 ; Komatsu-Takaki, 1995, Eur. J. Biochem. in press ). In this report, it will be shown that Lys168, Lys222, and Lys231 of the ,/subunit change thier reactivities depending on AttH+.

P-10-027 EXCHANGE OF TRINITROPHENYL-ADP

(TNP-ADP) TIGHTLY

B O U N D T O C H L O R O P L A S T C O U P L I N G F A C T O R 1 (CF1) F O R MEDIUM NUCLEOTIDE J. G. Di~el & R. E. McCarty Dept. of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA The fluorescence of the A D P analog TNP-ADP is enhanced - 8 times upon binding to CF1. The exchange of TNP-ADP tightly bound to CFI depleted in e with medium nucleotide was observed as a decrease in TNP-ADP fluorescence. Rates of exchange initiated by ADP, ATP, and AMP-PNP addition, were found to be similar, and exchange to consist of at least two phases. Free M g 2+ inhibited exchange. Exchange initiated by the addition of M g 2÷ together with ADP was faster than in the absence of M g 2+. Exchange for A T P was not similarly enhanced by M g 2÷. The presence of Pi in the exchange medium with A D P effects exchange rates in a way which is dependent on M g 2+ concentration, showing little effect in the absence of M g 2+, and a 4-fold increase in rate when added together with l m M M g 2÷ and 5mM ADP. Exchange is fastest under conditions which favor ATP synthesis, even in the solubilized enzyme. This work was supported by the NSF.

P-10-028

P-10-025 C H A N G E S IN CHARACTERISTICS OF THERMOLUMINESCENCE BANDS IN ISOLATED THYLAKOIDS INCUBATED WITH ATP. J. F a r i n e a u SBE/DBCM, C E A / S a c l a y , 91191 Gif sur Y v e t t e Cedex, F r a n c e . C h a n g e s in c h a r a c t e r i s t i c s of flash-induced t h e r m o l u m i n e s c e n c e (TL) glow c u r v e s in thylakoids of l e t t u c e following incubation of the o r g a n e l l e s with ATP, u n d e r illumination or in t h e dark, was i n v e s t i g a t e d . TL bands w e r e induced by 1 or 2 flashes fired a t - 1 0 ° C in t h y l a k o i d s suspended in g l y c e r o l - t r i c i n e b u f f e r , pH 8.0. TL curves in c o n t r o l thylakoids: d a r k - a d a p t e d or s u b m i t t e d to an illumination w i t h o u t A T P c a n be d e c o n v o l u t e d as the sum of one single B b a n d and m i n o r c o n t r i b u t i o n s as bands n e a r 0°C and 4245°C. In thylakoids i n c u b a t e d for 2 min with 0.5 mM ATP, e i t h e r u n d e r light or in t h e dark ( a f t e r a 2 rain preillumination), bands p r e s e n t e d c o m p l e x shapes; a f t e r deconvolution, l.hey a p p e a r e d c o m p o s e d of a B b a n d with a low Ea (0.6 as c o m p a r e d to 0.75 in control), and a band p e a k i n g a t 5°C (2 fl) or 12-15°C (1 fl). The band a t low t ° was suppressed by low c o n c e n t r a t i o n s (10-20 riM) of v a l i n o m y c i n e , n i g e r i c i n e or F C C P as well as by 10 mM NH4CI, leaving b a n d s with the s a m e c h a r a c t e r i s t i c s as in c o n t r o l m a t e r i a l . Finally with higher nigericine c o n c e n t r a t i o n s bands a r e single B bands w i t h high Ea (0.9). These c h a r a c t e r i s t i c s would define 3 d i f f e r e n t e n e r g i z e d s t a t e s for thylakoids.

P-10-026 ANION AND CATION CHANNELS IN THE THYLAKOID MEMBRANE I.I. Pottosin & G. SchOnknecht Lehrstuhl Botanik I der Universit~it W0rzburg, Mittlerer Dallenbergweg 64, D-97082 Wiirzburg, Germany Light-driven accumulation of IT inside the thylakoid lumen is coupled to the C1- uptake and efflux of Mg2+ and K+. We studied the properties of single ion channels, facilitating anion and cation transloeation across the thylakoid membrane (TM), by application of patch-clamp technique to the native TM. The voltage-dependent anion channel with an ideal CI-/K + selectivity ratio was observed in TM from different plant species. In spinach TM the other ion channel was also characterized, which was permeable for K + as well as for M g 2+ and Ca 2+ but not for CI-.

118

2',3'-O-(2,4,6-trlnitrophenyl)-ADP

has covalentIy labeled t w o different

s i t e s o n t h e c o u p l i n g factor ATPase b o u n d o n t h y l a k o l d m e m b r a n e u n d e r the photosynthetic condition

Hisabori, T. I, a n d Strotmann, H. (Inst. Plant Biochem., Heinrich-Heine Univ. Duesseldorf, Germany; IPermanent address: Tokyo Inst. Tech, Japan] Irradiation of Isolated chloroplast thylakolds with TNP-ADP results covalent incorporation of a reaction p r o d u c t of TNP-ADP formed b y p h o t o s y n t h e t i c reduction into the s o - c a l l e d "tight" ADP b i n d i n g site of CF I. This covalent incorporation i n d u c e d the partial inhibition of the p h o t o p h o s p h o r y l a t i o n activity a n d the m e m b r a n e b o u n d ATPase activity. In experiments with TNP[14C]-ADP, radioactivity w a s detected almost equivalently on a l p h a a n d beta s u b u n i t s (Hiaabori et al., 1993, J.blnchem. 114, 3 2 4 - 3 2 8 ) . P y r o p h s p h a t e , known as a specific competitor against ADP on non-catalytic binding sites, inhibited the covalent incorporation of TNP-ADP a n d r e d u c e d the enzyme inhibition. Although the 'tight' ADP h a s been known to be important for the maintaining of enzyme conformation, incorporation of covalent TNP-ADP did not affect the digestion pattern of isolated CF I by trypsin.

P-10-029 INFLUENCE OF LUMEN pH ON THERMOLUM1NESCENCE EMISSION IN COUPLED THYLAKOIDS OR LEAVES T Miranda, A. Toulouse, J.M. Ducruet Section de Biotnerg&ique, BS.t 5352, 1NRA / CEA Saclay, 9119i Gif-sur-Yvette cedex, France In coupled thylakoids, a transthylakoidal proton gradient can be generated by ATP hydrolysis in the dark, by PS-1 electron transport under far red light or by PS-II + PSI electron transport under white light. Thermoluminescence was recorded above 0°C after l to 6 flashes. The acidification of the lumen favors the $2 -> St and $3 -> $2 reverse transitions, thus inducing a shift towards low temperatures of the B and Q bands, suppressed by nigeriein, not valinomycin. Far-red illuminations (718 nm, 30 s) also generate a new TL band in leaves or intact chloroplasts, with a maximum peaking between 40°C and 55°C, depending on plant species and on the TL heating gradient This AG band is suppressed by uncouplers, by PS-II inhibitors or by freezing the sample. We demonstrate its homology to the afterglow rise in luminescence decays at constant temperatures after far-red illumination, previously observed by several authors in leaves or algae This delayed luminescence emission is strongly accelerated by temperature increase, so that it can be optimally resolved as a sharp and intense AG TL band under a 0.3°C/s to 0.5°C/s heating rate. This AG band is also observed in some batches of dark-adapted unfrozen leaves in the absence of far-red, after 1 to 6 flashes

Poster

P-10-033

P-10-030 ATP synthesis by co-reconstituted proteoliposomes BacteriorhodopsinlH*-ATPases. B. Pitard. P. Richard and J.L. Rigaud CEA centre d'(~tudes de Saclay, DBCM, SBE, 91191 Gif sur Yvette, France Co-reconstitutions of BR (bacteriorhodopsin) with H+-ATPase from thermophilic bacillus PS3 (TFoF1) or spinach chloroplast (CFoF0 were performed in order to study the ATP synthesis mechanism. ATP synthesis activity of 500 nmol ATP/min.mg were routinely obtained. Although the ATP synthase activities are low compared to the in vivo efficiencies due to the limitation in the amplitude of the ApH attainable using BR as AIJH+ generator, the present co-reconstituted systems are powerful tools to analyze thermodynamic, kinetic and enzymatic parameters. Cholesterol was found to induce a 4-fold increase in ATP synthase activity (CFoF1 and TFoF1) with a concomitant 65 % decrease in the K M for ADP suggesting that cholesterol can modulate events occurring in F1 part. The TFoF~, which was essentially free of bound nucleotides after isolation and purification, synthesized ATP in two different kinetic phases; an initial slow phase accelerated to a final 2-3 times faster steady state rate, It was shown that the state of occupancy of specific sites by ATP regulated the synthetic activity of TFoF~.

P-10-031 ANTIMYCIN INHIBITS QrQUENCEIING BY A PROTONOPHORIC MECHANISM

ChristineT. Yfrkes and A. R. Crofts, Program in Biophysics,U. ofIUinois,Urbana, IL 61801 W e have investigatedthe inhibitionof qE-quenching by antimycin and D C C D , and the enhancement by pr~illumination to form zeaxanthin. The effects of antimycin on qE-quenching can all be mimicked by classicaluncoupling agents at low concentration.The antimycin titreis dependent on rate of generation of

EFFECTS OF TENTOXlN ANALOGS ON A T P A S l C PROPERTIES OF C F 1 AND T F 1 (3. Gir~t~lltt, E. Pinet 1, S. Pezennec 1, L. Dubart t, F. Andr6 x F. Cavelierz, & J. Verdueci z tCEA-Saclay, DBCM/SBE, B~tt. 532, 91191 Gif-sur-Yvette Cedex, France 2URA-CNRS 0468, Universit6 Montpellier H, France

Tentoxin, a natural cyclic tctrapcptide produced by the phytopathogenic fungus Alternaria alternata, is known to be an energy transfer inhibitor of sensitive plants ATP synthetases. Two analogs o f this toxin were synthesized. 1 ~ A mutation of Ala to Ser produced a cyclic inhibitor o f CF]-e ATPasic activity as efficient as the natural product (10 "s M). Contrary to tcntoxin which is inefficient on TF I (the ATPase o f the thermophilic bacteria PS3), this analog also inhibits TF 1 ATPasc activity (10"610 "4 M). 2 ~ The Ala to Glu(OtBu) mutant inhibits CFI-s and activates TF 1 ATPasie activities. At high concentration (10 "4 M), tentoxin is known to reactivate CF~-s ATPasic activity. This effect was not observed for none of the two analogs.

P-10-034 ACTIVATION-INDUCED CHANGES TO METAL-NUCLEOTIDEBINDING SITES IN T H E C H L O R O P L A S T FI.ATPase.

Andrew L. P. Houseman, RussellLoBrutto, and Center for the Study of Early events in Photosynthesis and the Department of Botany, Arizona StateUniversity,Tempe, A Z 85287-1601.

the quenched state (an earlier literature has demonstrated that antimycin is an uncoupler, with a concentration dependence which varies with the rate of electron transport). Valinomycin potentiates the effect of antimycin in a synergistic and K+-dependent fashion. We conclude that antimycin inhibits by a protonophoric mechanism, not my specific binding to LHCII. We have observed that the zeaxanthin-enhanced fraction of qE-quenching, and that associated with non-cyclic flow, are more sensitive to antimycin than that driven by an artificial cyclic system, even when account is taken of the different rates of onset, possibly due to a gradient in I-I* activity between a site at which antimycin acts on the qE-quenching mechanism, and the bulk phase. DCCD inhibits qE-quenching only after preincnhation, but does not uncouple, end likely reflects binding at acidic residues of the minor CPs buried in the hydrophobic phase. Light-scattering (ALS) is not a reliable probe for Apii across the chloroplast membrane; under some conditions, quenching and ALS and not kinetically correlated. We will discuss the mechanism of qE-quenching in terms of s model suggested by Crofts and Yerkes, 1994, FEBS Lett. 352, 265-270.

The effects of activation on the chloroplast F1-ATPase (CF1) were investigated by EPR spectroscopy of the Mg 2+ analogue, VO 2+, at the catalytic and noncatalytic metal-nuclootide-binding sites. Upon activation of CFI with dithiothreitol, the equatorial coordination of the metal at the M2-N2 site (aDp in the crystal structure) changed from monodentate ATP with 3 equatorial aquo ligands to bidentate ATP with hydroxylate ligands (possibly aT176 and aS272 in the bovine mitochondrial sequence). This ligand set is consistent with that proposed in the crystal structure (2 phosphates from ATP and one hydroxylate from aT176). A change in the metal ligation at the M3-N3 ([~Tp) catalytic site from 2 H20, 1 RCOO', and 1 RO- (possibly [~D256 and 13T163) to 2 phosphates from ATP and 2 RO- (possibly ~T163 and ¢~$344) was also observed during activation. The new ligand environment matches that at the activated noncatalytic sites, except that the second hydroxylate at the catalytic site, (xS344, appears to come from a neighboring subnnit and would be present only in ~TP. A mechanism for ATP synthesis is proposed whereby (xS344 displaces the ¢~-phosphate of tridentate ATP in the ~DP, switches the site to ~TP, prevents back reaction, and favors dissociation of ATP over ADP.

P-10-032 THE METAL BINDING SITES OF CHLOROPLAST F1-ATPASE STUDIED BY PULSED EPR SPECTROSCOPY

P-10-035 C A T A L Y T I C F U N C T I O N OF THE CARBOXYLATE.RICH REGION O F T H E BETA SUBUNIT O F T H E C F t - A T P A S E F R O M C.

C. Buy, G. Girault & J.-L. Zimmermasn CEA-Saclay, SBE/DBCM, B~tt532,91 191 Gffsur Yvette, France The proton ATPase from thylakoid membranes catalyses the synthesis of ATP from ADP and phosphate by using the proton gradient across the membrane. Isolated CFI, the soluble part of this enzymatic complex catalyses the hydrolysis of triphosphate naclootides after activation with divalent metals such as Mg2+ or Mn2+. A total of 6 metal sites has been estimated to exist on CF 1 (Hiller & Carmeli, 1985, J. Biol. Chem., 260, 1614-1617). The role of these metal sites in the enzymatic process and their protein environment have been addressed by the ESEEM technique of pulsed EPR spectroscopyby using Mn2+ as a paramagnetic probe. When latent CF 1 containing ~ 2 nucleotides/CFI and depleted of metals by dialysis with EDTA is incubated with MnCI2, the 3 pulse ESEEM spectrum at 3240G reveals a pattern of frequencies with peaks at ~2.2, 3.4, 8.1 and 13.8MHz. Based on magnetic field effect studies, and similarities with ~ of Mn2+ sites coordinated with nitrogen donors, together with studies of isotope effects, the peaks at - 2.2 and 3.4 MI-Iz are tentatively attributed to coupling with one or more 14N nuclei. The peak at 13.8 MHz originates from weakly coupled protons. The origin of the 8.1 MI-Iz~pT~akis under investigation. The 14N frequencies in the 3 pulse spectrum are detected with Mn2+ concentrations as low as 0.2 Mn/CFI, suggesting that the metal site detected in this study correlates with a strong affinity metal binding site. The ESEEM data suggest that under these experimental conditions, this metal cation is coordinated by a nitrogen ligand from a protein residue.

REINHARDTII. ~ , Lola Morgan, Andrew N. Webber and Wayne D. Frasch Center for the Study of Early Events in Photosynthesis, Department of Botany, Arizona State University, Tempe, A Z 85287-1601 The mutations E204Q, D211N, and E204Q:D211N of the [3 subnnit of the chloroplast Ft-ATPase were made by biolistic transformation. The mutations affected niether the yield or circular dichrosim spectra of the CFt-ATPase. Photuantotrophic growth of Chlamydomonas sWains containing E204Q was virtually abolished, and decreased about 1.5 fold in the D211N mutant. The effect of the mutations on the light-driven ATPsynthase activity catalyzed by purified thylakoids was comparable to the changes in the photoautotrophic growth rate. Although the mutations significantly affected the kca~:~d of the Mg2+-ATPase activity of the purified CF1-ATPase, no significant effect on kc= was observed with any of the mutants compared to wild-type. No significant changes in the ability of Mg2+ or Mn2+ to serve either as a cofactor or as an inhibitor of ATPase activity were observed in the mutants relative to the wild -type CFt-ATPase suggesting that these residues do not serve as ligands to the metals at the catalytic sites. The data support a role for the carboxyl groups in the carboxyl-rich region (residues 204-218) in supplying E204 at the catalytic sites of the CF I-ATpase with protons used to make a phosphate oxygen a good leaving group and facilitate ATP synthesis.

119

Poster P-10-036 A D P - V A N A D A T E IS A T R A N S I T I O N S T A T E I N H I B I T O R O F CF1-ATPASE Irit Sagi, Yehosua Hochman, and Chanoch Carmeli Departments of Biochemistry,Tel Aviv University, Tel Aviv 69978, Israel The structure of vanadate which was suggested to function in the presence of ADP and divalent cation as a transition state inhibitor of CF1-ATPase was investigated by x-ray absorption. Analysis of the vanadium K-edge was used for determination of the structure of vanadate bound to CF1ATPase in solution. There was a decrease in the intensity of the pre-edge a n d o f two other shoulders at the edge region on binding of vanadate to CF1 in the presence of MnADP. The changes were due to alteration in the structure of vanadium from tetrahedral to five-coordinated trigonal bipyramidal geometry. 51V NMR m&asurements indicated that an anhydride between ADP and vanadate is the processor for the bound vanadium. The Ki for vanadate inhibition of activity decreased more than 20 fold and the K d for binding decreased thousand fold in the presence of CaADP. In the absence of added nucleotides vanadate binds to a single site in the enzyme but it binds to three sites which show positive cooperative interaction in the presence of CaADP. Since there is a positive interaction among the three active sites of the enzyme it is suggested that ADP-vanadate binds to these sites as a pentacoordinated vanadium having trigonal bipyramidal geometry. This structure is analogous to the transition state of the phcxsphate during the synthesis and the hydrolysis of ATP by CFI.

P-10-039 REFINED MEASUREMENT OF THE H/ATP RATIO AT THE ATP-SYNTHASE OF CHLOROPLASTS S. Berry & B. Rumberg Max-Volmer-lnstitut for Biophysikalische und Teohnische Universit&t, 10623 Berlin, Germany

Physikalisohe

The H/ATP ratio is determined kinetically by comparing ATP synthesis and proton flow across the ATP-synthase in spinach thylakoids under conditions of continuous illumination. Net proton flow at steady state is zero, therefore the initial efflux in the dark is taken. Conventionally, the proton flow is determined graphically by the tangent method. In this case the basal flow has to be measured independently and subtracted from the total flow. Here, a new way of kinetic analysis of the relaxation of the pH gradient is presented, allowing direct determination of phosphorylating and basal proton flow at the same time. Both methods give a H/ATP coupling ratio of 4. Several special details have to be considered: 1) The diffusion potential has to be suppressed by the K+-ionophore nonactin. This is checked by following the dependence of proton flow on ionophore concentration and also by direct measurement of the diffusion potential. 2) The total flow has to be corrected for the nonphosphorylating basal flow. 3) The influence of the dark phosphorylation on the proton efflux has to be taken into account.

P-10-037

A T P S~4'I~.ASEMUTANTOF C H L A M r D O M O N A H REINHARDTII P R O V I D E S • V I D E ~ C E F O R I N C L U S I O N B O D I E S F O R M A T I O N IN C H L O R O P L A S T S A N D F,%RLy I N T E R A C T I O N B E T W E E M THE a AND ~ SUBUNITS D U R I N G THE CF I B I O G B N B S I B. D. D r a o i e r I, S.L. K e t c h n e r 2, J. O l i v e ~, S. G a u d r i a u l t I, J. G i r a r d - e a s e o u I and F.A. W o l l m a n i. (t)Service de Photosynth~se, IBPC, Paris, France-(2)University of California, Berkeley, CA, USA.(3)Institut Jacques Monod, Paris, France. Two n e i g h b o u r i n g m i s s e n e e m u t a t i o n s in the c o d i n g region of the c h l o r o p l a s t atpA g e n e of m u t a n t FUDI6, lead to two aminoacid s u s t i t u t i o n s (Ile184/Asn and Asn186/Tyr) in the a subunit of t h e A T P synthase, in a well c o n s e r v e d r e g i o n located next to the n u c l e o t i d e b i n d i n g fold. The m u t a n t assembles o n l y 3% of Bwm~brane-bound CFI but it a c c u m u l a t e s e x t e n s i v e l y m e m b r a n e - f r e e a g g r e g a t e s of the a and ~ subunits, w h i c h form t y p i c a l i n c l u s i o n b o d i e s in the c h l o r o p l a s t stroma. The two suhunits show i n c r e a s e d rates of synthesis in the mutant. This effect, as w e l l as i n c l u s i o n b o d i e s formation, are lost in the d o u b l e m u t a n t s w h e r e the m u t a t e d a eubunit is s y n t h e s i z e d in n u c l e a r contexts a l t e r i n g the synthesis of the a or ~ subunit. We d i s c u s s t h e s e o b s e r v a t i o n s in light of a b i o g e n e s i s p a t h w a y for CF1 a s s e m b l y w h i c h depends on an early i n t e r a c t i o n b e t w e e n the two subunits.

P-10-040 KINETIC ANALYSIS OF THE PROTON TRANSLOCATING ATP-SYNTHASE FROM SPINACH O.P&nke & B. Rumberg Max-Volmer-lnstitut for Biophysikalische und Physikalische Chemie, Technische UniversitY.t, D-10623 Berlin The rate of both ATP synthesis and hydrolysis oatalysed by the thiol-modulated and activated ATP-synthase is measured in dependence on all substrates including H+i.. We observe: 1) sigmoid kinetics with respect to H+inl 2) hyperbolic kinetics with respect to ADP, ATP and phosphate, with a decreasing KM for phosphate upon increasing VMAX,3) competition between ADP and ATP as well as between phosphate and ATP. Simulation of the complete set of experimental data is obtained by a kinetic model featuring Boyer's binding change mechanism. The details are: 1) random binding of ADP and phosphate and competitive binding of ADP and ATP as well as phosphate and ATP at the open binding pocket, 2) simultaneous closure and opening of two out of three binding pockets by ApH-induced conformational changes, taking into account oooperativity with respect to all three c¢l~subunits, 3) spontaneous conversion between ADP and ATP within the tightly closed pocket, 4) translocation of four H+ across CFo per each ATP produced or hydrolysed, 5) conformational coupling between CFo and CFI.

P-10-041

P-10-038 Photophosphorylation in presence mined by electron flow but not by

Henrik Laasch Institute of Ecological H e i n e - U n i v e r s i t ~ t , D-40225

of prooaine is d e t e r ~ transthylakoid ApH.

Plant Physiology, HeinrichDUsseldorf, Germany.

A new m e t h o d for the d e t e r m i n a t i o n of ApH was d e v e l o p e d w h i c h makes use of the pH d e p e n d e n t a c t i v i t y of v i o l a xanthin de-epoxidase (VD) in the t h y l a k o i d lumen as a natural pH indicator. Application of the VD m e t h o d r e v e a l e d the well known linear r e l a t i o n s h i p of rate of p h o t o p h o s p h o r y l a t i o n , Vp, and ~pH when e l e c t r o n flow was controlled by light flux or by the electron flow inhibitor DCMU. In contrast, Vp increased despite a d e c l i n e of ApH, when ~pH was i n h i b i t e d by the l i p o p h i l i c amine procaine. I n h i b i t i o n of e l e c t r o n flow by DCMU in the p r e s e n c e of procaine, then d e c r e a s e d Vp w i t h o u t any change of ~pH. Obviously, Vp was related to the rate of e l e c t r o n flow, Vet but not to ~pH over a wide range of V e. I n d e p e n d e n c e of Vp and ~pH was only o b s e r v e d when linear e l e c t r o n flow p r o c e e d e d via the c y t o c h r o m e - b 6 / f complex. The data i n d i c a t e d that the d e l o c a l i z e d (bulk) ApH is not the d r i v i n g force of p h o t o p h o s p h o r y l a t i o n in the p r e s e n c e of the l i p o p h i l i c amine procaine.

120

Chemie,

REGULATION OF THE Q-CYCLE TRANSPORT OF GREEN PLANTS

IN PHOTOSYNTHETIC

S. Berry & B. Rumberg Max-Volmer-lnstitut for Biophysikalische und Technische Universit&t, 10623 Berlin, Germany

ELECTRON

Physikalische

Chemie,

Along the linear path of photosynthetic electron transport two protons are pumped across the thylakoid membrane into the lumen for every transported electron. According to the concept of the Q-cycle, the proton to electron ratio is raised to a value of three by means of the re-reduction of plastoquinone across the cytochrome b6/f complex. Our experimental results on the proton to electron ratio in spinach thylakoids show that the Qcycle is not obligatorily in action. Stimulation occurs at subsaturating light intensity and/or increased membrane permeability. However, a unique correlation between Q-cycle activity and the pH gradient is not observed. Also, a transmembrane electrical potential gradient seems to exert no control over the Q-cycle. These results are irrespective of the electron acceptor used (ferricyanide or NADP+flerredoxin). A reaction model is presented that allows simulation of all experimental results. The main feature of the model is the regulation of the Q-cycle being primarily dependent on the redox state of plastoquinone and additionally on the lumenal pH.

Poster

P-10-042 STRUCTURE-FUNCTION STUDIES ON A THIOL-MODULATED ATP SYNTHASE FROM A MUTATED CYANOBACTERIAL STRAIN. Riekv van Walraven, Marijke Schohs, Bea Krenn & Ruud Kraayenhof. IMBW, BioCentrum, Vrije Universiteit Amsterdam, The Netherlands. Cyanobacterial ATP synthase (FIF0) from Synechococcus 6716 shows, in contrast to the chloroplast enzyme (CFIF0), no influence of the redox state on activation by a proton gradient (A/irt+), nor any effect on ATP hydrolysis by treatment with thiol agents, heat or methanol. Functionally the FIF Ocorresponds to the reduced form of CFIF0 in that the ~ H + required for activation (Ap.a) is low, and the active form is stable in the absence of ADP [1]. A stretch of 9 aminoacids including two eysteines in the 7 subunit of the CFI part, that is absent in cyanobacterial 7, is involved in these types of activation. Using membrane vesicles from Synechocystis 6803 mutants, with a chloroplast-like segment inserted into the 7 subunit of FIF0 [2], we show that upon reduction the Ap.ais lower than the z~lSta of the wild type, the untreated FIFOand the reduced CF1F 0. Both active forms of the mutated FIF0 are stable when ADP is not present. Furthermore, the mutation makes the F1F0 susceptible to treatment with thiol agents and heat. 1. Krab, K., Bakels, R.H.A., Scholts, M.J.C. & Van Walraven, H.S. (1993) Biochim. Biophys. Acta 1141,197-205. 2. Werner-Grtine, S., Gtinkel, D., Schumann, J. & Strotmann, H. (1994) Mol. Gen. Genet. 244, 144-150/

P-10-043

P-10-045 MEMBRANE PERMEABILITY AND ENERGY COUPLING Y. de Kouchkovskv Biosyst~mes Meml~ranaires, ISV, CNRS, F-91198 Gif-sur-Yvette, Fz:ance Not all protons translocatex] by photosynthetic or respiratory chains flow back across phosphorylating ATPases: some enzymes may be inactive, yet leaky, and membrane is always permeable, naturally or due to organelle extraction or uncoupler addition. A proper definition of the ATP/electron stoichiometry (P/e') should take into account all pathways (cf. S.J. Ferguson, 1986: TIBS 11,351353). An analysis of the different membrane conductances, with or without ionophores, and of the resulting phosphorylating yield p (P/e'measured = p P/e'mechanistic) is presented and illustrated in the case of mitochondria (with P. Diolez) and thylakoids (with C. Sigalat). The correct determination of energetic ratios requires that "state 4" non-phosphorylating rate, when statistically ATPases are "closed" - not basal "state 2" - is subtracted from the phosphorylating "state 3" and that all these redox rates are taken at identical AIStR+ (with moreover AW and ApH both interfacial or interbulk, but convieniently here, only ApH: thylakoids, or AW: mitochondria, practically exist). Indeed, when ADP is added, ATPases open, proton gradient lowers and membrane conductance decreases. The highly non-ohmic behaviour of basal leaks in mitochondria (potato tuber) makes however that "total oxygen" may be considered: at AW in "state 3", membrane conductance becomes negligible. This is different in thylakoids, where the background permeability is higher and the dependence of proton conductance towards ApH less steep.

P-10-046

ApH+-REGULATION OF FoF1 ATPase IN CHLOROPLASTS AND MITOCHONDRIA.

UNISITE ATP SYNTHESIS AS FUNCTION OF pHin AND pHout

~

Dominique Razaka and Peter Griiber, Institut ~ r Physikalische Chemic, UniversitiR Freiburg, Albertstr. 23a, D-79104 Freiburg

1, p. Diolez 2, B. Chernyak3, M. Valerio 1, J. Velours4, M. and C. Sigalat 1. 1CEA Saclay, DBCM-SBE, 91191 Gif sur Yvette Cedex, France; 2UMR 9991, CNRS, Universitd de Bordeaux II, 33077 Bordeaux Cedex, France; 3A. N. Belozersky Institute, Moscow State University, Moscow 119899, Russia; 4UPR 9026, CNRS, Universit6 de Bordeaux II, 33077 Bordeaux Cedex, France; 5LNSA, INRA, 78352 Jouy en Josas Cedex, France. Ap'H+-activation and uncoupler-induced deactivation of FoF1 ATPase was studied in chloroplasts and mitochondria. This process seams to be universal, ApH+-activation being insensitive to the F0 inhibitor venturicidin. Catalytic turnover is required for ATPase deactivation in uncoupled mitochondria, the probability t o deactivate at each cycle depending on organisms and tissues. In potato tubers and pea seeves, ATPase deactivates in a few turnovers, as in oxidised chloroplast ATPases. In mitochondda from pea leaves, rat liver and yeast, deactivation occurs in several thousands of turnovers, as in thiol-reduced chloroplast ATPase. All these mitochondrial ATPases lack the regulating disulfide bridge present in CF1 y-subunit. By different approaches, we checked if initial ATP hydrolysis was kinetically limited by ATPase or ATP/ADP antiporter. Our studies also make possible to titrate activated vs. total FoF1 ATPases in whole cells.

P-10-044 TIGHT PROTON COUPLING IN MEMBRANE-BOUND CFoCF 1 REACTIVATED BY HIGH CONCENTRATIONS OF TENTOXIN. C. Siaalat, B.Pitard and F. Haraux. CEA centre d'~tudes de Saclay, DBCM, SBE, 91191 Gif sur Yvette, France The effect of tentoxin (-ITX) at high concentrations was investigated on membrane-bound chloroplast ATPase. In DTT-treated thylakoids, uncoupler-triggered ATP hydrolysis, completely inhibited at 2 ,uM TIX, was recovered up to about 15 % of the control activity at 200 pM TTX. This activity was inhibited by venturicidin. ApH measurements showed that the futile proton flow through CFoCF1 (in the absence of ADP) was partially inhibited by TTX at high concentrations, and kept its property of being 100 % blocked by ADP. Light-induced ATP synthesis, fully inhibited by TTX in the pM range, was also restored by higher concentrations of TTX, but to only a few percents of the control. TTX-modified enzyme had a higher driving-force requirement as the control to catalyse ATP synthesis. With CFoCF1 co-reconstituted with bacteriorhodopsin into liposomes, ATP synthesis was inhibited and reactivated in the same range as in thylakoids, except that the recovery at high TTX concentration was 25 % of the control. It is concluded that TTX-modified CFoCF1 differs from the native enzyme by its kinetic performances, but keeps the same mechanism, and especially a tight coupling between ATP synthesis / hydrolysis and proton pumping.

The H+-ATPase from chloroplasts was isolated and reconstituted into phosphatidylcholine liposomes. The rate of ATP synthesis was measured under uni-site conditions after energization of the proteoliposomes by an acid-base transition as a function of pHin and pHout. At any given pHout, the rate increased with increasing internal H÷ concentration for all pHout values between 9.2 and 7.5. The dependence on internal H÷ concentration can be described similarly as under multi-site conditions (Possmayer F. and Gdtber P. (1994) J. Biol. Chem. 269, 1896-1904). A reaction cycle under uni-site conditions is proposed.

P-10-047 PROTON RELEASE ASSOCIATED WITH PHOTOSYSTEM 1I IN A MUTANT OF ARABIDOPSIS THALIANA LACKING TRANSHEXADECENOIC ACID N1-J. Delrieu & A. Tremoli~res lnstitut de Biotechnologie des plantes, URA 1128, Bat. 630, Universite Pans XI, 91405 Orsay, France. The lipid phosphatidylglycerol (PG) and especially the one containing the fatty acyl group A3-tran,~-hexadecenoic acid (trans-C~6:~-PG)is specifically found in the light-harvesting chlorophyll a b complex of photosystem II (LHC II) and probably implicated in its trimeric form. In a preliminary analysis of antenna function and PS II activity, MeCourt et al (1985) were unable to establish a difference between the mutant lacking D'anLV-Cl~, I but having a compensating increase in palmitic acid (16:0) and the wild type. In agreement with these conclusions, we observed that the O: yield and flash-induced fluorescence yield were very similar in the mutant and the wild type. However, the extent of flashinduced pH transients revealed that the protolytie reactions in the mutant thylakoids were much less pH dependent than in the wild-type, in the range from 6 to 7.5. Oscillations of proton release in the mutant were much smaller than in the wild-type. It has been proposed that LHC lI shields the site of proton production and channels protons into the lumen (Jahns and Junge, 1992). Our results suggest a possible role for trans-C~6:~-PG,in the conductive mechanism of protons, from the water-splitting complex to the lumen.

121

Poster

P-10-048 THE DRIVING FORCE FOR THE ACTIVATION OF THE H+-ATPASE IN CHROMATOPHORES IS NOT CORRECTLY ESTIMATED BY THE CAROTENOID ELECTROCHROMIC RESPONSE. B. A. Melandri V. Fregni, A. L. Altimari and M. Crimi Department of Biology, University of Bologna, Italy. ATP hydrolysis induces the activation of the H+- ATPase in chromatophores of

Rhodobacter capsulatus supplemented with nigericine and 50 mM K + (i.e. when ApH < 0.2 units). The value of A~H+ expected for this activation exceeds 140 mV. The value oftransmembrane electric potential (A(o) driving this activation was measured using three different approaches: carotenoid electrochromisrn, uptake of SCN- and the responses of the dye oxonol VI. The osmotic volume of chromatophores was measured with the EPR-probe tempone, and was found to be 16 4- 3 ktl (ktmole bchl )-1. On this basis, the value of Atp calculated from the SCN- uptake was about 140 mV, while that indicated by the electrochromic signal, calibrated with K*-diffusion potentials, ranged between 35 and 70 mV. Only the value indicated by SCN" distribution is consistent with the energetic requirement for the activation of H+-ATPase. Electrochromism thus appears to underestimate grossly the A~ induced by ATP-hydrolysis in chromatophores.

P-10-049 THE EFFECT OF PROTONS ON PS2 PHOTOCHEMISTRY Giovanni Finazzi and Giorgio Forti. Centro C.N.R. per la Biologia Cellulare e Molecolare delle PianteDipartimento di Biologia. Universit/t degli Studi di Milano Via Celoria, 26 20133 Milano, ITALY Preillumination of isolated thylakoids with single turnover flashes of 6Hz frequency inhibited Qa reduction measured as the fluorescence induction rise upon poisoning the thylakoids with DCMU and hydroxilamine. The spontaneous relaxation of ApH in the dark or its dissipation with the uncoupler nigericin (but not NH4CI) abolished the inhibition, in agreement with the previous reports on the inhibition of PS2 limited rate of NAPD reduction. The inhibition was detectable after few flashes (10 c.a.) were fired. Quantitative analysis of the fluorescence induction curves reveals that PS2 efficiency is reduced by the protons generated during preillumination, while the other relevant parameters (fluorescence yield and cooperativity) are not effected

P-10-050 Molecular Characterization of atpA and atpB Deletion Mutants Induced in Chlamydomonas reinhardtii cw.15 by Chloroplast Transformation L

.

I

I

+

2

S. Leu, J. Schleslnger, D. Hu, H. Fledler, H. Strotmarm2 and N. Shavit' Ben Gunon Umverslty of the Negev, Beer Sheva, Israel, Hemnch Heine Universit~it DUsseldorf, Germany !

.

.

.

2

,

.

•

The Chlamydomonas reinhardtii chloroplast genes atpA and atpB, encoding the a and 13subunits of ATP synthase, were deleted using the strain cw-15. The aadA cassette, conferring spectinomyein resistance when expressed in the chloroplast, was cloned into the atpA or atpB genes, replacing most of the open reading frames of the corresponding genes. The resulting constructs were used to transform cw-15 by vortexing in the presence of glass beads. Spectinomycin resistant clones were grown at low light in the presence of acetate and spectinomycin to obtain strains homoplasmic for the aadA gene. These strains were not able to grow photoautotrophically, and no signals for the deleted gene sequences were detected by PCR or by Southern blot analysis. No ATP synthase subunits were detected when the mutants were analyzed by protein biochemical techniques, indicating that both subcomplexes, CFI and CF0, are assembled only when the a and I~subunits are expressed.

122

Poster

P-I 1-003 Structural organisation of higher plant thylakoids: Distribution and function of protein complexes after reductive activation of LHCII-kinase. Poster

session

11

$imone Andr6e. Marius Spittel & Engelbert Weis Institute of Botany, Univ. Miinster, D-48149 Mtinster (Germany) Dissociation of LHCII-P from PSII is a well known phenomenon. Here we investigate two problems in conjunction with the activation of the LHCIIkinase: (1) What are the consequences for structure and function of core complexes of PSII? (2) Is there a functional association of LHCII-P to different subpopulations of PSI? Phosphorylation of thylakoid proteins was followed by 32p-labelling; Protein complexes were separated from grana and stroma lamellae and characterized by native gel electrophoresis, gel filtration and immunoblotting. Pigments and redox-components were determined spectroscopically or by HPLC. PSIL" LHCI/phosphorylation is followed by interconversion of PSH-cores from its dimeric into a monomeric form, which then becomes inactivated in the light. PSI-antenna: After disconnection of LHCII-P from PSII and its subsequent migration, the antenna size of PSI in the stroma region increased by about 30%, whilst little change was seen in the grana. We derive a thylakoid model which incorporates distribution and density of different membrane proteins and we discuss its flexibility in relation to redox-controlled protein phosphorylation. The implications for the electron transport will be discussed in a separate poster (Spittel et al.).

Organisation of the photosynthetic apparatus P-11-001

- P-11-058

P-11-001

P- 11-004

EFFECT OF A SELEC~VE DEPLE~ON OF ACYL L~PIDS ON THE LIGHT AND DARK EffOSPHORYLATIONIN SPINACH ~ O I D R~IBRAI~S

Control analysis of electron t r a n s p o r t in higher plant thylakoids: evidence for functional microdomains.

P.A. S i e s e n t h a l e r & J . V a l l i n o Laboratolre de P h y s i o l o g i c v 6 g 6 t a l e , U n i v e r s i t 6 de N e u e h ~ t e l , Rue Emile-Argand 13, CH-2007 N e u e h ~ t e l , Switzerland

Helmut Kirchhoff & Engelbert Weis Institute of Botany, Univ. Mtinster, D-48149 Miinster (Germany)

S e v e r a l p h o t o s y n t h e t i c f u n c t i o n s a p p e a r to depend on the presence, the i n t e g r i t y and the t o p o l o g y of c e r t a i n a c y l l i p t d s i n the t h y l a k o i d membrane (TM). I n t h i s i n v e s t i g a t i o n , we have s t u d i e d the e f f e c t of a s e l e c t i v e d e p l e t i o n of e i t h e r m o n o g a l a c t o s y l d i a c y l g l y c e r o l (MGDG) or p h o s p h a t i d y l g l y c e r o l (PG) i n the o u t e r , then i n the i n n e r TM monolayer on the l i g h t and dark p h o s p h o r y l a t i o n (PP). To t h i s end we have used specific lipase treatment (e.g. the l i p a s e from arrhlzus and the phospholipase A 2 from Naja n a j a). A d e p l e t i o n of PG m o l e c u l e s ( a b o u t 69Z of the t o t a l PG) i n the o u t e r TM monolayer did not a f f e c t s i g n i f i c a n t l y the d a r k PP w h i l s t a d e p l e t i o n of the o u t e r MGDG m o l e c u l e s ( a b o u t 65Z) o b l i t e r a t e d almost c o m p l e t e l y t h i s a c t i v i t y . These r e s u l t s w i l l be d i s c u s s e d i n terms of the r o l e of l i p i d s i n t h y l a k o i d membrane functions and of the possible e f f e c t of lipid depletion a n d / o r h y d r o l y s i s p r o d u c t s on the TM i n t e g r i t y which might c o n t r i b u t e to p r o t o n l e a k a g e and t h e r e f o r e to a d e c r e a s e d PP a c t i v i t y .

P-I 1-002

The distribution of control of photosynthetic e- transport among different redox reactions is analyzed. Flash-induced redox-kinetics of plastoquinone (PQ), cytochromes (cyt), plastocyanine (PC) and P700 were ananlyzed. From inhibitor experiments, we conclude that there exists a surprisingly tight redox coupling between PSII and cyt b/f complexes. We also see a concerted inactivation of PSII and cyt b/f under conditions' where the 6pH across the thylakoid membrane exeerts control on electron transport. Only 1-2 PQ are involved in the e- transport between these complexes, while the turnover of a larger pool of PQ (about 6 per PSII) is slow and not likely to be involved in linear electron transport. This is in contrast to the idea of free exchange of PQ. We assume the existence of transient microdomains formed in the grana region by PSII, LHCII and cyt b/f where PQ diffusion is restricted. In agreement with this concept, we can demonstrate that the tight redox coupling between P S I / a n d cyt b/f, seen in stacked membranes, is broken upon destacking. In contrast, the e- transport from cyt b/f to PSI(P700), localized in distant stroma lamellae and 'exposed membranes', seems to be mediated by free exchange of 2-3 PC. The implications of this functional organisation of thylakoids for the control of e" transport during photosynthesis is discussed.

P-11-005

PHOTOSYSTEM II STRUCTURE INVESTIGATED BY ELECTRON MICROSCOPY AND SINGLE-PARTICLE AVERAGING

Structural organization of higher plant thylakoids: Control of electron transport by reductive activation of L H C I I kinase

Eobert J. Boekema 1, Ben Hankamer 2, Jon Nield 2 and James Barber 2

Marius Spittel, Helmut Kirchhoff, Simone Andr6e, & Engelbert Weis Institute of Botany, Univ. MUnster, D-48149 Mtinster (Germany)

1Biophysical Chemistry, University of Groningen, Nijenborgh 4, Groningen, The Netherlands; ~Volfson Laboratories, Imperial College of Science, Technology & Medicine, London, UK

From spinach a large dimeric Photosystem II particle was isolated by sucrose gradient centdfugation IBoekema et al., 1995|. We characterized this so-called supercore complex by analyzing 2000 single particle projections from electron microscopy. The dimensions in the membrane plane are 268 x 123 )l,, with a height of 90 A in the center and 60 A at the outer ends. From a comparison with other PS II particles the location of PsbC and PsbO subunits can be deduced. The projected structure of the supercore complex further suggests that two LHC-II trimers are present, each of them linked to an inner core part consisting of subunits PsbA-F and PsbO by other protein densities. Two of these connecting masses have the size of a LHC-II monomer and are possibly CP26 and CP29. There is some space left for other other connecting masses, but when they are present they should be much smaller than CP26 and CP29. Boekema E.J., B. Hankamer, D. Bald, J. Kruip, J. Nield, A.F. Boonstra, J. Barber and M. R6gner (1995) Proc. Natl. Acad. Sci. USA 92, 175-179

From biochemical data a structural model of the thylakoid membrane is derived which incorporates the stoichiometry, distribution and density of PSI, PSII, LHCI and -II and cytochrome (cyt.) bf complexes. The distribution and functional state of these complexes is specifically altered due to protein phosphorylation (see separate poster, Andr6e et al.). Here we investigate the consequences of protein phosphorylation for linear electron transport. Flash-induced kinetics of redox components (PQ, cyt., plastocyanine and P700) are analyzed in membranes before and after redoxdependent activation of LHCII kinase. We assume that "microdomains" are formed in stacked grana regions by PSII, LHCII and cyt. bf complexes. Diffusion of PQ within these domaines is restricted and only 1-2 PQ are involved in linear electron transport. Upon phosphorylation of LHCII, (dimeric) PSI/-LHCII supercomplexes are broken followed by inactivation of PSII cores. Due to tight functional coupling to cyt. bf inactivation of PSI] complexes leads to a decrease in the capacity of linear electron transport to PSI. At the same time the absorption cross section of one subpopulation of PSI located in stroma lamellae increases. A model of functional organization of the electron transport system is presented.

123

Poster i i

P-U-006 MEMBRANE ASSOCIATION OF CALVIN CYCLE ENZYMES AND FERREDOXIN-NADP+REDUCTASE IN P L A S T I D S OF BARLEY MUTANTS D E F I C I E N T IN T H Y L A K O I D MEMBRANE PROTEINS

Karl-Heinz Siiss, +Isabella Prokhorenko, Christoph Arkona, M a r k u s Teige a n d Klaus Adler I n s t i t u t e of P l a n t Genetics a n d Crop Plant Research, 06466 Gatersleben, Germany, a n d +Institute of Soil Science a n d Photosynthesis, Pushtchino 142292, Moscow Region, Russia T h e in situ-localization of Calvin cycle e n z y m e s a n d ferredoxinN A D P + r e d u c t a s e (FNR) was studied in barley viridis and chlorina m u t a n t s deficient in different thylakoid m e m b r a n e proteins. Calvin cycle e n z y m e s a n d FNR accumulate in m u t a n t plastids a n d are associated with thylakoid m e m b r a n e s even if devoid of either PS I, PS II, or LHC-II. E n z y m e association is lost, however, if t h y l a k o i d m e m b r a n e s do not posses H+-ATP synthase. This s u g g e s t s that (i) H+-ATP synthase m a y be involved in m e m b r a n e - b i n d i n g of watersoluble p h o t o s y n t h e t i c e n z y m e s a n d (ii) that Calvin cycle a n d !thylakoid m e m b r a n e e n z y m e s are organized into complexes in situ.

P-11-009 ELUCIDATION OF THE ROLE OF SOME LOW MOLECULAR MASS POLYPEPTIDES OF PHOTOSYSTEMS 1 AND 2 AS LONGWAVELENGTH CHLOROPHYLL-BINDING PROTEINS M.A.Ismailov, I.S.Zulfugarov and A.A.Asadov Institute of Botany, Azcrbaijan Academy of Sciences, Patamdar shosse, 40, Baku, 370073, Azcrbaijan Republic The true number, spectralcharacteristics(maxima, intensity,polarization) orientation and localizationof the most longwavelenght (LW) forms of chlorophyll (Chl) a of photosystcm (PS) I and 2 of higher plants, green algae and cyanobactcria arc under investigation. Synchronization of appearance of L W forms of PS 1 (Chl a forms at 705, 710 and 720 nm) and PS 2 (Chl a forms at 682 nm) with the formation of P S l core (A A 700) and PS 2 core (variable fluoresccnce, A F) was revealed. There are contradictory data on the localizationof L W forms of Chl a of PS 1 and PS 2 in differentproteins of P S l and 2. During the last years a number of research groups have rcvealed thc existence of low molecular mass proteins in 4-I0 kDa range, which can bc conncctcd with core proteins of P S I and 2. Moreover, functions of the most of these proteins are unknown. In this connection the possibilityof binding by low molecular mass proteins of the most L W forms of Ch a of P S l and 2 is undcr consideration.

P-11-010

P- I 1-007 SPECTRAL CHARACTERIZATIONS OF LANTHANIDE INTER. ACTIONS WITH THYLAKOID MEMBRANE SURFACES

O~.~O~.Ol~'TJr~ l ~ l n l ~ 'm ~ 01' CL1E.~aOI~.tdUF~ • 'BOM l~&~ AO Tn DIlIIATURATIOH l~lT/kll~l OIF ....

K. IC Karukstis. M. Y. Kao, D. A. Savin, R. A. Bittker, K. J. Kaphengst, and N. R. Nalto, Department of Chemistry, Harvey Mudd College, Claremont, CA 91711, USA

E. ADOS'bOIov&, M. B~sheva Se N. Tsvetkova Institute of Bioplxvsics,Bulgeaqa~ Acetlemy of Sciences, Acs~1. G. Bonchev St,r, BI. 21, Sofia 1113, Bulgaria

Lanthanide cations (Ln 3+) neutralize exposed segments of protein complexes on the thylakoid membrane surfaces of plant chloroplasts. We have used two spectral techniques to monitor Ln3+-membrane interactions and to characterize cation effects on membrane organization. The fluorescence emission of the extrinsic probe 2-p-toluidinonaphthalene-6sulfonate (TNS) reveals a dependence on Ln 3+ size and total orbital angular m o m e n t u m L value. TNS fluorescence is significantly enhanced by Ln 3+ ions with greater charge density (small size &/or low L value), suggesting a heterogeneity in the number and type of TNS binding sites and a variable extent of membrane appression. Chlorophyll absorbance increases in the 500.660 nm region, attributed to light scattering arising from membrane structural reorganization, also depend on Ln 3+ size. The data suggest that larger lanthanides with smaller enthalpies of hydration induce more extensive membrane appression.

Thylakoid membranes from wild type, OB67 and LK3 A.~bido~i~ t27atfar~a fatty acid deeaturation deficient mutea~ts were studied by measuring the chl0rophyll fluorescence. Low temperatxu'e (77K) emission spectra showed differences in the energy transfer between chlorophyll-protein complexes in the wild type and mutant membl-anes. However, both the chlorophyll fluorescence at room temperatuI~ and at 77K in presence and in absence of ce,tions show slight differences in cation-dependent lateral rearrangement of chlorophyn protein complexes. The fluorescence induction data show that the amount of PSH~. increase in the mutants when compared to the wild type. The observed differences in the fluorescent characteristics of wild type and mutants are discussed in term of the changes in the fatty acid composition.

P-11-008

~ r a z a a x a O~)

P-11-011

MAIZE

CHANGES IN THE U L T R A S T R U C T U R E AND DYNAMICS OF THYLAKOID MEMBRANES

P. Nyitrai, E. SLrvfiri, and F. Lkag Department of Plant Physiology, E6tv6s University, H-1445, Budapest P.O.B. 330, Hungary

Tobv D. Flint#, John W. Ford#, Robert C. Ford:~ and Andreas Holzenburg#*

EFFECT OF KINETIN ON MESOPHYLL THYLAKOIDS

THE

ORGANIZATION

OF

Cytokinins have an important regulatory role in the development of plants including chloroplast biogenesis. They effect on the synthesis and/or stability of some nuclear encoded m-RNAs increasing the level of the corresponding polypeptides (Parthier 1989, BPP 185, 289). We have investigated the effect of kinetin on the development and organization of maize mesophyll thylakoid membranes greened under different light regimes. Kinetin treatment promoted the synthesis and/or assembly of some components of thylakoid membranes (chlorophyll, light-harvesting chlorophyll a/b protein complex I and II) allowing a more developed s~ucture (i.e. more complete light-harvesting system) and higher efficiency of photosynthetic function, as CO 2 fixation. Kinetin facilitated the normal formation of thylakoids under extreme low-light illumination compensating partly the absence of higher light intensity, and it strongly promoted the further greening of intermittently-light grown seedlings under higher light condition. In conclusion, structure and function of hormon-treated thylakoids seemed to be in a more advanced developmental stage reflecting the synergic co-operation of the light and hormonal factor during the thylakoid biogenesis.

124

# Dept. Biochem. & Mol. Biol. and *Dept. of Genetics, University of Leeds, Leeds LS2 9JT, UK ~:Dept. Biochem. & Appl. Mol. Biol., UMIST, Manchester M60 1QD, UK Adaptation to different light regimes has been studied in higher plants using transmission electron microscopy (in conjunction with digital image processing) of negatively stained thylakoids following (i) growth of spinach (w.t.), barley mutants (lacking photosystem I) and Arabidopsis thaliana mutants (exhibiting a reduced membrane fluidity) under far red light of low intensity, (ii) contolled induction of phosphorylated/dephosphorylated states in photosystem II - enriched membrane preparations, and (iii) a combination of conditions (i) and (ii). Our findings are discussed and related to the proposed mechanisms for controlling the distribution of light excitation energy in higher plant thylakoid membranes which maximise photosynthesis.

Poster

P-11-012

P-I1-015 MEMBRANE

CHARACTERIZATION OF THE LIGHT-INDUCED REVERSIBLE STRUCTURAL CHANGES IN GRANAL THYLAKOID MEMBRANES

HIGH PERFORMANCE PROTEINS

A. Istokovics, V. Barzda, F. Lajkt, L Simidjiev and G. Garab Institute of Plant Biology, Biological Research Center, Szeged, Hungary

J. Vater, K. Heinze, B. Kablitz, K. Friedrich & J. Sainikow Institut fOr Biochemie und Molekulare Biologle, Technische Universitat Berlin, Franklinstr. 29, D-10587 Berlin, Germany.

By means of circular dichroism (CD) spectroscopic techniques we have earlier shown that in granal thylakoid membranes photosystem 2 particles are assembled into large chirally organized macrodomains. These macrodomains have been shown to be capable of undergoing light-induced reversible structural changes, detected as ACD. Earlier it was proposed that the structural changes in the antenna macroorganization were driven directly by the electron transport processes which generate transmembrane and/or local ApI-I. However, in this work we show that the ability of the macrodomains to undergo structural rearrangements, albeit facilitated by ApH, does not depend solely on the photochemical activity of the samples: (i) ACD occurs only under stacking conditions, (ii) the rate of ACD cannot be saturated with light intensities that saturate the electron transport, (iii) the structural changes can be inhibited by agents, such as some quinone antagonists, which do not affect the electron and proton transport, but may resist some inhibitors of the linear electron transport chain. It is suggested that the ability of structural changes is inherent in 3-D lamellar macroarrays of the particles and plays a role in the regulation of the energy migration and dissipation pathways in the antenna.

SEPARATION

It has been previously shown that, during zygote formation in C. relnhardtil, thylakold membranes originating from each parental gamete fused thereby allowing a lateral redistribution of transmembrane proteins. Using two mutant strains which were respectively devoid of the Photosystem II (PSII) subunit CP43 or of the PSII subcomplex CP47~I-D2, we got ultrastructural evidence for the assembly of the two sets of PSII subunlts in the stacked membranes of the zygote. Such a result was obtained using the freeze-fracture technique: zygotes displayed large EFs particles similar to those observed in WT, whereaseach gamete displayed smaller EFs particles. This showed that, when present in different parts of a membrane, the two sets of polypeptides were able to assemble upon membrane diffusion thus supporting our previous conclusions of a two step-model for PSII assembly where formation of a CP47-DI-D2 subcomplex is followed by assembly with CP43 subunlt. However, the fluorescence induction kinetics did not allow us to observe a partial recovery of photosynthetic activity in zygotes, either because the amount of active assembled PSII centers remained below detection or because some cofactor required for PSII function was lacking in such PSII complexes.

P-II-014 THE ELECTRICAL PROPERTIES OF THE THYLAKOID MEMBRANE AND ITS PARTITIONS ('BRUSH BORDERS') IN RELATION TO ENERGY SUPPLY W_L Vredenlmr~, A.A. Bulychev~, T. van Voorthuysen and J.F.H. Snel, Dept of Plant Physiology WAU, Arboretumlaan 4, NL 6703 BD Wageningen, the Netherlands, ~Dept Biophysics, Moscow University, Moscow, Russia Study of light- and electrically stimulated thylakoid responses (potential, current, impedance) with patch (voltage)-clamped chloroplasts of Peperomia metallica has enabled the kinetics of flash-induced potential (current) generation and of changes in conductance to be measured with high precision in individual intact chloroplasts. The present contribution will focus in particular on the light-dependent changes in conductance of resistive pathways in the chloroplast. A major part of these reversible conductance changes arc likely to originate from the partitions ('brush borders') of the folded thylakoid system. The changes are extremely sensitive to agents that modify ion transport and exchange processes in the membrane and its adjoining interphases. The patch clamp technique is unique for quantifying these conductance changes in intact chloroplasts on a ms time scale.

PSII

PSII membrane proteins (BBY particles) from spinach were separated by high resolution chromatographic and electrophoretic procedures. a) After dissociation of the PSII complex with SDS its protein components were fractionated by high performance electrophoresis chromatography (HPEC) using an Applied Biosystems model 230A HPEC-System. This technique combines the inherent resolving power of gel electrophoresis with the convenience of continuous sample elution and automatic sample collection. b) The PSII complex was dissociated by acidification with 0.1% trifluoroacetic acid and extracted with isopropanoFwater mixtures). At 50°/5 isopropanol the 3 extrinsic proteins (16; 23 and 33 kDa), the 10 kDa protein and ¢ytochrome bss9 were released from the complex. At 60% isopropanol the LHC11 subcomplex was extracted. The residual material contained the DI and D2 protein, CP43 and CP47. PSII membrane proteins were separated by reversed phase HPLC on a cyanopropyl-Nucleosi1300-7CN column.

P-11-016

P-11-013 J. Ollvel, M. Recouvreurl, J. Girard-Bascou2 and F.A. Wollman2 11nstltut Jacques Monod, Unlverslt~ D. Diderot, 75005 Paris, 2 Instltut de Biologle Physico-Chimlque, 75005 Paris, France.

OF

OROW£Ii, FLUORESCENCE ~ PI~NT LJ~rr~mRLL abyssa//s (PHEOPHYTA).

ANALYSIS OF THE B~OWN ALGA

K.Reis~, y.yoneshigue-Valentin I, D.O. Hall2 and C.P.aantoil. iDept de Bioquimica-I.Q.-U.F.R.J.-Rio de Janeiro-Brasil 2Biosphere Division-King's College-London-U.K. Light saturation curves were constructed using modulated fluorescence in wild fronds of Laminaxia abyssalis, after growth in the lab for a few months. The ascending slope of this curve (~)was utilized as an indicative of the quanttun yield of the Photosystem II and showed an increase from 0.011 near the stipe to 0.018 near the apex. Pigment content was evaluated by HPLC along the frond, and their contents expressed by square centimeter. The content of chlorophyll_c increased 40% near the apex, whereas the other pigments did not change in relation to the stipe region. The cell size also increase from the stipe to the apex and the length of the largest chloroplast changed from 4.4 to 7.5 ~zn, despite their number been approximately constant (63-68.105/cm2). Our data suggests that the increase in the photosynthetic efficiency near the apex on this plants is due to the increase of the size of the light-harvesting complex. (supported by CAPES,BRASIL and EEC contract- CI/0770)

P-11-017 EVIDENCE FOR A D I ] ~ R I C ORGANIZATION OF ]PHOTOSYTEM H IN VIVO: "['lie PHOTOSYNTHETIC bIEblBRANE OF ]~ANTONIELLA SOUAMATA

B. Hecks 1, C. Wilhelm2 and H.-W. Trissl1 ~Abteilung Biophysik, University, Barbarastr. 11, D--49069 Osnabrfick 2 Botanisches Institut, University, Johannisallee 19-21, D-04031 Leipzig The organization of photosystem I 0aS I) and photosystem II (PS 11) as well as their light-harvesting complexes (LHC) in the thylakoid membrane of Mantoniella squamata was characterized by measurements of fluorescence induction kinetics and picosecond fluorescence decay kinetics. This microalga possesses a homogeneous thylakoid membrane system in which the two photosystems are somehow mingled. Here we addressed the questions whether PSI and PS 1I are in such a close contact that PS I drains excitation energy from PS II as suggested by Trissl and Wilhelm (Trissl, H.-W. & Wilhelm, C. TIBS 18, 415-419) and whether the PS II photosynthetic units themselves are in excitonic contact. From the relative high ratio of maximal Fm/Fo = 3.2 we conclude that the excitonic contact between PS II and PSI is weak. However, the sigmoidieity of the fluorescence induction curve indicates significant excitonic contact between PS 11 units that is best described by a domain theory for excitonically well coupled dimers.

125

Poster III

P-I1-018 T H ~ A B S O R P T I O N S P E C T R U M O F DI-D2-CYT-Bs59 R E A C T Z O N CENTERS : TIIPERATURE DEPENDANCE, PIGNENT ASSIGE~ENT A N D ZNIIOMOGENROU8 B R O A D E N I N G

Lars K o n e r m a n n a n d A l f r e d R. H o l z w a r t h

Max-Planck-Institut f~r Strahlenchemie, Stiftstr.34-36, D-45470 MOlheim, Germany In this s t u d y w e c o m p a r e the a b s o r p t i o n s p e c t r a of photosystem-II reaction centers from samples with d i f f e r e n t c h l o r o p h y l l / p h e o p h y t i n ratio. A m o d e l is p r e s e n t e d to a s s i g n the i n d i v i d u a l p i g m e n t s to t h e i r s p e c t r a l p o s i t i o n s in the r e a c t i o n c e n t e r spectra. This model is based on theoretical calculations of the inhomogeneously broadened p i g m e n t spectra, t a k i n g into a c c o u n t e l e c t r o n phonon coupling. It d e s c r i b e s the substantial t e m p e r a t u r e d e p e n d a n c e of the s p e c t r a in the r a n g e b e t w e e n i0 K and 277 K v e r y well. B y u s i n g this spectral model we present simulations of picosecond-time resolved fluorescence measurements.

P-11-021 BACTERIOCHLOROPHYLL A IN GREEN SULFUR BACTERIA Christof Francke and Jan Amesz Department of Biophysics, Huygens Laboratory, University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands The identity of the bacteriochlorophyU (BChl) a present in the green sulfur

bacteria: Prosthecochlotis aestuarii, Chlorobium phaeovibrioides, Cb. tepidura and Cb. vibrioforme, was determined by means of normal and reversed phase HPLC. In contrast to what was recently suggested by Trom'ud and Matthews (1993, In: Norris J and Deisenhofer J (ads) The Photosynthetic Reaction Center, Vol 1, pp 1321. Academic Press, San Diego, CA) the FMO-proteins of these bacteria contain solely BChl a esterified with phytol, h was also found that about 1% of the ehlorosomal pigments consisted of BChl ap. Since the esterifying alcohols of the other ehlornsomal pigments (BChls c, d, and e) are different (mainly fame.sol), esterifieation, and perhaps also the synthesis, of the BChls in the interior of the chlorosome and of the BChls in the "baseplate" must be spatially and genetically separated processes.

P-II-019 ON STRUCTURALALTERATIONSOF THE CPI- ANDLHCPI-COMPLEXESIN N1COTIANA TABACUMIN DEPENDENCEONTHECO2-CONTENTOF AIR. A. Makewicz, A. Radunz and G. H. Schmid Universitat Bielefeld, Fakult/it fur Biologic, Lehrstuhl Zellphysiologie, Posffach 10 01 31, D-33501 Bielefeld, Deutschland. According to our investigations the quantitative composition of the peptides of photosystem I does not remain constant when plants are grown under high CO2 (700 ppm). In particular the molar ratio between peptides, lipids and carotenoids appears to be changed. In plants grown for 3 weeks under 700 ppm CO2 the CPI portion increases by 15% and the LHCPI complex decrases by 18% when compared to plants grown under the same light and temperature conditions but under the normal COrcontent of air. Correspondingly,the chlorophyll a/b ratio in these plants increases from 1.6 to 2.1 with the portion of carotenoids increasing stronger in the CO2-plants than the chlorophylls. The "CO2-plants" exhibit a photosystem I activity which is incrased by 12%. The changes in molecular ratios between peptides and lipids or carotenoids were comparatively analyzed with monospecificprotein, lipid and carotenoid antisera in the Western Blot-procedure. PSI analyses after further growth under these conditions will show whether the peptide composition changes further.

P-11-022 SUBUNIT COMPOSITION OF THE IBOLATED FROM PHOTOSYSTEM

PHOTOSYSTEM X-DELETED

SYNECHOCYSTIS P C C 6803. ~asahiko IkeuchiI & Wim Vermaas 2 ~Dept Biology, University Qf Tokyo, Meguro, Tokyo 153 Japan; ZDept Botany, Arizona State University, Tempe, AZ 85287-1601, USA For functional analysis af PSII complex, it is essential to identify all subunit pLob:ins. Since PSI is a major component of thylak~ds in cyenobacberJa, w e isolated the PSI/ oomplex from a P S I - d ~ mutant of Synechocystis PCC 6803 b y step density gradient centrifugation afar solubilization with dodecylmal~oside- The complex thus obtained retained most pSI/ prohMns and contained additionally ii kDa and 13 kDa p,-uteins. Based on comparison Of the fractlonation pra£11e with that from DI- mutant, it was dearly demonstral~_d that these pLo~_ins a s s e m b l e into the complex. B y N - t e r m i n a l a m i n o acid sequencing, it was revealed that (I) a 4.1 kDa region contained two proteins, PSI/-M and "4.1 kDa p ~ " , the latter of which has been detected in higher plants' PSI/ as nuclear-encoded; (2) 4.5 kDa region contained a gene product Of ycf~ (3) ii and 13 kDa protsins are novel PSI/ components. A h o m o k ~ u e Of the latter is present in higher plants.

P-11-023

P-11-020 MOLECULAR STRUCTURES AND OPTICAL PROPERTIES OF A G G R E G A T E D F O R M S OF C H L O R O P H Y L L S A N A L Y Z E D BY M E A N S OF M A G N E T I C C I R C U L A R D I C H R O I S M M. Kobayashi 1, T. Nozawa 1, Z.-Y. Wang 1, H. Konami 2 & M. Mimuro 3 ~Dept of Biochemistry & Engineering, Faculty of Engineering, Tohoku University, Sendal 980-77 Japan; 2Research Institute for Reaction Science, Tohoku University, Sendal 980-77 Japan; 3National Institute for Basic Biology, Myodaiji, Okazaki, 444 Japan Magnetic circular dichroism (MCD) spectra were measured on chlorophylls in different aggregation states formed in organic solvents and in pigmentprotein complexes. An index expressed as a ratio of MCD intensity to that of absorption (A~M/E) is introduced, which reflects the magnetic dipole contribution of a specific transition. The absolute value of the index is higher in monomeric Cht a than that in monomeric BChl a. This index changes depending on the aggregation state of (B)Chl; in the case of dimer of BChl a in pigment-protein complex and BChl d in organic solvents, the value was nearly the half of that for the respective monomer, and it decreases further for a higher aggregate of BChl d. Based on the index, a molecular structure of the primary electron donor in reaction center complex is discussed.

126

II C O M P L E X MUTANT OF

P I G M E N T - P R O T E I N C O M P L E X E S OF THE B L U E - G R E E N A L G A SYNECHOCYSTIS P C C 6803 R. Barbato F. Rigoni and G.M. Giacometti Department of Biology - University of Padova, via Trieste, 75, 35121 Padova, Italy The resolution of Photosystem I and II by Deriphat-PAGE from thylakoid membrane of the cyanobacterium Synechocystis PCC 6803 is reported. Characterization of the photosystem preparations has been carried out by some biochemical and biophysical methods. In addition, a carotenoid binding protein has been isolated and partially characterized. The effects of photosynthetically inactivating light on Photosystem II organization have also been studied.

Poster

P-11-027

P-11-024 CROSS-LINKING STUDIES OF PHOTOSYSTEM II IN GRANA MEMBRANES

COVALENT BINDING OF ATP AND GTP TO THE PsbP-PROTEIN OF THE OEE COMPLEX

Richard 1~ Collins*, Toby D. Flint#, Andreas Holzenburg//+ and Robert C. Ford*. *Dept. of Biochem. & Appl. Mol. Biol., UMIST, PO Box 88, Manchester M60 1QD, UK. #Dept. of Biochem. & Mol. Biol., and ~Dept. of Genetics, University of Leeds, Leeds LS2 9JT, UK.

A. Gal ], H. ZerJ, B. Andersson 2, R.G.Herrmann 3 and I. Ohad I 1Dept of Biological Chemistry, The Hebrew University, Jerusalem, Israel; 2Dept of Biochemistry, Stockholm University, Stockholm, Sweden and 3institute of Botany, Ludwig Maximilian University, Muenchen, Germany As part of our work on the purification of the LHCII-kinase from spinach thylakoids we have tried to identify trinucleotide binding polypeptides by covalent labeling of proteins in kinase active fractions with 32p-ct-ATP under UV light in the cold. As a result of these experiments it was found that a 26 kDa polypepetide is specifically labeled by both ATP and GTP. The UVdependent labeling by these trinucleotides was competed by unlabeled di- and trinucleotides. Further identification studies of the nucleotide binding protein using immunodetection with specific antibodies and N-terminal amino acid sequencing analysis revealed that this protein is identical with the PsbP-protein of the OEE (Oxygen Evolving Enhancer) complex. This phenomenon could also be observed in soluble fractions obtained by Tris washing of spianch and Chlamydomonas thylakoids that contained also the 23 kDa OEE component. The functional aspects of these phenomena are now under investigation. (Supported by SFB-184 grant awarded to I.O and R.G.H.)

Photosystem ii (PSII) -enriched thylakoid membranes were prepared from spinach chloroplasts and then treated with the zero len~th bifunctiohal cross linker, l-ethyl-3-(dimethylamino provyl/ ca~pdiimide (EDC) in an attempt to improve the quantity a n d quality of ordered 2D arrays of PSII. Cross-linking was monitored by sodium-dodecyl sulphate polyacrylamide gel eleetropboresis. Preliminary results from electron microscopy of controland cross-linked membranes suggests that the cross-linking does improve the quality of the arrays as well as stabilising the PSII extrinsic proteins against chaotropes such as 1M NaC1 and 1.5M CaCt2. Evidence for cross-linking between membrane sheets was also obtainea.

P-11-028

P-11-025 FREE CHLOROPHYLL EFFECTS ON VARIABLE FLUORESCENCE Jonathan 1~ Marder .Varda Caspi and I~ctor I. Rask~ Dept. Agricultural Botany, The Hebrew University of Jerusalem, Rehovot, Israel

Chlorophyll fluorescence from leaves has been considered formerly to indicate the state of photosystem II (PSH), with lowest yield (F0) occurring when the PSII population is maximally "open", the highest yield (Fmax) representing 100% closure, and the transient "variable fluorescence" (i.e. Fmax minus F0) representing PSII performance. This is valid only ifPSII (open or closed) is the only source of fluorescence detected. We have tested this by analysing the fluorescence lifetime profiles in intact leaves using a phase modulation technique. In green barley leaves, the main component had a 0.25 ns lifetime with a less intense 1.7 ns component which represent open and closed PSII respectively. The shorter component is abolished by adding DCMU, which causes complete PSH closure. However, in early greening, we also see a 5.6 ns component which later disappears and is probably emitted by uncoupled chlorophyll. By assuming a constant fluorescence yield from this component during variable fluorescence measurements, we predict its likely effect on the variable fluorescence and find this consistent with actual measurements. Conversely, we can estimate free chlorophyll from the measured Fmax:F0 ratio, and will demontrate this for various abiotic and biotic stress situations.

CHARACTERIZATION BY CRYO-ELECTRON MICROSCOPY OF TWO-DIMENSIONAL CRYSTALS OF THE C P 4 7 - D I - D 2 CYTOCHROME b-559 COMPLEX OF PHOTOSYSTEM II K. Nakazato 1'4, C. Toyoshima 2, I. Ermmia & Y. Inoue4, IPRESTO, JRDC, 2Inst. of Molecular and Cellular Biosci. Univ. of Tokyo, Tokyo 113, aDept. Biol., Science Univ. of Tokyo, Tokyo 162, 4Solar Energy Res. Orp., The Inst. of Phys. and Chem. Res. (RIKEN), Wako, Saltama351-01, Japan Two-dimensional (2I)) crystals of the CP47-D1-D2 and cytochrome b-559 complex of spinach photosystem II (PSII) were prepared from nheptylthioglucoside-solubilized PSII complexes by a convensional dialysis method. The crystals were flattened cylindrical vesicles of approx. 1-2 IJmby 36ore. Images of ice-embedded crystals were recorded using low dose microscopy and analysed by digital image processing. The symmetry of the crystals wasp22121. The unit cell consists of 4 monomers (two face-up and two face-down) and has dimensions of 16.7 x 15.3 nm. The projection map at 1.5 nm resolution showed that each monomer has an asymmetrical shape with a maximal length of 8.1 um and consists of four areas of density. Two high density areas of each monomer are closely linked and form a roughly rectangular core (40 x 65 nm in projection). We propose that this core area corresponds to the D1/D2 heterodimer and the remaining large area surrounding the half of the core corresponds to the CP47 of PSII complex.

P-11-029

P-11-026 IRECONSTITUTION OF CROSS-LINKED COMPLEX OF PHOTOSYNTHETIC REACTION CENTER AND HORSE HEART CYTOCHROME C INTO LIPOSOME Takao Ueno, Masayuki Hara*, Jun Miyake* and Takaaki Fujii Department of Bioresorees Chemistry, Chiba University, 648 Matsudo, Matsudo, Chiba 271, Japan. *National Institute of Bioscience and HumanTechnology, I-1-3 Higashi, Tsukuba, Ibaraki 305, Japan Cross-linked complex between (RC) from Rhodobacter spheroides R-26 and cytochrome c (eyt c) from horse heart was prepared to examine the assembly of the protein. Cyt c in the complex was covalently conjugated to the surface of H subtmit of RC. In this work, we tried to reconstitute the complex into the liposome to organize the RCs. Orientation of the complex was studied. Heterobifunctional reagent N-sueeinimidyl 3-(2-pyridyldithio) propionate (SPDP) was used for the cross-linkage. The direction of the RCs in the liposome was confirmed by using 3 methods. At first, the efficiency of electron transfer from reduced cyt c to the RC in the complex in the liposome was measured. It was depressed to 8% meaning that about 90% ofcyt c binding site of RC in the complex faces to the inner side of the liposome. At second, the recovery of l.eavaged cyt c by treating the complex liposome with dithiothreitol was counted. indicated that about 80% ofeyt c in the complex was in the outer side of liposome. At last, the productivity of membrane potential were compared between RC liposome and the complex liposome. These results suggested that about 90% of RCs in the complex were organized to the liposome with same direction.

CHARACTERISATION OF THE 12.5 kDa PHOSPHOPROTEIN OF SPINACH THYLAKOIDS M. Lindahl. I. Carlberg, W.P. Schrrder and B. Andersson Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-I06 91 Stockholm, SWEDEN Light-induced phosphorylation of spinach thylakoid proteins in vitro results in a strong labelling of LHC II as well as some of the PS II core proteins, such as D1, D2, CP 43 and the psbH gene product. We have characterised one additional heavily labelled polypeptide, which migrates at 12.5 kDa on SDS-PAGE. Its phosphorylation /dephosphorylation behaviour closely follows that of LHC lI the pattern of which is distinct from that of the PS II core subunits. The N-terminal sequence, which is not identical to any other known protein, was determined. The sequence contains both basic and acidic residues and in addition there is at least one threonine which is a putative phosphorylation site. In contrast to other thylakoid phosphoproteins, this 12.5 kDa phosphoprotein can be recovered in the supernatant after washing thylakoids at high salt concentrations and is therefore the only thylakoid phosphoprotein which is not an integral membrane protein.

127

Poster

P-I1-030 C H A R A C T E R I S A T I O N OF A NEW NUCLEAR E N C O D E D POLYPEPTIDE IN SPINACH PSII REACTION CENTER Lan-Xin Shi. Klaus-Dieter Irrgang l, Christiane Funk l and Wolfgang P Schrtder Department of Biochemistry, Arrhenuis laboratories for Natural Science, Stockholm University, S-10691 Stockholm, Sweden 1. Permanent address: Max-Volmer-Institut, TU Berlin, Strasse des 17. Juni 135, D-106 23 Berlin, Germany A nuclear encoded polypeptide of 6.1 kDa was identified in isolated PSII reaction center from Spinacia oleracea. The hydrophobic membrane protein easily escapes staining procedures such as Coomassie or silver staining, but it is clearly detected by immunodecoration with peptide-directed IgG. This additional subunit was found to be present in PSII reaction centers previously known to contain only the DI/D2/Cytb559 proteins and the psbI gene product. Furthermore, cross linking experiments using EDC (1-(3-dimethylaminopropyl)3-ethylearbodiimide) showed that the nearest neighbours were the D1- and D2- proteins and Cytb559. The 6.1 kDa protein was purified by immune affinity chromatography. Nterminal sequence analysis of the isolated protein confirmed the identity of the 6.1 kDa protein. Using ELISA in combination with thylakoid membrane preparations of different orientation, N-terminus of the protein is suggested to be exposed at the lumen side of the membrane. The structure and topology of the 6.1 kDa vrotein in relation to the PSII reaction center will be discussed.

P-11-031 F U N C T I O N A L STUDIES ON THE NEWLY DISCOVERED 6.1 K D A PROTEIN F R O M SPINACH THYLAKOIDS

P-11-033 PHOTOSYSTEM II: THREE-DIMENSIONAL (3D) STRUCTURE AND OXYGEN EVOLUTION

Andreas Holz~nbure#:[ :, Richard P. Collins*, Toby D. Flint#, Ashraf Kitmitto*, Paul McPhie#, Mark F. Rosenberg*, Fiona H. Shepherd#, Svetla Stoylova#:~ and Robert C. Ford* #Dept. Biochem. & MOl. Biol. and :~Dept. Genetics, University of Leeds, Leeds LS2 9JT, UK *Dept. Biochem. & Applied Mol. Biol., UMIST, Manchester M60 1QD, UK Electron microscopy of negatively stained 2D crystals has been used to determine the 3D structure of (i) native photosystem II (PSII) and (ii) Tristreated PSII devoid of the three extrinsic polypeptides (33, 23, and 17 kDa) comprising the oxygen evolution-enhancing complex (OEC) at 3 and 1.8 nm resolution, respectively. A comparison between the two structures reveals the location of the OEC and, for the first time, provides unambiguous proof for the existence of a special environment for oxygen evolution which takes the shape of an intramolecular cavity. The absence of this cavity in Tris-treated PSII correlates well with the reduced oxygen evolution rates observed. The location of each indivudual extrinsic polypeptide has been determined by comparing the 3D map of Tris-treated PSII with Fourier projection maps of PSII devoid of either (i) the 17- and 23-kDa or (ii) the 17-kDa polypeptide only, permitting relationships between PSII architecture and oxygen evolution to be established.

P- 11-034 SPATIAL LOCALISATION AND SPECIFICITY OF PROTEIN KINASE AND PHOSPHATASE ENZYMES IN THYLAKOID MEMBRANES

Christiane Funk, Lan-Xin Shi and Wolfeane P. Schr6der Department of Biochemistry, Arrhenius Lab. for Natural Sciences, Stockholm University, S - 106 91 Stockholm, Sweden

Kleoniki Gounarls Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, U.K.

The n e w l y discovered 6.1 kDa protein of higher plant h a s been studied with respect to its function in p h o t o s y s t e m II. Developmental studies have been performed to investigate the behaviour of the protein d u r i n g g r e e n i n g of s p i n a c h plants. F u r t h e r m o r e , a g e - d e p e n d e n t accumulation on etiolated plants a n d organ-specific regulation of the protein in the plastid m e m b r a n e s have been investigated. It h a s been s h o w n earlier that the 6,1 kDa protein can be crosslinked to cytb559 a n d the D1 a n d D2 proteins of p h o t o s y s t e m II in m a t u r e green plants. Here we investigate the cross-linking behaviour during the greening process. The i n v o l v e m e n t a n d function of the 6.1 kDa protein d u r i n g a s s e m b l y of p h o t o s y s t e m II will be discussed. However, also a role within Photosystem I can be considered.

Studies on protein kinase and phosphatase activities in thylakoid membranes have led to conflicting evidence as to the number of enzymes involved in the reversible phosphorylation of thylakoid proteins. Furthermore, the type of protein phosphatase(s) responsible for the dephosphorylation process has not been unequivocally determined. We have used a series of protein phosphatase inhibitors in an attempt to classify the phosphatase enzymes in the thylakoid membranes. The data indicate the presence of multiple enzymes with different substrate and inhibitor specificity. We employed mechanical fractiormtion of thylakoid membranes and studied both the protein kinase and phosphatase activities in the granal and stromal fractions. We used such membrane fragments to investigate phosphorylation and depho~phorylation of endogenous and exogenous substrates, and by the use of inhibitors studied the specificity and requirements of the enzymes. Evidence will be presented on the spatial localisation of protein kinase and phosphatase activities and their specificities.

P-11-032

P-I 1-035

CRYO-ELECTRON MICROSCOPY OF PHOTOSYSTEM H

P U R I F I C A T I O N OF T H Y L A K O I D KINASE A C T I V I T Y AND CYTOCHROME btf BY PERFUSION CHROMATOGRAPHY

Svetla Stovlova#:~, Paul McPhie#, Toby D. Flint#, Robert C. Ford* and Andreas H01zenburg#'¢ #Dept. Biochem. & Mol. Biol. and :~Dept. Genetics, University of Leeds, Leeds LS2 9JT, UK *Dept. Biochem. & Applied Mol. Biol., UMIST, Manchester M60 1QD, UK

A. Gal l, H. Zer 1, H. Fulgosi2, R. G. Herrmann2, B. Andersson3 & I. Ohad l IDept. of Biological Chemistry, The Hebrew University, 91904, Jerusalem Israel; 2Institute of Botany, Ludwig-Maximillian University Munchen, Germany; 3Dept. of Biochemistry, Stockholm University, Stockholm, Sweden

We are carrying out cryo-electron microscopy studies of ordered twodimensional (2D) arrays of photosystem II (PSII). Specimens are observed unstained, and in their hydrated state, i.e. embedded in vitreous water at -160 °C. Under these conditions, the structural integrity of PSII is preserved and structural detail m potentially atomic resolution can be resolved. Analysis of preliminary data has suggested that: (i) negative staining procedures as employed for conventional transmission electron microscopy studies do not induce array formation, (ii) any significant shrinkage of PSII under negative staining conditions can be excluded, and (iii) the overall structural features such as the central intramolecular cavity surrounded by four anisotropically distributed domains (giving rise to a pseudo-twofold symmetry in projection perpendicular to the thylakoid membrane) are preserved and are not artefacts of the negative staining technique.

The redox-controlled phosphorylation of LHCII is the underlying mechanism of state transition and regulation of energy distribution between the two photosystems. The control of this kinase activity was found to be tightly linked to the redox state of the cytochrome b6f complex. Despite extensive attempts toward the purification of this enzyme, its identity remains still obscure. We have resolved crude cytochrome b6f preparations containing kinase activity by rapid perfusion chromatography (15 min) using the anion exchanger POROSPI. Active fractions enriched about 150 fold in protein phosphorylation activity toward histone and exhibiting self phosphorylation activity were obtained. These preparations still contain cytochrome b6f components. Kinetic studies of autophosphorylation activity reveal the initial phosphorylation of a 64 kDa polypeptide together with that of a 12 kDa as well as that of a 20 kDa band identified as the Rieske Fe-S protein. This activity is inhibited by NaF and by quinone analogs. Further purification of kinase activity and its identification by molecular techniques are now in progress. (Supported by grant SFB-184).

128

Poster P-I 1-036 PHOSPHORYLATION OF MEMBRANE PROTEINS OCCURRING IN ISOLATED SUB-THYLAKOID VESICLES DERIVED FROM DIFFERENT STRUCTURAL DOMAINS OF THE SPINACH CHLOROPLAST THYLAKOID. H. Steffinsson, L. Wollenberger, S.G. Yu and P.-/~. Albertsson Dept. of Biochemistry, Chemical Center, University of Land, P.O. Box 124, S-221 00 Lund (Sweden) Sub-thylakoid vesicles, representing the different structural domains of the thylakoid membrane, stroma lamellae, grana lamellae, grana core and grana margin, were examined for their properties following an incubation in the presence of light and ATP. Phosphorylation of membrane proteins including LHC II and PS II associating polypeptides was observed in membrane fractions deriving from the appressed region but not in the fractions representing the grana margins or stroma lamellae. Three unidentified polypeptides of 15, 20 and 22 kDa became weakly pbosphorylated in the stroma lamellae fraction but no polypeptides became phosphorylated in the vesicle fraction representing the grana margin. The results are discussed with respect to bow the structure of the thylakoid relates to the function of the electron transport system.

P - I 1-037 LIMITED PROTEOLYSIS OF RECOMBINANT MANGANESE S T A B I L I Z I N G P R O T E I N O F SYNECHOCOCCUS ELONGATUS M, HIRANQ 1, A. MOTOKI l, T. SHIMAZU 1 and S. KATOH 2 lToray Res. Center, Inc., Kamakura 248; 2Dept. Biology, Toho University, Funabashi 274, Japan, The Mn-stabilizing protein (MSP) is an extrinsic protein of about 33kDa that binds to the lumenal sidc of photosystem II (PS ID reaction center complexes and stabilizes Mn ions which play an important role in water splitting reaction in plants, algae and cyanobacteria. The psbO gcne from the thcrmophilic eyanobactcrium Synechococcus elongatus [BBA, 1172. 357 (1993)] was expressed in Escherichia coli. Several proteases attacked the recombinant MSP (r-MSP) preferentially in the two regions, Phe156-Gly163 and Arg184Serl91, where are predicted to form a loop and a turn structure exposed to the outer phase, respectively. The first region is particularly well conserved among 10 different MSPs from plants and cyanobacteria. The protein with a nick between Phe156 and Leu157 largely retained to native secondary structure but failed to bind to PS II complexes and reactivate oxygen evolution. It is suggested, therefore, that the peptide region is involved in the binding of MSP to PS II complexes. This study was supported by New Energy & Industrial Technology Development Organization.

P- 11-038

P-11-039, E X P O S U R E O F VARIOUS T H Y L A K O I D P R O T E I N E P I T O P E S TO TRYPSIN AND TO M O N O C L O N A L ANTIBODIES Christopher Plambeck, Rill A. Reuter and Steven P. Bert, Biology Department, Winona State University, Winona, MN 55987 Monoclonal antibodies (Mabs) specific for various thylakoid proteins were prepared and used to determine the exposure of their respective epitopes. Epitope exposure was determined directly by using a monoclonal antibody dependent, Staphylococcus aureus (Cowan) mediated immunoprecipitation o f rightsideout (RSO) or mixed RSO and insideout (ISO) tbylakoids. Epitope exposure was indirectly determined by briefly exposing RSO and ISO thylakoids to trypsin followed by immunoidentifieation of the epitopes after LDS-PAGE (urea) and western blotting. The epitopes for the Mabs MLH-I (found on the 29 kDa peptide ofLHCII), FC8 (found on the 28 kDa peptide CP26) and HB7 (found on the 22 kDa peptide CP24) were found to be exposed on the stroma surface of the thylakoid. The epitopes for the Mabs HF3 (found on all three peptides of LHCII), JB2 (aspl 11-arg154 of cytochrome f), FCC4 (found on the 17 kDa extrinsic peptide associated with PSII) and FAAC4 (found on the 34 kDa extrinsic peptide associated with PSII) were found to be exposed on the lumen surface of the thylakoid.

P-I1-040 PHOTOINDUCED SURFACE CHARGE DENSITY CHANGES IN PEA AND Chlamydomonas reinhardtii TH~LAK01DS BY THE MODIPICATIO~ 0F P H A - MOLECULES Virj inia Do Itchinkova Dept Biophys & R~diohiol, Biol l~ao, Sofia Univ, Bulgari& Pea and Ohla~domonas reinhardtii %hylakoid membranes are characterized by the method of particle mieroeleo%ro phoresis using an "OPTON" Cytopherometer. Lectin activation of thylakoid stacking processes serves as a model to study the blophysioal mechanisms underlying staeSs formation and aggTega%ion. Phytohemagglutinin - M (PHA) interacts speoifioal~ with pea th~lakoid membranes leading to surface charge effects in wide r a n g e (from -19% at 8 nK PHA/ml in reaction medium, pH 8.0 up to 3 ~ at 3 ng PHA/ml, pH 7.5). Photoinduoed effects of 0.1 and I u g PHA/ml doses on Chlsa~domonas reinhardtil thylakold membranes at low salt concentrations are detected. A ml nimum effect at 0.75 ~g PHA/ml thylakoid suspension is registered. The relationship between the funotlonal activity of thy la~oids and photo- and lectim induced changes in their surface charge densities is studied.

P-I 1-041

MUTAGENESIS OF THE PUTATIVE PHOSPHORYLATION SITE OF THE D2-PROTEIN IN CHLAMYDOMDNAS REINHARDTI!

PROTEIN PHOSPHATASE ACTIVITIES OF SPINACH THYLAKOIDS: PURIFICATION, ASSOCIATION WITH CYTOCHROME BF AND REGULATION

M. M. Fleischmann & J.-D. Rochaix, Depts of Molecular Biology and Plant Biology, University of Geneva, CH 1211 Geneva, Switzerland

[k Andersson. A. V. Verier, and I. Carlberg Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden

The D1- and D2-proteins of photosystem II (PSII) are monophosphorylated in higher plants in a light-dependent fashion. However, the function of this phosphorylation is less well understood than phosphorylation of the light harvesting complex II (LHCII) proteins. In order to acquire information about the role of photosystem 1I phosphorylation we mutated threonine 2 of D2-protein in Chlamydomonas reinhardtii, as D l-protein is not phosphorylated in this organism (Delepelaire P., 1984, EMBO J. 3, 701-706). Threonine in position 2 of D2-protein is homologous to the phosphorylation site in spinach, Mutant genes in which the threonine 2 codon of D2 has been changed into alanine, valine, glycine, proline, aspartate or glutamate have been constructed and introduced into (7hlamydomonas retnhardtti by particle-gun mediated transformation. Replacement of the threonine by an uncharged amino acid showed no distinct phenotype compared to the wild type, as far as photosynthetic growth, fluorescence induction, PSII accumulation, state transition and photosensitivity are concerned. However. mutant strains carrying acidic amino acids in position 2 showed highly reduced amounts of stable PSII. This result indicates that the charge in the N-terminal part of D2 is crucial for PSI1 stability and function. How this correlates with PSII phosphorylation remains to be elucidated.

In an effort to identify and purify the protein phosphatase(s) in the thylakoid membrane we have designed an assay for enzymatic analyses based upon radioacively labelled peptides obtained by proteolytic cleavage of phosphorylated thylakoid membranes. Solubilizatinn of thylakoids by non-ionic detergents and subsequent fractionation by FPLC resulted in co-purification of the phosphatase activity with the cytochrome bf complex while other thylakoid membrane complexes were devoid or lacked this activity. Even cytochrome bf complexes isolated by conventional procedures was found to contain the phosphatase activity. The identity of the polypeptides associated with the protein phosphatase activity will be discussed. Furthermore, we have found that the LHCII dephosphorylation is influenced by the redox state of thiol-groups as judged by the opposing effects of dithioerythritol and iodobensoate. This observation may suggest that the ferredoxin-thioredoxin system is involved in the regulation of thylakoid protein dephosphorylation.

129

Poster

P-11-042 THREE-DIMENSIONAL CRYSTALLISATION EXPERIMENTS ON CP43, DIMERIC PSII CORES AND PSII-LHCII SUPERCORE COMPLEXES J.Nield, B.Hankamer, E.Morris & J.Barber Biochemistry Department, Imperial College, Imperial College Rd., London SW7 CP43, oxygen evolving PSII dimers and dimeric PSII-LHCII supercore complexes were subjected to microbatch crystallisation experiments. The oxygen evolving dimers consisted of CP47, CP43, the 33kDa extrinsic polypeptide, D2, D1, the a and 13subunits of b559, as well as a number of low molecular weight subunits. The PSII-LHCII supercore complex contained Type I and Type II LHCII, CP26 and probably CP29, in addition to the subunits associated with the PSII dimer. The automated microbatch system was used to dispense 2gl droplets containing protein, precipitant, detergent, amphiphiles and additives into an oil filled well (Chayen et al. (1990) J. App. Cost. 23: 297302). The oil layer ensures that water evaporates from the droplet in a conu'olled manner. Small, needle-like crystals having dimensions of about 30 x 30 x 2001ma were obtained from CP43. The PSII-LHCII supercore complexes gave 40 x 40 x 1200txm crystals. These crystals were fixed with glutaraldehyde, embedded in resin and sectioned for electron microscopy in order to gain unit cell information. The crystals often grew from green, presumably protein-detergent rich, phases. Various amphiphiles were screened in an attempt to reduce this phase separation.

P-11-045 LOCALISAIION OF TIlE EXTRINSIC PROTEINS OF P H O I O S ~ S T E M !1 BY ELECTRON MICROSCOPY Mark F Rosenbere*. Tob'~ D Flint=, Fiona H. Shepherd=. Andreas Holzenburg~ and Robert C Ford*. *Dept. of Biochem. & Appl. Mol. Biol.. UM1ST, PO Box 88, Manchester M~0 1QD, UK. SDept, ofBioehem. & Mol. Biol. and Dept. of Genetics. UnA ersity of Leeds. Leeds LS2 9JT, UK. The locations of the extrinsic oxveen enhancin,, proteins of ~hotosvstem il have been studied'by electron na'icroscopv Ordered ~D arrays of the complex in th~ ]akdid men.branes were "studied after re~mvaI and rebindine of the extrinsic pohpeptides. Rernmal of the proteins was manife,~ted by the disappearance of the typical four-domain structure in prqiectfon with the loss of the lum~nal intramolecular cavity ~ hlch we predict has a ~ ital role in photosvmhetic ox~ eX~ne~ olution. Rebindin~ of the 33.23 and 17kD !3roteins led't~ a restoration of the ori~_,inal structure which was concomitant with a stimulation of the o-x\~en e~ olution activity of the preparation. Rebindin~ was alio'monitored by labellihg the exmnsic proteins with ~ 1.4 nm Nanogold particle

P-11-043 CHARACTERISATION OF MONOMERIC AND DIMERIC PSII CORE COMPLEXES B. Hankamer, E. Morris, J. Nield, D. Zheleva & I. Barber Biochemistry Department, Imperial College, Imperial College Rd, London SW7 Oxygen evolving monomeric and dimeric PSII core complexes (240 and 445kDa respectively) containing CP47, CP43, D2, D1, the c~ and !8 subunits of cytb559 and the 33kDa polypeptide have been isolated. No apparent difference was observed in the complement of low molecular weight subunits associated with these two PSII complexes, The dimeric complexes are able to support higher rates of oxygen evolution than their monomeric counter parts. Fluorescence measurements showed the dimers to be associated with higher levels of functional QA. Together with the finding that the dimeric complexes are associated with slightly higher levels of plastoquinone, chlorophyll b and carotene, these results support the hypothesis that PSII may exist as a dimer in its most active in vivo form. Gentle solubilisation of BBY type membranes yielded LHCI/-PSI/supercore complexes. Single particle analysis of negatively stained complexes of this type showed them to consist of a central dimeric PSII core, flanked by two sets of antenna components consisting of CP26/CP29 and type I /II LHCII trimers (Boekema eta/., (1995) PNAS 92:175-179). Interpretation of the electron micro-graphs supports the concept that LHCII forms trimers in vivo and that type III LHCII is only associated with the outer, more loosely bound PSII antenna. The detergent solubilised PSII dimers were supplemented with lipid and were induced to form two-dimensional arrays by detergent removal.

P- 11-046 INWESTIGATION OF THE PROCESS OF ORDERED 2D ARIL,*~YFOR.'~L~tTION IN SPINACH PHOTOSYSTEM I1 - ENRICHED GRANA Ashraf Kitmitro*. Andreas Holzenburgy~r~ and Robert C. Ford*. *Dept. ofBiochem & Appl. Mol. Biol.o UMIST, PO Box 88, Manchester M60 1QD, UK. #Dept. of Biochem & Mol. Biol., and *Dept. of Genetics, Universi~" of Leeds, Leeds LS2 9IT, UK. Various experiments have been carried out in order to understand and characterise the process of ordered 2D array fi)maoation hv photos','stem I1 in spinach grana n~embranes. " Arrays are dbservedbv electron microsc?opv in about 1% to 5% ofthe m'ana mem[~ranes isolated from spinach chloroplasi's, and these arrays vary considerably from one preparation to another in tel-ms of'their abundafiee, size and cu'stallinitv. The process of nucleation and the stability of the ordered ar-/a'¢shas been esal ualed with preparations o~tained fro n plants ~rown under various re~imes and i~lated using different protocols. The affect of detere.ents, butters. illuminanon rea,ime and mowth tempe?ature on array formation is discussed.

P-11-044 PHOSPHORYLATION OF AN ENDOGENOUS 64-kDa PROTEIN BY A THYLAKOID PROTEIN KINASE

P-11-047 P H O T O S Y S T E M II A C T I V I T Y OF T H Y L A K O I D S A N D W H O L E C E L L S IN L O W W A T E R S Y S T E M S

H. L. Race and G. Hind, Biology Dept., Brookhaven National Laboratory, Upton, NY 11973, USA

A. D a r s z o n t A. Srivastava 2 a n d R.J. Strasser 2 , 1Institute o f Bioteclmology, U N A M , Cuernavaca, Morelos 6227, Mexico, 2Laboratory o f Bioenergetics, University o f Geneva, Jussy, CH1254, G e n e v a , Switzerland

Solubilization of spinach thylakoids with octyl-/3-D-glucoside releases serine/threonine protein kinase activity, which was reported to originate in a 64-kDa protein (Coughlan & Hind (1986) J B i o l Chem 261, 11378-11385). The N-terminal sequence of a putative 64-kDa protein kinase was later determined (Gal etal. (1992) FEBS Lett. 298, 33-35). Using a novel fractionation technique, fluid phase isoelectric focusing, together with an antibody to the documented N-terminal sequence, we found that the 64-kDa protein can be resolved from the active kinase fractions. Active fractions catalyze the phosphorylation o f several endogenous proteins including the 64-kDa protein o f Gal et al. Our results indicate that this 64-kDa protein is not the protein kinase responsible for the phosphorylation o f the photosystem II lightharvesting complex. [Supported by the U. S. Dept. of Energy]

130

W h o l e leaves, unicellular organism like C h l a m y d o m o n a s remhardtii or thylakoid m e m b r a n e s in pure organic solvents or in HexadecaneT w e e n - S p a n (HTS) low water s y s t e m s h o w different OJIPfluorescence rise than in a q u e o u s medium. In cells long chain hydrocarbons preserve the primary photochemistry over hours or even days. The cells and the thylakoid m e m b r a n e s loose their variable Chl a fluorescence in the HTS s y s t e m which can be restored by the addition o f water. M o s t probably H T S induces the physical separation o f antenna c o m p l e x e s from the PS II reaction center.

Poster

P- 11-048 CHARACTERIZATION OF CHL-PROTEIN C O M P L E X E S ISOLATED FROM THE RED ALGAE G R A C I L A R I A VERRUCOSA Gianna Casazza 1, Roberto Barbato 2& Maria Sperandei 3 1Stazione Zoologica "A. Dohrn" - Napoli, c/o ENEA. CRE Casaecia, Dip. Innovazione, Set. Biotecnologie e Agricoltura; 2 Dipartimento di Biologia, Universith di Padova. via Trieste 75, 35121 Padova; 3ENEA, CRE Casaccia. Dip. lnnovazione, Set, Biotecnologie e Agricoltura, Via Anguillarese 30l, 00060 S.Maria di Galeria, (Roma), Italy The photosynthetic apparatus from macrophytic red algae, has been, so far, poorly investigated. Here we report a preliminary characterization of chlorophyll-protein complexes from thc photosynthetic membranes of the rhodophycea G r a c i l a r i a verrucosa. The thylakoid membranes were solubilized, and fractionated by sucrose gradient and ion exchange chromatography. The fractions were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and absorption spectroscopy. Immunoblotting assays were performed with antibodies raised against PSI and PSII proteins from higher plants and cyanobacteria. The results are compared to those on PSI and PSII complexes, obtained from other photosynthetic organisms.

P-11-049 ON THE HETEROGENEITY OF PHOTOSYSTEM II :

CHARACTERIZATION OF "IHREE PS H POPULATIONS BY STUDIES OF FLUORESCENCE INDUCTION AND PHOTOSYNIHt>, TIC ELECTRON TRANSPORT B. B6thin-Geis, W. RQhle, M. Richter and A. Wild Institute of General Botany, Johannes Gutenberg-University, 55099 Mainz, Germany The amount of different PS II populations and the'tr rate constants of Qxreduction were determined by fluoreseanee induction of DClVlU-poisoned spinach thylakoids before and after different light treatments. Two slow exponential phases and a fast sigmoidal phase were found in untreated spinach thylakoids. To record the very slow T-phase, the duration of measuring time was very important and depended on the chosen actinic light intensity. Linear correlation of the rate constants of all three phases with the actinic light intensity suggested that all phases represent different PS II centers. A comparison of effects on fluorescanee induction and on the ferrieyanide mediated oxygen evolution after different light treatments supported the hypothesis that only the a- and ~-PS II centers are involved in Qa-dependent linear electron transport.

P- 11-050 HOMOGENOUS BUNDLE SHEATH AND MESOPHYL THYLAKOIDS CAN BE ISOLATED FOLLOWING MECHANICAL TREATMENT OF

MAIZE LEAF TISSUE Harry Teicher & Henrik V. Seheller Department of Plant Biology, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg, Copenhagen, Denmark Homogenous, active bundle sheath and mesophyl thylakoids have been succesfully isolated from the CA. species maize (Zea mars) and sorghum (Sorghum bieolor), following mechanical trealanent of leaf tissue. Homogeneity and quality of the preparations were determined using SDS- polyaerylamide gel-elec~ophoresis, by comparing the ratios of chlorophyl a/b, chlorophyl/P700, chlorophyl/cytoehrome f, and by comparing the respective rates of Photosystem I and II photosynthetic electron transport. This method of mechanical isolation effectively permits the isolation of relatively large volumes of homogenous thylakoid stock, minimizing subsequent experimental variabilty, and thereby optimizing the possibility of observing differences between treatments. In addition, mechanical isolation permits a more rapid isolation of thylakoids than is possible using enzymatic isolation, at temperatures lower than the optima for most digestive enzymes, including proteases.

P-I1-051 STABILIZATION OF C Y A N O ~ PIK)TOSYSTEM II BY MEANS OF GLYCINEBETAI~

J.Mik~ovsk~, B.Sopko, D.Sofrov~, Biochemistry Dept.,Charles University, CZ-128 40 Prague, Albertov 2030, Czech Republic Glycinebetaine stabilizes oxygen evolution and 2,6-dichlorophenol indophenol photoreduction during 6h incubation at room temperature. This effect is even more distinct if glycinebetaine is present in a cone. of I mol/dm 3 during isolation of thylakoid membranes from the cyanobaoterium Synechococcus slsn~atu_s. Three detergents were employed for extraction of PS II particles from thylakoid membranes: N-dodecyl-N,N-dimethylammonio-3-propane sulfonate (SB 12), octyl-~,D-glueopyranoside (0G) and dodecyl~,D-maltoside (DM). The effect of glycine betaine added to the isolation media of PS II particles was not unequivocal. In the case of SB 12 and OG, photochemical activities increased slightly (approx. 50%). However, the DM extract primarily contained biliproteins and only a minimum amount of chlorophyll-protein complexes} moreover, mainly PSI.

P- 11-052 FUNCTIONAL PHOTOSYSTEM I WITHOUT LONG-WAVELENGTH FLUORESCENCE EMISSION IN A LOW-LIGHT G R O W N GREENING MUTANT OF THE GREEN ALGA SCENEDESMUS OBLIQUUS

H. Schiller, M. Hahn, S. KlingelhOfer, H. Dan and H. Senger FB Biologie/Botanik, Philipps-Universitttt, 35032 Marburg, Germany The greening mutant CP-Ia-13-y3 o f the unicellular green alga Scenedesmus obliquus was grown heterotrophically in the dark for three days and then transferred to either low fight (0.1 Win-2), medium light (30 Win-2) or strong light (100 Win-2) for one day. The weak-light culture was characterized by unusual low chlorophyll content, but relatively high amount o f carotenoids. Using native green gel electrophoresis, in vitro Hill reactions and EPR-spectroscopy we could show that in all three cultures P S I as well as PS II were present and active. In contrast, low-light grown algae show no 77K fluorescence emission at 715 nm, whereas this long-wavelength peak o f the strong light culture is higher than in the wild type. Western-Blot analysis showed a different expression of LHC I proteins in all three cultures. These studies indicate that long-wavelength pigments are not necessary for the function o f PS I. Using different light conditions during greening of this mutant the amount of long-wavelength-pigments can be manipulated.

P-I 1-053 ISOLATION AND CHARACTERIZATIONOF GRANAAND STROMA LAMELLAEMEMBRANALFRACTIONS.POSSIBLE ROLE FOR PHOTOSYSTEM II IN THE STROMA LAMELLAE Kopf Zohar & Malkin Shmuel The Weizmann Institute of Science, Biochemistry department, Rehovot, Israel. A new method for isolation of grana and stroma lamellae from Pisum sativum produced both pure and active fractions, in an easy reproducible way. When performing SDS-PAGE followed by immunoblot analysis and detection with D1 antibody, the presence of a degradation product migrating around 22-23Kda, was located mainly in the stroma lamellae. This finding is consistent with the repair cycle model of photosystem II, that is not necessarily correlated to photoinhibtion but rather takes place also in steady state conditions, since the plants were grown in relative moderate light intensities (200-900 I.tE m-2sec-1).

131

Poster

P-11-054 Investigating the nature of phosphoproteins in P S I I cores and reaction centers O.Kruse, J. Sharma and J. Barber PhotosynthesisResearchGroup, Departmentof Biochemistry,ImperialCollegeLondon LondonSW7 2AY ,~?,lI.thylakoids were isolated and illumiuted with 1500pE" s-I "m"2for 15rain in order to phosphorylate proteins with inorganic Pn-pyrophosphate. Phosphorylated and nonphosphorylated PSII cores in their monomeric and dimerie forms were isolated by sucrose gradient centrifugation after octylglueoside solubilization of BBYs. The various preparations showed differences in their absorption spectra which seem to reflect different levels of CP26 and/or CP29 as confirmed by SDS-PAGE. In particular the phospborylated forms were more enriched in these components which in themselves were phosphorylated. Furthermore four phosphorylated peptides with molcular weights less than 10kDa could be detected in the autoradiogram. The isolation of reaction centers from PSII-monmners using nationexchange clu'omatography led to a preparation in which the D1 and D2 proteins were in their phosphorylated states hut also Which contained a number of breakdowu prodocts of these proteins. Low molecular weight components, including fragments, from these phosphorylated reaction centers and also from raonomeric cores were separated by Y,_PLCreversed phase chromatography. In some of these HPLC-fractions, P32-radioactivity could be detected in the scintilation counter indicating the presence of phosphorylated forms. One particular non-phosphorylated fragment having an apparent molecular mass of 18.5 kDa was identified as a C-terminal I32 fra[,maentand has been investigated by FAB-mass spectroscopy.

P-11-055 LOCALIZATION O F SUBUNITS IN PS1, PS2 A N D A PS2/LIGHT-HARVESTING-SUPERCOMPLEX

J. Kruip1, D. ~ 1 , B. Hankamer 2, J. Nield 2, A.F. aoonstra 3, J. Bafoer~, E.J. Boekema3 & M- Rd~_ner~; llnstituts of Botany, Unlvemity of Miinster, Schloasgartsn 3, D-48149 MOnster, Germany; 2Dept. of Biochemistry, Imperial College, London, UK; aBIOSON Research Institute, Univemity of Groninge~, The Nethadands Tdmeric photosystem 1 (PS1) has been Isolated from the cyanobactedum Synechocyst/s PCC 6803 and from alte-dlracted mutants. Purification by several HPLC-steps enables a detailed structural investigation by electron microscopy (EM). Computaraided image reconstruction procedures show the localization, size and shape of the following PSl-subunits: The stmma-exl:x)sed subunits PsaC/D/E, the membrane-embedded PsaJ and the lumen-exposed subunit PseF. The data indicate one trans-membrane ¢-halix for both PsaJ and PsaF at the outer edge of the thrner, and allow structures from X-ray d l ~ of PS1 cwstals to be assigned to specific subunits. Photeaystem 2 (PS2) has been isolated and pudfled from the thermophilic cyanobactshum Synechococcuselongatusand tom spinach as a dlrnedc, oxygen evolving core complex. EM, in combinal~on with image analysis, revealed an antiparallel organization of the monomers within these complexes surrounding an area of low protein density. The dlmaric particles (17x10x9 nm) al'~w 4 protein densities and the PshO-subunit at very similar position in cyanobecteda and spinach, indicating an identical overall structura of PS2. In spinach we could characterize a large PS2/LHC complex with 26.8x12.3 nm and a max. height of 8.8-9.3 nm. Comparison with the Isolated PS2 core complex shows the dlmar as the center of the symmetric supercomplex in which each monomer is connected to one trimedc LHC2 by 3-4 smaller ChlJoindlng proteins.

P-U-056 THE ELIMINATION OF POLYUNSATURATED LIPIDS AFFECTS THE STRUCTURE OF PHOTOSYNTHETIC MEMBRANES B. Szalontai 1, Z. Gombos 1, O. Zsiros 1, Y. Tasaka2, N. Murata 2 1Biol. Res. Centre Hung. Acad. Sci., Szeged, P.O.Box 521, Hungary 2National Institute for Basic Biology, Okazaki, Japan Cyanobacterial strains can be used as a simple model system to study the role of polyunsaturated fatty acids in structure of biological membranes. Recently we developed a transformation system by using Syncchocystis PCC6803 strain in that we eliminated defined steps of fatty-acid desaturation producing transformant cell line which completely missing polyunsaturated fatty acids. In this transformant the genes encoding A6 and A12 fatty-acid desaturases were disrupted by the insertion of Cm r and Km r cartridges, respectively. The lack of polyunsaturated fatty acids decreased the capability of the transformant cells to grow at temperatures lower than 30°C. W e isolated the thylakoid membranes from wild type and the transformant cells grown at 25, 30 and 35°C. Measuring the temperature dependence of the symetric vibration of the CH 2 groups in the fatty acyl chains of the lipid molecules yields information about the phase properties of membranes. Fourier transform infrared spectra of thylakoid membranes both from wild type and transformant cells revealed that the membranes without polyunsaturated fatty acids are more rigid at temperatures lower than the growth temperatures. This indicates that the cells are not able to compensate the loss of polyunsaturated fatty acids in the respect of membrane structure.

132

P-11-057 REDOX DEPENDENT PROTEIN PHOSPHORYLATION IN VITRO IN CHROMATOPHORES FROM THE PURPLE PHOTOSYNTHETIC BACTERIUM RHODOSPIRILLUM RUBRUM AND IN THYLAKOIDS FROM THE CYANOBACTERIUM S Y N E C H O C Y S T I S SP. P C C 6 8 0 3 Andr6 Stru~lics and John F. Allen, Plant Cell Biology, Lund University, Box 7007, S-220 07 LUND, Sweden Both ct and 13 polypeptides of the LH I light-harvesting complex were phosphorylated in the chromatophores from the purple photosynthetic bacterium Rhodospirillum rubrum S1. Phosphorylation was done using ['¢-32p]ATP as the phosphate group donor, and involved a protein kinase whose activity was under redox control, as indicated by effects of redox reagents. In contrast to the chloroplast LHC II kinase, the purple bacterial LH I kinase appears to be activated by the oxidised form of an electron carrier in the vicinity of the quinone pool or cytochrome b/c complex of the photosynthetic and respiratory electron transport chains. Data is presented on potentiometric redox titration of chromatophore protein phosphorylation and on the functional effects of phosphorylation. Data is also presented on redox dependent protein phosphorylation in thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803.

P-11-058 CARBON DIOXIDE FIXATION AND PHOTOEVOLUTION OF HYDROGEN AND OXYGEN IN A MUTANT OF CHLAMYDOMONAS LACKING PHOTOSYSTEM I * E. GreenbaumJ J. W. Lee,j (2. V. Tevault,~ S. L. Blankinship, ~L. 3. Mets 2 1Chemical Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6194; 2Department of Molecular Genetics and Cell Biology, University of Chicago, 1103 E. 57th Street, Chicago, IL 60637 Although mutant 114 ofChlamydomonas reinhardtil lacks Photosystem I, it is capable of pbotoantotrophic assimilation of atmospheric carbon dioxide and sustained simultaneous photoevolutinn of molecular oxygen and hydrogen. At saturating light intensities, carbon dioxide reduction was stable under anaerobiosis but unstable in air. At lower light intensities, carbon dioxide reduction was stable in both atmospheres. The data indicated that Photosystem I is not necessary for autotrophic photosynthesis. One interpretation of these results is that oxygenic photosynthesis developed as a single-fight-rcaction process, presumably from a pheophytln-quinone-reaction-center bacterium but became unstable as oxygen in the earth's atmosphere accumulated. PS I was the second light reaction, added to confer stability in oxygen-containing atmospheres. Viewed from this perspective, the well-known Z-scheme of modern photosynthesis is seen as a specialized adaptation for performing low-potential rednetlve photochemistry in oxygen-containing atmospheres, but is not an irreducible necessity for satisfying the thermodynamic and mechanistic requirements of carbon dioxide photoreduction using water as the source of reductant.

Poster

P-12-003 INTERACTION BETWEEN PHOTOSySTEM l I b~.,DIATED AND RESPIRATORY ELECTRON TRANSPORT IN WILD TYPE AND MSP-FREE MUTANT CEI.J~ OF SYNECHOCOCCUS PCC7942 AND SYNECHOCYSTI$ PCC6803

Poster

session

Dlrk IL Eneels & EIfriede K. Platorlus Biologic VIII: Zellphysiohigie, Universitat Bielefeld, Postfach 100131, D-33501 Bielcfeld, FRO

12

Alternative electron transfer pathways and regulation P-12-001

- P-12-038

P-12-001 BIOGENESIS OF CYTOCHROME-COMPLEXES IN RHODOBACTER CAPSULATUS

Previously, we were able to demonstrate that in MSP (manganese stabilizing protein) free mutant cells of Synech~cus PCC7942 and Syn¢chocyatis PCC6803 the water oxidizing enzyme (WOE) became totally inactive during extended dark periods and that this inactivation could be reversed by photoactivation. In the corresponding wild type cells, oxygen evolution was nearly lmaffeeted by dark adaptation (Engels, Lott, Schntid and Pistorins, 1995, Photosynth. Res. 42, 227.244). In the presont work we show that dark inactivation of the WOE in photoactivated MSP-free mutant cells of Synechoeoccua PCC7942 and S.vnechocyati~ PCC6803 is greatly redue~ in cells which had been carbon starved by dark cultivation - indicating that this inactivation is e consequence of electron donation from the respirator T chain to the WOE. In late logarithmic wild type cells of Syaechococcua PCC7942 and - even more pronounced - in the e~'esponding, photonchvated mutant, the flash pattern of oxygen evolution exhibits a poor or no oscillation. Here we dcmonstrata that this deviation from a normal Kok pattern is diminished after carbon stm'vatinn. As a eoneequonee, in dark cultivated wild type ceils the flash pattern alrproaches a normal Chlorella-sequance. Addition of DCBQ eliminates the depondimce of the Synechococcus.flash pattern on eultare conditions, inducing a Chlorella-like sequonce. Inhibition of the respiratoW chain by KCN in dark cultivated cells leads to a modification of the flash pattern which indicates the formation of highly ~duced states of the WOE. From these results we conclude that in Synechococcus PCC7942 PSII mediated electron transport is strongly affected by the activity of NAD(P)H.-dehydrogenases which feed eleemms from the breakdown of carbohydrates into the photosynthetic plastoquinone pool. In late logarithatie cells, a highly reduced plasmquinone pool could severely impair the efficiency of eh,,trge separation in PSI1, leading to the observed irregularities of the flash pattern. Electron donation from the PSn-acceptor side to the WOE may also contribute to these irregularities as well as it may cause the dark inactivation of the WOE in the MSp-free mutants of Synechococcus PCC7942 and Synechocysti3 PCC6803.

P-12-004 THE PARTICIPATION OF CYCLIC ELECTRON TRANSPORT IN THYLAKOID MEMBRANE ENERGIZATION

Hans-Georg Koch, Michael Grooms and Fevzi Daldal. University of Pennsylvania, Dep. Biology, 204 Mudd Building, Philadelphia, PA 19104, USA. The facultative phototrophic bacterium Rhodobacter capsulatus is capable of growth in a wide range of environmental conditions using a highly branched electron-transfer chain. During respiratory growth of this organism reducing equivalents are conveyed to oxygen via two terminal oxidases, the previously purified cyt cb oxidase (Cox) and the as yet unebaracterized quinol oxidase (Qox). Complementation of three different Cox-negative mutants, M4, M5 and MTG, yielded 2 different plasmids p4A and p5T. The latter, which complements M5 and M7G contains at least part of the structural genes of eyt cb oxidase. An insertion mutation located in the 32 kDa subunit of eyt cb oxidase was constructed in vitro and introduced into the chromosome of an otherwise wild-type strain by gene transfer-mediated genetic cross. The obtained mutant MG1 is NADI-, lacks both membrane bound c-type subunits of the eyt eb ox~lase and can be complemented by plasmid p5T. On the other hand p4A, which complements M4 but not M5, MGI or MTG carries as yet unidentified genes required for biogenesis or regulation of this enzyme. Further studies axe underway to characterize these genes.

P-12-002 E V I D E N C E F O R AN A L T E R N A T E E L E C T R O N C A R R I E R B E T W E E N T H E C Y T O C H R O M E B 6 / F C O M P L E X A N D PS I IN SYNECHOCYSTIS 6803: IS I T CYTOCHEOME M ?

S.U. Metzger l, H. Pakrasi 3 and L Whitmarsh 1,2,4 lCenter of Biophysics and Computational Biology and 2Dept of Plant Biology, University of Illinois; 3Dept of Biology, Washington University, St. Louis, Me; 4photosynthesis Research Unit, USDAJAgrieultural Service, Urbana, IL 61801 Mutant strains of Synechocystis 6803 lacking either plastocyanin (PC) or cytochrome c6 (cyt c6) can be grown so that both carriers, PC and cyt c6, are absent. The cells grow photoautotrophically and sustain normal rates of oxygen evolution and dark respiration suggesting the presence of an alternate carrier (Zhang et al., 1994, JBC, Vol. 269, pp. 5036-5042). The oxidation kinetics of cytochrome f and the reduction kinetics of P700 have been measured spectroscopically revealing that electron transfer occurs at a 4-6 times slower rate in these mutant cells than in wild-type. We created a double mutant that lacks cyt c6 and the recently discovered cytochrome M (Malakhov et al., 1994, J. Plant Physiol., Vol. 144, pp. 259-264) to investigate the possibility that the latter could act as an electron carrier between the cytochrome b/f complex and Photosystem I.

Boris Ivanov], Gerald Edwards 2. ~Institute of Soil Science & Photosynthesis, Pushchino, Russia. 2Botany Dept., Washington State University, Pullman, USA. The influence of Antimycin A on processes associated with thyiakoid membrane energization was investigated in the intact mesophyll chloroplasts of maize which catalyze the ATP dependent conversion of pyruvate to phosphoenolpyruvate (PEP) and oxaloacetate (OAA) reduction to malate. The presence of 1 ~aM Antimycin A inhibited induction of 02 evolution during illumination of chloroplasts in the presence of pyruvate, Pi, and PEP carboxylase (PEPC) without added OAA as an electron acceptor. When chloroplasts were illuminated with pyruvate and OAA (in the absence of PEPC) addition of 1 ~M Antimycin A caused a decrease in the ratio of PEP/malate Antimycin A addition increased the rate of OAA dependent O2 evolution in the absence of pyruvate up to two times and did not change the rate of Oz uptake with methyl viologen as accepter. Under the latter conditions only during OAA reduction does the light-induced quenching of 9-aminoacridine fluorescence decrease significantly. These results show that cyclic electron transport energized the thylakoid membrane concurrently with the noncyclic transport

P-12-005 STUDIES O N THE RELATIONSHIP BETWEEN PHOTOSYNTHETIC ELECTRON TRANSPORT A N D CARBON METABOLISM DZ Habash ~, M] Paul~ , MA] Parry ~, SD Driscoll I , DW Lawlor ~, A] Keys~& ] Gray2. ~Dept. of Biochemiscr'/and Physiology, IACR-Rothamsted, H arpenden, AL5 5TP, UK. 2Dept. of Plant Sciences, Univ. of Cambridge, CB2 3EA. Simultaneous measurements of chlorophyll fluorescence and CO 2 gas exchange on attached leaves of wheat grown at 700pmol mol ~ CO 2 showed an increase in the rates of carboxyladon and linear electron transport and a reduction in the rate of phocorespiradon. The increase in electron transport was due to feedback from carbon metabolism and the higher carboxylation was due to a larger [CO2] at the carboxyladon site and an increase in the extractable activity of Rubisco. The enhancement of carbon assimilation was maintained when measurements were done under ambient CO2 poindng to positive acclimation (Habash et al. 1995, Planta, in press). Tobacco transformed with antisense gene constructs to reduce the activity of phosphoribulokinase by 85-95% (Paul et al. 1995, The Plant ]., in press) showed a reduced net carbon assimilation reflecdng a lowered rate of carboxylation. The photosynthetic system adjusted by reducing the rate of total linear electron transpor~ and photosystem two activity (a lower photochemical quenching, quantum yield of pho[osystem two electron transport, and a higher non-photochemical quenchingof fluorescence) and by lowering the rate of photorespiration (a lower vo/vc).

133

Poster P-12-006

P-12-009

PHOTOSYNTHESIS 1N LEAVES: ROLE OF pH REGULATION AND OF PHOTOSYSTEM I-DEPENDENT CYCLIC ELECTRON TRANSPORT Heber, U., Gerst, U., and Hauser, M., Julius-von-Saehs-Institut, Universitat Wt~rzburg, D-97082 Wtirzburg, Germany

PARTITIONING OF ELECTRONS BETWEEN THE ALTERNATIVE AND CYTOCHROME RESPIRATORY PATHWAYS DURING DEVELOPMENT OF PHOTOSYNTHETIC FUNCTION IN SOYBEAN COTYLEDONS.

Proton concentrations play a central role in maintaining optimum conditions for enzyme catalysis in photosynthesis and in controlling the activity of photosystem 11 of the chloroplast electron transport chain. A proton gradient across the thylakoid membrane is the main energy source for chloroplast ATP synthesis. In leaves, the pH in the chloroplast stroma is regulated by effective proton/cation exchange across the chloroplast envelope and across the tonoplast. A relatively small transthylakoid pH gradient can satisfy the ATP requirements of carbon reduction. A somewhat larger transthylakoid proton gradient is necessary for a protonation reaction in the thylakoid lumen which effectively controls the activity of photosystem II under excess light providing protection to the chloroplast electron transport chain against photoinactivation Formation of this increased proton gradient is not possible during the linear electron flow to carbon dioxide or to oxygen in photorespiration. Proton transport coupled to photosystem 1-dependent cyclic electron transport is proposed to be important in controlling photosystem II under light and water stress.

S. A. Robinson 1, M. Ribas-Carbo2, L. Giles2 , J. N. Siedow 2 & J. A. Berry3. 1Research School of Biological Sciences, Institute of Advanced Studies, The Australian National University, ACT 2601, Australia. 2Dept of Botany, Duke University, Durham, NC 27708-1000, USA. 3Carnegie Institution of Washington Department of Plant Biology, 290 Panama Street, Stanford, CA 94305-1297, USA. In mitochondria the cytochrome and alternative oxidases discriminate against 180 to different degrees. Stable isotope methods can therefore be used to differentiate between electron flux through the 2 pathways. We have investigated changes in respiratory partitioning during photosynthetic development of soybean cotyledons. Alternative pathway flux was low in etiolated cotyledons but increased rapidly after 6 h in the light. After 12 h light treatment the alternative pathway accounted for 50% of the total, uninhibited respiration and by day two this had risen to 70% of the total flux. The relationship between alternative pathway activity and photosynthetic function will be discussed.

P-12-007 BOROXAZOLIDONE ACT AS HILL'S REACTION OR ENERGY TRANSFER INHIBITOR TO PHOTOSYNTHESIS. J. Fernandez 1, B. Lotina-Hennsen 1 , G. Farfdn2 and D. Castillo 1Divisi6n de estudios de posgrado, Fac, quimica UNAM, Ciudad Universitaria, Coyoac~n 04510, M6xico D. F. fax 6 22 53 29. 2Departamento de quimica CINVESTAV, I:P:N.,A P.14-740, Mexico, D. F. 07000 M6xico. It is known that some boroxazolidones shown to have practical aplications due to their broad insecticidal, fungicidal, and herbicidal activities. In this research various derivatives were synthetized and assayed their effect on photosynthesis. We found that diphenyl (iysinate -O, N) borane, inhibited on pea (Pisum sativum) chloroplasts the basal, phosphorylating, uncoupled electron transport, ATP synthesis and H+-uptake. by 42%, 51%, 79%,100% and 100% respectively therefore act as Hill's reaction inhibitor localized at water spliting enzyme level. However diphenyl (glutamate -O, N) borane, diphenyl (glycine -O, N) borane, diphenyl (threonine -O, N) horane, diphenyl (aspartate -O, N) borane acts as energy transfer inhibitors. Partially suported by DGAPA IN204493.

P-12-008 T H E p H - D E P E N D E N T R E G U L A T I O N OF PHOTOSYNTHETIC ELECTRON TRANSPORT

G. Sch0nkneeht, S. Neimanis, U. Gerst & U. Heber Lehrstuhl Botanik I, Univ. WOrzburg, Mittlerer Dallenbergweg 64, D-97082 W ~ z b u r g , Germany U n d e r conditions (500-2000 ttl/1 CO2; 1 % 02) which allow high rates o f photosynthesis, chlorophyll fluorescence and carbon assimilation were measured at different light intensities. Using 3 A T P / C O 2 and the known relationship between ATP synthesis rate and trans-thylakoid ApH we calculated the light-dependent p H gradient across the thylakoid membrane (ApH) in intact leaves. These ApH values were correlated with the photochemical (qp) and non-photochemical (q~) quenching o f chlorophyll fluorescence, and with the quantum yield o f PS II (q~sn). At ApH > 2.1 all parameters (qp, qN, tI~sn) changed very steeply, following hexacooperative titration curves with slightly different p i ca values. In Chl b-less mutants o f barley (chlorina.f2 2800) the dependence on ApH was less steep compared to the wild type.

134

P-12-010 IS THE FERREDOXIN:NADP ÷ REDUCTASE BOUND TO THE PSI-E SUBUNIT OF PHOTOSYSTEM I INVOLVED IN CYCLIC ELECTRON TRANSPORT? Hanne Linde Nielsen, Birgitte Andersen & Henrik Vibe Scheller Plant Biochemistry Lab, Dept of Plant Biology, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark Two different forms of ferredoxin:NADP+ oxidoreductase (FNR) have been isolated from barley (Hordeum vulgate L.) thylakoids. One isolated form is a free protein in solution while the other form is bound to the PSI complex. The PS I- bound form of FNR has about ten times lower V,~, than the free form in assays for diaphorase and NADP+ photoreduction activity whereas the two forms have almost equal Vm~ in ferredoxin dependent cytochrome c reduction. I ~ for NADPH, NADP+, and ferredoxin is higher for the bound form. ATP increases the K~ for the substrates but these effects require high concentration of ATP and are probably of little importance in vivo. Both the bound and free form of FNR catalyze the reduction of soluble quinones using NADPH as electron donor. The observed rates are sufficient to account for rates of cyclic electron transport in thylakoids. The hydrophobic quinones decyl-plastoqdinone and vitamin K~ do not function as electron accepters. Rather, these compounds are shown to strongly inhibit diaphorase activity.

P-12-011 H202.INDUCED CYT B-$59 OXIDATION IN INTACT SPINACH CHLOROPLASTS IN THE PRESENCE OF ASCORBATE $, Heimann & U. Schreiber, Lehrstuhl Botanik I, Universit~t Wiirzburg, Mittlerer Dallenbergweg 64, D-97082 Wfirzburg, Germany Cyt b-559 absorbance changes in intact chloroplasts were deconvoluted using the LED-array-spectrophotometer (Klughammer et al.1990,PRES 25:317-327). When intact chloroplasts are isolated in presence of ascorbate, approx. 15% of the total cyt b-559 can be oxidized by 200pM H202 (KM at 30/aM). The H202oxidizable fraction is considerably increased (up to 40% of total cyt b-559) by heat-pretreatment, which is known to induce HP---~LP-transformation. The 15% fraction displays unusual redox properties. Following oxidation by fen-icyanide, it cannot be reduced by ascorbate, thus qualifiing as an LP-form. On the other hand, ascorbate promotes it's rereduction following H202-comsumption, probably involving the ascorbate radical created by the ascorbate peroxidase. The same fraction can be oxidized by H202 in the light or just by strong light in the presence of a potent acceptor like nitrite. As the here identified cyt b-559 is in the reduced state in intact chloroplasts, which contain 10-20 mM ascorbate, these findings should be relevant for the interpretation of previously reported cyt b-559 oxidation in leaves upon strong illumination.

Poster

PSI FLUORESCENCE AT ROOM TEMPERATURE: EFFECTS ON QUENCHING COEFFICIENTS

P-12-012 POSSIBLE

P-12-015 THE FUNCTION OF ASCORBIC ACID IN PHOTOSYNTHETIC PHOSPHORYLATION

E. P~ndel Institut ~ r Pflarmengenetik und Kulturpflanzenforschung, Corrensstrafle 3, D-06466 Gatersleben, Germany

Giorgio Forti and Gianluea Elli Centro C.N.R. per la Biologia Cellulare e Molecolare delle PianteDipartimento di Biologia. Universit/t degli Studi di MUano Via Celoria, 26 20133 Milano, ITALY

Chlorophyll fluorescence of PSI can introduce errors in calculation of PS II quenching coefficients and yield (G-enty et al., 1990, Photosynth Res, 26 133139). To assess inaccuracy of PSlI parameters, PSI contribution to total fluorescence as measured by a PAM 101 fluorimeter was estimated. This was done by recording normalized variable fluorescence (Fv/Fm) of dark-adapted leaves and low temperature (77K) emission spectra of leaf powder of 12 species and 2 hybrids of Flaveria. The genus Flaveria includes species with C3 photosynthesis and C4 species (NADP-ME), the latter with elevated PSI/PSlI ratios, and C3-C4 intermediate-type species. When species were compared, Fv/Fm decreased with increasing ratio of 77K fluorescence at 735 nm to fluorescence at 685 rim. A simple model yielded Fv/Fm of 0.88 for pure PSII. PSI contribution to total fluorescence ranged from 5% for C3 to 12% for C4 species. PSI-effects on quenching parameters will be discussed.

Ascorbete is oxidized to the free radical monodehydroascorbate (MDA) by 02 and H202 formed in the Mehler reaction. MDA is an efficient electron aceeptor at the reducing end of PS1, competing with NADP for electrons. The 1-I20 to MDA electron transport system is coupled to ATP formation with a ratio ATP/2e" of 1. In the presence of ascorbate the 02 uptake due to the Mehler reaction is quantitatively balanced by the Oz evolution due to MDA reduction, and the net oxygen exchange is zero. The system ascorbate-MDA performs two functions: the scavenging of O2 and H2Oz, and the electron transport coupled to ATP synthesis. Such a system could perform the important role of balancing the ratio NADPIVATP required for CO2 assimilation.

Z8 THI FLJkSB-ZNDUC'D XNlZZBZTZOM CHLORORISPZI~TORYPATHNAyZN ~ 8

OF

P-12-013 RISPZIU~TZO! LZlI'KZD TO T~l RSZNZtRDTZZ ?

Pierre Gans* and Fabrice Rebeill~ + *IBS-LRMN. 41 Avenue des Martyrs. 38027 Grenoble CEDEX, France. +LPCV/DBMS C E N Grenoble. 17 Avenue des Martyrs. 98054 Grenoble CEDEX. France. In C. r e i n h a r d t i i , transient inhibition of respiration is observed in the presence of saturating flashes (Peltier et al, 1987, BBA, 893, 83-90). This phenomenon was attributed to an inhibition of chloroplastic respiration on the basis of the sensitivity of the transient inhibition to classical mitochondrial inhibitors (cyanide, mixothiazol or antimycin)(Ravenel & Feltier, 1991, Photosynth. Res., 28, 141-148) Here, we have investigated the reduction state of thePQ pool in wild-type strains under conditions where the transient inhibition was no longer observable. First, we mesured the maximal fluorescence in the wild-type w h i c h depends on the nttmber Of phosphorylated LHCII associated w i t h the PSI and thus is linked to the reduction state of the PQ pool. we investigated the amplitude of the b phase of the 520 n m electrochromic shift, w h i c h has b e e n also proposed to be dependent on the presence of PQH2. Last, we determined the rerec~/ction t~/n8 after one flash of cyt c6, an electron carrier located between the cyt b6f and the PSI in a plastocyanine-devoidedmutant AC208, . None of these three phenc~nena have been found to be different in presence of CN, AM or MX from a control, whereas conditions in w h i c h m i t o c h o n d r i a l r e s p i r a t i o n was i n h i b i t e d i n d u c e d v e r y large v a r i a t i o n s in these phenomena. These experimental conditions clearly indicate that under the conditions where the chlororespiration was inhibited (i.e. w h e n the flash-induced transient inhibition of oxygen uptake was no longer observed), the PQ pool was still oxidised, a r e s u l t in d i s a g r e e m e n t w i t h the c u r r e n t m o d e l of chlororespiration.

P-12-016 FERREDOXIN-DEPENDENT AND ANTIMYCIN A-SENSITIVE REDUCTION OF CYTOCHROME B-559 IN MAIZE THYLAKOIDS: PARTICIPATION OF A MENADIOL--REDUCIBLE CYTOCHROME B-559 IN CYCLIC ELECTRON FLOW

~

U. Schreiber 1, H. Miyoshi2 and K. Asada ood Sci. Kyoto Univ., Uii,2Kyot9, 611, JAP.A/)I.. 1Le~stuhl Boranik I Univ. Wiirzburg, GERMANY, Faculty ot ,agriculture, r-_yoto univ., JAPAN. We measured the redox state of Cyts in thylakoids from maize mesophvll chloroplasts which show a high activity of cyclic electron flow [Asada et ~l. Plant Cell Physiol. (1993) 34: 39], and identified a Fd-reducible and antimycin A (AntiA)-sensltive Cyt/?-559 (Fd), using an LED array spectrophotometer [Klughammer et al. Photosynth. Res. (199.0) 25:3171. The absorbance peaking at 554 nm was decreased by far-red lfgh~, showmg the photooxidation of Cytf. However, on addition of 5 pM F-d an increase in absorbance with a peak at 560 nm was observed, indicating the reduction of Cyt b-559 by the photoreduced Fd. Thephotoreduction of Cyt b-559(Fd) was inhibitea by I/~M AntiA, but not affected by ascorbate or dioxygen, showing that Cvt b-559(Fd) is not autooxidized. The Cyt b-559(Fd) was reduced-by men~diol (E~ -10 mV), but not by ascorbate (F£, +80 mV), being different from the Cyt b-559 in PS II. AntiA showed the parallel concentration dependent inhibitions of the photoreduction of Cyt b-559(Fd) by far-red light and the Fd-dependent cyclic electron flow. Fourteen derivatives of AntiA also show the similar structural responses in the both reactions. These observations indicate that a menadiol-reducthle Cyt b-559 is a mediator in the Fd-dependent cyclic electron flow.

P-12-014 A NOVEL TEPH)OPTERIN OF GREEN SULPHUR B A C F E ~ CHLOROBIUM TEPIDUM

P-12-017 CYCLIC ELECTRON TRANSPORT MEDIATED BY THYLAKOIDBOUND, NADPH-SPECIFIC PYRIDINE NUCLEOTIDE DEHYDROGENASE IN CYANOBACTERIA

Seung-Hyun Cho, Jong-Uk Na, Hwan Youn and Dept. of Microbiology, College of Natural Sciences, Research Center for Molecular Microbiology, Seoul National University, Seoul 151-742, Republic of Korea

H. Mi, T. Endo, T. OgawaI and K. Asada The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan and lBioscience Center, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan

A novel pterin compound, designated as tepidopteril% were detected from a thermophilic photosynthetic green sulphur bacteria, Chlorobium tepidum, by analytical high performance liquid chromatography. By means of spectroscopic (UV-visible, fluorescence, and circular dichroism spectrophotometry, NMR spectroscopy and mass spectrometry), enzymatic (~N-acetylglucosamidase), and chemical (acid hydrolysis and periodate oxidation) methods, its structure was determined as 1-O-(L-threobiopterin-2'-yl)-~N-acetylglucosamine. The content of tepidopterin in the cell were calculated to be about 1.79 I.maol~ g of dry weight. Of three different oxidation states of tepidopterin, 96.7% of tetrahydro-fom~ 2.9% of dihydroform, and 0.4% of fully-oxidised form occurred in the cell, which were determined by differential iodine oxidation.

Participation of pyridine nucleotide dehydrogenase complex (NDH) in the cyclic electron flow around PSI has been shown in cells and spheroplastsof cyanobacteria [Mi et al. Plant Cell Physiol.33: 1099, 1233(1992), 35: 163(1994)]. Thylakoids isolated from Synechocystis PCC6803 showed an increase of Chl fluorescence upon the addition of NADPH and also the NADPH-dependent photoreductionsof dioxygen and 1'700+ in the presence of DCMU. These NADPH-dependent reactions were inhibited by the inhibitors of NDH such as rotenone and mercurous chloride, but not by antimycin A. The thylakoids from the ndhB-less mutant cells did not show the above NADPH-dependent reductions of the plastoquinones in the intersystemchain. Simultaneousdeterminations of Chl fluorescence and photoreduction of NADW in the presence of ferredoxin (Fd) indicate the donation of electrons to the plastoquinonesfrom the photoreduced NADPH. The thylakoidbound NDH catalyzes also the reduction of the plastoquinone by the photoreduced Fd in PSl. Thus, the NADPH-specific NDH-complex is bound to the thylakoids and catalyses the NADPH- as well as Fd-mediated cyclic electron flows around PSI.

135

Poster

P-12-018 NON-PHOTOCHEMICAL QUENCHING OF CHLOROPHYLL FLUORESCENCE INDUCED IN THE COURSE OF TEMPERATURE SHIFT AND ACETATE ASSIMILATION IN Chlamydomonas reinhardtii. T.,_P,,ada1, U. Schreiber2 and K. Asada1 *The Research Institute for Food Science, K~,oto University, Uji, Kyoto 611, Japan; ZLehlstuhl ffir Botanik I, Universit~t Wiirzburg, Mittl'erer ]Sallenbexgweg 64, 97082 Wfirzburg, Germany On transfer of Chlamydomonas reinhardtii cells from 28°C to 38°C, nonphotoch.emical queaching of Chl fluorescence (qN) was induced. This quenching was cancelledby addition of the proton6phore nigericin, indicating .tha3. ~ e qN was ApH dependent (enexgy quenching, qE). The quenching moucuo.n ~pr(~e¢l ia pararell with the increase in temperature of the cell suspeasmn, the induction of qE was followed by a much slower induction of the uncoupler-~sensitive quenching attributable to the transition from state 1 to state 2: ln.duction of the uncoupler insensitive quenching was associated with me r~luetaon oI .me etectron .c~iel"s in the photosynthetic intersystem chain. t ngoa[tr.respirataot], was also s t i m ~ . ~ , by the temperature abil~ from 280C to ~t~hC. It m pruposea tt2at ehloror~pirafion ts associated with the induction of qN at lgh temperam~ Simunr pattern or ovetrapped induction of Fro' qnenehing as upon the tetnl m m t ~ , shift was ob.served whea acetic acid, a respira~ry ~aeu~ commomy u.s~. for heterotrophie culture of (7. einlmrfftii, was added to celt culture. Addition of acetic acid also stimulated the dark respiration and reduelao.n of the rote[system, electron carriers. These results indicate intrinsic assocaaUon of the dark respiration and the induction of qN.

P-12-019 T S E P R E S E N C E O F n d ~ B AE~D ~ J ~ P R O O U C T S I N B L A C K PINE CHI~OPI~%STS SU~a~TS A MIGRATION OF z~ 6 E N E S T O T H E I~CLE~JS

S. Corneille, s. Cuing, G. Guedeney, M. Havaux & G. Peltier CEA, Laboratoire d'Ecophysiologie de la Photosynth~se, D P ~ , Centre de Cadarache, F-13108 Saint-Paul-lez-Durance, France It has been recently reported that the chloroplastic genome of the black pine (Pinus thunbergii) is lacking of all the chloroplastic ndh genes (Wakasugi et al. 1994 Proc. Natl. Acad. Sci. USA 91, 9794-9798). By using antibodies raised against synthetic peptides deduced frown conserved sequences of the chloroplastic genes ndh B and ndh J, we show however the presence of the corresponding gene products in black pine chloroplasts. Non-denaturing gel electrophoresis of Triton X-100 solubilized thylakoid membranes of black pine chloroplasts followed by staining with nitroblue tetrazolium revealed the presence of different NAD(P)H dehydrogenase activity bands. By another way, studies of dark rereduction kinetics of PT00+, show that electrons can reduce the intersystemphotosynthetic transport chain in Pinus needles and in potato or tobacco leaves. We conclude from these results that ndh gene products are present in black pine chloroplasts where they participate to the donation of electrons to the plastoquinone pool. These results are interpreted by the probable migration of the corresponding genes from the chloroplast to the nucleus during the evolution of plastids.

P-12-020 B ~

~

J ~

PR~OUCTS ARE ~U~u~ITS OF A CRLO~PLASTIC

~D(P)H DI~xD~I~IllaUlE OONPLEX G. Guedeney, S. Corneille, S. Cuin6 & G. Peltier CEA, Laboratoire d' Ecophysiologie de la Photosynth~se, DPVE, Centre de Cadarache, F-13108 Saint-Paul-lez-Durance, France Sequencing the chloroplast genome of different higher plant species has revealed the presence of ii genes showing homologies with genes encoding for subunits of the mitochondrial NADH dehydrogenase coraplex (Sugiura, 1992 Plant Mol. Biol., 19, 149~ 168). Although direct evidence are lacking, these higher plants genes have been assumed to encode for subunits of a thylakoid NAD(P}H dehydrogenase, involved in chlororespiration and/or in cyclic electron flow around P S I . In the present study, we used antibodies directed against conserved sequences of two ndh genes (ndh B and ndh J) to show that the corresponding gene products (MW of 32 and 18 kDa, respectively) are present in thylakoid membranes of potato chloroplasts. Using non-denaturing gel electrophoresis and staining with nitroblue tetrazolium, we report the existence of two NAD(P)H dehydrogenase activity bands in potato thylakoids; one of the activity bands is recognized by both anti-ndh B and anti-ndh J antibodies. We conclude that ndh B and ndh J gene products are subunits of a chloroplastic NADH dehydrogenase complex likely involved in chlororespiration and/or cyclic electron flow around PS I.

la¢

P-12-021 ~RESPIRATI~

IN U N I C E L L U Z d % R G ~ E E N A I ~ S A E

G. Peltier & J. Ravenel CEA, Laboratoire d'Ecophysiologie de la Photosynth~se, DPVE, Centre de Cadarache, F-13108 Saint-Paul-lez-Durance, France Fluorescence studies in unicellular algae have shown that the redox state of the plastoquinone (pQ) pool could be modified by O 2 and by respiratory inhibitors; this was interpreted by the existence of a chloroplastic respiratory chain interacting with photosynthesis (Bennoun, 1982, PNAS 79, 4352-4356). However, the redox state of the PQ pool has been reported to be mainly affected by changes in mitochondrial activity (Gans & Rebeill~, 1989, Plant Physiol., 1015, 150-155), electrons entering chloroplasts via metabolic shuttles and reducing PQs by a chloroplastic NAD(P)H dehydrogenase. Evidence for a chlororespiratory activity has independently been obtained by studying flash-induced inhibition of a respiratory process (Peltier et al., 1987, BBA 893, 83-90). In order to check whether this phenomenon is due to an inhibition of chlororespiration, we studied Chlamydomonas mutants defective in mitochondrial cytochrome oxidase. We report that the flashinduced inhibition of respiration occurs in such mutants and is insensitive to inhibitors of the alternative mitochondrial respiration. We conclude that the flash-induced 02 signals are not due to an inhibition of mitorespiration, but to an inhibition of chlororespiration.

P-12-022 PHOTODYNAMIC ACI'ION OF HYPERICIN ON CYANOBACTERIA S. S. Brody l, K. Alygizaki-Zorba2 and G. Papageorgiou 2 JDept. Biology, New York University, New York, USA 2NRC 1~emokritos', Biology, Athens, Greece The plant pigment hypericin has photodynamic activity. Its action arises mainly from its production of singlet oxygen in light. Hypericin was prepared from hypericum perforatum. The effect of hypericin on photosynthesis and respiration was monitored in 5~nechocystis and Anacystis nidulans. An oxygen electrode was used to measure net oxygen evolution or consumption. Photosynthesis was completly inhibited in Synechocysas andAnacystis, when the mol ratio of chlorophyll to hyperiein was about 38/1 and 160/1, respectively. Hyperiein rapidly inhibites photosynthesis before inhibiting respiration. This appears to be related to the intemal organization of the cyanobaoter. In Synechococcus sp. both photusystemI reaction center and ATP synthase proteins are located mostly in the outermost thylakoid, toward the periphery of the call.

P-12-023 RESPIRATION AND THE SLOW APPARENT 0 2 RELEASE UNDER FLASHING LIGHT IN SYNECHOCYSTIS SP. PCC 6803 Pascal C. Meunier 1, Robert L. Bnmap2 & L. A. Sherman1, l:Department of Biological Sciences, Purdue University, West Lafayette, IN 47906, USA; 2:Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA We investigated the slow apparent 02 release under brief light flashes by using PSII mutants of Synechocystis sp. PCC 6803 lacking PsbC and PsbA. The slow apparent 02 release was present at higher amplitudes in the mutants. It was inhibited by starving the mutants of glucose (>90%), by 10 mM NaN3 (85%) and by boiling samples for 2 min (100%). The combination of DBMIB (2,5-dibromo-3-methyl-6-isopropyl-pbenzoquinone) and HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) also inhibited 95% of the signal. This demonstrates that two pathways are available for the oxidation of respiratory intermediates by PSI. Rotenone had little effect even in spheroplast preparations. The kinetics of the wildtype signal was deconvoluted as a positive deflection due to the inhibition of respiration by PSI activity, and a negative deflection due to the stimulation of respiration by electrons originating from PSI/. We found no evidence of a "meta-stable $3" in Synechocystis sp. 6803 that could contribute to the slow apparent 02 release. We propose a calculation to remove the contribution of the slow signal from the true photosynthetic 02 signal.

Poster

P-12-024 OCCURRENCE OF AN ELECTRON CARRIER PROTEIN IN CHLOROPLAST ENVELOPE MEMBRANES Y. Murata 1, Y. Saijoh2, Y. Arii2, A. Hatanaka2 & M. Takahashi1 1Dept. of Appl. Biochem., Coll. of Agr., Osaka Pref. Univ., Gakuen-Cho, Sakai, 593, Japan; 2Dept. of Biol., Konan Univ., Okamoto 8-9-1, •FIigashinada, Kobe 658, JAPAN Isolated chloroplasts reduce exogenous cytochrome (Cyt) c on illumination at an initial rate of ca. 5 gtmoles (mg of Chl)"1 h "1. From the resistance of this reduction of Cyt c to superoxide dismutase and no production of a disproportionation product from 02" outside the intact chloroplasts, 02_" generated by illuminated chloroplasts is not a direct reductant for the Cyt c. Difference spectrum reveals the presence of a stable oxidant in the envelope membranes isolated from spinach and pea chloroplasts when the envelopes were mixed with reduced Cyt c. Phospholipid vesicles that were reconstituted with spinach envelope membrane proteins carried electrons to extravesicular Cyt c from an electron-donating system entrapped in the vesicles. Redox potential of the envelope electron transfer protein is estimated to be ca. 150 mV although n was altered when envelope mebranes were lyzed by octyl-f~-D-glucoside (OGP). Above findings evidence that chloroplast envelope has an activity to transfer electrons of photosynthetically generated reductants to a cytoplasmic electron acceptor.

P-12-027 CYCLIC E L E C T R O N TRANSPORT IN CYANOBACTERIA Robvn Cleland Division of Biochemistry and Molecular Biology, Australian National University, Canberra, ACT 0200, Australia The photosynthetic and respiratory electron transport systems of cyanobacteria share several components, providing a diversity of pathways for energy transduction. Cyanobacteria operate several forms of cyclic electron transport, these include Photosystem I to NAD(P)H dehydrogenases, and Photosystem I directly to the plastoquinone pool by ferredoxin plastoquinone reductase ('FQR). Although electron flow through these pathways is small compared to the total flux, they provide a significant mechanism for modulating the NADPH/ATP ratio and may be important under stress conditions. There are distinct differences between FQR activity in higher plants and cyanobacteria (Manasse and Bendall, 1994, Biochim. Biophys. Acta. 1183: 361-368) Cyclic electron transport was studied in peas, Synechocystis PCC 6803, Syncechococcus and Anabena variabilis PCC 7120.

P-12-028

P-12-025 OXIDATIVE ELECTRON TRANSPORT THROUGH CYTOCHROME AND ALTERNATIVE PATHWAYS ESSENTIAL FOR PHOTOSYNTHESIS IN MESOPHYLL PROTOPLASTS OF PEA (Pisum sativum L )

CYCLIC ELECTRON FLOW, NAD(P)H-DEHYDROGENASE, RESPIRATION AND SALT STRESS IN Synechocystis PCC6803

~ A,S. Raghavendra & K. Padmasree Sch Life Sci, Univ ofHyderabad, Hyderabad 500 134, INDIA. The essentiality o f cytochrome and alternative pathways for photosynthesis was evaluated in pea mesophyll protoplasts. Photosynthesis was suppressed by inhibitors o f oxidative electron transport/phosphorylation and not by those o f glycolysis or tricarboxylic acid cycle. Oxidative electron transport through both cytochrome and alternative pathways was important in optimizing photosynthesis. There was a steep decrease in intracellular ATP and an increase in ADP in presence o f oligomycin or antimycin A, but only a marginal change in ATP or ADP with salicylhydroxamic acid. We suggest that oxidative electron transport plays a much more significant role than the oxidative phosphorylation in benefitting photosynthesis.

1, M. Havaux2, T. Ogawa3, F. Joset I & H. Matthijs4 CNRS, 13402 Marseille Cedex 20, France; 2 CEA, 13108 St Paul-lez-Durance, France; 3University Bioscience Center, Chikusa, Nagoya 464-01, Japan; 4Lab. Microbiol., Nieuwe Achtergracht 127, 1018-WS Amsterdam, Netherlands. A NADH-dehydrogenase deficient mutant of Synechocystis PCC6803 (mutant M55) was used to study cyclic electron flow around PSI and photophosphorylation. In the absence of this plastoquinone reduction pathway, sensitivity towards antimycin A and myxothiazol was appreciable. The wildtype could also be rendered susceptible to these inhibitors provided experiments were done in the presence of N-ethylmaleimide or after starvation of the cells. Growth of the mutant in high salinity gave rise to a stimulation of cyclic electron flow and of respiration, both essential in these stress conditions but insufficient to support photoheterotrophic growth. This increase was correlated to the presence of a new NAD(P)H dehydrogenase activity located in the thylakoids, only slightly sensitive to antimycin A and myxothiazol. The different pathways involved will be discussed.

P-12-029

P-12-026 ENHANCEMENT IN THE EFFICIENCY OF PHOTOSYSTEM II P H O T O C H E M I S T R Y IN SYNECHOCOCCUS PCC 7942 DUE TO PROLONGED PARTIAL INHIBITION OF PHOTOSYSTEM II 1 ~ , Prasanna Mohantv2 and Salil Bose 1 ~Centre for Biotechnology and 2School o1~Life Sciences, Jawaharlal Nehru University, New Delhi, India

Growth of cyanobacterium Synechococcus PCC 7942 in the presence of sub-lethal levels o f P h o t o s y s t e m II (PSII) inhibitor 4 - c h l o r o - 5 dimethylaminoo2-phenyl-3(2H) pyridazinone (SAN 9785) leads to a prolonged imbalance in the rate of electron transport through the two photosystems. Such a stress was observed to increase the efficiency of light energy utilization by PSII as evidenced by an increase in the variable fluorescence in SAN 9785 grown cells. Relative quantum yield of PSII was also estimated to increase using polarographic measurements. The nature of changes that effect partitioning of energy in favour of PSII and an enhancement in its photochemical utilization appear to be independent of mechanism that operates during state transitions as the cells retain their ability to undergo these transitions,

POLYPHASIC CHLOROPHYLL A TRANSIENT IN L E A V E S E X P O S E D CONDITIONS

FLUORESCENCE TO ANAEROBIC

P. Haldimann, A. Srivastava and R.J. Strasser. Bioenergetics Laboratory, University o f Geneva, 1254-Jusssy, Geneva, Switzerland The effects o f anaerobiosis on the photosynthetic apparatus was analysed in leaves o f Pisum sativum L. flushed with pure N2 gas either in the dark or in the light (95 ~tmol m -2 s l ) b y measuring the chlorophyll (Chl) a fluorescence induction transient. Leaves kept in air showed a typical O-J-I-P induction transient. Gassing the leaves with pure N2 in the dark induced a marked increase in the J level. After 2 hours in N2, the induction curve resembled that obtained for leaves lreated with D C M U . The Chl a fluorescence induction curve returned to the normal pattern when the leaves were allowed to recover in air. Anaerobiosis in low light affected the J level to a lesser extent but induced a decrease in the I level. The variable Chl a fluorescence yield (Fv) decreased during the N2 treatment but recovered in air.

137

Poster P-12-030 THE FAST FLUORESCENCE TRANSIENT OF RHODOSPIR I L L U M R U B R U M IS P O L Y P H A S I C O F T H E T Y P E O - J - I - P Reto J. Strasser and Robin G-hosE Laboratory o f Bioenergetics, University o f Geneva, CH-1254 Jussy-Lullier, Geneva, Switzerland The facultative, non-sulphur purple bacterium RhodospJrJllum rubrum expresses large amounts o f specialired photosynthetic membranes under anaerobic photoheterotrophic or semi-aerobic, chemoheterotrophic conditions. These membranes contain all components o f the photosynthetic apparatus necessary for cyclic electron transport. W e show that the fast fluorescence rise at 900 nm is polyphasic exhibiting the steps we call O-J-I-P (similar to higher plants and cyanobacteria. R.J. Strasser, A. Srivastava and Govindjee. Photochem. Photobioi. 61: 32-42, 1995). The first step F0 to J seems to reflect directly the photochemical reactions. The other steps, J to I to P, are linked to further redox states. Thus, this technique can be used for biosensing o f photosynthetic bacteria in biotechnological applications and to characterize the physiological behaviour o f different mutants.

P-12-031 NUMERICAL SIMULATION OF THE FLUORESCENCE T R A N S I E N T IN P L A N T S U N D E R I N V I V O C O N D I T I O N S A.D. Stirbet 1, Govindjee 2, and R.J. Strasser3. University of: Bucharest, Romania1; Urbana (IL), U S A 2, and Geneva, Switzerland 3 The time-dependent chlorophyll a (Chl a) fluorescence o f a photosynthetic system in vivo, the fluorescence transient, is a noninvasive tool to investigate the sequence o f events when the photosynthetic apparatus starts to work. Our goal is to simulate and to fit the experimentally measured Chl _a fluorescence kinetics on the basis o f well defined biochemical reaction schemes. Using recently available specialised software (GEPASI version 2, available as free software on the Internet), and numerical integration o f the Livermore Solver Ordinary Differential Equations with method switching (LSODA), w e have simulated for the first time fluorescence transients as they were measured under m vivo conditions. Effects o f changing heterogeneities, pool sizes and rate constants o f various PSI! redox reactions are tested systematically and will be presented.

P-12-032 CYCLIC ELECTRON FLOW INDUCED BY MIGRATION OF CYTOCIIROME BJF COMPLEXES DURING STATE TRANSITIONS D. Braun, V. Bolle, W. Riihle and A. Wild Institute of General Botany, Johannes Gutenberg-University, 55099 Mainz, Germany Some authors have set up the hypothesis that the migration of cytochrome b6/f complexes into stroma thylakoids during state 2 increases cyclic electron flow. It was tried to proof this aspect by measuring kinetics of cytochrome f oxidation and reduction with photosystem I light in stroma thylakoids from spinach with dual wavelength spectrometry. Isolated plastocyanine and ferredoxin were used to restore a loss in interaction at the oxidizing and reducing site of photosystem I. 9-Aminoacridine fluorescence was used to monitor the cyclic electron flow in phosphorylated and non-phosphorylated membranes.

138

P-12-033 IN VIVO ESTIMATION OF CO2/O 2 SPECIFICITY FACTOR OF

RUBISCO AT VARYING TEMPERATURE J. Ghashghaie & G. Comic Lab. Ecologie V6getale, Bat. 362, Univ. Paris-Sud, 91405-Orsay, France Photosynthetic electron transport rate and partitioning of electrons between CO 2 and 02 reduction were estimated in vJvo at different temperatures using simultaneous measurements of leaf gas exchange and chlorophyll fluorescence emission on intact leaves of both hairy-willow herb (Epilobium hirsutura L.) and french bean (Phaseolus vulgaris L.). Carboxylation (Vc) and oxygenation (Vo) rates of RuBP calculated from these measurements were used to estimate the CO2/O 2 specificity factor of RubisCo (S) from (Vc/Vo)=S([C02]/[02]) where, [CO2] and [02] are concentrations, at a given temperature, of CO2 and 02 dissolved in the leaves. The results showed that for both species and within a wide range of temperature the specificity factor of RubisCo estimated in vivo is similar to the in vitro values (reported in the literature) obtained using spinach enzyme. Our results suggest that carboxylation and oxygenation of RuBP are the main processes consuming photosynthetic electrons, the processes of 02 reduction other than photorespiration being negligible.

P-12-034 STATE TRANSITIONS IN THE UNICELLULAR RED ALGA RHODELLA VIOLACEA : PSI] FLUORESCENCE QUENCHING E. Delphin, D. Kirilovsky, J.C. Duval, A.L. Etienne URA CNRS 1810, ENS 46 rue d'Ulm 75230 PARIS cedex 05 In many photosynthetic organisms a rapid response to changes in illumination conditions is known as the state transitions. This regulatory process allows to adjust the distribution of excitation energy between PSI and PSII, upon preferential excitation of either one. We studied the state transitions in the unicellular red marine alga Rhodella violacea. The 77 K fluorescence emission spectra showed that the dark-adapted state was very close to state 1. Cells adapted to far red light, that preferentially excites PSI, were converted in state 1. Green light brought the cells to state 2. The deconvolution of these spectra indicated that only the photosystem II fluorescence bands at 685 and 695 nm were affected by the transitions. The effect of DCMU of DBMIB suggested that the oxidoreduction state of plastoquinone was implicated. The fast kinetics (sec) of state transitions and preliminary results using inhibitors of phosphatase and kinase suggested that protein phosphorylation of the phycobilisome antenna and/or the reaction center of PSII was not implicated in this regulatory process in red algae.

P-12-035 NAD(P)H:PLASTOCHINON OXIDOREDUCTASEAS ADDITIONAL ELECTRON DONOR FOR PHOTOSYNTHESISIN A CHROMOPHYTEALGA, PLEUROCHLORISMEIRINGENSIS W. Seidel-Guyenot, J. Franke, C. Biachel Institute of General Botany, University of Mainz, 55099 Mainz, FRG In isolated chloroplasts of Pleurochlor~s meiringensis (Xanthophyceae) we measured a NAD(P)H:plastochinon oxidoreductase (NPO). activity clearly distinguishable from ferredoxin:NADPH oxidoreductase action. NPO, reported to be involved in chlororespiration, oxidised both NADH and NADPH with pH optima at pH 9 and pH 7.5, respectively. The km for NADH was 7.3 laM. Under optimal conditions, i.e. saturating light intensity and NADH concentration, this enzyme donated 8 gmol e-/mg Chl • h to the electron transport chain measured as light dependent, methylviologen mediated oxygen uptake upon the addition of DCMU. Recording of the P700 oxidation under far-red light revealed the in-vJvo importance of NPO In whole cells, even high intensities were not sufficient to saturate the oxidation of P700, although acceptor side limitation could be ruled out. Cyclic electron transport was demonstrated not to be responsible for this phenomenon. The P700 oxidation was 20% slower when only electron transport from photosystem II was inhibited in comparison to additional inhibition of NPO. The influence of this chlororespiratory enzyme on regulation of photosynthesis will be discussed

Poster I

I

P-12-036 CHARACTERIZATION OF ELECTRON CARRIERS, INCLUDING IRON-SULFUR PROTEINS, IN CHLOROPLAST ENVELOPE MEMBRANES P. J~iger-Vottero, A.J. Dome. J. Jordanov, R. Douce & J. Joyard Laboratoire de Physiologie Cellulaire Vtg&ale, URA CNRS 576, Dtpartement de Biologic Moltculaire et Structurale, Centre d'Etudes Nucltaires de Grenoble et Universit6 Joseph Fourier, 38054 Grenoble cedex 9, France. The desaturase activities (i.e. phytoene and fatty acid desaturases) observed in plastid envelope membranes imply oxidoreduction reactions. Quinones are generally involved in such reactions and the presence of these compounds (plastoquinone-9 and a-tocopherol) has been demonstrated in both chloroplast envelope membranes. The redox behavioor of envelope quinones was investigated by EPR spectroscopy, under strong chemical oxidation and reduction conditions. In these conditions, EPR envelope spectra exhibit several signals: a semiquinone, a 4Fe-4S cluster and an unknown Fe-S cluster, It was possible to modulate these signals with NADPH. An additional 2Fe-2S cluster was detected upon reduction with NADH. We have also demonstrated that envelope membranes contain free flavins (FAD and FMN), but are devoid of detectable cytochrome. All together, these results strongly suggest the presence of functional electron-transfer chains in the chloroplast envelope.

P-12-037 ELECTRON TRANSFER IN RHODOCYCLUS TENUIS Laure M e n i n Pierre Parot, Barbara Schoepp, Pierre R i c h a u d and Andr~ V e r m t g l i o CEA-DPVE/LBC-C.E. CadamcJae 13108 Saint Paul-lez-Durance, Cedex France

Rhodocyclus tenuis contains, like most of photosynthetic bacteria, a tetrahemic cytochrome subunit bound to the reaction center. This subunit possess two high potential hemes (liP, a-band centered at 554 and 558 nm) and two low potential heroes (LP, a-band centered at 552 nm). Titration o f these hemes has been performed to characterize them in terms o f midpoint potentials. Many periplasmic eleclron carriers are present and consist o f : a HiPIP (300 mV), a high potential cyt c551 (405 mV), a low potential cyt c549 (-200 mV) and a c' type cyt. Reconstitution experiments with purified membranes supplemented by these electrons carriers of R. tenuis have been carried out under conditions o f poised redox potentials. The reduction o f the HP heine(s) is fast and performed by the HiPIP as observed in Ru. gelatinosus. EPR and absorption spectroscopy experiments carried out on whole cells confirmed that the HiPIP of R. tenuis is the immediate electron donor to the bound tetrahemic cytoehrome.

P-12-038 GAS EXCHANGE AND MODULATED CHLOROPHYLL FLUORESCENCE TECHNIQUES FOR IN VIVO ASSESSMENTS OF ALTERNATIVE ELECTRON TRANSFER F. Fredrick & R. Lemeur Dept of Applied Ecology and Environmental biology, University of Ghent, Coupure links 653, 9000 Ghent, Belgium. Gas exchange measurements in photorespiratory conditions (i.e. 21% 02) allow to estimate the electron requirements associated with the photosynthetic carbon rednctive (PCR) and oxidative (PCO) cycles. Relevant relations are presented. Modulated chlorophyll fluorescence measurements allow to assess linear electron transport rates. Both techniques were applied to assess at various CO2 levels the excess of electron transport in well irrigated and water stressed Schefflera arboticola plants. The results corroborated the premise that excess of electron transport is considerably higher in water stressed plants.

139

Poster P-13-003 CONSTRUCTIONOF A REcA DELETIONSTRAIN OF RHODOPSEUDOMONASV/R/DIS AS A NEW TOOL FOR SITE-DIRECTED MUTAGENESIS I-Penn CHEN and Hartmut Michel Max-Planck-lnstitut for Biophysik, Heinrich-Hoflmann-Str. 7 60528 Frankfurt am Main, Germany

Poster session 13

Expression and regulation prokaryotes

A stable propagation of homologous genes in Rhodopseudomonas (Rps.) v/r/dis is the prerequisite of a successful homologous expression of modified proteins. The recA gone product is known to play a crucial role in homologous recombination, which should be avoided. For the construction of a recA deletion strain, first, the recA gone from Rps. v/r/dis has been cloned from a 2.6 kb Pstl fragment identified by colony hybridization. Sequence analysis revealed an open reading lrame of 1047 bp with high homology to the recA gone from Rhizobium meliloti. Second, the Rps. viddis rocA gone was disrupted by replacing its 5' terminal Sphl fragment with a Tn5 kanamycin cassette. The resulting lips. viddis recA deletion strain shows a common recA-phenotype fog. more sensitive to alkylating agents than the wild type). In order to express modified proteins in the Rps. v/r/dis recA-strain, plasmids containing the promoter region of Rps. v/r/dis puf operon and the gone to be modified were constructed. Furthermore, a C-terminal aflinity tag was added to facilitate purification of the modified proteins.

of genes:

P-13-001 - P-13-031

P-13-004

P-13-001 THE MUTATIONS IN DNA DYAD SYMMETRY AND IIIF-B1NDING SITE UPSTREAM OF THE pttc PROMOTER AFFECT OXYGEN REGULATION OF TIlE puc OPERON IN

Rhodobuctcr sphaeroldea

Tn5469 MUTAGENESIS OF CHROMATIC C A L O T H R I X SP. S T R A I N P C C 7601

ADAPTATION GENES IN

Y.Q. Wu ~~. S, Kaplan 2 a ShanghaiInstitute of Plant Physiology, AcademiaSJnica, Shanghai 2 0 0 0 3 2, China ; 2 D'ept, of Microbiology and Molecular Genetics, University of Texa~ Health Science Center at Houston, Houston. TX 77225, USA

K. Kahn 1, D. Mazel 2, J. Houmard 2, N. Tandeau de Marsac 2 and M. Schaefer t I U n i v e r s i t y o f Missouri, Kansas City, M O 64110, U S A ; 2Institut Pasteur, P h y s i o l o g i c Microbienne, 7 5 7 2 4 Paris, France

To identify the e/s-acting sites required for tile regulation of pac gone e×prcssion by Oz, seven mutations and five 10-bp deletions were constructed in the 5j noncc~lingregion of the pac operon by bt v~o manipulation of cloned pac upstrcana sequence with a ii~odifiedPolymerase Chain Reaction (PCR) and oligonucleotide-diremed mutagcnesis. Transcriptional fusions of the !eecupstream region containing different c/~-mutations or I 0-bp deletions were constructed by utilizing the reporter molecule encoded by htcZ, The role of the target site controlling the ~ expression were investigated by analysis of the effects of the mutations and deletions on the ~galaetosidase(fa,-gal) activity resulting from the fusions when present in wild type R. ~;ehaero/des 241 grown chemoheterotrophically and fermematively. The [3-gel activity of PCFtl 1 with A to G transition mutation at -12 within a region of dyed syramctry was increased about 7, 8 fold compared with that of the wild type fusion construct in the presence of O,. The pu¢ upstream sequence of PCFK carrying lhrce mutations at - 197. - 193 and-190 between l'~sitions -199 to -185 within another dyed symractry GCAGCN:GCTGC produced 6.3-fold more fS-gal activity that did the wiM type. Mutation~ located at the integration host factor (IHF) -binding site either at -113 or at - I 15 a~ ~ ell as at I 19/- I I 0 of the y,w operon resulted in a two- to threefold increase ill ti~e level of [~-g~ll aclivity relative to wild type under aerobic condition, indicating that IHF bitlding is necessary for mm~criptional regulation by O~. Binding of IHF is believed to induce a DNA be~ld which was proposed to facilitate interaction between the O~-regulatory represser and the tx~o regions of dyed symmetry.

T h e c y a n o b a c t e r i u m Calothrix sp. strain P C C 7601 chromatically adapts to c h a n g e s in l i g h t q u a l i t y by altering the p h y c o b i l i p r o t e i n c o m p o s i t i o n o f the light-harvesting phycobilisomes. Green light promotes transcription from c p e B A and c p e C D E , r e s u l t i n g in p h y c o b i l i s o m e s e n r i c h e d f o r the p h y c o b i l i p r o t e i n p h y c o e r y t h r i n (PE). E l e c t r o p o r a t i o n o f w i l d - t y p e c e l l s yielded mutant strain FdR1 which is locked in the green light regulatory mode and e x h i b i t s c o n s t i t u t i v e synthesis o f PE. T h e FdR1 p h e n o t y p e is due to i n a c t i v a t i o n o f the r c a C gone by an e n d o g e n o u s t r a n s p o s o n d e s i g n a t e d Tn5469. E l e c t r o p o r a t i o n o f F d R I cells y i e l d e d a c o l l e c t i o n o f s e c o n d a r y p i g m e n t mutants, several with phenotypes correlating to Tn5469 transposition. T w o o f these, F d R 1 E I and F d R 1 E 4 , harbor an additional copy o f Tn5469 in the u n c h a r a c t e r i z e d c p e Y Z l o c u s d o w n s t r e a m o f c p e B A and e x h i b i t s i g n i f i c a n t l y reduced l e v e l s o f PE. A w i l d - t y p e g e n o m i c D N A f r a g m e n t c o n t a i n i n g c p e Y Z c o m p l e m e n t s the F d R 1 E 1 and F d R 1 E A p h e n o t y p e , s u p p o r t i n g a role for c p e Y Z in P E synthesis or assembly. T n 5 4 6 9 a c t i v i t y correlates to the phenotypes o f 15% o f the primary and secondary p i g m e t t t mutants in our strain collection.

P-13-005

P-13-002 GENE

IDENTIFICATION OF A "REDOX RESPONDING" SIGNAL TRANSDUCTION SYSTEM CONTROLLING PHOTOSYNTHESIS GENE EXPRESSION IN RHODOBACTER CAPSULATUS

Uni-

C.S. Mosley. J.L. Kouadio, K. Indue, C.E. Bauer Indiana University, Department of Biology, Bloomington, Indiana 47405.

We have constructed a s i t e - s p e c i f l c D1 polypeptide mutant strain LC (de1227-246) of S y n e c h o c y s t i s PCC 5803. In LC, the core p o l y p e p t i d e s of photosystem If, i n c l u d i n g the reaction center polypeptide D1 itself, are almost entirely absent. However, the psbA2 transcript level of LC is h i g h e r t h a n that of the control. T h i s is d u e to d i f f e r e n c e in t r a n s c r i p tional activity, since T x / 2 of p s b A 2 t r a n s c r i p t s of b o t h c o n t r o l a n d LC w e r e m e a s u r e d to be 15 m i n u n d e r all light i n t e n s i t i e s studied, psbA2 transcripts of wild-type Synechocystis cells have been reported to be quite stabile in the dark. O n the contrary, psbA2 t r a n s c r i p t s of LC are not dark-stabilized. We are testing the h y p o t h e s i s that the d o w n s t r e a m sequence of the p s b A 2 gene, w h i c h is i n t e r r u p t e d in m u t a n t s t r a i n LC, c o d e s for a regulatory protein responsib l e for s t a b i l i z a t i o n of t h e psbA2 t r a n s c r i p t .

In Rhodobactercapsulatus, the apoproteins of the light harvesting (LH) and reaction center (RC) are encoded by the structural genes puf, puc and puh. Molecular analysis has unveiled several regulatory genes involved in controlling the expression of these structural genes in response to the redox poise of the cell. Two of these regulatory genes, regA and regB, have been shown to be members of the family of eubaeterial signal transduction proteins. RegA and RegB have sequence similarity to response regulators and h/st/dine protein kinases, respectivelyil.2). Mutational analysis of regB demonstrates that it has a similar phenotype to regA, thereby indicating that RegB and RegA are cognate partners in a signal transduction cascade that controls LH and RC gene expression in response to anaerobiosis. Interestingly, autophosphorylation and phosphotransfer in vitro arc still redox dependent. We are currently using molecular and biochemical techniques to investigate the mechanism of the signal transduction cascade in which RegA and RegB are effected by redox, the cofactors implicated in their activation and die interaction with their putative target sites.

P O S S I B L E ROLE OF DOWNSTREAM SEOUENCE ON DARK-STABILITY OF THE TRAMSCRIPT P. Mulo, E-M. A r o & P. M ~ e n p ~ D e p t of P l a n t P h y s i o l o g y a n d M o l e c u l a r v e r s i t y of Turku, BioCity, 20520 Turku,

140

OF p e b A 2

Biology, Finland

Sganga and Bauer, (1992) Cell 68:945 Mosley et al. (1994) J. BacterioL 176:7566

Poster

P-13-006

P-13-009

TRANSFORMATION OF THE THERMOPHILIC CYANOBACTERIUM SYNECHOCOCCUS ELONGATUS

TRANSCRIPTIONAL FACTORS WHICH CONTROL HYDROGENASE SYNTHESIS RIIODOBACTERCAPSULATUS.

U. Mfihlenhoff 1 & Franek Chauvat 2 1Biologisches Institut H, Universit~t Freiburg, D-79104 Freiburg, FRG; 2SBGM, CEA Saclay, F-91191 Gif-Sur-Yvette, France

De Sury d'Aspremont R., Toussaint B., Elsen S., Colbeau A., Vignais P.M. CEA / Biochimie Microbienne (CNRS URA 1130) / DBMS / CENG 17 rue des Martyrs, F-38054 Grenoble cedex 9

IN

Rhodobacter capsulatus contains a cluster of 20 genes necessary

Axehie cultures of the thermophilic cyanobacterium S. elongatus were transformed by electroporation using genomic fragments carrying genes of PS I subunits as integrative vectors. Antibiotic marker genes are stably integrated into the genome by homologuons recombination, thus generating the first specific PSI mutants o f this organism. Chloramphenicol, kanamycine, streptomyeine and specfinomycine are useful for the selection of transformands. This set of marker genes appears to be sufficient for in vivo site-directed mutagnenesis. In addition, lncQ broad host-range plasmids, introduced by electroporation or conjugation with E.coli, are stably maintained. These vectors may be employed for complementation and protein expression in this strain, which otherwise lacks low molecular-weight plasmids.

P-13-007 A D D I T I O N OF P H Y T O E N E S Y N T H A S E G E N E S M O D I F I E S PHOTOS~SIS IN S Y N E C H O C O C C U S P C C 7 9 4 2

THE RATE OF

~ , St. G a t z e k & P. B ~ g e r Lehrstuhl f~r P h y s i o l o g i e und Biochemie der Pflanzen, U n i v e r s i t ~ t Konstanz, D-78434 Konstanz, G e r m a n y

for the biosynthesis of the membrane-bound [NiFe]hydrogenase (the structural hupSL genes encoding the small (34kDa) and the large (65kDa) subunits, resp., genes necessary for metal insertion at the active site, for the maturation of the enzyme and regulatory genes. The product of hupR, a response regulator of the NtrC subfamily and R. capsulatus IHF are transcriptional activators. HupT, of the superfamily of sensors, participate to the repression of hydrogenase synthesis. HypF is involved in derepression in presence of H z. The HupU and HupV proteins play a role in the control by 02 and by n 2. The role of these factors is being studied by the use of mutants in which the corresponding genes have been inactivated. Successive deletions of the upstream region of hupSL (promoter region) are also used to determine where the transcriptional factors bind and how their function is regulated by H 2 and/or 02.

P-13-010 OLIGONUCLEOTIDE-DIRECTED COMPLEMENTATION OF A LETHAL DELETION WITHIN THE LARGE HYDROPHILIC DOMAIN OF CP47 Shannon Clarke and Julian J. Eaton-Rye Dept. of Biochemistry, Otago University, Box 56, Dunedin, New Zealand

The f o r m a t i o n of phytoene, an i n t e r m e d i a t e product of c a r o t e n o i d biosynthesis, is c a t a l y z e d b y the e n z y m e phytoene synthase. P h y t o e n e synthase genes o r i g i n a t i n g from two d i f f e r e n t organisms, the h e t e r o t r o p h i c b a c t e r i u m Erwinia uredovora and the c y a n o b a c t e r i u m Synechocystis PCC6803, were cloned downstream the n e o m y c i n p h o s p h o t r a n s f e r a s e gene of t r a n s p o s o n T n 5 and s u b s e q u e n t l y ins e r t e d into an " i n t e g r a t i o n platform" r e s i d i n g in the c h r o m o s o m e of the c y a n o b a c t e r i u m Synechococcus PCC7942. The r e s u l t i n g t r a n s f o r m a n t s e x h i b i t e d d i v e r g e n t phenotypes in respect to the control strain: I n s e r t i o n of the Erwinia gene led to an increase of c a r o t e n o i d b i o s y n t h e sis a c c o m p a n i e d b y an i n c r e a s e d rate of p h o t o s y n t h e s i s . In the t r a n s f o r m a n t carrying an a d d i t i o n a l cyanobacterial gene, on the o t h e r hand, the level of total car o t e n o i d s a n d the rate of p h o t o s y n t h e s i s were decreased.

The oxygen-evolving photosystem II (PS lI) core complex contains at least six intrinsic proteins: the D1 protein, the D2 protein, the c~-and 13-subunitsof cytochrome b559, two intrinsic chlorophyll a-binding proteins, CP47 and CP43, and the extrinsic PSH-O (or 33-kDa) protein. The CP47 protein possesses a large hydrophilic domain of approximately 190 amino acids that is predicted to be located in the thylakoid lumen and may have a structural or functional role in the oxygen-evolving complex. A deletion from Gly-351 to Thr-365 within the large hydrophilic domain of CP47 produced an obligate photoheterotrophic mutant of the cyanobacterium Synechocystis PCC 6803 (Eaton-Rye and Vermaas (1991) Plant Mol. Biol. 17:1165-1177). This strain was constructed by deleting 45 nucleotides within psbB, the gene encoding CP47, which created an Eco RI site that replaced the coding region for the deleted amino acid residues. We have designed oligonucleotides that contain site-directed mutations within the original deleted sequence or essentially random sequences that have been reinserted into the Eco RI site. Synechocystis 6803 strains able to grow photoautotrophically and obligate photoheterotrophic mutants have been obtained. Characterization of these strains will be presented.

P-13-008 EXCITATION OF ELECTRON TRANSPORT REGULATES D1 EXCHANGE IN SYNECHOCOCCUS

P-13-011 E XPRESS1ON OF THIOREDOXIN GENE ( t r x A ) IN THE CYANOBACTERIUM SYNECHOCYSTISSP. PCC 6803 IS REGULATED BY LIGHT.

D. Campbell, A.K. Clarke, G. Zhou, P. Gustafsson and G. Oquist Department of Plant Physiology, University of Ume~, S-901-87 Ume~, Sweden. tel.: 46 90 16 52 89 fax: 46 90 16 66 76 emaih [email protected]

Francisco Navarro, and ~ . Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla-CSlC, Apdo. 1113; 41080-Sevilla, SPAIN.

The cyanobacterium Synechococcus alters PS II function under excitation stress through loss of psbAI message and expression of psbAII and AIII. This results in transient replacement of the constitutive D1:1 protein (encoded by psbAD with an alternate, functionally different form, D1:2 (encoded by psbAII/AIII). D1:1/D1:2 exchange is triggered by excess light, but also by lowering the temperature or inhibiting electron transport. These treatments all increase excitation pressure on PS II relative to energy dissipation processes. Therefore, psbA expression and D1 exchange responds to excess excitation of photosynthetic electron transport, and not specifically to light intensity or quality per se. This modulation of IX3 II activity through differential expression of D1 isozymes contrasts sharply with light acclimation strategies in plants.

The gene encoding the thioredoxin of the facultative heterotrophic cyanobacterium Synechocystis sp. PCC 6803, trxA, has been cloned by heterologous hybridization using as a probe the corresponding gene from the cyanobacterium Anacystis nidulans. The deduced amino acid sequence of trxA predicts a protein of Mr 11,750 and reveals a strong identity with its cyanobacterial counterparts and others m-type thioredoxin of photosynthetic eukaryotes. The trxA gene is transcribed as a single transcript of 450 nt. and it is strongly regulated by light through a transductional system that requires an active electron transport, being trxA transcript accumulation almost absent in darkness. A drastic decrease was also observed at high ligth intensity. However. thioredoxin transcript stability was similar in all the conditions tested. Glucose is able to mimick light effect, providing the electrons through its metabolization. In a Synechocystis mutant strain lacking photosystem II, glucose starvation promotes a strong decline in the level of trxA transcript. An essential role of thioredoxin not only in photosynthetic but also in heterotrophic growth conditions is discussed.

141

Poster ii

P-13-015

P-13-012 A SIGMA70-L1KE FACTOR AFFECTS CIRCADIAN EXPRESSION OF THE PSBAI GENE IN SYNECHOCOCCUS SP. STRAIN PCC 7942 N. F. Tsinoremas1, M. Ishiura2, K. Tanaka 3, Y. Liu4, H. Takahashi 3, C. H. Johnson4, T. Kondo2 and S. S. Golden 1 tDept, of Biology, Texas A&M Univ., College Station, TX 77843, USA; 2Natl. Inst. for Basic Biology, Okazaki 444, Japan; 3Inst. of Mol. and Cellular Biosciences, Univ. of Tokyo, 113 Japan; aDept, of Biology, Vanderbilt Univ., Nashville, TN 37235, USA We developed a mutagenesis procedure for Synechococcus sp. strain PCC 7942 that tags the sites of insertion into the genome for easy cloning of the relevant loci. By this strategy we obtained mutants affected in circadian expression of the psbAl gene (encodes Form I of the D 1 protein). Among these was a mutant in which bioluminescence from a psbAl::luxAB reporter gene exhibits a low amplitude circadian rhythm. We identified the disrupted locus as the rpoD2 gene, a member of a gene family that encodes sigma70-1ike transcriptional factors in Synechococcus. We further confirmed the role of the rpoD2 gene in affecting the circadian rhythm amplitude by interposon mutagenesis. The rpoD2" strain showed no growth defects, suggesting that the gene product belongs to the class 2 thmily of sigma70 factors, whose members are not essential for normal growth. We suggest that the rpoD2 gene product is a component of an output pathway of the biological clock that regulates the expression of a subset of genes in Synechococcus,

REGULATION

OF D1 SYNECHOCYSTIS 6803

POLYPEPTIDE

SYNTHESIS

T. Tyysti~trvi, P. Mfienpaa & E.-M. Aro Plant Physiology and Molecular Biology, Univ of Turku, BioCity A, Tykist6katu 6, FIN-20520 Turku, Finland The modified degradation rate of the D1 polypeptide in site-directed DI mutants CAI [deI(E242-E244);Q241H)], E243K and E229D has provided a tool to determine whether D1 polypeptide synthesis is directly regulated by lightqntensity-dependent factors or whether the light-dependent degradation of the D1 polypeptide controls its own synthesis. Accumulation of sSsmethionine in vivo into D1 polypeptide was found to correlate with D1 degradation rate rather than with incident irradiance, suggesting that factor(s) generated during DI degradation regulate the, translation of psbA mRNA. Althought amount of the psbA transcript increased very rapidly with increasing light intensity in all strains, up to 10-fold differences in the psbA mRNA levels under growth-light conditions between mutant strains suggest that psbA gene transcription is not solely under light control. To further understand the translational control of psbA mRNA, we are now analyzing the amount of translatable psbA mRNA in mutant strains under different light conditions.

P-13-013 COMPLEMENTATION

OF

MUTANT OF GLUCOSE-TOLERANT STRAIN OF DEFECTIVE IN CO 2 TRANSPORT B Y A 154-bp O N L Y I N GLUCOSE-SENSITI~rE STRAIN

A

SYNECHOCYSTIS PCC6803 NUCLEOTIDES pRESENT

A. K~toh I, K.Lee 2, H. Fukuzawa 2, K.Ohyama 2 & T.Ogawa 1,3 iBiochem. Regulation, 3BioScience Center, Nagoya Univ, Nagoya 464-01, Japan and 2Lab of Plant Mol Biol, Dept of Agr Chem, Kyoto Univ, Kyoto 606-01, Japan Mutants (SC1, SC2) defective in CO 2 transport were isolated from glucose-tolerant strain of Synechocystis PCC6803. A gene (designated cotA) that transforms both mutants to the wild-type (WT) phenotype was cloned from a genomic library of WT Synechocystis. The gene is homologous to cemA and its homologues in chloroplast genomes and encodes a protein of 440 amino acids with four transmembrane domains. There was a single bp substitution in cotA of SCI and deletion of 7 bp nucleotides in SC2. In addition to cotA, a 154-bp DNA fragment that complements SC1 but not SC2 w a s isolated from a genomic library of glucose-sensitive strain of Synechocystis PCC6803. This D N A fragment was present in the glucose-sensitive strain but not in the glucose-tolerant strain. The comple/nentation was observed as a result of insertion of the nucleotides to the genomic DNA of SCI. The role of this DNA fragment on CO 2 transport is now being studied.

IN

P-13-016 Requirement of the the PufX protein for photosynthetic activity in an LH1 -deficient strain of Rhodobacter sphaeroides. Fuleher, T.K., Hunter, C.N. and Jones, M.R. Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S l 0 2UH. The pufX gene of the purple bacterium Rhodobacter (Rb.) sphaeroides is transcriptionally-linked to the genes that encode the reaction centre L and M subunits and the c¢ and ~ subunits of the LH1 antenna complex. The PufX protein has been shown to be essential for photosynthetic growth in strains of Rb. sphaeroides with RC+LHI+LH2 + and RC+LHI+LH2- phenotypes, but is not required for photosynthetic growth of antenna deficient strains. It has been established that in the absence of the core LH1 antenna complex, excitation energy can be rapidly and efficiently transferred from the peripheral LH2 antenna complex to the reaction centre. In this report we examine whether the PufX protein is required for the photosynthetic growth of such LH1-deficient strains, and the effects of deletion of the pufX gene on assembly of RC/LH2 complexes.

NITROGEN REGULATION OF THE GENES INVOLVED IN NITRATE AND CARBON ASSIMILATION IN THE CYANOBACTERIUM SYNECHOCOCCUS sp. PCC7942.

P-13-017 Construction and Characterlsation of a Light-Harvesting Complex II (LH2) deficient Strain of R h o d o s p i r i l l u m molischianum DSM 119

I. Suzuki, T. Sugiyama, T. Omata Dept. Applied Biol. Sci, Sch. Agric. Sci., Nagoya Univ., Furo, Chikusa, Nagoya, 464-01, Japan

and Ha~nut Michel Max-Planck-Institut ftlr Biophysik, Heinrich-Hoffmann Strafie 7, 60528 Frankfurt, Germany

In Synechococcus PCC7942, six genes involved in uptake and reduction of nitrate form an operon (nirA operon). We found that ammonium, the product of nitrate reduction, represses the n J r A operon and activates the rbcL operon. Inhibition of ammonium fixation (i.e. the synthesis of Gin) or of transfer of amide nitrogen from Gin to other compounds abolished the effects of ammonium, showing that accumulation of a nitrogenous compound(s) synthesized from Gin acts as a metabolic signal for regulation of the two operons. One of the rnetabolites of Gin, carbamyl phosphate, and its decomposition product, cyanate, were found to repress the nirA operon and to activate the rbcL operon. The effects of cyanate were not ascribable to the ammonium formed by decomposition of cyanate, because inhibition of ammonium fixation did not abolish the effects of cyanate. These findings suggest that Synechococcus sp. PCC7942 responds to the increase in nitrogen availability by sensing the increase in the level of cyanate in the cells.

The photosynthetic bacterium Rhodospirillum molischianum (Rs. molischianum) DSM 119 contains two light-harvesting complexes, namely a LH1 and LH2 complex. We have constructed a LH2 deficient strain of Rs. molischianum by replacing the corresponding wild type genes with a kanamycin resistence cassette using the conditionally lethal sacB gene for selecting for double crossover mutants. The LH2 deficient strata was characterized spectroscopically and by Southern blot analysis. With a significant rate the LH2 deficient strain expresses a B800-820 complex demonstrating that the wild type contains silent LH3 genes. This fact is also manifested by Southern blot analysis which indicates that the genome contains at least three sequences homologous to the LH2 genes. One of those has been sequenced. The expressed B800-820 complex was investigated for its biochemical properties.

P-13-014

142

Poster P-13-018 ALTERATIONS OF THE PHOTOSYNTHETIC APPARATUS IN A SYNECHOCYSTIS SP. PCC 6803 ORF184 MUTANT Hendrik Schubert 1, Annegret Wilde 2, Heiko H~irtel2 & Thomas Btrner 2 1FB Biologie, Universitat Rostock, 18055 Rostock; 2institut ftir Biologie, Humboldt-Universitiit, 10115 Berlin, Germany

We cloned and sequenced a gene of the unicellular cyanobacterium

Synechocystis 6803 that is homologous to the conserved chloroplast open reading frame (ORF) 184. The function of the putative gene product remained unknown till present. Synechocystis orfl84 mutants were constructed by insertional inactivation and deletion. We have investigated the effects of this mutation on kinetics and composition of components of the linear electron transport chain. Besides extensive changes in photosystem (PS)I- to PSII ratio, antenna size and quantum yield, we found also alterations in kinetic parameters as light dependent slope of PSII fluorescence yield and PSI donor and acceptor limitation, respectively. Further analyses concerning effects of photosynthetic and respiratory inhibitors as well as excitation energy transfer efficiency elucidated the role of the putative ORF184 protein in photosynthesis.

P-13-019 SIGNAL TRANSDUCTION DURING CHROMATIC ADAPTATION David M. Kehoe and Arthur R. Grossman, Carnegie Institution of Washington, 290 Panama Street, Stanford, CA 94305 USA

P-13-021 Several Methodsfor Isolationand Characterlsaflonof PlasmidDNA in Cyanobacterla WeiqunLu, E. HilaryEvans,S. McColland V. Sounders Schoolof BiomolecularScience, LiverpoolJohnMooresUniversity,By~m street. I.dvexpool L3 3AF,U.K. Plasmid distribution amongfour different eyanobscteriawas studied using six simplified methodwhichinvolveddirectagarosegel electrophoresisof heat-trealed,ethanol-pranipitated. plasmid preparations from the cleared lysales without requiring ultranenlrffugat~0nand plasmidscreeningfromdifferentgrowthperiodculture.Twoof thesemethods,basedon lysis bylysozymeand lysisby SDS,Weresuccessful.Thelattermethodprovedmoresensitive.All of strainsexaminedpossessedone or two plasmids,and mid-exponentialphaseculturewas optimal for all plasmidsand lurge plasmid yield obtained. These procedurespermitteddie selective isolationof plasmidsDNAthat can be useddirectlyin electrophureticanalysisand restrictionendonucleaseanalysis.

P-13-022 GENERAT~ON AND CHARACTERIZATION OF A PHOTOHETEROTROPttlC MUTANT OF Syneehoe~tis sp. PCC 6803 .H. Kim 1, J.S. Choi l, Y.H. Chung l, L. Bogorad 2 & Y.M. Park 1 Biomolecule anal group, Korea Basic Science Institute, Taejeon, Korea; Dept of Cell Development Biol, Harvard University, Cambridge, USA

We have used a combination of site directed mutagenesis (SDM) and complementation of novel mutants to extend our understanding of the mechanisms controlling complementary chromatic adaptation (CCA). Found in a number of cyanobacteda, CCA is an adjustment of the phycobilisomes' composition in response to changes in ambient light quality. Previous work demonstrated that one of the components controlling CCA, RcaC, shows strong sequence similarity to response regulator proteins of two component regulatory systems. The activity of such proteins is controlled by the reversible phosphorylation of an aspartate (D) residue found within a domain of highly conserved amino acids. RcaC contains two such putative D phosphorylation domains, at the amino- and carboxy-terminal ends. SDM of the D within each of these domains demonstrated that the amino-terminal D is primarily responsible for the control of CCA and established the relationship between the phosphorylation state of this D and the physiological state of the ceils. We have also gained insights into photoperception and signal transduction during CCA by complementing a number of new mutants.

Cyanobacterium Synechocystis sp. PCC 6803 performs oxygenic photosynthesis analogue to that of higher pla:~s. The reaction center of phot.osystem I (PSI) is composed of a heterodimer of homologous polypepticleS, PsaA and PsaB. In order to investigate the structure and function of the PSI, cartridge mutagenesis technique was used to inactivate the /~aB gene which encodes subanit Ib (PsaB protein) of PSI. The psaB- mutants were generated by transforming wild type cells with the psaB gene of Synechococcus 7002 which was interrupted by a ehlorampherdcol acetyltransferase gene. Unexpectidly, many kinds of mutants having different color were obtained. One of the mutants, B2, was selected for further dissection. The 132 mutant would not grow photoautotrophically, which is the common characteristics of photosystem mutants. The amount of chlorophyll of the mutant was greatly reduced. Oxygen l~ptake rate of the mutant was ca. 60% of wild type. lmmonodeteetion analysis indicated that the mutan has reduced amount of PsaA/PsaB, but a normal range of PsaC and PsaD. This mutant will be used to study structure]function i~elationship and biogenesis of PSI.

P-13-020 CLONING AND CHARACTERIZATION OF SEC AND FFH GENES FROM THE CYANOBACTERIUM Synechococcus PCC7942

S O M E S T U D I E S W I T H M U T A N T S T R A I N S IN W H I C H S P E C I F I C GENES ENCODING SUBUNITS OF PHOTOSYSTEM I WERE INACTIVATED.

Masato Nakai,'Tetsuya Nohara, Daigo Sugita, and Toshiya Endo Department of Chemistry, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan

Hitoshi Nakamoto Dept. of Molecular U r a w a 338, J A P A N

Cyanobacteria have an internal thylakoid membrane network in addition to the cell envelopes that consist of the outer and the inner cytoplasmic membranes. Distinct protein compositions between the cytoplasmic and the thylakoid membranes suggest the presence of a mechanism of protein sorting between the two membranes in cyanobacterial cells. We have cloned secY and secA gene homologs from the genome of cyanobacterium Synechococcus PCC7942; SecA, a peripheral membrane protein, and SecY, an integral membrane protein, are known to play essential roles in the protein translocation across the cytoplasmic membrane in E. coli. The cyanobacterial sec genes are likely single-copy genes and their gene products are localized in both the cytoplasmic and thylakoid membranes, suggesting that the same Sec proteins probably mediate protein translocation across both the cytoplasmic and thylakoid membranes in the cyanobacterium. Furthermore, we have cloned a cyanobacterial ffh gene which encodes a component of putative bacterial homolog of signal recognition particle (SRP). lntracellular localization of the Ffh protein and its possible involvement in protein transport in the cyanobacterial cells are under investigation.

In o r d e r to c l a r i f y f u n c t i o n s of s u b u n i t s of p h o t o s y s t e m I c o m p l e x (PSl), we have c o n d u c t e d b i o c h e m i c a l a n a l y s i s of PsaD-, E-, F-, and I - l e s s m u t a n t s t r a i n s of S y n e c h o c ~ s t i s sp. PCC6803. We o b s e r v e d slow p h o t o a u t o t r o p h i c g r o w t h of P s a D - l e s s m u t a n t at all t e m p e r a t u r e s e x a m i n e d and of P s a E - l e s s mutant u n d e r 22°C. W e s t e r n blot analysis s h o w e d the absence PsaC in t h y l a k o i d membranes from P s a D less m u t a n t . Thus, P s a C is u n s t a b l e in PSI in the a b s e n c e of PsaD. Photoa u t o t r o p h i c g r o w t h of P s a D - and P s a E - l e s s m u t a n t s were severely inhibited at h i g h light i n t e n s i t y (1200 u E / m " / s ) . This p h o t o i n h i b i t i o n was f u r t h e r increased under low temperature. Possible f u c t i o n s of P s a D and P s a E in r e l a t i o n to p h o t o i n h i b i t i o n will be d i s c u s s e d .

P-13-023

Biology,

Saitama University,

143

Poster

P-13-027

P-13-024 The

Esterification

of

Bacteriochlorophyll

a

in

Rhoc~bacter

sphaeroides H. A. A d d l e s e e & C. N. Bunter Dept. of M o l e c u l a r B i o l o g y & Biotechnology, University Sheffield, Firth Court, W e s t e r n Bank, Sheffield Sl0 2UH, UK.

of

All of the known genes essential for, and specific to, b a c t e r i o c h l o r o p h y l l b i o s y n t h e s i s in Rhodobacter sphaeroides lie within the 45kb p h o t o s y t h e s i s gene cluster. Analysis of a p r e v i o u s l y u n s e q u e n c e d region of the cluster has confirmed the presence of genes involved in the esterification of b a c t e r i o c h l o r o p h y l l a (BChl a). Open reading frames in this region have been subjected to localized transposon Tn5 mutagenesis, and the e x t r a c t e d pigments analysed by HPLC. Mutations in a bchG h o m o l o g u e led to pale, n o n - p h o t o s y n t h e t i c mutants, lacking the ability to esterify bacteriochlorophyllide. Those in a bchP homologue produced weakly-photosynthetic mutants, which accumulated BChl a esterified with the phytol precursor, geranylgeraniol. These results support the p r o p o s e d p a t h w a y for the synthesis of BChl a in R. sphaeroides.

CHARACTERIZATION OF THE PETH GENE AND ITS PRODUCT FERREDOXIN-NADP + OXIDOREDUCTASE (FNR) IN THE CYANOBACTERIUM SYNECHOCYSTIS PCC 6803 J.J. van Thor, H.C.P.Matthijs, L.R. Mur and K.J.Hellingwerf Department of Microbiology, University of Amsterdam. Nieuwe Achtergracht 127, 1018 WS Amsterdam, the Netherlands. In addition to its role in NADP + reduction, FNR has been shown to also function as a NADPH:quinone oxidoreduetase. This dehydrogenase function is instrumental in poising of resp'watory and cyclic-PSI electron transfer. We question whether different forms of the FNR enzyme determine its functional role and in what way NADPH/NADP + reduction activities are mutually regulated. Sequence analysis revealed an enlarged NI-I2-terminus of the Synechocystis PCC 6803 FNR in comparison to the plant FNR, as has been previously found for the Synechococcus sp. 7002 (Sehluchter, W. & Bryant, D., 1992) and in the Anabaena variabilis FNR (Fillat, M. et al., 1993). We report on attempts to segregate an insertional inactivation ofpetH and on NH2-terminal truncation. This third example of petH enlargement in yet another cyanobacterium, illustrates the probable significance of this sequence information.

P-13-028

P-13-025 CYANOBACTERIAL MUTANTS IMPAIRED IN BICARBONATE UPTAKE

MOLECULAR CHARACTERIZATION OF CYANOBACTERIAL RNA-BINDING PROTEINS

M. Ronen-Tara2i, D. J. Bonfil, J. Lieman-Hurwitz & A. Kaplan Dept. of Botany, The Hebrew University, Jerusalem, Israel

M. Sugita & M. Sugiura Center for Gene Research, Nagoya University, Nagoya 464-01, Japan

Mediated bicarbonate uptake is an essential constituent of the CO2concentrating mechanism of cyanobacteria but the genes encoding the components involved were not identified. Several Synechococcus sp. PCC 7942 mutants defective in HCO3- uptake were isolated with the aid of an inactivation library composed of small genomic fragments. The mutants were obtained after single cross-over recombination. Therefore, they contain the interrupting vector which is being used to map the mutation. They require high CO2 concentration for growth since they are unable to induce bicarbonate uptake ability when exposed to low CO2. Northern analysis indicated that the genomic regions interrupted in the mutants are transcribed in the wild type but we were not able to clone the genes from the genomic EMBL3 clones, unless they were first inactivated. Sequence analysis is in progress.

We have recently described cloning and characterization of the genes encoding eukaryotic RNP-consensus sequence (RNP-CS) type RNAbinding proteins, 12RNP1 and 12RNP2, from the unicellular cyanobacterium S vnechococcus PCC 6301 (Sugita & Sugiura, 1994, Nucleic Acids Res., 22, 25-31). This is the first example of typical RNP-CS proteins in prokaryotes. Gent-disruption experiments using Synechococcus PCC 7942 indicated that deletion of 12RNP1 gent is lethal while 12RNP2 is not essential under normal growth condition. Expression of the 12RNP1 gent is regulated in a growth-dependent manner and is enhanced by coldshock. The possible role of 12RNP proteins along with their biochemical properties will be discussed.

P-13-026 Comparison of mutants of Synechocystis with psbO and psbH genes deleted Friso. G and Barber, J. Photosynthesis Research Group, Biochemistry Department, Imperial College of Science, Technology & Medicine, London SW7 2AY, UK Mutants of Synechocystis 6803 with the psbO or psbH genes deleted show higher sensitivity to photoinhibition than the wild type [1]. Despite this similarity they have different modifications in their function. ThcpsbO mutant has defects on its donor and accepter sides [2] while the deletion of the psbH gent leads to a change in the transfer of electrons between QA and QB, with the equilibrium shifted towards QA- [3]. Both impairments give rise to increased rates of photoinacdvation of PSI1 activity. In the case of the psbOless mutant the increased vulnerability to photodamagc is accompanied by an increased rate of D1 turnover. For the other mutant the repair process seems to be partially inhibited leading to a low rate of D1 synthesis. Northern blot analyses indicates that this distinct difference is reflected in the expression of the psbA genes. [1] Komenda, J. & Barber, J. (1995) Biochemistry in press [2] Vass, I., Cook, K.M., DeAk, Z., Mayes, S.R. & Barber, J. (1992) Biochim. Biophys, Acta 1102, 195-201 [3] Mayes, S.R., Dubbs, J.M., Vass, I., Hideg, E., Nagy, L. & Barber, J. (1993) Biochemistry 32, 1454-1465

144

P-13-029 Modification of the psbA gene family in Synechocysffs6803

Gaza F. Salih, Ronney Wiklund & Chdster Jansson Department of Biochemistry, The Arrhanius Laboratories, Stockholms University, S-106 91 Stockholm, Sweden

The photosystem II reaction center protein D1 is encoded by the psbA gent. In higher plants and algae ~ normally exists as a single copy in the plastid ganome. In cyanobacteda, on .the o~.erhand,.there are two or more copies of the PsbA pane. In uynecnocystisuuo3 me pst~.g.ene . family contains three copras, psbA1, psbA2 and psbA3. The p s i aria psbA3 genes are nearly identlca.I and.highly express_ed, RNA an~.yses have revealed that under normm conoitions, aoout ~/% or me tot~u pSOA transcript levels originate from p .sly_ an.d therest from psb~.. The psbA1 gene is silent and differs from pst:~z ann pso/~, DOra m cooing ano noncodin~, regions, and, were it expressed, w.o.uldencode a D1 po.l.~0eptide that differs from the normal form in several interesting aspects, we nave constructed several mutations in the promoter region of both psbA1 and psbA2 to medi.fy their expression and regulation .and hopefully an.s~v.erthe following quesbons: is psbA1 subjected to regulatory repressaon7 How would PSII activity and structure be afJected by exchanging the psbAt product for the psbA2/psbA3product?

Poster P-13-030 LIGHT REGULATED TRANSCRIPTION OF THE psbA GENE FAMILY IN THE CYANOBACTERIUM SYNECHOCYSTIS 6803.

,~[JhLQU~~

I, Jan Eriksson 1, Pirkko M~ienp~2, Taina Tyystjt~b'vi2 and Chdster Jansson1. 1Dept of Biochemistry, The Arr'nenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden. 2Dept of Biology, University of Turku, BioCity A, FIN-20500 Turku, Finland.

Light-stimulated transcription of the psbA gene-family in Synechocystis 6803 is dominated by the psbA2 gene that produces more than 90% of all psbA transcripts under laboratory conditions. Less than 10% originates from psbA3 and no transcript have been observed for psbA1. Both psbA2 and p.sbA3 encode an identical D1 protein whereas psbA1 would encode a signdiantly different form. The direct repeat TTGTTA-Ns-TTGTTA responsible for complementory chromatic adaption in Calothrix7601 is also present in the Synechocystis6803 psbA3 gene overlapping the -10 and -35 promoter elements. The sequence GG-Ns-AAC or GA-Ns-AAC, proposed to be a light-responsiv element in Synechosystis6803, is found overlapping the Shine-Dalgarno sequence and at a number of positions in the ORF of both psbA2 and psbA3. Preliminary gel shift assays indicate one or more DNA binding proteins for the pst~.gene. Computer searches for direct repeats revealed the sequence TTACAA-N4-TTACAA in close vicinity to the -10 and -35 promoter element of psbA2 and psbA3 and a CAAAAT-Ns-CAAAAT sequence in the psbA1 gene.

P-13-031 In Vlvo DEGRADATION PA'rFERN OF THE psbA TRANSCRIPT IN THE CYANOBACTERiUM SYNECHOCYSTIS 6803. Jan Eriksson. Halle Ghebremedhin and Christer Jansson. Dept of Biochemistry, The Arrhenius Laboratories, Stockholm University, S- 106 91 Stockholm,Sweden. Steady state levels of the Synechocystis6803 psbA gene family originatas to ,90% from pst~2 and to ,10% from psbA3. Transcription o f ~ e psbA1 gene has so far not been observed. Both the psbA2 and the psoP,u genes have -10 and -35 promoter elements as well as a Shine Dalgamo region that conform both m sequence and position to E. Coficonsensusmotifs. The remarkable dark stability of the psbA transcript in darkness is accompanied by the accumulation of a 0.9 kb degradation product. A similar stability and degradation pattern was observed in light provided the photosynthetic electron transport was shut down. The pnma~ cleavage site has been mapped to the 5' end of the pst~A2gene. No aegradation product has been detected for the less stable psbA3 transcript and we argue that the 5' noncoding sequence is involved in psbA transcript degradation process. The sequence divergence upstream of the Shine Dalgarno region in the otherwise well conserved psbA2 and psbA3 genes supports this hypothesis.

145

Poster

P-14-003 Developmental regulation of light-independent transcription of nuclear- and plastid-eneoded photosynthetic and chlorophyll synthesis genes in Scots pine. Poster

session

B, Karpinska, S. Katpinski, G. Wingsle and J-E. H~illgrea

14

Department of Forest Genetics and Plant Physiology. Faculty of Foreslry, Swedish University of Agricultural Sciences, S-901 83 Umeh, Sweden.

Expression and regulation of genes: eukaryotes P-14-O01

-

P-14-046

We have quantified transcription and mRNA levels for photosynthetic genes, nuclear and plastid encoded during pine plastid development in the dark. Pinus sylvestris was cultivated in the dark at different temperatures at 5°C, 7°C, t0°C, I6°C and 25°C to the similar developmental stage of seedling. Cotyledons were collected in the dark for plastid isolation and RNA isolation. The transcription of several photosynthetic genes were studied by slot-blot hybridisation and correlated with the chlorophyll content and chloroplast gene dosage. RadiolabeUed transcripts of plastid run-on transcription assays were extracted and hybridised to fragments of cp DNA containing portions of plastid genes,The results are discussed in relation to the differential expression of nuclear- and plastid-encoded genes.

P-14-001 REGULATION OF PSBD-PSBC TRANSCRIFFION BY BLUE L I G H T A N D ULTRAVIOLET-A R A D I A T I O N IN HIGHER PLANT CItLOROPLASTS D.A. C h r i s t o p h e r Dept. of Plant Molecular Physiology, University of Hawaii, St. John 503, H o n o l u l u , H I 96822, USA W e are interested in the g e n e r e g u l a t o r y m e c h a n i s m s that m a i n t a i n the r e p a i r of the p h o t o s y s t e m II r e a c t i o n center (PSII) in p l a n t s e x p o s e d to h i g h light conditions. O n e of t h e s e m e c h a n i s m s is the blue l i g h t / U V - A - r e g u l a t e d e x p r e s s i o n of t h e psbD-psbC g e n e s , w h i c h e n c o d e D2 a n d CP43, a r e a c t i o n c e n t e r p r o t e i n a n d a c h l o r o p h y l l - b i n d i n g a n t e n n a p r o t e i n of PSII, respectively. W e h a v e p r e v i o u s l y s h o w n that the differential a c c u m u l a t i o n of b l u e l i g h t / U V - A - i n d u c e d psbD-psbC m R N A s d u r i n g b a r l e y chloroplast m a t u r a t i o n is controlled at the level of transcription f r o m a phylogenetically c o n s e r v e d chloroplast p r o m o t e r , d e s i g n a t e d as blue l i g h t / U V - A responsive p r o m o t e r . In o u r c o n t i n u i n g efforts to d e f i n e t h e b l u e p h o t o s e n s o r y p a t h w a y ( s ) i n v o l v e d a n d to isolate the genetic e l e m e n t s a n d p r o t e i n s that m e d i a t e b l u e l i g h t / U V - A - a c t i v a t e d psbD-psbC t r a n s c r i p t i o n ~,e h a v e been u s i n g three approaches: (1) A n a l y z i n g psbD-psbC expression in (2) H e t e r o l o g o u s e x p r e s s i o n of h i g h e r p l a n t chloroplast p r o m o t e r s in the g e n e t i c a l l y a m e n a b l e c y a n o b a c t e r i a l s y s t e m . U s i n g this s y t e m w e h a v e identified a n e g a t i v e e l e m e n t w i t h i n the p r o m o t e r ; (3) A l t e r i n g chloroplast transcription in v i v o w i t h inhibitors of signal transduction. W e will describe o u r recent results f r o m these a p p r o a c h e s in o u r poster.

P-14-004 POSSIBLE FUNCTION OF CND41, A NOVEL CHLOROPLAST-NUCLEOID DNABINDING PROTEIN. AS A NEGATIVE REGULATOR IN CHLOROPLAST GENE EXPR ES S ION T. Nakano*, S. Murakami, T. Shoji, Y. Yamada and F. Sato Dept. of Agric. Chem.. Kyoto Univ., Kyoto, 606-01, Japan *present address: The Inst. of Phys. and Chem. Res. (RIKEN), Wako, Saitama, 351-01. Japan A 41 kD DNA-binding protein (CNI~I) detected in tobacco chloroplast-necleoids is a novel protein that contains a putative helix-ttma-belix motif which was abounded in Lys and a zinc finger motif. Heterulogoas expression of CND41 in E. coli showed that the Lys-rich region was essential for the DNA-bieding activity and CND4I bound to chloroplast DNA nonspecifieally. To examine the function of CND41, we analyzed the accumulation of CND41 in plant tissues. Western analysis showed that CND41 accumulated in stem more abundantly than in leaf and root. Furtherroore, C N I ~ I accumulation in photomixotrophie and heterotruphic cells eultered on the medium containing sucrose was much higher than photoautotrephicculturedenU andplant tissues. Furtheranalyses of psbA and focL transcripts indicated the negative correlation of psbA and rbcL mRNA and CND41 protein. The results suggest the possibility of CNIMI function as a negative regulator in chloroplast gean expression. The induction of CND41 accumulation by sucrose and glucose was also observed in excised leaves. On the other hand, the accumulation of CND41 in salt-adapted green cells was lower than unadapted green ceils. These results suggested that CND41 expression was regulated by sugar and salt. The more detailed analysis of CND41 accumulation and photosynthetic gene transcription a e going.

P-14-005

P-14-002 Differential redox regulation by glutathione of glutathione reductase and CnZn-superoxide dismutase gene expression in Pinus sylvestrls L. needles

CHARACTERIZATION O F GENES I N D U C E D CHLAMYDOMONAS REINHARDTII

Stanislaw Karpiaski and Guanar Wingsle

~L,y. Chen, M. Burow and J.V. Moroney Department of Plant Biology, Louisiana State University, Baton Rouge, LA 70803, USA

Dept of Forest Genetics and Plant Physiology, Faculty of Forestry, Swedish University of Agricultural Sciences, S-901 83 Ume~, Sweden. To get information about hypothetical redox regulatory mechanisms controlling Gor and Sod gene expression we artificially enhanced the levels of GSH and GSSG in Pious sylvestris L. needles. Scots pine shoots were placed for 12 hours in beakers containing 5 mM GSH, 5 mM GSSG or water. Increased levels of both GSSG and GSH were observed in the GSSG-treated needles after 3 h. In contrast, only the GSH level was increased by the GSH treatment. Thus, the GSH/GSSG ratio i n e r ~ up to 15-fold during the GSH treatment and decreased approximately 2-fold during the GSSG treatment. The GR activity was significantly higher (60%) when GSSG was applied, without any apparent change in the amount and isoform population of GR or transcript accumulation of Gor gene. This indicates that the GR activity increased per se in the GSSG treatment. The cyt CuZn-Sod transcript level was decreased significantly by the GSH treamtent without any change in enzyme activity. The cp CuZn-Sod gcoe generally showed a more stable transcript level in the different treatments. However, a similarity between the eyt and cp CuZn-Sod transcripts levels could be observed in different treatments. This suggest, that the redox state of glutathione plays a significant role in the in vivo regulation of CuZn-Sod gene expression in plants.

LOW

CO 2 IN

Unicellular algae grow well under limiting CO 2 conditions due to the carbon concentrating mechanism (CCM) which allows algae to efficiently deliver inorganic carbon to Rubisco. In C. reinhardtii this mechanism is inducible and is only present in cells grown under low CO 2 conditions. To identify genes that code for components of the CCM we have constructed a cDNA library from cells that were adapted to low CO 2 and then screened for cDNA's present in low CO2-adapted cells but not present in ceils left on elevated CO 2. We have isolated six classes of low CO2-inducible clones, three of which have been sequenced; a periplasmic carbonic anhydrase, a membrane spanning protein which has no homology to any previously identified gene, and an alanine aminotransferase (AAT). We have confirmed the inducible nature of A A T through immunoblots and enzyme activity assays. We will present the results of our experiments concerning the physiological role of A A T and its relationship with the CCM. We will also present our sequencing data as well as our latest results concerning the identities of the other genes induced by low environmental CO 2 levels. Supported by the National Science Foundation

146

BY

Poster

P-14-006 INVESTIGATION INTO THE ROLEOF THE PSBHPOLYPEPTIDEIN PSII

Amanda Cheater, Helen O'Connor, Stuart Ruffle, Saul Purton & Jonathan Nugent. Dept. of Biology, University College London, Gower St. London WCIE 6BT, U.K. The psbH gene product is an important polypeptide in the photosystem II complex. The gene is located in the chloroplast genome of algae and plants. The gene product is one of the major phosphorylated polypeptides (the 9 or lOkDa phosphoprotein) suggesting a regulatory role for this protein. We are studying psbH in the alga. Chlamydomonas reinhardtii and have characterised and sequenced the gene. A deletion mutant has been constructed and this results in a greatly reduced PS2 content. We have also constructed mutants with s i t e directed changes at the phosphorylation site, which in C. reinhardtii is threonine 2. We w i l l describe the properties of PS2 in the mutants and discuss the role of the psbH gene product.

P-14-007 REDOX REGULATION SYNTHESIS

OF C H L O R O P L A S T

PROTEIN

Lilting Chew, and John F. Allen Plant Cell 13iology, Lund University, Box 7007, S-220 07 Lurid, Sweden Different subsets of chloroplast proteins are synthesized under different redox conditions [I]. Our recent results are consistent with this report. The effect of DCMU and redox reagents on chloroplast protein synthesis also depended strongly on the ATP concentration. The possibility that the ATP plays a role as a redox buffer is discussed. The protein synthesis patterns of psbA and petA obtained by immunoprecipitation under changing redox conditions showed that the DCMU significantly blocked the synthesis of these two proteins at 1 mM ATP concentration. Protein synthesis in the dark is less than in the light, even in the presence of reducing agents. [1] Carol A. Allen, Gunilla HAkansson and John F. Allen (1995) Redox conditions specify the proteins synthesised by isolated chloroplasts and mitochondria. Redox report 1, 119-123.

P-14-008 RYE CHLOROPLAST GENES, ENCODING POLYPEPTIDES OF PSI AND PSI!

V.L Kolosov& M.T. Clmika Institute of Photobiology, Belarussian Academy of Sciences, 220072 Mmsk, Skorma str. 27, Belams To date as many as five genes (psaA, psaB, psaC, psal, psaJ) of higher plant chloroplast DNA, encodingpolypeptides of photosystemI, and not fewer than 12 genes - polypeptidesof photosy~m II are known. We located most of them on rye chloroplast DNA by blot hybridization and Bam HI and Pst I fi'agments comprisingthese genes were clenod directly into plasnuds. Oligonucleotides used as the hybridizatien probes were synthesized in accordance with the known gructares of the analogousgenes of other plants. The total length of the sequenced fragmentsof DNA comprises about 12000 bp. All the clened genes are in the same Bam HI and PstI fragments as those from wheat. The identity of the pictures of agarose gel electropheresis of rye and wheat chloroplast DNA digested with various restriction e~donucleases enabled a suggestion of their similarity. ~he structures of psbE, psbF, psbl genes of these cereals are entirely identical. The nontranslated regien longerthan 1200 bp betweenthe psbI and psbD genes differs by only severaldeletims ofmaxanum 16 bp (homology95%).

P-14-009 THEpsbE ANDpsbF GENES ARE S E P A R A T E L Y T R A N S C R I B E D IN CHLAMYDOMONAS REINHARDTII T.S. Mor 1, 2, O. Erez I, J. Hirschberg 3, R.T. Sayre4, I. Ohad I and H.B. Pakrasi 2 I Dept. of Biological Chemistry and 3Genetics, The Hebrew University, Jerusalem 91904, Israel; 2Biology Dept., Washington University, St. Louis MO, USA; 4Dept. of Plant Biology, Ohio State University, Columbus OH, USA The psbE and psbF genes encode the apoproteins of cytochrome b559, an essential component of photosystem II. Together with psbL and psbJ, these genes constitute a single operon in all photosynthetic organisms examined thus far. We have cloned and sequenced the psbE and psbF genes of C. reinhardtii. The predicted N-terminal domains of both polypeptides are more positive as compared to those of other organisms. Northern blot analysis demonstrated that psbE is transcribed into a 0.3 kb mRNA while transcription of psbF and psbL result in a 0.9 kb transcript. The splitting of the psbEFLJ operon into separate transcription units suggests a unique regulation mechanism for the expression of these genes in C. reinhardtii. As a first step towards site directed mutagenesis of psbF we have constructed a plasmid that contains a 2.6 kb fragment of the C. reinhardtii plastome including psbF and psbL with the aadA cassette inserted at a Bglll site upstream ofpsbF. Stable transformation of the C. reinhardtii plastome with this construct rendered the cells resistant to spectinomycin and streptomycin without altering their photosynthetic phenotype. (Supported by a BARD grant awarded to I.O. & H.P.)

P-14-010 M/NIMAL PROMOTER REGIONS SUFFICIENT TO MEDIATE C I R C A D I A N E X P R E S S I O N O F T O M A T O L H C G E N E S IN TRANSGENIC TOBACCO

Bir~it Piechulla, Nicole Merforth, Daniel Dreesmann*, Institut Fir Biochemie der Pflanze, Untere Karspiile 2, 37073 GSttingen, Germany; *present address: ETH Zilrich, Universit/itsstr. 2, 8092 Ziirich, Switzerland To dissect the molecular regulatory components of the 'circadian clock' machinery, the minimal promoter region necessary for conferring rhythmic mRNA accumulation was determined for four tomato Lhc genes. 5"deletion constructs were analysed in transgenic tobacco plants under light/dark and continuous dark conditions. The parallel study revealed regions between -150 and -300 upstream of the transcription start point that are sufficient to mediate circadian expression pattern. Comparison of the sequences so far does not exhibit a consensus 'clock mediating' sequence. In a second approach we try to isolate proteins, which bind to these particular 5'-upstream regions, e.g., by gel retardation experiments.

P-14-Oll THE PLASTOCYANIN GENE FROM SCENKDENMUSOBLIQUUS

Jeanette M. Quinn Dept. Chenx & Biochem, UCLA, 405 Hilgard Ave., Los Angeles, CA 90095, USA. The mechanism of copper-dependent regulation of plastocyanin mRNA accumulation in the eukaryotic ~ceen alga Scenedesmus obliquus is unknown. As a first step towards elucidating this mechanigm~ I am cloning the plastocyanin-encoding gene from Scenedesmus, with the ultimate goal of expressing this gene and studying its expression in the related and experimentally well characterized alga Chlamydomonas. Using the known protein sequence ofScenedesmus plastocyanin, I designed degenerate primers and used these primers to amplify Scenedesmgs genomic DNA usmg the polymerase chain reaction. A single PCR product was obtained, and sequencing of this product verified it was an amplification product of the plastocyanin gene. A sabgenomic library ~om Scenedesmus genomic DNA was constructed and screened using the PCR product. A single partial clone was isolated, lacking the 5' upstream and coding sequences. Sequence analysis revealed that the Scenedesmus plastocyanin clone contains a single intron, and that this intron is located in the same position relative to the protein coding sequence and the single intron in the Chlamydomonas plastocyenin ~ene, suggesting these genes are closely related, and that Chlamydomonas will be capable of expressing the Scenedesmus gene. The partial Scenedesmus clone is being used to screen a total library for a fuU-length clone that can be used in expression studies.

147

Poster

P-14-012 CIS AND TRANS FACTOR(S) OF A LIGHT-RESPONSIVE P R O M O T E R O F p s b D / C G E N E C L U S T E R IN W H E A T P L A S T I D S Z~ Tsunoyama, Y. Nakahira, T. Shiina and Y. Toyoshima Graduate School of Human and Environmental Studies, Kyoto Univ., Yoshida-Nihonmatsu-cho, Sakyo-ku, Kyoto 606-01, Japan The wheat (Triticum aestivum) psbD/C gene cluster has a conspicuous lightresponsive promoter (D/C-3 promoter). In this study, ~ve analyzed cis and trans factors involved in the activation of this promoter. Deletion of sequences upstream from -78 with respect to the transcription start site eliminated the transcription from the D/C-3 promoter in vitro. Gel shift assay and DNase I l~)t printing experiments using chloroplast extract demonstrated the presence of trans-acting factor(s) to a specific cis element bet~vcen -85 and -60. The sequences of this region were highly conserved in several higher plants. We also identified putative transcription factor(s) invoh'ed in light-induced activation of the D/C-3 promoter. The extract from chloroplasts was subjected to a hydrophobic column and a fraction eluted by a low concentration of ( N H a h S O 4 found to activate the transcription from the D/C-3 specifically. This transcription factor(s) may be related to the trans-acting factor(s) which binds to the specific cis element of the DfC-3 light-responsive promoter.

P-14-015 R~h~LYSIS OF THE 5'-UPS~RE~4 RNA POLEMEP~SE GENE (r~B )

~GION

OF

~Y~IE C H I ~ ) R O P I ~ T

H. Inada I, M. Seki 2, H. Morikawa 2, M. Nishimura I & K. Iba 1 iDept of Biol,

Fac Of Sci, Kyushu University

, Fukuoka 812-81,

Japan, 2Grad Dept of Gene Sci, Fac of Sci, Hiroshima University, Higashi-Hiroshima 724, Japan. We analyzed the 5 ' - u p s t r e ~ region of the chloroplast-encoded RNA polymerase gene (rpoB) from Arabidopsis thaliana by a transieat expression system using pneumatic particle gun. A chloroplast expression vector containing the B-glucuronidase (GUS) gene, placed under the control of the 5'-upstream region of rpoB (pSIlll), was introduced into cultured tobacco cells (BY-2). The plastid-specific expression of phil11 in BY-2 cells was confirmed by the experiment using the specific inhibitors for nuclear or plastid gene expression. The chloroplast expression vectors containing a series of 5'-deletions of the rpoB 5'upstream region were constructed, and were introduced into BY-2 cells. Comparison of the 5'-upstream regions of rpoB in several plant species, together with the deletion analysis, revealed several regulatory regions for the rpoB expression containing consensus sequences.

P-14-013 A SYSTEM FOR RANDOM MUTAGENESIS OF THE PSAA AND PSAB GENES ENCODING THE MAJOR PHOTOSYSTEM I SUBUNITS IN CHLAMYDOMONAS REINHARDTIL

P-14-016 EXPIV~SSION

OF

THE

~

DEVELOPMENT I S D ~ I ~ E R T

IN

GF~E ~ ESSENTIAL

AN FOR

IF~M~L¥

STAGE

RICE

K. Kusumi, Akiko Mizutam, Mitsuo Nishimura and Koh Iba Dept of Biology, Faculty of Science,Kyushu University,Fuku0ka812-81, Japan.

Kevin Redding and Jean-David Rochaix, Department of Molecular Biology, University of Geneva. In an effort to understand better the contribution of the PsaA and PsaB proteins to the various energy transfer and electron transport processes occurring within Photosystem I (PSI), we have created a system to randomly mutate large segments of the psaA and psaB open reading frames (ORFs) by polymerase chain reaction (PCR). We have constructed libraries of mutations in various regions of the genes and used them to bioballistically transform Chlamydomonas strains lacking almost the entire OFF of either gene. Transformants expressing mutant PSIs are detected by altered fluorescent induction transients. Those strains that assemble PSI yet have a PSIphenotype are detected by immunoblot screening with an antibody we have raised against PsaA. Using this method, we hope to create a large collection of mutant PSIs that are capable of assembling, yet are inefficient in one or more steps of electron transport.

The rpoB gene encodes the 13-subunit of plastid RNA polymerase, which is thought to be the catalytic center of the core enzyme. Northern and in situ hybridization analyses revealed that in rice the rpoB transcript was highly accumulated at a strictly limited stage of early leaf development (between stages })4 and PS) prior to the expressions of other chloroplast-encoded genes (psbA, rbcL, 16S rRNA). Northern analysis on a temperature-sensitive chlorophylldeficient mutant, virescent (Vl), indicated that such stage-specific accumulation of rpoB transcript was missing and the accumulation occurred at later developmental stages (P5 to P6) in the mutant seedlings when grown at restrictive temperature. In such mutant seedlings, normal chloroplast development was disturbed and the expressions of the other chloroplast-encoded genes were blocked. These results suggest that the expression of rpoB is one of the primary events of chloroplast formation and that the regulation of rpoB expression plays an important role in the initiation process of chloroplast differentiation associated with leaf development.

P-14-017

P-14-014 CHARACTERIZATION OF PARAQUAT-RESISTANT MUTANTS OF CHLAMYDOMONAS REINHARDTII

RE,GULATION OF THE ARABIDOPSIS

~ , Masahiko Kitayama and Robert K. Togasaki Dept. Biology, Indiana University, Bloomington, IN 47405, USA

T. Nishiuchi, H. Kodama, M. Nishimusa & K. Iba

The bipyridinium herbicide pamquat can mediate the transfer of electrons from reducing sites of the electron tmnspo~ chain to molecular oxygen, causing the production of toxic superoxide anions. To investigate the mechanisms by which plants respond to the photooxidative stress produced by paraquat, paraquatresistant mutants of the green alga Chlamydomonas reinhardtii were isolated and characterized. One highly resistant mutant (PAR19) was found to have elevated Fe superoxide dismutase (Fe SOD) enzyme activity and higher levels of Fe SOD mRNA. A second mutant (BI-1) exhibited higher Mn SOD enzyme activity and mRNA accumulation. The behavior of these mutants towards a variety of oxidants was exarmned and found to be consistent with previous data indicating that Fe SOD localizes to the chloroplast and Mn SOD localizes to the mitochondria. The level of ferredoxin-NADP+ reductase (FNR) mRNA accumulation and Fe SOD mRNA accumulation correlated in all mutants examined, suggesting that FNR and Fe SOD gene expression is co-regulated.

148

OF

CHLOROPlJSST

TRANSGENIC

FAD7

GENE PROMOTER IN

TOBACCO PLANTS

Dept of Biol, Fact of Sci, Kyushu University, Fukuoka 812-81, Japan The Arabidopsis FAD7 geue encodes a chloroplast ¢o-3 fatty acid desatmase that catalyzes the desaturation of lipid-linked dienoic fatty acids (18:2 aud 16:2). An 825 bp FAD7 promoter fragment upstream from the transcriptional start point contained several short sequences which were homologous to the cis-eleraents (box II and G-box etc.) conserved in many light-responsive genes. We introduced the FAD7 promoter fused to the gluenronidase (GUS) or the Iueiferase (LUC) reporter gene into tobacco plants. The -825 pronaotarsequenceeonfen'ed a tissue-specific and light-resIxmsive expression to both these reporter genes in transgenic tobaeen, indicating that these regulation chracteristies of the FAD7 gent occur mainly at the transcriptional levd. Hislx~chcmiealGUS staining showed that the activity of the FAD7 promoter is restricted to the cells of chloroplast-oonmining tissues although the staining was exceptionally absent in the chlot'oplast-enntaining cells associated with vascular systems. The 5' deletion experiments of the promoter revealed that the -362/-167 region, containing two putative box II sequences, was responsible for the tissue-specific and light-responsive expression of the FAD7 gcus.

Poster P-14-018 The chloroplastie isozyme of CuZn-superoxide dismutase in Scots pine is encoded by at least four different genes which are differentially expressed.

P-14-021 TISSUE-SPECIFIC REGULATION OF O-ACETYLSERINE ( T H I O L ) L Y A S E G E N E S IN Arabidopsis thaliana.

S. Karpinski, B. Karpinska, G. Wingsle and J-E. Hltllgmn Department of Forest Genetics and Plant Physiology. Faculty of Forestry, Swedish University of Agricultural Sciences, S-901 83 UmeS, Sweden. A Scots pine (Pinussylvestris L.) eDNA library was screened with homologous eDNA probe (PST13) encoding chloroplastic copper zinc superoxide dismutase (CuZn-Sod). Several positive full length clones for chloroplastic CuZn-Sod were isolated, subcloned, mapped and sequenced. Among these eDNA clones four different sequences have been found. N ~ blot hybridisation and quantitative RT-PCR analyses showed that, chloroplastic CuZn-Sod genes are expressed differentially in Scots pine needles during photooxidative anti chilling stresses. Additionally our results indicate that cp CuZn-Sod gone induction depend on developmental stage of chloroplast and is not light dependent. The results are discussed in relation to earlier observations of CuZn-Sod in plants and in to the putative signal transduction pathway regulating Sod gone expression.

C. Barroso, F. J. Cejudo, J. M. Vega, C. Gotor Instituto de Bioqufmica Vegetal y Fotosfntesis, Universidad de Sevilla y CSIC. Sevilla, Spain. The last step for L-cysteine biosynthesis is catalyzed by Oacetylserine (thiol)lyase (EC 4.2.99.8). Very recently, we have isolated a e D N A , Atcys-3A, encoding cytosolic O-acetylsefine(thiol)lyase from A. thaliana (FEBS Lett., in press). Atcys-3A gone expression and activity level are activated by sulfur limitation, requiring a carbon and nitrogen source for maximal activation. To get information on the tissue-specific expression pattern o f this gone, we have performed Northern blot analysis. The transcript was present in different organs o f mature Arabidopsis (e.g. roots, stems, young leaves, mature leaves, siliques and flowers), being substantially most abundant in roots. In situ hybridization analysis are currently in progress. W e have also analyzed the O-acetylserine(thiol)lyase activity level in the various organs. Activity was detected in all o f them but the maximun level was observed in stems and roots.

Supported by DGICYT Grant PB93-0735.

P-14-019 CIILOROPIIYLL

FLUORESCENCE

ASSAY

QUANTITATIVE METHOD FOR R E S I S T A N T TOBACCO PLANTS.

AS

A

SENSITIVE

SCREENING

AND

KANAMYCIN

P-14-022 POSSIBLE ROLES OFLIGHT-RESPONSIVEpsbPROMOTERS IN REGULATION OF TURNOVER OF PSII PROTEINS

Y . i - E u I, M. H. Lee 1, H. S. Changi,T~.H.. Rhew" & C.-H. Lee t rDept of Molecular Biology, :~Dept of Biology, Pusan National University, Pusan, 609-735, Korea

K. Baba, J. Sato, Y. Nakahira, T. Shiina & Y. Toyoshima Graduate School of H u m a n and Environmental Studies, Kyoto University, Yoshida-nihonmatu-cho, Sakyo-ku, Kyoto, 606-01, Japan

Efficiency of chlorophyll (Chl) fluorescence assay was tested for screening kanamycin resistant tobacco (Nicotiana tabacum cv. Xanthi) plants. By the treatment of 200 mg/L kanamycin (Km) to leaf discs from wild-type plants, (Fv)m (Fro minus Fo) was decreased, and Fo was increased significantly. Variations among samples could be reduced by using (Fv)m/Fo ratio as a screening parameter. By using this parameter, Km-treated samples could be distinguished from the control within 4 days after the treatment. Tobacco plants were transfurmed by using Agm)bacterium containing nptll gene. Seventy-five shoots were selected in 200 mg/L Km containing media and leaf discs were taken from them for Chl fluorescence assay. Among them, 40 plants were Km sensitive ('escaped'l. All the sensitive plants did not contain nptll gone in their chromosome as shown by polymerase chain reaction amplification of this gene and did not show any neomycin phosphotransferase activities also. All the resistant plants were proved as positive by these two tests. These results prove the efficiency of this non-destructive Chl fluorescence assay in screening Km resistant plants.

DI, D2 and CP43 in photosystem II (PSII) core complex undergo lightdependent rapid turnover. We compared the light response of chloroplast promoters encoding these proteins in mature chloroplasts in order to examine the involvement of transcriptional regulation in the light-dependent turnover. Dark treatment of light-grown wheat (Triticum aestivum) seedlings severely reduced the transcription activity of D/C-3 promoter which produces mRNAs encoding D2 and CP43, and quickly reactivated (within 1 hour) by light illumination. While, accumulation of the transcripts from the D/C-4 site (CP43-specific) was reversibly reduced in response to the dark -* light shift depending on the light intensity. The psbA promoter encoding D1 protein scarcely responded to the dark -* light shift in mature chloroplasts. Quick light-dependent change in the accumulalaon of the transcripts from the D/C-3 and D/C-4 sites may suggest the involvement of transcripUonal regulation in the turnover of PSII core proteins responding to the light condition.

P-14-020 DEVELOPMENT OF A NOVEL SELECTION METHOD FOR PHOSPH1NOTHRICIN-RESISTANT PLANTS ACCORDING T O ITS EFFECTS ON PHOTOSYNTIIESIS AND PHOTORESPIRATION B.._C. (;hung ~, J.-D. Song 1, M. H. Lee l, It. Y. l.ee :~ & C~-tL Loci_ _ IDept of Molecular Biology, Pusan National University, Pusan 609-7,35, Korea; 2Depl of Resource Plant, Suncheon National University, 540-742, Korea The effects of a herbicide, phosphinothricin (PPT), on photosynthetic apparatus of tobacco leaves were investigated by using chlorophyll (Chl) fluorescence assay. Fm/Fo and qQ, parameters related to photosynthetic efficiency, were decreased and other parameter, Fv and qNP, were increased by the treatment of PPT. Under this light condition (30 IJmol • m ~ "s t), Fm/Fo value from wild type tobacco leaf discs were decreased in half by the treatment of 100 mg/L PPT for 12 hrs. However, similar effects could be observed only 1-3 hrs after the treatment under high light (60 tirnol "m :~ "s ~). No similar changes in these fluorescence induction parameters was observed from tobacco plants transformed with the bar gene coding for the PPT-acetyl transferase (PAT). All the plants transformed and screened by Chl fluorescence assay showed PAT activities. Within 48 hrs nf the tmalment, sympturns of PPT toxicity in the leaf discs were hardly detectable by naked eyes. These results suggest that bar gene containing plants could be screened very quickly by employing Chl fluorescence assay under high light condition.

P-14-023 E F F E C T O F A G E I N G O N E X P R E S S I O N OF PHOTOSYNTHESIS-RELATED G E N E S IN P I N E N E E D L E S .

Kenji Shinohara, Forestry and Forest Products Research Institute (FFPRI), P.O. Box 16, Tsukuba Norin Kenkyu Danchi-Nai, Ibaraki 305, Japan. Effect of ageing on expression of photosynthesis-related genes was studied in current, 1-year-old and 2-year-old needles of pine (Pinus thunbergii). Total amount of RNA prepared from 1-year-old and 2-year-old needles were about one-fourth of that from current needles, on a fresh weight basis. Composition of rRNA was not drastically changed in three preparations. When an equal amount of RNA was used for northern blot, levels of cab and rbcS m R N A s did not change significantly with progress of needle-age. In contrast, psbA and rbcL transcript levels increased andpsaA-psaB transcript levels decreased gradually. Judging from the recovery of total RNA, psbA transcript levels remained high in 2-year-old needles. Continued synthesis ofpsbA gene product (D 1 protein) correlates with maintenance of transcript levels. The decline in psaA-psaB transcripts may parallel the decline in synthesis of PS1Chl apoproteins with increased needle-age. These results suggest that the expression of photosynthesis-related genes on nuclear and chloroplast genomes is differently regulated with progress of needle-age, and that the regulation may be associated with the developmental stage of plastids in the needles.

149

Poster II

P-14-024 DIFFERENTIAL EXPRESSION OF GENES FOR THE NAD(P)HPLASTOQUINONE-OXIDOREDUCTASE IN MESOPHYLL AND BUNDLE SHEATH C H L O R O P L A S T S OF THE C4-PLANT SORGHUM BICOLOR SUGGESTS A FUNCTIONAL ROLE OF THE ENZYME IN CYCLIC ELECTRON TRANSPORT A. Kubicki, E. Funk, P. Westhoff & K. Steinmiiller Institut ftir Entwicklungs- und Molekularbiologie der Pflanzen, HeinrichHeine-Universittit, Universitiitsstra~ 1, 40225 DUsseldorf, FRG The plastid genomes of angiosperms contain eleven genes (ndhA-K) that encode subunits of a putative NAD(P)H-plastoquinone-oxidoreductase, however the function of the enzyme in plastid metabolism is unknown. One possibility is, that the enzyme operates in a cyclic electron Iransport pathway around photosystem I, because it has been shown that two subunits (NDH-H and -K) are located on the stroma thylakoid membranes in close neighborhood :o photosystem I. To test this hypothesis, we compared the expression of ndhgenes in mesophyll and bundle sheath chloroplasts of the C4-plant Sorghum ~i¢olor. because bundle sheath plastids establish a high rate of cyclic electron xansport to generate ATP for CO2 fixation. We found that the levels of :ranscripts for all ndh-genes are increased 2-4fold in bundle sheath plastids :ompared to mesophyll plastids. Western blot analysis of protein levels showed :hat the differences in the concentrations of three subunits (NDH-H, -J and -K) ire even higher (5-10fold).

P-14-025 Chlamydomonas reinlmrdtii lhc-LiS18 mRNA accumulation is promoted by light and the circadian clock and peaks several hours before that of the lhea and lhcb mRNAs. Savard, F. and Guertin. M. D6partement de Biochimie, Facult~ des Sciences et de G6nie, Universit6 Laval, Qu6bec, Canada Expression of the nuclear genes encoding the light harvesting complexes (lhc) in plants and unicellular algae like Chlamydomonas reinhardtii is under the circadian clock control with a peak in mRNA levels by the middle of the day. Up to day, exceptions to this timing of expression have been restricted to the ELIP genes which encode distant related Ihc proteins, the early light inducible proteins (ELIP). ELIP genes are turned on very early during the greening of etiolated plants well before the lhca and Ihcb genes. In young green plantiets, ELIP genes are governed by the circadian clock and expressed coordinately with Ihca and Ihcb genes. We report here the cloning and the characterization of the C. reinhardtii Li818 gene encoding a new member of the Ihc gene family. Under light/dark regimes, Li818 mRNA starts to accumulate several hours before the light period concomitantly with Ihca and Ihcb mRNAs hut peaks 5-6 hours before Ihca and lhcb mRNAs. Upon illumination, Lig18 mRNA increase rapidly by 10-20 fold through activation of nonphotosynthetis photoreceptors while the kinetics of lhca and lhcb mRNAs are unaffected.

P-14-027 EXCITATION PRESSURE, NOT IRRADIANCE, REGULATES LHCH APOPROTEIN AND CAB mRNA ABUNDANCE IN GREEN ALGAE D.P. Maxwell, D.E. Laudenbach, and N.P.A. Huner Dept.Plant Sciences, Univ.Western Ontario, London, Ont., Canada, N6A 5B7 Excitation pressure, which reflects the redox poise of intersystem electron transport and carbon metabolism was estimated in vivo by the fluorescence parameter 1-qP. To equalize the chlorophyll (Chl) cell-1 and Chl a/b ratio of cells grown at 13°C and 20 PPFD, Dunaliella salina was grown at 30°C but at 150 PPFD. These two growth conditions resulted in cells which were exposed to equally low excitation pressures. Similarly, to match the Chl cell-1 and Chl a/b ratio of cells grown at 13°C and 150 PPFD, D. salina was grown at 30°C but at 2500 PPFD. Theses cells were exposed to high excitation pressure. Both growth regimes at high excitation pressure resulted in cells with a lower abundance of both LHCII apoprotein and cab mRNA relative to cultures grown at low excitation pressure. Thermodynamic relaxation of high excitation pressure, at constant irradiance, resulted in a 6 and 8-fold increase over a 12 hour period in LHCII apoprotein and cab mRNA abundance, respectively. Our results do not support the current view that irradiance regulates cab gene expression, but rather that expression is under redox control

P-14-028 CHLOROPLAST GENOME STRUCTURE OF GREEN ALGA CHLORELLA VULGARIS C-27.

UNICELLULAR

,1. Tsudzukil, K. Nakashima 1, T. Tsudzuki2, M. Horihata3, K. Satoh3, K. Yoshinaga4, T. Wakasugi5, T. Nagai5, M. Kapoor5 and M. SugiuraS. 1Sugiyama Jogakuen Univ., Nagoya, 464 JAPAN; 2Aichi-Gakuin Univ., Nisshin, Aichi, 470-01 JAPAN; 3Okayama Univ., Okayama, 700 JAPAN; 4Shizuoka Univ., Shizuoka, 422 JAPAN; 5Center for Gene Research, Nagoya Univ., Nagoya, 464-01 JAPAN. Eighty percent of the chloroplast genome (154 kbp) of Chlorella vulgaris C-27 (formerly C. eUipsoidea C-27) have been sequenced. This genome contains one copy of rDNA and no large invereted repeat. Over 80 genes/ORFs have been found, many of which are shared with land plants or some (chll, rpsg, rpl5, rp119, tufA) with algae, but for a mind gene homologue which is unique in ChloreUa and is possibly involved in the chloroplast division. No ndh genes has been found similarly in other algae and conifers. Several genes (atpF,, clpP, petB, D, rps12, rpl2, rp116, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnV-UAC) in this genome lack introns which are common in land plants, whereas 23SrDNA, trnL-UAA and chlL contain group-I introns. The intron found in chlL is the first ease for all the chloroplast genomes examined.

P-14-029

P-14-026 DNA-BINDING P R O T E I N S MEDIATE I N T E R A C T I O N O F N U C L E O I D S WITH E N V E L O P E M E M B R A N E IN D E V E L O P I N G P L A S T I D S N. Sato ~, O. Misumi~, J. Joyard =& R. Douce 2 'Lab. of Life Science, Tokyo Gakugel University, Koganei, Tokyo 184, Japan; =CENG and University Joseph Fourier, Grenoble, 38041 France

In developing plaatids, most of the nucleoids are found in close contact with the envelope membrane, whereas the nucleoids in mature chloroplasts are dispersed in the interior of plastid. We previously discovered a 130 kDa DNA-binding protein (PEND protein) in the envelope membrane of developing pea plastids. PEND protein was shown to bind selectively to several AT.rich regions of plastid DNA This protein was purified partially by column chromatography on heparin-Sepharose, Superdex 200 HR and Resource Q as well as affinity purification with DNA- magnetic particle conjugate, and was found to be oligomer of ca. 600 kDa in native state. We also obtained a cDNA encoding another DNA-binding protein of 87 kDa (PD1). Isolated envelope membrane and isolated nucleoids were both found to contain PEND protein and PDI. Since isolation of envelope is accompanied by dissociation of nucleoids, and isolation of nucleoids needs selubilization of envelope, this result suggests that the two DNA-hinding proteins mediate binding of nucleoids to envelope membrane. It seems likely that the plastid envelope provides a solid support for the functioning of plastid genome.

150

Pdk GENE EXPRESSION IN TRANSGENIC C4 PLANTS E. Rosehe, J.A. Chitty and W.C. Taylor CRC for Plant Science, CSIRO Plant Industry, Canberra 2601, Australia The enzymes of C4 photosynthesis accumulate in a cell-specific pattern in two specialised cell types. Pyruvate, orthophosphate dikinase, present in mesophyll cells, is encoded by a single gene, Pdk, in the C4 dicot Flaveria trinervia. This gene encodes two transcripts, a prevalent 3.4 kb mRNA coding for the chloroplastic form in mesophyll cells and a shorter mRNA coding for the mature form of the protein which is detected at low levels in stems and roots. Transcription of the shorter Pdk mRNA is controlled by a promoter located in the long first intron of the gene and is increased in the dark. Accumulation of the longer transcript is induced by light. To determine the mechanisms regulating the expression ot the Pdk gene, we are using our recently developed stable transformation system for the related C4 plant F. bidentis to study the expression of promoter regions in transgenic plants. A 1.5 kb region from the 5' end of the Pdk gene has been fused to the gusA reporter gene and shown to confer high level expression in leaves. Progress in defining sequences responsible for light regulation and mesophyll expression will be reported.

Poster

P-14-030 MOLECULAR STUDY OF A LIGHT-HARVESTING APOPROTEIN OF A CHRYSOPHYCEA: GIRAUDYOPSIS STELLIFER C. Passaquet & C. Lichtld Lab. Photordgulation et D y n a m i q u e des Membranes vdgdtales, Ecole Normale Supdrieure, 46 rue d'Ulm 75005 Paris, France. A gene for a chlorophyll a / c binding protein (named cac for Chl a./c_.by homology with cab for C_.hl a/b._) of a Chromophyte alga has been isolated from a library of nuclear DNA and sequenced. The cac genes are nuclear encoded and part of a multigenic family. The comparison of the deduced amino-acid sequence with other Chl a / c and Chl a / b binding protein sequences s h o w s that structural and functional features, i.e. the arrangement "en X" of the two A and B t r a n s m e m b r a n e helices and the Chl a binding sites are shared by both Chromophyta and Chlorophyta. In contrast to Chlorophyta, a very strong identity is found among Chromophyta in the C helix suggesting a main function associated to this specific region. Nevertheless, the primary structure of the apoprotein does not seem affected by the pigment composition in Chromophyta. The cac genes are light-induced at a transcriptional level.

P-14-033 GENERIC ~ I Q N C@"~ (VIGNA I.IBGUI(XR~T&L.N~M,lP) BY /K]RQB~CI"~II.]MT ~ A C I ~ I S USINGGOIlfLI~CtlS AS KIUR./tI~. A. Gnanam, B. Muthukumar, Marimmm t4~xanen & K. Veluthambi Department of Plant Sciences, School of Biological Sciences, Madarai Kamaraj University, Madurai 625 021, India Cotyledons were excised from 2-3 day old cowpea seedlings and cultured on B5 basal medium containing varying concentrations of BAP. Maxirftnnntmi3er of shoot formation (365) occurred in the meditma with 8 x 10 "6 kt BAP. Shoots emerged from the callus at the proximal end of the cotyledon within 15 days. Histological analysis revealed the de novo foz~ation of meristematic centres at the proximal end of cotyledons. Cotyledons with intact proximal end were preincubated for 24 hr, inoculated with A. tumefaciens ffJCD2614 carrying 1N.JCD2340, co-culticated for 48 hr and transferred to hygromycin (25 mg/1) containing shoot induction meditrn. Approximately 15-19~ of the explants produced shoots on the selection medium. The elongated shoots were subsequently rooted in growth regulator free B5 basal medium containing hygro rnycin. The transgenics were later established in soil. The presence of hptll gene in the transgenic plants was confirmed by southern hybridization analysis.

P-14-031 TISSUE-SPECIFIC ALTERNATIVE SPLICING OF H-PROTEIN OF GLYCINE DECARBOXYLASE IN C4 FLAVERIA SPECIES

P-14-034 The construction and analysis of deletion and site directed mutants in psbH of Chlamydomonas reinhardtii.

S. Kopriva, R. Cossu, C.-C. Chu, & H.Bauwe Institute of Plant Genetics and Crop Plant Research, Corrensstrasse 3, 06466 Gatersleben, Germany

Ruffle, S.V., O'Connor, H,, Cheater, A.J., Purton, S. and Nugent, J.H.A. Photosynthesis Research Group, Department of Biology, University College London, Gower Street, London, WC1E 6BT. U.K.

Alternative splicing is a well-known posttranscriptional regulatory mechanism but there are only very few reports on alternative splicing in plants. The analysis of cDNAs encoding H-protein of the glycine decarboxylase from the C4 species Flaveria trinervia revealed the presence of two transcripts differing in insertion of 6 nucleotides. The single copy gene has been cloned and sequenced. The extra 6 base pairs origin from the 3"end of the first intron and code for additional two alanines very close to the N-terminus of mature H-protein. The quantification of the relative amounts of both transcripts in different organs revealed that the alternative (longer) mRNA is prevalent in leaves and stems whereas the "normal" one dominates in roots. The alternative splicing, with the same ratio between the two mRNAs, was detected also in other C4 species of the genus but not in any C3 or C3-C4 intermediate ones.

The psbH gene in Chlamydomonas reinhardtii encodes for a 9.3 kDa

P-14-032 TRANSLATIONAL REGULATION OF PEA PSBA GENE BY CHANGES IN THE REDOX POISE OF THE CELLS

P-14-035 Nuclear Control over the Biogenesis of the Cytochrome b J Complex.

phosphoprotein component of the photosystem II complex. H-protein sequences all contain a threonine residue close to the N-terminus which is a good candidate for the site of phosphorylation. Mutants lacking the psbH gene have been generated using insertion inactivation. Deletion of the gene yields mutants which appear to have no photosystem II activity as measured by loss of variable fluorescence, oxygen evolution and the EPR signal associated with the tyrosine "D" radical. Site-directed mutants at the threonine residue at position 3 in the polypeptide has been created. A change to alanine, which can not be phosphorylated, shows photosystem II activity similar to the wild type with respect to EPR, oxygen evolution and fluorescence. Data is presented that shows photosystem II activity is lost in C. reinhardtii when the psbH gene product is deleted, but the lack of a site of phosphorylation is not the primary cause.

Krassimir Alexciev Plant Cell Biology, Lund University, Box 7007, 220 07 Lund, Sweden

M. Turner, S. Purton, N. Gumpel, L.Ralley, J. Girard-Bascou* and F-A Wollrnan*. Biology Department, University College London, London WC1E 6BT, UK. and *Institut de Biologic Physico-Chemique, Paris 75005, France.

Photosynthesis is performed by dynamic structures in the thylakoid membranes of chloroplasts and photosynthetic bacteria. The components of these membranes are rapidly changing in amounts and ratios depending on the external stimuli which suggests tight control of their expression. Changes in the intensity and quality of light or oxygen, for example, lead to changes in the redox poise of the cells. It has been suggested that such changes control the transcription of chloroplast-encoded proteins. It is more likely, however, that this redox signalling affects mainly mRNA processing and/or translation of the messages into protein species. When isolated chloroplasts from pea were 35Slabelled after pre-treatment with different redox agents (either oxidising or reducing) the proteins syntbesised de novo show drastically changes. Some of these changes ave preserved even when the chloroplasts are pre-treated with Actinomycin D. One of the proteins which show such behaviour is DI, the product of psbA gene. The quantitative analysis of mRNA levels in pea chloroplasts treated with different redox agents shows no difference in the amounts olD1 mRNA. The results show that the redox state of the chloroplasts changes the translation ofpsbA message into protein molecules (D 1 protein). In this way the protein amounts are adjusted to the changes in the environment.

In order to identify specific nuclear encoded factors involved in expression and assembly of the cytochrome bJcomplex a tagged insertional mutagenesis approach has been used to generate nuclear mutants of Chlamydomonas reinhardtii. The ARG7 marker has been used as the insertional mutagen, and nuclear mutants that are unable to assemble a functional cytochrome b J complex have been isolated. Of these, mutants have been identified that are affected at the level of mRNA stability of cytochrome f or cytochrome b6 transcript, and another mutant is defective in beam cofactor attatchment to cytochrome be. Genetic analysis of these three mutants has identified cosegregation of the functional ARG7 gene and the mutant phenotype, and detailed genetic maps of the site of ARG7 insertion in the nuclear gennme have been constructed and DNA flanking the insertion has been cloned. The screening of wild-type cosmid libraries is now enabling clones carrying the affected genes to be isolated, and the mutations to be complemented.

151

Poster

P-14-036 Response of th) lak01d membrane proteins during shade-adaptatlon in Silerre d/olca. ..~arlin P. VinneIl. Chris|the A. Raines and Nell R. Baker. Department of Biolo~', Uni~ersily of Essex, Colchester, CO4 3SQ, U.K. Silene dioica is a "~oodland plant that competes successfully and exhibits similar photosynthetic efficiency in both open and heavily shaded habitals. Tile molecular basis of this highly successful adaptation to open and shade euvironments is not know,at. Irmnunological analyses of the changes that occur ill a range of thylakoid l~roteins ~hen plants grown in an open glasshouse environment were transferred to (i) a natural haa',), shade environment produced by sunlight filtration through a layer of Lemna minor and (it) an artificial shade environmeot similar ill PPFD to tile natural shade em~ironmentbut enhanced in the blue region of tile spectrum. On transfer 1o both shade euvironments tile components of the core PSII complex increase, LHCII shows no change, whilst components of PSI and ~'tochrome bsf complex decrease. Interestingly, on transfer to the natural shade the 33 kD extrinsic protein associated with PSII did not change despite the increases in the PSII core proteins. However, this 33 kD protein was found to increase in plants transferred to the artificial shade ~ith blue Iight enhancement. This response of the 33 kD protein to the artificial shade would appear to be mediated by blue-light The changes in the thyl,'&oid proteins on transfer to these shade environments were complete after 18 days. When plants were transferred back to tile open glasshouse enviranment after 18 days in the shade, the levels of all proteins decreased mlggesting the onset of chloroplast seneseence had been triggered. Northern blot analyses of total RNA using specific heterologous cDNA probes together with 35S-methionine pulse-chase experiments have been used to examine the molecular basis of the shade-indnced changes in thylakoid proteins.

P-14-037 NITRATE INDUCES REDUCTASE-BINDING PROTEIN IN ROOT PLASTIDS R.H. Vallejos, S. Pessino & G. Sosa,CEFOBI (Centro de Estudios Fotosint@ticos y Bioqu~micos), Suipacha 531, 2000, Rosario,Argentina. Maize and spinach root plastids are shown to have a membranebound diaphorase activity that is induced by pretreatment with nitrate.Antibodies against spinach chloroplast reduetase and a eDNA of pea chloroplast reductase reacted with a root plastid nitrate-inducible reduetase in Western and Northern blots. Similarly,antibodies against the chloroplast rednctase binding protein and its eDNA from maize leaves reacted with nitrate-induced maize and spinach root plastids suggesting the presence of the binding protein. Both the reductase and the binding protein seem to be forming a complex in root plastids similar to that described in chloroplasts (Vallejos e t a l . , 1984) since the reductase eopreeipitated with the binding protein and its eleetrophoretic mobility changes in conditions that affects the complex.

P-14-038 The Effect of Different Li£ht Intensities on the Transcript Level of Proteins Involved in Photosynthesis of Mustard Plants L. Wild-Peters, K. Kehl, I. Teuber, and A. Wild Institute of General Botany, Johannes Gutenberg-University 55099 Mainz, Germany The influence of light quantity on the steady-state levels of plastid encoded transcripts was examined during the development of primary leaves from mustard plants. RbcL mRNA (LSU of Rublsco), petA mRNA (Cyt f), psbA mRNA (D1 protein), 25 S rRNA, and 16 S rRNA were investigated in nigh-light (HL) and low-light (LL) leaves. The light regime had a remarkable influence on the absolute levels of 25 S rRNA, 16 S rRNA, rbcL mRNA and petA mRNA: Parallel to the amount of total RIGA, the content of the four transcripts per unit fresh weight was more than twice higher in HI., leaves than in LL leaves at the beginning of leaf development. The content of psbA mRNA per unit fresh weight was somewhat nigher in HL than in LL leaves and did not decline significantly during HL and LL leaf ontogenesis. The comparison of the absolute levels of rbeL mRNA, petA mRNA, and psbA mRNA with the contents of the corresponding proteins indicates that the transcript levels principally contribute to the differences in the protein contents between H L and LL leaves.

152

P-14-039 EFFECTS OF REDOX POTENTIAL ON THYLAKOID PROTEIN PHOSPHORYLATION AND ON CHLOROPLAST AND MITOCHONDRIAL PROTEIN SYNTHESIS John F. Allen. Carol A. Allen, Ltiling Cheng, Gunilla H~tkansson, and Dalibor Stys, Department of Plant Cell Biology, Box 7007, Lund University, S-220 07 LUND, Sweden Thirteen pea chloroplast thylakoid phosphoproteins show redox-dependency of phosphorylation, with Em= 40 + 10 mV, n = 1. This result is consistent with a primary site of redox control at the Qi site of the cyt b6/fcomplex, at the QB site of the PS II reaction centre, or at cytochrome b559LP. All thylakoid phosphoprotein phosphatase reactions are strictly redox-independent and thus light-independent. NMR spectroscopy shows that phosphorylation changes the 3-D structure of N-terminal LHC II fragments, consistent with the "molecular recognition" hypothesis, by which phosphorylation causes a structural change in LHC II that decreases its affinity for PS II while increasing its affinity for PS I. In intact pea chloroplasts and mitochondria, 35S-methionine incorporation reveals that different proteins are synthesised in the presence of external redox reagents and in the presence of different electron transport innibitors. Redox sensors and response regulators may therefore regulate gene expression in chloroplasts and mitochondria, explaining longterm adaptations such as adjustment of PS stoichiometry, and the evolutionary requirement for chloroplast and mitochondrial genomes to encode those proteins whose synthesis must respond to changes in redox potential.

P-14-040 N U C L E A R C O N T R O L O F T H E E X P R E S S I O N O F T H E CHLOROPLAST P E T GENES ~ , Y. Choquet t, N. Gumpe 12, D. Culler~, S. Purton 2, S. Merchant 3, F. Laquerri~re t, F.-A. Wollman'. qBPC, 13 rue P. et M. Curie, 75005 Pads, France; 2UCL, London, UK; sUCLA, Los Angeles, USA To define and characterize the nuclear genes required for the biogenesis of the cyt b6f complex, we studied a large collection of nuclear mutants defective in b6f complex activity. The mutants were generated either by traditional mutagenesis or by the gone tagging technique. To assess the primary defect in these mutants, we studied the expression of chloroplast petA, petB and petD genes at the mRNA and protein synthesis levels, and the defects in cytb6f complex assembly. For each class, a genetic analysis was carried out to define complementation groups and tight linkages. Several classes of mutants were observed: (1) mutants affected in the stability of pet mRNA, (2) mutants deficient in translation of pet mRNA, (3) mutants deficient in heine attachment. For each pet gene, we found only one nuclear gone controlling mRNA stability while several genes were found for the other classes. Tagged mutants were found in classe 1.

P-14-041 RNA EDITING IN ATPA, ATPF AND RPSI4 TRANSCRIPTS FROM TOBACCO: OCCURENCE OFSILENT EDITING IN CHLOROPLASTS T. Tsndzukil~ T. Hirose2, H. Fan2, J.Y. S ~ 2 , T. Wakasugi2, H. K6ssel3 and M. Sugiura2. 1Aichi-Gakuin Univ., Nisshin, Aichi 470-01 JAPAN; 2Center for Gene Res., Nagoya Univ., Nagoya 464-01 JAPAN; 3Institut fin" Biologie 1II, Univ. Freiburg, D-79104 Freiburg, GERMANY; Four new C to U RNA editing in tobacco chloroplast atpA, atpF and rpsl4 transcripts have been identified. Three of them lead to amino acid substitutions to restore the conserved amino acids. However, one editing in atpA transcripts was found to take place partially at the third letter of a serine codon (CUC to CUU), leading to no amino acid substitution. This is the first report on silent editing in chloroplasts. An additional editing site was found 4 nt upstream from the silent editing site in the atpA transcript and some direct repetitive sequences are present around the two sites. The extent of silent editing depends on plastid stages and light conditions but not depend on the size of expanded leaves. The upstream editing takes place constitutively even in bleached white seedlings grown in the presence of speetinomycin. In pea and spinach, no silent editing at the corresponding tobacco site was found in spite of the same sequences around the site. This observation indicates that silent editing is species-specific.

Poster ii

P-14-042

P-14-045

ORGANIZATION OF THE LIGHT-RESPONSIVE CISELEMENTS OF TOBACCO psaDb GENE

GENES FOR Mg-CHELATASE SUBUNrrs IN WILDTYPE AND MUTANTS OF BARLEY (HORDEUM VULGAREI

M. Nakamura, Y. Kondo, Y.Y.Yamamoto & J. Obokata Graduate School of Environmental Earth Science, Hokkaido University,Sapporo 060, Japan

B.L Petersen, P.E. Jensen, R.D. Willows*, U. Vothknecht*, C.G. Kannangara*, B.M. Stummann, D. yon Wettstein* and K.W. Henningsen. Royal Veterinary and Agricultural University, 1870 Frederiksberg C, *Carlsberg Laboratory, 2500 Valby, Copenhagen, Denmark.

psaDb is a nuclear gene encoding the PSI-D subunit of photosystem I in Nicotiana sylvestris, and its expression is induced by light. We studied the organization and structural characteristics of the light responsive elements (LRE) of psaDb, by use of transgenic tobacco system and transient expression assay with particle bombardment. The obtained results indicate that psaDb has two different types of LREs, namely upstream LRE and internal LRE. The upstream LRE is located between 170 and -1 relative to the transcription start site, and contributes to the light-responsive transcriptional activation. The internal LRE is located in the transcribed region, between +1 and +861. The internal LRE is utilized in seedlings but not in developed leaves. Hence, psaDb is likely to be subjected to complex lightresponsive and tissue-specific regulation through distinct types of c/s-acting LRE. Further characterization of the upstream LRE is also discussed,

The Olive gene from Antirrhinum and the CH-42 gene from Arabidopsis may encode two subunits of Mg-eheLatase, which inserts Mg 2+ into protoporphyrin IX during chlorophyll biosynthesis. A genomic and a eDNA clone for each of these genes have been isolated from barley. Chlorophyll deficient xantha (xan) mutants at the loci Xan-f, -g and -h accumulate protoporphyrin IX and have no detectable Mg-chelatase activity. Northern and western analysis show that the mutants Xan-fl' and ../,o are deficient in Olive mRNA and protein while Xan-h ~ -h ~ and -h ~7 are deficient in CH-42 mRNA and protein. Southern analysis shows that the mutant Xan-h ~ has a deletion in the Xan-h gene. It is concluded that the isolated Olive and CH-42 genes from barley are identical to the Xan-fand Xan-h genes and encode two subunits of the Mgz+ chelatase complex.The expression patterns of the Xan-fand Xan-h genes in wildtype barley have been characterized for greening etiolated seedlings and for green plants grown in a light/dark regime.

P-14-046

P-14-043 REDOX REGULATION OF TRANSCRIPTION IN CHLOROPLASTS

I N VITRO ANALYSIS OF CIS. AND T R A N S . A C T I N G REGULATORY ELEMENTS FOR TRANSLATION OF PHOTOSYNTHETIC GENE TRANSCRIPTS IN TOBACCO CHLOROPLASTS

A. Tullbero, G. Hgtkansson and J. F. Allen Dept. of P/ant Cell Biology, University of Lund, Box 7007, S-220 07 Lurid, Sweden.

T. Hirose. M. Sugita & M. Sugiora Center for Gene Research, Nagoya University,Nagoya 464-01, Japan In chloroplasts, translation is one of the important regulatory steps and there are some unique feature~ distinct from those in prokaryotes and eukaryotes. Several chloroplastmRNAshave no SD-like sequence at the appropriate position, suggestingthat chloroplastribosome recognizes the correct initiation site via other sequence elements. To elucidate molecular mechanismsof translation initiation and its regulation, we attempted to develop an in vitro translation system using tobacco chloroplast lysetes. After optimization of multiple conditions for preparation of chloroplast lysates and translation reaction, we succeeded in developing an in vitro system in which several chloroplastmRNAscan be accurately translated. Using this system, we have investigated the c/~-acting element for translation initiation of three photosyntheticgene transcripts ~sbA, atpB and rbcL). In vitro translation analysis using various mutant mRNA constructs revealed that multple specific sequenceelements contribute to translation initiation in a gene specific manner. Furthermore, competition experiments of in vitro translation and gel retardation assay suggestedthat trans-acting factor(s) interacts with the 5'UTR ofpsbA mRNA.

Chloroplasts were isolated from peas (Pisum sativum), and incubated with different redox agents. Then, run-on transcription was performed, and the isolated transcripts were hybridized to filters with single genes from the tobacco (Nicotiana tabacum) chloroplast genome. The chloroplasts incubated under oxidizing conditions (with potassium ferricyanide) showed an enhancement in transcriptional activity compared to the ones incubated under reducing conditions (with dithiothreitol). Efforts are underway to characterize the regulatory proteins involved in this process.

P-14-044 psaA TRANS-SPLICING: INVESTIGATING THE ROLE OF CHLOROPLAST AND NUCLEAR ENCODED FACTORS IN CHLAMYDOMONAS. M. Goldschmidt-Clermont, F. Diirrenberger and J.-D. Rochaix. Depts of Plant Biology and of Molecular Biology, University of Geneva, Sciences II, 1211 Geneva 4, Switzerland. In Chlarnydomonas reinhardtii, thepsaA gene is composed of three exons scattered around the choroplast genome. They are transcribed separately as precursors which are then assembled by a process that requires two steps of splicing in trans. T h e split introns share many of the conserved structural features of group II introns, but an additional small chloroplast RNA (product of tscA) is required for trans-splicingof the first split l~ntron. At least 14 nuclear loci are required for one, the other, or both transssplicing steps. One nuclear mutant (L135F) appears to be defective for the production of tsc.A RNA from a polycistronic precursor. We have constructed a "c/f' version of the first intron, encoding in a single transcript the precursor of exon 1, tscA and the precursor of exon 2. This "cis" intron undergoes splicing in vivo. However the "c/s" transcript is efficiently processed, and its parts can be spliced in trans to the endogenouspsaA transcripts. The effect of nuclear mutations on the splicing of this construct are also being determined. We have used transformation to generate tagged mutants and thus cloned one of the nuclear loci required forpsaA trans-splicing.

153

Poster

TRANSLOCATION OF CYTOCHROME CHLOROPLAST THYLAKOID MEMBRANE. Poster

session

R. M. Mould I, S. High 2 and J. C. Gray 1 1Dept of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K.; 2School of Biological Sciences, University of Manchester, Manchester M13 9PT, U.K.

15-16

Protein translocation and assembly P-15-16-001

-

P-15-16-038

P-15-16-001 DIFFERENCES IN THE LOCATION OF EXPRESSION-RELEVANT REGIONS IN PLANT LEADER SEQUENCES C. Bolle, R.G. Herrmann, R. O¢lmi~llcr Botanisches Institut der Ludwig-Maximilians-Universi~t,Menzingerstr. 67, 80638 Miinchen, Germany Deletion of sequences for 5"-untranslated leader regions in chimeric GUS gene fusions with 5"-flanking regions from genes for thylakoid proteins completely abolishes GUS gene expression in transgenic tobacco. Closer inspection uncovered that the crucial region in the PetE leader is relatively close to the transcription start site ( + 4 / + 15), while that of the AtpC leader is located in vicinity of the translation start site ( + 1 3 3 / + 1 7 3 ) . This region contains a CT-rich sequence (TITCTCTCTCCT); the same or slightly modified sequence is also present in 214 plant genes deposited in the EMBL Databank. 40% of these genes harbor this sequence in their 5'-untranslated leaders. The importance of CT-sequence for AtpC expression was confirmed by site-directed mutageneses which resulted in a 2- to 3fold reduction of GUS gene expression in vivo. If the complete AtpC leader is inserted between the 35S RNA CaMV minimal promoter (-90/+3) and the GUS gene, the GUS activity and mRNA level increased more than 10-fold. These results suggest that plant leader sequences can contain essential cis-elements for expression.

P-15-16-002 MOLECULAR CHARACTERIZATION OF PsbW, A NOVEL AND THE ONLY NUCLEAR-ENCODED COMPONENT OF THE PHOTOSYSTEM I1 REACTION CENTER COMPLEX IN SPINACH Z. J. Lorkovi~ l, W. P. Schroeder 2, H. B. Pakrasi3, K.-D. lrrgang4, R.G. Herrmann I , R. Oelmiiller1 IBotanisches lnstitut der LMU, Menzingerstr. 67, 80638 Miinchen, Germany; 2Department of Biochemistry, Stockholm University, S-10691 Stockholm, Sweden; 3Depamnent of Biology, Washington University, St. Louis. MO 63130, U.S.A.; 4Max-Volmer-lnstitut, Technical University of Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany We describe the isolation and characterization of cDNAs encoding the precursor polypeptide of the 6.1 kDa polypeptide associated with the reaction center core of the photosystem II complex from spinach (gene: PsbW). The gene is present in a single copy per haploid genome. The mature polypeptide with 54 amino acid residues is characterized by a hydrophobic transmembrane segment, and, although an intrinsic membrane protein, it carries a bipartite transit peptide of 83 amino acid residues, which directs the N-terminus of the mature protein into the chloroplast lumen. Thylakoid integration of this polypeptide requires neither a ApH across the membrane nor is it azide-sensitive, suggesting that the polypeptide chain inserts spontaneously or in an as yet unknown way. The PsbW mRNA levels are lightregulated. Similar to cytochrome b559 and PsbS, but different from DI, D2, CP43 and CP47, PsbW is present in etiolated spinach seedlings.

154

P-15-16-003 F ACROSS THE

Cytochrome f is an integral component of the cytochrome bf complex in the chloroplast thylakoid membrane. It is synthesised with an N-terminal signal sequence and is inserted into the thylakoid membrane co-translationally. Most of the polypeptide is translocated across the membrane and the signal sequence is cleaved off in the thylakoid lumen. A C-terminal region acts as a 'stop transfer' signal. Several pathways have been elucidated for the post-translational translocation of nuclear-encoded thylakoid proteins into, or across, the thylakoid membrane. We have demonstrated that a chimeric protein consisting of the presequence of Rubisco SSU fused to turnip cytochrome f is imported by isolated pea chloroplasts and that cytochrome fdelivered in this manner integrates into the thylakoid membrane post-translationally. This insertion is not dependent on a transthylakoidal ApH but is sensitive to azide which is a potent inhibitor of SecA activity. The signal sequence of cytochrome f can be cross-linked to both mammalian and stromal SRP54 proteins suggesting that SRP54 is involved in the translocation pathway of this protein.

P-15r16-004 ISOLATION

OF SecAAND

SecY eDUAm FROM HI--PLANT8

A. Kanazoulou, S. R. Haward and J. C. Gray. Department of Plant Sciences, University of Cambridge, Street, Cambridge, CB2 3EA, UK.

Downing

Proteins destined for the thylakoid membrane or lumen of h i g h e r p l a n t c h l o r o p l a s t s m u s t be t r a n s l o c a t e d a c r o s s two m e m b r a n e systems: the chloroplast envelope and the t h y l a k o i d membrane. Little is k n o w n about the c o m p o n e n t s of the thylakoid protein-transport apparatu s • T h e r e is g r o w i n g evidence that a s e c system homologous to the bacterial proteine x p o r t s y s t e m o p e r a t e s for at least a subset of t h y l a k o i d proteins. A p r o t e i n h o m o l o g u e of b a c t e r i a l s e c A has been isolated from pea chloroplasts and shown to be involved in the t r a n s p o r t of p l a s t o c y a n i n and the 33 kD p o l y p e p t i d e of the oxygen evolving complex. Homologues of the bacterial s e c A and secY genes have b e e n found in the c h l o p r o p l a s t g e n o m e s of various chromophytic algae but these genes are not present in chloroplast DNA from higher plants. We have isolated a 3.4 kb secA cDNA from pea, using a PCR approach based on c o n s e r v e d regions of the known s e c A p r o t e i n sequences. An A r a b i d o p s i s s e c Y cDNA has been isolated using an Arabidopsis EST probe and both cDNAs have been characterized by s e q u e n c i n g . The information obtained is being used to overexpress the cDNAs and p r o d u c e a n t i b o d i e s to the c o r r e s p o n d i n g proteins for luther characterization of the thylakoid transport apparatus.

P-15-16-005 PROTEIN TRANSPORTACROSSTHE THYLAKOIDMEMBRANE Alexandra Mant 1, Ralf B. KI6sgen2 and Colin Robinson 1 IDept. of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK; 2Botanisches Institut der LMU, Menzinger Strage 67, 80638 MUnchen, Germany At least two distinct mechanisms transport nuclear-enceded proteins across chloroplast thylakoid membranes, one of which is See-dependent, requiring a stromal protein factor and ATP. Although the thylakoidal ApH has appeared unimportant in the Sectype mechanism, we show that it plays a critical role in the translocation process, depending on the identity of the passenger protein and the level of ATP present. Dissipation of the ApH almost blocks transport of spinach 33kDa photosystem II protein (33K) across the thylakoid membrane of intact chloroplasts, but has no effect upon plastocyanin (PC). The 33K presequenee directs the transport of mature PC by a ApH-independent mechanism, whereas a ApH is essential for the reciprocal construct. Using standard assays for the import of proteins by isolated thylakoids, we have examined the roles of ApH and ATP for the translocation of wheat 33K. In these assays, the proton ionophore, nigericin, reduces import efficiency five-fold when the ATP concentration is 120 ttM, and abolishes import when ATP is reduced to around 5 I.tM. In both cases, additional AlP, but not non-hydrolysable ATP analogues, can rescue import efficiency to a significant level, suggesting overlap in the roles of ATP and ApH.

Poster P-15-16-006 THE HETEROLOGOUS IMPORT OF A PRECURSOR-PROTEIN INTO ISOLATED CHLOROPLASTS DIRECTED BY A BIPARTITE TRANSITSEQUENCE Peter Brandt. Institut fiir Pflanzenphysiologie und Mikrobiologie, Freie Universitiit Berlin, K6nigin-Luise-Stral3e 12-16a, D-14195 Berlin and Robert KochInstitut, Wollankstral3e 15-17, D-13187 Berlin, Germany The nuclear gene barley mutant viridis-zb *~ has been shown to lack completely photosystem I-activity (1). This PS I-deficiency is caused by a disordered assembly procedure (2). Isolated chloroplasts of this mutant, however, are competent to import an in-vilxo synthesized precursor-protein for the PSI subunit IV from tomato. The precursor-protein of about 28.5 kDa is processed to the mature form of about 22 kDa. A specific endoprotease-activity is present in the stroma of the mutant-chloroplasts, which processed the precursor-protein to an intermediate form of about 23.5 kDa. The heterologous import of the precursor-protein from tomato into the isolated chloroplasts from the barley mutant viridis-zb 63 depends on the presence of a fraction of cytoplasmic proteins isolated from the mutant. (1) Hiller et al., 1980, Carlsberg Res Commun 45:315-328; (2) Brandt, 1993, Plant Physiol (Life Sci Adv) 12:183-192;

P-15-16-009 Recognition signal for processing protease on D1 precursor protein of PSII reaction center Y.Yamamot01 , F.Taguchi 1 and K.Satoh ~'~ 'Dept. of Biology, Okayama University, Okayama 700, 2NIBB, Okazaki 444, Japan The D1 subunit of PSII RC is synthesized as a precursor ( p D l ) with C-terminal extension consisted of 8 to16 amino acids, which is cleaved off by the action of processing protease. The cleavage process is absolutely essential to the manifestation of 02 evolution in PS#1, if the C-terminal extension is present in p D l . In the present study, substituted C-terminal oligopeptides corresponding to the sequence of pD1 were subjected to the enzymatic analysis, both as substrata and as inhibitor, in order to understand the mechanism of interaction between thylakoid-embeded substrata (pD1) and the protease in lumenal space. The result indicated that the structure in the vicinity of cleavage site of pD1 formed by linkage b e t w e e n two parts of the protein molecule, i.e., C-terminal extension and C-terminal moiety of D1 mature protein, is important in the interaction and that the specific amino acid side chain (Leu-343) plays a crucial role in the recognition. The result also indicated that amino acid on theC-side of the cleavage point (+1 position) markedly influences the reaction rate ; Phe, Ala, Ser, Cys > Gly > V a l > > Pro=0. The conclusion derived from these in vitro analyses will be discussed in comparison with those obtained by in rive study using site-directed mutants of Ch/am),domonas (Takahashi et aLL

P-15-16-007 CHLOROPLAST IMPORT AND PROCESSING FERREDOXIN-NADP + REDUCTASE PRECURSOR

OF A RECOMBINANT HOLOPROTEIN

P-15-16-010 BARLEY MUTANT C H L O R I N A - 1°4 CANNOT IMPORT LHCA 2 INTO CHLOROPLASTS UNDER RESTRICTIVE GROWTH

E. Serra, A. Krapp, J. Ottado, E. Cecarelli & N. Carrillo Dto. Cs. Biol6gicas. Fac. de Bioqufmica. Universidad de Rosario. Suipacha 531. 2000 Rosario. Argentina

D.U. Meyer. J. Knoetzel & L.H. Grimme Institute for Cell Biology, Biochemistry and Biotechnology, University of Bremen, Leobener Str./NW II, 28334 Bremen, Germany

The precursor of the pea chloroplast flavoprotein ferredoxin-NADP + reductase (preFNR) was expressed in Escherichia cell and purified to homogeneity. Isolated preFNR contained bound FAD but had very low levels of activity. Proteolysis of the transit peptide rendered a functional FNR core displaying enzymatic activity. The FAD-containing precursor was transported into isolated pea chloroplasts and processed to mature size, both inside the plastid or by incubation with stromal extracts. Import required the presence of ATP, and was strongly stimulated by the addition of leaf extracts. Unfolding of preFNR with urea enhanced import, but not binding to the plastid envelope. Binding and kinetic parameters were determined under different conditions, indicating the existence of a single type of translocation sites and of a v e r y efficient import mechanism.

The barley mutant chlorina- T M lacks LI-ICA 2, the LHCI-680Bprotein, and LHCH under restrictive growth conditions of low temperature (15°C) and high fight intensity (220 p E m "2 s'l). When shifted to permissive conditions (22°C, 75 pE rn"2 sq) the mutant returns to wild-type phenotype (Knoetzel & Simpson, 1991, Plama 185, 111-123). The precursor of barley LHCA 2 is imported into mutant and wildtype chloroplasts The nucleotide sequence of mutant L h c a 2 is identical to the sequence of the wild-type gene. As expected, chloroplasts isolated from clo- t°4 and wild-type grown under permissive growth conditions import the LHCA 2-precursor, and the mature protein is found in the thylakoid membranes. Chloroplasts isolated from c/o-1°4 grown under restrictive growth conditions are not capable to import LHCA 2. These growth conditions had no influence on the import of LHCA 2 into wild-type chloroplasts.

P-15-16-011

P-15-16-008 SPECIFICITY C~DOMONAS

OF

PROCESSING

ENZYMES

IN

CHLOROPLASTS

OF

REINHARDII

A. ROfenacht & A. Boschetti. Institut for Biochemie, Universit~t Freiestrasse 3, CH-3012 Bern, Switzerland

Bern,

From the stroma of isolated chloroplasts of Chlamydomonas reinh~rdii three proteases were partially purified, each of which processed either the precursor of the small subunit of ribulose-bisphosphate carboxylase or the precursor of GEE1 of Chlamydomon~s specifically at different sites; i.e. several processing enzymes specific for different precursors exist. Furthermore, the precursors of three proteins of photosystem I, of cytochrome c552 and of plastocyanine from Chlamvdomonas were also synthesized i~ vitro. Binding to and import into isolated chloroplasts of Chlamvdomonas are shown and the specificity of in vitro processing with stromal enzymes was studied. A protocol for the import of these five proteins into isolated thylakoids of Chlamvdomonas is presented. All five precursors incorporated in a thermolysin insensitive form in thylakoids, though two of them with somewhat reduced efficiency.

t67W in Loop C of the CP47 Protein is required for the stable Assembly of Functional Photosystem II Centers Jituo Wu and Terry M.Bricker Department of Plant Biology, Louisiana State University, Baton Rouge, LA70803 The substitution of arginine for tryptophan at position 167 in the CP 47 protein by chemical mutagenesis resulted in the loss of PS H activity and photoantotrophic growth(Ermokova, S.Y.et al, 1993, Photosynthesis Research, 37: 139-146). Using site-directed mutagenesis we have changed :eTW to 1e7S. The W167S mutant exhibited a 70% loss in oxygen evolution activity( H 2 0 to DCBQ ) and grew extremely slowly in the absence of glucose. Immunoblotting analysis indicated that the.D2, CP 47, and CP 43 intrinsic proteins and the 33 kDa extrinsic protein accumulated to nearly normal l~vels. The amount of the D1 protein, however, was greatly decreased. These results suggest that 167Wof the CP 47 protein is important for the assembly and/or stability of the PS II reaction center.

155

Poster II

P-15-16-012 CARBOXYL-TERMINAL PROCESSING PRECURSOR PROTEIN IN SPINACH

PROTEASE

P-15-16-015

FOR D1

STRUCTURE-FUNCTION STUDIES ON THE D1 POLYPEPTIDE IN SYNECHOCYSTIS 6803

iN. Inagaki, 2H. Mori, 3S. Fujita, 3y. Yamamoto and 1,3K. Satoh (1Nati. Inst. Basic Biol., Okazaki, 2Nagoya Univ., Nagoya, 3Okayama Univ., Okayama, Japan)

Ronnev Wlldundl, Gaza Salth 1, Catherine Gerez 2 and Chrlster Jansson 1 1Department of Biochemistry, The Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden and 2LEDSS V-B~t Chimie Recherche, Universit~ Fourier, BP 53x 38041 Grenoble Codex, France

In all oxygen-evolving photosynthetic organisms so far analyzed, except for

Euglena gracilis, the D1 precursor protein (pD1) of PSII RC is furnished with a C-terminal extension, and its excision is absolutely essential in the manifestation of water-oxidizing function. The protease involved in the cleavage was purified from spinach and identified to be a monomeric protein of about 45 kDa. Based on the amino acid sequence, spinach eDNA clones coding the enzyme were screened and sequenced, which code a protein consisting of 539 amino acids. By comparing with the N-terminus of purified enzyme, the protease was deduced to be synthesized as a precursor containing a N-terminal extension, which is characterized as transit and signal sequences, consistent with the conclusion derived from biochemical analysis. The mature protein has no characteristic motif for major classes of proteases. However, the sequence shows a significant homology with that of a gone (ctpA) identified in Synechocystis, by a genetic complementatinn analysis using a mutant deficient in the function of PSII, proving that ctpA actually is the gone for C-terminal processing protease.

P-15-16-013 DETECTION OF THE P-SUBUNIT OF THE ClpPROTEASE IN CHLOROPLASTS C. Weiss-Wichertt, U. Altenfeld2 & ~ , ~ , ~ j ~ l 1 Biocbemie der Pflanzen, Albeft-Ludwigs-Universitgt, Schinzlestr. 1, 79104 Freiburg, Germany; z Biochemie der Pflanzen, Rohr-Universit~t, Universitatsstr. 150, 44780 Bochum, Germany Cells have the ability to recognize and degrade defect proteins using ATP-

dependent proteases. Procaryotes like E.coli possess the well known Clp Im34eaze consisting of CIpP subunits with proteolytic activity and regulatory ClpA subunits with ATPase activity and chaperone function. In eucaryotes, a similar degrA4a~ve system may exist: higher plants encode ClpP-like proteins on their chloroplast genome and ClpA-like subonits with chloroplast transit sequences on their nuclear genome. To investigate the Clp protease in chloroplasts, we analysed CIpP expression at the protein level in higher plants and algae. By overproducing the wheat clpP gane in bacteria we raised antibodies against CIpP. They crossreact with proteins of the size expected from the DNA sequence. ClpP is enriched in chloroplast extracts where it appears to be partly associated with the membrane fraction.

P-15-16-014 PROTEIN ENGINEERING OF THE N-TERMINAL REGION OF THE PHOTOSYSTEM II REACTION CENTER PROTEIN D1 Christer Jansson. Gaza Sallh & Ronney Wlklund Dept. of Biochemistry, The Arrhenlus Laboratories, Stockholm University, S-106 91 Stockholm, Sweden In chloroplasts, the Photosystem II reaction center protein D1 is synthesized on thylakoid-bound polysomes as a ~33.5 kDa precursor with a C-terminal extension. It is integrated in the stroma lamellae and subsequently transported to the grana stacks where it assembles in the PSII complex. During this process the D1 protein is processed and covalently modified. A prominent modification is phosphorylation of amino acid T2. In cyanobacteria, which lack grana lamellae, the sequence of events is less clear. The thraonine at position 2 is conserved in the cyanobactedal protein and is often followed by two additional thraonines at positions 3 and 4. Using site-directed mutagenesis of the psbA2 gone in the cyanobacterium Synechocystis 6803 we are studying the role of the threonine cluster at the N-terminal end of the D1 protein.

156

Photosystem II (PSll) is a multiprotein complex with more than 20 different polypeptides. PSII contains a heterodimer of the twopolypeptides D1 and D2, which carries most of the cofactore necessary for PSII photochemistry, in our work we are using site-directed mutagenesis to probe structure-function relationships of the D1 polxpeptide in Synechocystis 6803. Around 30 mutants are at present in d=fferent stages of analyses. We are particulady interested in studying how the D1 polypeptide is integrated into the thylakoid membrane and how PSII is assembled. To address these problems, we are using immunochemical and pulse-chase techniques, protease topology mapping and different activity measurements.

P-15-16-016 RELATIONSHIPS BETWEEN TRANSPORT OF PROTEINS ACROSS THE THYLAKOID MEMBRANE AND THYLAKOID ENERGIZATION S ~ ~ Tecr and Steven M. Theg Section of Plant Biology, University of California, Davis, CA 95616, U.S.A. Translocation pathways for moving proteins across the thylakoid membrane vary in their dependence on the proton motive force (pmO. While the energy source for translocating some proteins is provided completely by a pmf, other proteins utilize energy from ATP hydrolysis in addition to a pmf, still other proteins are imported into thylakoids in the absence of a pmf. Details of how pmf energy is harnessed to provide vectorial movement of polypeptides are unknown. We are investigating import pathways that utifize energy from a ApH in isolated thylakoids. Using subunits of the oxygen evolving complex synthesized both in vitro from cloned genes and by overexpression in bacteria, we are examining the relationships between thylakoid membrane energedcs and protein translocation. Specifically, our experiments correlate the extent of thylakoid membrane energization and the rate of protein translocation, as well as the reciprocal effect of translocation on the energized state.

P-15-16-017 THE PROTEIN TRANSLOCATION SYSTEM OF CYANOBACTERIA A. C. Barbrook, J.C.L. Packer and C.J. Howe - Department of Biochemistry, Cambridge University, Tennis Court Road, Cambridge, CB2 1QW, UK. Cyanobactefia have the ability to target proteins to more than one membrane. This makes their protein translocation machinery especially interesting since it may be responsible for this targeting. We have investigated components of the machinery. We have developed an assay system for leader peptidase in vitro and have used this to charactedse the activity of the protein. We have also cloned and sequenced the leader peptidase gene from the thermophilic cyanobactefium Phormidium laminosum. Both the biochemical analysis and the cloning and sequencing suggest the leader peptidase is present as a single copy. We have also isolated a secA gone from P. laminosum. This also appears as a single copy. Using this sequence and sequences from a number of other photosynthetic organisms we are interested in the secA gene as a phylogenetic marker. We are continuing our work to encompass more aspects of the translocation machinery and have detected a cyanobacteriat homologue of the 54kDa SRP protein.

Poster P-15-16-018 PRESEQUENCE INDEPENDENT IMPORT OF THE PsaF PHOTOSYSTEM I PROTEIN FROM CHLAMYDOMONAS REINHARDTII INTO BOTH CHLOROPLASTS AND MITOCHONDRIA. Lars-Gunnar Franzdn 1, Marie Hugosson2, Ghasem Nurani 2 & Elzbieta Glaser2 1Dept of Plant Physiology, Gtiteborg University, Carl Skottsbergs gata 22, S-413 19 G0teborg, Sweden, and 2Dept of Biochemistry, Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden It has previously been shown that presequences of nucleus-encoded chloroplast proteins from Chlamydomonas reinhardtii share structural features with mitochondrial presequences. We have tested if two chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii can be imported into spinach mitochondria in vitro. We found that the PsaK precursor protein was not imported by spinach mitocliondria, but the PsaF precursor protein was imported with high efficiency in a membrane potential dependent manner. Furthermore, if the presequence of the PsaF protein was truncated or deleted by in vitro mutagenesis, the protein was still imported with approximately the same efficiency as the full length precursor. Interestingly, the mutant PsaF proteins were also imported into C. reinhardtii chloroplasts. Our fesalts show that the PsaF protein has unusual properties and can be imported by both mitochondria and chloroplasts even in absence of the presequence. Possibly, the mature PsaF protein is able to interact with membrane lipids and/or the chloroplastic and mitochondrial protein import systems.

P-15-16-019 THE ASSEMBLY OF PsaD A PERIPEIERAL SUBUN1T OF PHOTOSYSTEM 1 Limor Minai I, Yuval Cohen I, Parag R. Chitnis 2 and Rachel Neehushtal 1 l Department of Botany, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; 2 Division of Biology, Kansas State University, Manhattan, KS 66 506, USA Photosystem 1 (PSI) which functions as plaatocyanth-ferredoxirl oxidoredu~'tase, is composed of at least twelve subunits. In plants and algae, about half of these subunits are encoded in the nucleus, synthesized in the cytoplasm as precursors which posses a leader pepride in their amino terminus. The precursors are post-translationally imported into the plastids, where their processing and assembly into the thylakoids take place. In the present work, we studied the mattmatioll and assembly of PsaD, a peripheral suhimit of PSI situated at the stromal side of the thylakoids. In spthaeh, the precursor of psaD (pre-PsaD) is synthesized as a 232 KDa protein Within the chloroplast, the 5.3 KOa leader peptide is removed and the 17.9 KDa mature PsaD is obtained. PrePsaD/PsaD, produced in vitro, was introduced to isolated chloroplasts and/or thylakoids. To follow the assembly of the protein into the thylakoids, the melnbranes were treated with a cheotropic agent (NaBr) or subjected to proteolyti¢ digestion (by thermolysin or trypsin). The results indicated that both the precursor and the mature form of PsaD assembled directly iato the PSI complex, in a manner resistant to NaBr wash. The pre-l~aD present in the thyl~koids was susceptible to proteolytie digestion by thermolysin or trypsin. However, following its processing PsaD became resistant to the proteolytic digestion; thermolysin removed only the typical 2KDa fragment at the N.terminus of the protein. This resistance to proteolysis of the newly assembled mature PsaD was similar to that observed for PsaD present in the thylakoids in situ. To better characterize the difference in sensitivity to proteolysis of pre-PsaD and PsaD and to verify whether they bind to PSI differently, the assembly of the two forms was performed in increasing concentration of salts. The data indicated that while the assembly of pre-PsaD was only slightly affected, high eoneenWations of salts decreased the ability of the mature form to assemble into the thylakoids. Taken together, we propose the following two steps model for the assembly of PsaD into the thylakoid membranes: pre-PsaD binds to PSI in an extended conformation that probably induces its susceptibility to proteolysis. Then, the processing to the mature PsaD occurs. The latter is probably accompanied by a conformational change that allows the formation of electrostatic interactions between PsaD and other subunits in PSI. These intra-complex interactions probably embed and stabilize the assembly of PsaD. In addition they protect PsaD from proteolytie digestion. Mature PsaD is resistant to proteoly~is only when present in FSI.

P-15-16-021 SPINACH POLYPHENOL OXIDASE IS A PHOSPHORYLATABLE PROTEIN OF THE THYLAKOID LUMEN G. Hind & J. W. Davenport*, Biology Dept., Brookhaven Natl. Laboratory, Upton, N ¥ 11973; *Biology Dept., Univ. o f Memphis, Memphis, TN 38152 A 64-kDa protein identified (Gal et al., (1992) FEBS Lett. 298, 33-35) as a protein kinase with specificity toward the photosystem II lightharvesting complex was cloned and sequenced. Its transit peptide has an N-terminal chloroplast import signal and a C-terminal domain that predicts import into the thylakoid lumen by a pathway shared with the 16and 23-kDa proteins o f the oxygen-evolving complex. The mature protein sequence is ca. 50% identical to several polyphenol oxidases (PPOs); canonical protein kinase or ATP-binding site motifs are absent. The protein is efficiently phosphorylated by an unidentified protein kinase in detergent extracts o f thylakoids. Partial trypsinolysis o f the phosphoprotein yields a peptide with intact N-terminus, comparable to a 42-kDa form of PPO, and a C-terminal domain containing a predicted 7-stranded antiparallel [3-barrel and the phosphorylation site. The significance o f the location of PPO and its phosphorylation is under study. [Supported by U. S. Dept. o f Energy]

P-15-16-022 Mutations in a signal sequence for the thylakoid membrane identify multiple protein transport pathways and nuclear suppressors. Brace D. Kohom. Benoit Baillet, and Phillip Hartzog. Box 91000. DCMB Duke University, Durham NC 27708 We have used a genetic approach to characterize the transloeation of cytochrome f, a chloroplast encoded protein that spans the thylakoid once, in Chlamydomonas reinhardtii. Mutations in the hydrophobic core of the cytochrome f signal sequence (ss) inhibit the ability of these cells to grow photosynthetically, and mutant strains fail to accumulate normal levels of cytochrome f. In vivo labeling and protease protection experiments with two hydrophobic core mutant swains show that the cytochrome f precursor is synthesized, but translocation is greatly reduced or eliminated altogether. One ss mutant also reduces the accumulation of two additional thylakoid membrane proteins LHCP and D1, but not those proteins, PC and OEC 33, that translccate completely across the membrane into the lumen. However, lumenal OEC 23/17 are increased in transport. Thus the hydrophobic core of the cyt f ss is required and is likely involved in one of three pathways into the thylakoid. We have selected extragenic suppressors called tip 1 and tip2 of two cyt f signal mutations, and these describe at least two nuclear genes whose products likely describe the translocation apparatus. Selected second site chloroplast suppressors further define regions of the cytochrome f signal peptide. We are currently cloning these nuclear genes by library complementation. Suppressors of OEC 23 and PC signals are also being isolated.

P-15-16-020 ROLES OF SEC PROTEINS IN PROTEIN TRANSPORT WITHIN CHLOROPLASTS

P-15-16-023 IN VITRO SYNTHESIS OF PSII CORE PROTEINS. THE D1 PROTEIN CAN BE INCORPORATED INTO PHOTOSYSTEM II

Toshiya Endo, Tetsuya Nohara, Akira Goto and Masato Nakai Department of Chemistry, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan

K.J. van Wi_ik1, S. Bingsmark 1, E-M Art 2 & B. Andersson 1 1Dept. of Biochemistry, Univ. of Stockholm, 10691 Stockholm, Sweden and 2Dept. of Plant Physiol., Univ. of Turku, 20520 Turku, Finland

Many chloroplast proteins are encoded by the genes in the nucleus, synthesized in the cytosol, imported into chloroplasts, and transported to their final destination within chloroplasts such as thylakoids. Since this last step resembles the intraeelldar protein transport process in cyanobacteria (possible chloroplast ancestors) and cyanohacteria have SecY and SecA proteins for protein transport across both the cytoplasmic and the thylakoid membranes, it is interesting to ask if chloroplasts have cyanobacterlal-type Sec proteins for protein transport within the organelles. Indeed recently, we have identified SecA in pea chloroplasts and cloned its entire eDNA. Pea secA eDNA encodes a polypeptide of 1,011 amino acids and shows high sequence similarity with cyanobacteriai SecA. Pea SecA was synthesized as a larger precursor and was imported into isolated chloroplasts in vitro. The purified pea SecA, which was expressed in Escherichia coil cells, stimulated the in vitro import of the 33-kI)a protein of the oxygen-evolving complex into thylakoids. These results indicate that higher plant chloroplasts contain a bacterial-type SecA-dependent system for the intraorganellar protein uansport into thylakoids.

The D1 reaction centre protein of photosystem II (PSII) has a much higher turnover rate than the other PSI] proteins. Thus the D1 protein has to be replaced while the other PSI] components are not newly synthesized. Synthesis of chloroplast-encoded proteins was followed in isolated chloroplasts. The incorporation of newly synthesised products into complexes was analysed by sucrose gradient centrifugation of dodecy. 1 maltoside solubilized thylakoid membranes. The sucrose gradient analysis allowed identification of the release of the nascent chains from the ribosomes and of at least four potential assembly steps. We will show that in isolated chloroplasts i) newly synthesized D1 protein is incorporated into existing PSII complexes, and ii) the PSI/core proteins D2, CP43 and CP47 are also synthesized and released from the membrane-bound ribosomes but substantial incorporation into PSII complexes occurs to a much smaller extent. Instead they accumulate predominantly as 'free' proteins in the thylakoid membrane. It can thus be concluded that the D1 protein can be replaced in PSI] complexes without the need for simultaneous import of nuclear encoded products.

157

Poster

P-15-16-024 MECHANISM OF D1 INCORPORATION INTO PHOTOSYSTEM II AND DEPENDENCE ON STROMAL FACTORS K.J. van Wiik. 1 B. Andersson 1 & E-M Aro2 1De~t. of 13iochemistry, Univ. of Stockholm, S-10691 Stockholm, Sweden and ZDept. of Plant Physiol., Univ. of Turku, 20520 Turku, Finland Translation of chloroplast encoded proteins was followed in isolated chloroplasts and in a homologous run-off translation system, consisting of isolated thylakoids with attached ribosomes. In the latter system, no biosynthesis of chlorophyll or other cofactors could take place, nor were soluble stromal factors available. By application of pulse and chase translation experiments in both systems, the process of elongation and release from the ribosomes and subsequent incorporation of newly synthesized products into complexes was followed. A stepwise incorporation of the newly synthesised D1 protein into PSII subcomplexes was observed. An initial fast cotranslational incorporation step, in parallel to a post-translational incorporation step was detected, while later steps were clearly posttranslational and sequential. Based on the comparison between the two translation systems, steps dependent on stromal factors or biosynthesis of cofactors in the chloroplast were found. Accumulation of intermediates indicated that cytosolic components(s) are needed for complete incorporation of DI into PSII. A scheme for the mode of replacement of the DI protein is presented.

P-15-16-025

P-15-16-027 CATIONS CONTROL THE ASSOCIATION OF THE STROMA PROTEASE TO THYLAKOIDS. Involvement of the proteolytic mechanism in "low-salt"-induced srana unstacking end supramolecular pigment-protein complex organization. J.H Argyroudi-Akoyunoglou and L Tziveleka Institute of Biology, NCSR "Demokritos", Athens, Greece. High proteolytic activity against LHCII is detected in the stroma fraction after lysis of intact chloroplasts in cation free medium. The activity is drastically reduced when the lysis medium is supplemented with Mg++; the loss in stromal activity parallels a concomitant gain in activity of the respective thylakoid fraction. This suggests that the association of the stromal protease to thylakoids is under cation control. Benzamidine (BA) the serine type protease inhibitor, when added to isolated "low-salt" chloroplasts, inhibits grana unstacking. Similarly, BA addition to thylakoids, prior to solubilization in SDS, enhances the organization of oligomeric supramolecular pigmentprotein complexes, and inhibits the Mg++-induced oligomer to monomer dissociation (Argyroudi-Akoyunoglou, 1980, Photobiocbem. Photobiophys. 1, 279-287). The effects are fully reversed upon washing of BA. The results suggest that a proteolytic mechanism may affect grana unstacking, as well as the organization of the supramolecular pigment-protein complex assembly, and that for the latter organization a critical monomer concentration is required, below which the oligomers dissociate.

P-15-16-028

INHIBITION BY Cu 2+ OF THE IMPORT OF POLYPNENOL OXIDASE INTO CHLOROPLASTS

ISOPRENYLATED PROTEINS IN THE THYLAKOID MEMBRANE

A. Sommer 1, E. Ne'eman 1 , S. Koussevitzky1, M.D. Hunt 2, J.C. Steffens 2, A.M. Mayer I , EJclareJ 1 Botany Department, The Hebrew University, Jerusalem, Israel 1 and Plant Breeding Department, Cornell University, Ithaca, N.Y., U.S.A. 2

Cathie Shinton, Ingela Parmryd, Gustav Dallner and Bertil Andersson Biochem. Dept., Arrhenius Laboratories, University of Stockholm, Stockholm, S-10691, Sweden.

Polyphenol oxidases (PPOs) are plastidial copper enzymes of a yet undetermined function. The nuclear-coded PPOs are routed to the thylakoid lumen by the conventional two-step mechanism. Cu 2+ is not required for PPO import or processing. Binding to the thylakoid membrane and chelation of Cu 2+ take place after completion of import and processing. Furthermore, translocation of the precursor (pPPO) across the plastid envelope is prevented by low concentrations (1-5 H,M) of Cu 2+. Translocetion of the envelope-bound pPPO is resumed when Cu 2+ is removed by cyanide. The stromal processing peptidase (SPP) which catalyzes the first step of pPPO routing is also inhibited by Cu 2+. The mechanism(s) of SPP and import inhibition are being investigated using mutant pPPOs in which the Cu binding domains have been modified by replacement of histidyl residues or deletions.

P-15-16-026

Protein isoprenylation in vivo has been demonstrated in spinach seedlings using 50 ~tCi/plant [3H]mevalonate and an incubation time of 24 h. The total protein labelling was increased by a 20 h pretreatment of the seedlings with 30 ttlvl mevinolin, an inhibitor of HMG-CoA reductase. Approx. 30 radiolabelled bands could be observed by SDS-PAGE and autoradiography of lipid-extracted proteins. By hydrolysis of the bound isoprenes from total plant protein, it was possible to identify thioether-linked famesol, geranylgeraniol and phytol, as well as longer chain isoprenoids which were covalenfly attached via another bond type. Cellular subfractionation revealed the presence of isoprenylated proteins in all fractions studied, most prominently in the nuclei and mitochondria. However, at least three labelled bands were observed in the chloroplast fraction with apparent molecular masses of 28, 24 and 10 kDa. Preliminary data indicates that they could be photosystem II-related. These proteins are not soluble stromal proteins, but are associated with or integral to the thylakoid membrane. The identity of the isoprenylated proteins of the thylakoid membrane and their modifying groups are investigated.

P-15-16-029

PXSUM S 4 T Z ~

A C C U M U L A T I O N OF T R A N S L A T I O N A L I N T E R M E D I A T E S OF D1 PROTEIN IN PEA CHLOROPLASTS IN THE D A R K .

N Magnin I, A Hunt I, R ca~illeri I, P Thomas 2, S Ridley 2 and ~ . xSchool of Biological Sciences, Royal Holloway, University of London, Egham, TW20 0EX; =Zeneca Agrochemicals, Jealott's Hill Research Station, Sracknell, RGI2 6EY, UK.

H. K u r o d a I a n d K. S a t o h 2,3 IThe G r a d u a t e School of N a t u r a l Sci. a n d Tech., 2Dept. Biol., Fac. Sci., O k a y a m a Univ., O k a y a m a 700, 3NIBB, Okazaki 444 J a p a n .

The D1 protein of photosystem II (PSII) in pea is synthezised with a 9 amino acid extension at the carboxyl terminus which is removed by a specific protease (DPP) prior to ligation of the water-splitting manganese complex. DPP has been extensively purified from an extract of sonicated pea thylakoids by chromatography on celite (to remove lipid), hydroxyapatite and reactive blue-2 coupled to sepharose CL-6B, and size exclusion HPLC. Inclusion of ethylene glycol in the elution buffers significantly improved chromatographic resolution of DPP. The apparent molecular weight of DPP from size exclusion HPLC was 32.5kDa, but the only protein band in SDS-PAGE profiles which co-eluted with DPP activity was at 40kOa, close to the reported molecular weight of the corresponding protease from Syneohocystis 6803 (Anbudurai et al, p N A S g l (1994) 8082-8086). We hope to verify the identity of this band as the DPP protein by N-terminal sequencing and immunoblotting using antibodies raised to the cloned DPP gene (ctpA) from Synechocystis. Pea DPP has an unexpectedly high pH optimum of 7.5 and is not significantly inhibited by PMSF, pepstatin, leupeptin or TLCK.

By p u l s e - l a b e l i n g w i t h L-~SS-methionine, isolated p e a c h l o r o p l a s t s a c c u m u l a t e p r e c u r s o r a n d m a t u r e f o r m s of DI p r o t e i n in t h e light, b u t n o t in t h e dark. By t h e a d d i t i o n o f ATP i n t h e dark, h o w e v e r , 22- a n d 24-kDa t r a n s l a t i o n a l i n t e r m e d i a t e s a c c u m u l a t e as m a j o r b a n d s . T h e a c c u m u l a t i o n of t h e s e i n t e r m e d i a t e s was o b s e r v e d e v e n in t h e l i g h t in t h e p r e s e n c e of ATP w h e n t h e i n h i b i t o r of p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t , s u c h as DCMU, w a s a d d e d to t h e r e a c t i o n mixture. On t h e o t h e r h a n d , by pre-illuminating c h l o r o p l a s t s f o r S m i n at 25 °C, t h e a c c u m u l a t i o n of f u l l - s i z e d DI p r o t e i n was o b s e r v e d e v e n in t h e d a r k in t h e p r e s e n c e o f ATP, s u g g e s t i n g t h a t stable f a c t o r ( s ) f o r m e d b y t h e o p e r a t i o n of p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t r e g u l a t e s t r a n s l a t i o n o f D l p r o t e i n at t h e specific step(s) of p o l y p e p t i d e e l o n g a t i o n .

P U R I F I C A T I O N AND CHLqACTBRXSATZON UP THE ~MqnOXYL T B R N ~ N ~ PROCESSZNG P R O ~ E J ~ E OF THE D I pROTEIN OF P H ( Y Z O R ¥ 8 ~ I I FBON

158

Poster

P-15-16-033

P-15-16-030 POSTTRANSCRIPTIONAL PROCESSES OF LIGHT-HARVESTING COMPLEX I ASSEMBLY IN RHODOBACTER CAPSULA TUS

U G H T DEPENDENT RELEASE OF THE LARGE SUBUNIT OF RIBtKXNSE-

Anja Meryandini and Gerhart Drews Institute of Biology 2, Microbiology, Albert-Ludwigs-University Schaenzlestr. 1, 79104 Freiburg, Germany

Sibdas Ghosh~'2, Matthew D. Smith*, Erwin B. Dumbrofft'', John E. Thompe~m* *Department of Biology, University of Waterloo, Waterloo, ON, Canada N2L 3G1; =Department of BiologicolSciences, University ofW'mco~dn-Whitewater,Whitewater, WI, USA 53190; 'Kennedy-Leigh Centre for Horticultural Research, Fsculty of Agriculture, Hebrew University of Jerusalem, P.O. Box 12, Rehovot, Israel 761000.

The insertion and stable assembly of the light-harvesting (LH) polypeptides a and 13in the membrane is a multistep process (Drews 1991, Richter & Drews, 1991), which was investigated in a homologous cell-free translation system under variation of its components. It was shown that the chaperone DnaK is essential for effective translation. GroEL supported the stable insertion of LHlct13 into the membrane. In absence of GroEL the polypeptides were extractable from the membrane by 6 M urea. LHI ct and 13 interact with each other and with GroEL in the cytoplasm. The single polypeptides were not stably inserted in absence of the partner protein. Incorporation in membranes from cells mutated in formation of the pigmentprotein complexes were less efficient. The highest rate of LHIct13 insertion was with membranes from chemotrophieally grown cells.-Drews G (1991) in Sympos. Soc. Gen. Microbiol. 47: 249-274; Richter P, Drews G (1991) J. Bacteriol. 173: 5336-5345. The work was supported by DFG and DAAD.

1,5-BISPHOSPHATE CARBOXYLASE FROM THYLAKOID8

A class of llpid-protein particles (LPPs) was generated in vitro by incubating intact thylakoids under light (625/maoL m-a.ser-*)or dark for 1, 2, 3 or 4 hours at 210C. A 52 kDa polypeptide that co-migrated during SDS-PAGE with the purified large subtmit (RLSU)ofribulose-l,5-bisphospbate carboxylase (Rubieco)was identified only in the LPPs generated in the light. The identity of RI~U was confirmed by Western blot analysis. However, the Bubieco ~ l ! subnait was not detected t. the LPP fraction. Lipid-protein pertides containingRLSU were purified by s~e exckudon Sephaeryl column ehromategrapby. Laser scanning, together with tr-nmni~ion electron mieroeeopy,indicated that the LIPs e~ltAinin~ RJ.~U ~ fi'om 0.05 to 0.10 microns in radins. The fatty acid c~apoeitiorm of the L P P s and thylakoids are ~im!bar, However, the free to esterified fatty acid ratio of the L P P s was 0.595, whereas the

corresponding ratio for thylakoids was 0.068. The~ obeervations suggestthat RI~Us are eloaely asaoeiated with thylakoida and released into the stroma by light. This release may in turn be coordinated in situ with the assembly of Rubisco holoenzyme.

P-15-16-034

P-15-16-031 NEUTRAL AMINOPEPTIDASES IN DARK- AND LIGHTG R O W N AND GREENING EUGLENA GRACILIS B. Young, G. Erdos & Dept. of Physiology and Biophysics, University of Illinois, Urbana, IL 61801, USA Protein turnover is well-known during chloroplast (c0-biogenesis, but specific proteases involved are little known. Here we examine neutral aminopeptidase (AP) activities in Euglena: dark-grown (contain proplastids), greening (cts at early stage of differentiation) and light-grown (mature cts). Cells were sonicated and the 20-80% ammonium sulfate fraction (contains 90% of the AP activity) of the 15,900g supernatant was used with 7 amino acyl-13naphthylamides as substrates. Leu-NA gave the highest AP activity in all cases followed in order by ala-, met-, pro-, phe- and asp-NA. Greening cells showed the highest AP activities while, depending on the substrate, darkgrown ceils were greater than or equal to fight-grown cells in activity. Pepstatin A, DFP, PHMB, PMSF and CuSO4 similarly inhibit AP activities with individual substrates in all 3 cell-types indicating the presence of constitutive APs in Euglena including asp, ser and sulfhydryl enzymes. Bestatin, antipaln, leupeptin and EDTA are most inhibitory to fight-grown followed by greening Euglena and little inhibitory to dark-grown ceils. Specific ser/cys- and metallo-APs characterize cells with mature cts.

P-15-16-032 CAROTENOID-DEFICIENCY IN PLASTIDS AFFECTS THE TRANS-

LOCATION REACTION ACROSS THE ENVELOPE MEMBRANES BUT NOT BINDING OF PRECURSOR PROTEINS TO THE PLASTID SURFACE Clas Dahlin Dept of Plant Physiology, GOteborg University, Carl Skottsbergs Gata 22, S-413 19 GOteborg, SWEDEN Precursor protein binding and import into young etioplasts, isolated from Norflurazon-treated, carotenoid-deficient wheat seedlings (Triticum aestivmn L.), have been studied. These plastids exhibit a significantly lower import efficiency of nuclear-encoded plastid proteins compared to etioplasts with normal contents of carotenoids (Dahlin, 1993, Physiol. Plant. 87 410-416). It is shown that plastids from control and Norflurazon-treated plants contain the same amount of binding sites on the plastid surface. Binding assays performed in the dark at 4°C or at low concentrations of Mg-ATP, showed that only in control plastids did binding lead to import into the plastids upon restoring the plastids to import promoting conditions. This, together with the presence of translocation intermediates, suggest that low import activity of carotenoiddeficient plastids is due to an inactive translocation machinery across the envelope membranes, and not to an inability of the precursor to bind to the plastid surface. The amount of the assembly-related proteins, Hsp 60 and Hsp70, were not different between the two treatments, as shown by Western blot analysis. The possible role of the carotenoids in the import of nuclearencoded proteins across the envelope membranes is discussed.

33 AND 23 k D a C~I]?MIX

EXTRZNgZC SUIIJUNIT8 OF ~ ARE A S s w t r : I , IRD BY DZFFI~iDi'JL' NIK2IIANZS]I8

O ~ G E N lff4OLVZI~I

Akiko Hashimoto z, Yasusi yamamoto: & Stev~n M. Theg 2 iDepartment of Biology, Faculty of Science, Okayama University, Okayama 700, Japan; 2the Section of Plant Biology, University of California, Davis, CA 95616, U.S.A. Nuclear-encoded oxygen-evolving complex (OEC) subunits are synthesized in the cytosol, imported into the chloroplasts, translocated across the thylakoid membranes and then assembled to the PSII core. The assembly of two OEC subunits (OE23 and 33) was studied in isolated chloroplasts (in organelle) and in PSII particles (in vitro). In vitro synthesized and newly imported OEC subunits became localized both in the thylakoid membranes and in the lumen, paralleling the location of the endogenous subunits. Newly bound subunits could be washed off the membrane at the same salt concentrations that removed the endogenous subunits, suggesting that they had assembled correctly into OECs. Reconstitution experiments demonstrated that unassembled 0E23 present in the soluble lumen pool was still assembly competent. In contrast, 0E33 from this pool did not bind efficiently to PSII. Further, fractionation of the thylakoid membranes showed that the assembly of 0E33 occurred in the unappressed regions soon after import and then migrated to the appressed regions, while OE23 assembled to PSII only in the appressed regions.

P-15-16-035 C h l o r o p l a s t and n u c l e a r m u t a n t s deficient in c y t o c h r o m e b 6 h e m e a t t a c h m e n t in Chlamydomonas reinhardtii Y. Choque~, R. Kuras 1, S. Buaclden 1, D. Culler 2, S. Merchant 2, J. Girard-Bascou 1 and F.-A. Wollmanl 1) Inst. Biol. Phys.-Chim. 13 rue P. et M. Curie 75005 Paris, France 2) Univ. o f California, Los Angeles, USA We constructed transformants o f C. reinhardtii mutated for the histidines that ligand the b h and b 1 hemes of the cytochrome b 6 by chloroplast site directed mutagenesis of petB gene. These mutations result in the destabilisation of the whole b6f complex and alter in a specific way the synthesized polypeptide products o f the petB gene. W e also isolated nuclear mutants of C. reinhardtih specifically altered in petB gene expression. These mutants exhibit the same phenotype as the choroplast transformunts, suggesting that they are specifically altered in the binding o f b h and b I hemes to apocytochrome b6. Genetic analysis o f these mutants indicates that the products of at least four distinct nuclear genes are required for this process to occur.

159

Poster

i

P-15-16-036 RAPID DEGRADATION OF M I S T A R G E T E D OEE33 IN THE CHLOROPLAST STROMA Tainar Halperin & .Zach Adam Dept. of Agricultural Botany, The Hebrew Univ., Rehovot 76100, Israel The 33-kD subunit of the oxygen-evolving complex (OEE33) normally resides in the thylakoid lumen. To study the fate of mistargeted proteins in chloroplasts, we substituted the bi-partite transit peptide of OEE33 with that of CAB7, a thylakoid membrane protein. As a result, when imported into isolated chloroplasts, the mutant protein was targeted to the stroma instead of the thylakoid lumen. While the wild-type OEE33 was totally stable for at least 2 h, the mutant protein was rapidly degraded, with a half-life of 60 roin. Degradation of the mutant protein was stimulated by supplemented ATP, when intact chloroplasts were incubated either in the light or the dark. Degradation could also be observed in lysed chloroplasts, in an ATP-stimulated manner. When lysates were fractionated, the proteolytic activity was found associated with the stromal fraction. Characteristics of the proteolytic activity, with respect to nucleotide requirements, sensitivity to inhibitors, and temperature and pH dependence, will be presented.

P-15-16-037 THE CHLOROPHYLL a / b / c LIGHT-HARVESTING COMPLEX OF MANTONIELLA SQUAMATA E. Riehl, A. Randolf, W. Lange, E. M5rschel Fachbereich Biologie, Philipps -Universit&t, Karl - von - Frisch - Str., D 35032 Marburg cDNA species encoding four very homologous precursor polypeptides of the chlorophyll a / b / c LHC of Mantoniella sauamata were cloned and sequenced. The precursor polypeptides have molecular weights of 24.2 kDa and are related to the major chlorophyll a / b polypeptides of higher plants. However, the mature light-harvesting polypeptides lack an Nterminal domain of 30 amino acids, which in higher plants, contains the phosphorylation site of LHCII and simultaneously mediates membrane stacking. Therefore, the chlorophyll a / b / c polypeptides do not show any light-dependent preference for photosystem I or II. The lack of this domatn also indicates weak attractive forces between stacked thylakoids. The LHC is the dominating protein of the thylakoids and is fractured with the protoplasmic membrane face as a 7.5 nm particle. The particles form paracrystalline arrays with a purported hexagonal arrangement in native thylakoids and when reconstituted in liposomes.

P-15-16-038 IN VITRO IMPORT OF PRE-FERREDOXIN-NADP+-OXIDOREDUCTASE FROM CYANOPHORA PARADOXA INTO CYANELLES AND INTO PEA CHLOROPLASTS

,J, Jakowitsoh1, Y. Ma1, C. Neumann-Spallart1, H.E.A. Schenk2, H.J. Bohnert3 & W. L0ffelhardt1 llnstitut fnr Biochemie und Molekulare Zellbiologie der Universit~ltWien und LudwigBoltzmann-Forschungsstelle for Biochemie, A-1030 Vienna, Austria; 2Botanisohes Institut der Universit~it TQbingen, D-72076 Tnbingen, FRG; and 3Department of Biochemistry, University of Arizona, Tucson, AZ 85721, USA Cyanophora paradoxa is a photoautotrophic fresh-water protist harboring plastids (cyanelles) that have retained a prokaryotic cell wall between their two envelobe membranes. Cyanelles encode far more proteins than chloroplasts of higher plants and are considered to be ancient plastid types. In a heterologous system with pea chloroplasts as the recipient organelles pre-ferredoxin-NADP+-oxidoreductase (preFNR) from C. paradoxa was as efficiently imported as the FNR precursor from the higher plant M. crystallinum. Using a novel cyanelie isolation procedure translocation of the Cyanophore precursor across the peptidoglycen-containing cyanelle envelope was also observed in vitro. Less efficient but still detectable was the reverse heterologous import of the plant precursor into isolated cyanelles. Thus, the Cyanophora precursor is recognized by the import receptors of higher plant chloroplasts, is transloceted and processed and vice versa. These results support the conclusion derived from comparative analysis of plastid genome organization, that all plastids originate from a common semiautonomous endosymbiotic ancestor.

160

i

Poster

P-17-003 TEMPORAL EVOLUTION OF PIGMENT COMPOSITION AND CHLOROPHYLL a ' CONTENT IN GREENING COTYLEDONS Poster session

Tadashi Watsnabe & Akimasa Nakamura Inst. Industrial Science, University of Tokyo, Minato-ku, Tokyo 106, JAPAN

17

Biosynthesis of tetrapyrroles and lipid metabolism P-17-001

-. P-17-033

Visible illumination to dark-grown seedlings reduces protochlorophyllide a into chlorophyllide, followed by biosynthesis of differently esterified chlorophylls (Chls) and production of mature, phytylated Chls a and b which are then assembled into photosystem proteins. We have studied these events in cucumber cotyledons by means of acetone extraction/reversed-phase HPLC. The amounts of intermediate Chl a (GG-, DHGG- and THGG-forms) normalized by leaf weight exhibited a sharp maximum at about 50 min of illumination, probably reflecting the processing of protochlorophyllide a that had been accumulated during growth in darkness (5 days). The Chl a'percentage (against Chl a + a') was fairly low initially, increased steeply in ca. 30 min to a maximum value of 1.2%, and then leveled off, in 5-6 hours, toward a stationary value of 0.40-0.45%, which is generally found in mature plant samples and corresponds to a stoichiometry of Chl a'/P700 = 2 (Maedaet al., BBA, 1099, 74-80, 1992). These observations are, on our hypothesis that Chl a' is an integral component for the RC of PS I, well in line with a view that rapid formation of core RC complexes is followed by slow association of LHC proteins to them in the initial phase of greening.

P-17-004

P-17-001 Effects of Mg-chelatase on early steps of heine and chlorophyll biosynthesis J. Papenbrock, H.-P. Mock, E. Kruse & B. Grimm Institut fiir Pflanzengenetik und Kulturpflmmenforschung, Corrensstr. 3, 06466 Gatersleben, Germany Chelation of Fe2+ or Mg2+ with proto IX occurs at the branchpoint of heme and chlorophyll biosynthesis. Mg-chelatase consisting of several different subunits is of interest from structural, regulatory and enzymatic points of view. Tobacco and barley eDNA clones encoding two putative subunits of the Mg-chelatase are homologous to the cs and olive sequences of Arabidopsis thaliana and Antirrhinum majus, respectively. They have been isolated and used in expression studies. Both mRNAs are regulated by light, developmental state and the circadian clock. Transgenic tobacco containing copies of an antisense gene for the tobacco cs homolog were used to study the effects of reduced levels of Mg-chelatase on chlorophyll biosynthesis, photosynthesis and other metabolic pathways, mRNA, enzyme level and activity as well as pigment levels of chlorophyll, heine, tetrapyrrole precursors and carotenoids were analysed. It is hypothezised that the bleached phenotype is due to regulatory reduction of enzyme activity of early steps in chlorophyll biosynthesis.

P-17-002 EFFECT OF GROWTH LIGHT INTENSITY AND N 2 0 PHOTOSYNTHETIC APPARATUS IN CHLOROBIUM TEPIDUM

ON THE

DEREGULATION OF T~FRAPYRROLE S ~ AFFECTS THE D~SENSE SYSTEM AGAINSTOXIDATIVE STRESS H.-P.Meck, E.Kruse and B.Grimm Institutftir Pflanzengenetikund Kulturpflanzenforsehung,06466 Gatersleben,Germany Generation of reactive oxygen species is the dangerous consequence of aerobic metabolism. Plants evolved an efficient antioxidant defense system for protection against the toxic and potentially lethal effects of these reactive molecules. Tetrapyrroles, such as berne, chlorophyll and phytoehrome, are essential cofactors for many metabolic processes. Deregulation of tetrapyrrole biosynthesis leads to accumulation of photosensitive intermediates which subsequently photooxidize and cause the formation of reactive oxygen species. We used transgenic plants with reduced activity for uroporphyrinogen decarboxylase or coproporphyrinogen oxidase to study the induction of the antioxidative protection mechanisms. While the young leaves of the transformants still couM counterbalance destructive effects of accumulated uroporphyfinogen or coproporphyrinogen, mature leaves showed a variety of necrotic phenotypes. Lipid peroxidation and membrane lesions were observed in porphyric plants. Carotenoid and tocopherol contents were determined. Analysis of the superoxide dismutase activities revealed an iscenzyme specific induction. Spatial and developmental regulation of the expression of several detoxifying enzymes in wild-type and transformants is currently investigated.

P-17-005 REGULATORY MUTATIONS AFFECTING CHLOROPHYLL BIOSYNTHESIS IN CHLAMYDOMONA$ RF.INHARDTII.

P.D. Gerola 1, M. Miller 2 and R.P. Cox2 1Dipartimento di Biologia, Universit~ degli Studi di Milano, Milano, Italy 2Institute of Biochemistry, Odense University, Odense, Denmark

E,M. Chekunov#, E.B.Yaronskaya z, N.V.Shalygo 2, N.G. Averina 2, and A.S.Chunayev ~ ~Department of genetics of St. Petersburg State University, St. Petersburg, V-34, 199034 Russia; ~ Institute of Photobiology of the Belorassian Academy of Science, Minsk-72, ul. Skoriny 27, 220733, Belorussia.

The light intensity during growth has been reported to have little effect on the pigment composition of the Chlorobiaceae, in contrast to C h l o r o f l e x u s where changes of a factor of 10 are observed. However, it is known that gases such as N20 inhibit the synthesis of BChl c. We have investigated possible interactions between these effects by growing Chlorobium tepidum at a range of light intensities in the presence and absence of N20. The content of pigments and the spectra of chlorosomes and cell membranes were investigated after sucrose density gradient centrifugation, Increasing light intensity decreased the cellular content of chlorosomal BChl a and BChl c, and the BChl a associated with the membranes. There was a decrease in the BChl c / a ratio and in increase in carotenoids in both fractions. The spectral properties of BChl in both fractions were not changed by altering the light intensity. The inhibitory effect of N20 on BChl c levels was associated with an increase in BChl a associated with the membranes.

A double mutant chH rood-u-25 of Chlamydomonas reinhardtii have been obtained from mutant chlI, which accumulate protoporphyrin IX (PP). chH rood-u-25 accumulates 20 times more PP than chH. The regulatory mutation rood-u-25 causes 2-fold increase in 5-aminolevulinic acid (ALA)synthesizing activity in the double mutant as compared to original chll mutant and to the wild-type strain. On the background of wild-type allele CHL1, rood-u-23 mutation causes overproduction of ALA and 30% reduction in chlorophyll accumulation. The ALA-synthesizing enzymes from chll and rood-u-25 mutants, as well as from wild-type strain are equally sensitive to heme inhibition. The protoheme synthesis in the double mutant is not impaired, rood-u-25 mutation is inherited uniparentally in chloroplast gene manner ( Supported in part by RFFR grant 93-04-6781).

161

Poster ii

P-17-006 INFLUENCE

OF LIGHT, 5-AMINOLEVULINIC ACID, HOMOCYSE1NE AND DIPYRIDYL ON MAGNESIUM-CHELATASE ACTIVITY AND PORPHYRIN ACCUMULATION IN PLANTS N. G. AVERINA, E.B.YARONSKAYA, V.V. RASSADINA, N.V. SHALYC-O AND G. WALTER*

Institute o f Photobiology, Academy of Sciences, Skorina str.,27, MJnsk, Belarus. *Humboldt University, Institute of Biology, Philippstr., 13, Berlin, FRG The porphyrin accumulation and photodamage processes in intact green bean (Phaseolus vulgaris) and cucumber (Cucumis sativus) seedlings treated for 17 h in darkness with 5-aminolevulinic acid (ALA) and homocysteine were enhanced compared with action of ALA alone resulting in plant death. An inverse dependence between the magnesiumchelatase (MCH) activity and Mg-protoporphyrin IX (monomethyl ester) amounts, acounmlated from exogenous ALA and dipyridyl in green pea (Pisum sativum L.) leaves has been obseawed. It supports the idea about the regulative role of membrane-bound products on the activity of their enzymes m vivo. The illumination of intact predarkened for 17 h green pea leaves resulted in 1-2 h lag phase in the MCH activity with its following strong stimulation that was absent in leaves treated with cycloheximide. The results are discussed from the position of fight induced synthesis of MCH molecules de novo.

P-17-009 DETECTION OF T R I P L E T M O L E C U L E S OF CHLOROPHYLL PRECURSORS BEAN LEAVES T R E A T E D W I T H CHELATORS OF IRON

K . V . N e v e r o v I, N . V . S h a l y g o 2, N.G.Averina 2, A . A . K r a s n o v s k y Jr 1 IA.N.Bach Institute of Biochemistry, M o s c o w 117071, Russia; 21nstitute of Photobiology, M i n s k 220733, Belarus Chelators of iron such as 1,10-phenanthroline (Ph) and 2,2'dipyridyl (DP) are k n o w n to cause the accumulation of chlorophyll a (Chl) precursors in chloroplasts accompanied by the strong p h o t o d a m a g e of t r e a t e d plants. Excited porphyrin molecules in t r i p l e t state were considered to p h o t o s e n s i t i z e the photodestruction. The formation of triplet porphyrin molecules was studied by m e a s u r i n g of their low-temperature (77K) phosphorescence in d a r k - i n c u b a t e d P h / D P - p r e t r e a t e d green bean leaves. The long w a v e l e n g t h phosphorescence maximum at 980 nm c o r r e s p o n d i n g to Chl and a weak m a x i m u m of p r o t o c h l o r o p h y l l i d e (Pchl) at 880 nm were observed in normal leaves. The strongest p h o s p h o r e s c e n c e band at 820 nm was detected after 5-30 mM Ph/ DP treatment. The e x c i t a t i o n spectra showed it to b e l o n g to monomeric m o l e c u l e s of M g - p r o t o p o r p h y r i n (Mg-PP) accumulated in leaves under the action of chelators. The 880 nm band increased by s factor of 12. The short wavelength emission of Chl appeared as a shoulder at 955 nm. The data suggest that Mg-PP as well as short w a v e l e n g t h Chl and Pchl (P627) molecules app e a r e d due to partial d i s s o c i a t i o n of their long w a v e l e n g t h forms may to initiate the photodamage caused by Ph and DP. The action spectra of Chl p h o t o b l e a c h i n g in chloroplasts from treated plants indicate the key role of Mg-PP in this process.

P-17-007 THE PSH-S POLYPEPTIDE - A NON-TYPICAL CAB PROTEIN Christiane Funk]. 2, Wolfgang P. Schrbder 1, Beverley R. Green 3, Iwona Adamska4, Bertil Andersson[, Gernot Renger 2 1Dept of Biochemistry, Arrhenius Lab. for Natural Sciences, Stockholm Univ., S - 10691 Stockholm, Sweden; 2Max-Volmer Institute, TU Berlin, StraBe des 17. Juni 135, D - 10623 Berlin, Germany; 3Botany Department, University of British Columbia, Vancouver, BCV6T1Z4, Canada; 4Institute for Botany, Univ. of Hannover, Herrenh~iuser Stral3e 2, D - 30419 Hannover, Germany. The intrinsic PSII-S protein has recently been shown to be able to bind chlorophyll (Chl) a and b (Funk et al., 1994, FEBS Lett. 342: 261-266). Here we report the isolation of the PSII-S protein using non-ionic detergents and isoelectric focusing. The isolated Chl-protein complex has a Chl ~ ratio of 5+1 and an absorption maximum at 671nm. This indicates that the PSII-S protein is a member of the CAB (Chl _a/b_binding) family as previously indicated by sequence similarities. However, the PSII-S has four transmembrane helices, rather than the three helices found in other members of the CAB proteins. The isoelectric point (pI of 6.1) as well as the Chl/carotenoid ratio were found to differ from the other CAB proteins. 77K steady state fluorescence measurements, fluorescence decay measurements and CD spectra gave evidence for a weak coupling of the pigments. Furthermore, in contrast to the other CAB proteins, the PSII-S protein was found to be stable without pigments in etiolated plants as well as in pigment lacking mutants.

P-17-008 Formation of primary chlorophvllide forms at low temperatures in etiolated leaves, prolmnellar bodies (PLB) and prothylakoids (PT). O.Belyaeval_and C.Sundqvist 2 1Biology Department, Moscow State University, 119899 Moscow, Russia; 2Botanical Inst., Dept. of Plant Physiology, Univ. of G6teborg, Carl Skottsberg Gata 22, S-413 19 G6teborg, Sweden A comparative investigation of the first steps of chlorophyllide (Chld) formation from protochlorophyllide in leaves, PLBs and PTs was perfbrmed by measuring fluorescence emission spectra. It was shown, that the fornmtion of the first weak fluorescent chlorgphyllide tbnns (from non-fluorescent intermediates) is a complicated process including several dark reactions with different rates and dift~rent temperature dependence. Four primary chlorophyllide forms were found, with a weak fluorescence band at 690, 695, 705 and 684 nm, respectively. The first weak fluorescent chlorophyllide form Chld690 was observed when the temperature of leaves illuminated at 77 K was increased to 199 K. At the further rise of the temperature to 253 K two chlorophyllide forms, Chld695 and Chld684, were formed. Chld705 was lbund in isolated PLBs. Chld695 and Chld705 were transformed to the shortwavelength chlorophyllide forms during a prolonged dark exposition at 253K.

162

IN

P-17-010 EFFECT OF SUPEROXIDE A N D REDOX AGENTS ON LIPIDS PEROXIDATION IN PEA A N D BEAN THYLAKOID M E M B R A N E S .

Garstka M.. Kargul J. and Kaniuga Z. Institute of Biochemistry, Warsaw University, Poland. Our previous studies on lipids peroxidation in thylakoid membranes (Acta Physiol. Plant. 16, 1994, 337-344) indicated that formation of final aldehydic products during the first minutes of incubation was more effective in bean than pea thylakoids and was not dependent on illumination. Now the effect of both light-generated superoxide (02"') and dark-effect of photosynthetic acceptors (hydroquinone, HQ; DCIP; methyl viologen, MV) on peroxidation was studied. Formation of O2'" by the Mehler reaction or MV autoxidation in light did not affect peroxidation, while in dark MV (E ~ -430 mV) significantly stimulated peroxidation in pea and bean~hylakoids, Agents with potential higher than MV, DCIP( E 7 + 220 mV) and HQ (E _ + 2 8 0 mV) inhibited this reaction in dar~. These data seem to i~licate that initiation of lipids peroxidation in thylakoid membranes during the beginning of incubation is not related to O'- generation. The findings suggest that peroxidative reaction in thyla~,oids membranes was initiated by low potential and inhibited by high potential agents.

P-17-011 A NOVEL PATHWAY OF CHLOROPHYLL BIOSYNTHESIS, THAT OPERATES IN OE-ETIOLATED PLANTS OF ARABIDOPSIS THALIANA

N. Lebedev 1,2, B. van Cleve 1, G. Armstrong1, & K. Apel 1 1Inst.fur Pflanzenwissensehaften ETH-Zentmm, Universitatstr. 2, Zudch 8092, SWITZERLAND: 2A.N.Bakh Inst. of Biochemistry, Leninskii pr. 33, Moscow 117071, RUSSIA Chlorophylls and their derivatives play a fundamental role in the energy absorption and transformation activities of all photosynthetic organisms. Up to now the only known enzyme, that converts pmtochlomphyllide to chlomphyllide under action of light is protochtomphyllide oxidoreductase. Analysis of spectmseopic, polypaptide composition and gene expression data shows that etiolated plants have two enzymes and two pathways of pmtochlomphyllide photo-reduction, of which only one is present after illumination. In the present study we describe a new Arabidopsis thaliana mutant, that has only one of these two biosynthetic enzymes, the same, as in illuminated wild type plants, Analysis of chloroplast formation in the mutant and wild type plants indicates that the changes of the pathway of chlorophyll biosynthesis play a regulatory

role in the assembly of photosystem core complexes and light-harvesting chlorophyllproteins.

Poster

i

P-17-012 CHANGES IN THE BIOSYNTHESIS AND CATABOLISM OF INTRAPLASTIDIC POLYAMINES DURING THE CHLOROPLAST PHOTODEVELOPMENT IN ZEA MA YS. Anastasia Andreadakis and Dapar~ent of Biology, University of Crete, P.O.Box 1470, 71110 Heraldion, Crete, Greece. Polyamines (pAs) and activities of ornithine dvcauboxylase (ODC), arginine decarboxylase (ADC) and diamine oxidase (DAO) were found in isolated ¢tioplasts, chloroplasts and the intermediate plastids of the chloroplast photodevelopment. The isolated vtioplasts contain high amounts of polyamines [puU'escine (Put), spermidine (Spd), spermine (Spm)] and high activities of the main FAs biosynthetic enzymes ADC and ODC combined with very low activity of the main PAs catabolic enzyme DAO. During photodevelopment of efioplast to chloroplast a two step decrease of the intraplastidic PAs, ADC and ODC takes place. One very fast and an other more intense after very long exposure to the light. The almost zero DAO activity in ctioplasts increased rapidly and showed a maximum after an exposure for 911 to light. The correlation of these changes with the protochlorophyllide/ Chlorophyllide photocenversion and the development of the photosynthetic apparatus was discussed.

P-17-013 INDUCTION OF TETRAPYRROLE SYNTHESIS IN LEAVES AND ROOTS OF A BARLEY ALBINA MUTANT UNABLE TO ACCUMULATE tRNAGhi Crabv Walter. Antje Mfiller, Paul Hoffinann and Thomas BOrner Institute of Biology, Humboldt-University Berlin, Invalidenstr. 43, D-10115 Berlin, Germany White leaves of the barley mutant albostrians accumulate only about 2 % of tRNAgl u compared to green leaves.This deficiency leads to a diminished ability to produce 5-aminolevulinlc acid (ALA) which can be overcome by feeding detached leaves or roots of the mutant with exogenous ALA. Application of the metal chelator 2,2'-dipyridyl (DIP) stimulates an accumulation oftetrapyrroles also in absence of ALA. The anabolic reduction charge or ATP seem to be non limiting factors for induced tetrapyrrole synthesis in the mutant. In etiolated leaves of the mutant the main product is protoporphyrin IX like in roots. Illuminated leaves of the mutant accumulate additionally Mg-protoporphyrin under the action of DIP. DIP-induced accumulation oftetrapyrroles in leaves and roots can be blocked by 3 mM gabaculine. We conclude that, inspite of the low amount oftRNA Glu , tetrapyrrole synthesis occurs via the C5-pathway and there is a chanelling in the direction of Fe-containlng tetrapyrroles (heme).

P-17-015 INTEGRATION AND TURNOVER OF PHOTOSYSTEM rl PIGMENT

Victor I. Raskin. Dvorah Fleminger and Jonathan R Marder, Dept. Agricultural Botany, The Hebrew University of Jerusalem, Rehovot, Israel Green barley leaves were labelled with radioactive amino-levulinlc acid (ALA), a precursor for chlorophyll synthesis. Chloroplast membranes were prepared and fractionated via standard procedures. Specific activities of chlorophyll (chi) in different fractions were determined to~track the main entry points of newlysynthesized chl. From pulse labelling experiments, it was found that the specific activity of chl A in the purified reaction centre of photosystem II (PSII) lags behind the labelling of total chl A and of chl A in grana-enriched thylakoids. The labelling of pheophytin is more strongly delayed. In chase experiments, the radioactivity in PSI] reaction centre chl declined faster than chl A of either whole thylakoids or the granal fraction indicative of more-rapid turnover. These apparently contradictory data are consistent with PSII reaction centre renewing its chl with intermediate rate relative to other populations in the thylakoid. We suggest that this chl turnover may be associated with the well-established PSII assembly/diassembly cycle. The decline in pheophytin specific activity in the PSI] reaction centre was slower than for chlorophyll, indicating either that it is exchanged more slowly, or is released and reincorporated to a greater extent than for chlorophyll.

P-17-016 SHORT-WAVELENGTH PROTOCHLOROPHYLLIDE FORMS AND THEIR PHOTOTRANSFORMATION IN PEA EPICOTYLS B. B6ddi I B. Mc Ewen 2, M. Ryberg 2 & C. Sundqvist 2 ~ l ~ t Phys. E6tv6s Univ. M u z e u m krt 4]a H-1088 Budapest, Hungary; 2Dept. Plant Phys. Univ. G6teborg, Carl Skottsbergs Gata 22, S-413 19 G6teborg, Sweden The presence of protochlorophyllide (Pchlidel and the NADPH-Pchlide reductase enzyme was detected in epicotyls of dark-grown pea seedlings. 77 K fluorescence spectroscopy analyzes showed that the Pchlide is arranged there mainly into 629 and 636 n m e .mittin.~ forms. Flash illumination did not transform these terms out o o m terms showed a slow and temperature dependent transformation w h e n epicotyis were illuminatedwith continuous light of low irra~ance; Only one chlorophyU/ide form (emittin8 at 680 nm] w a s aemc~ea during this process. Disaggresating .effects ~ d not change the shape, of the fluorescence spectra of the eplcotyls. ~ moaet has Deen crea~ea suggesting that the 629 n m form is a pool of Pchlide molecmas successively tranlocating to the active sites of t h e e nz.yme creatin 8 the 636 n m active form. The 680 n m emitting chloropnylliae.Iorm removes from the enzyme permitting the re-entering of new Pctflide mole.cul.as. This Pchlide transformation can be a link between the similar processes of lower plants and those of leaves of higher plants.

P-17-017

P-17-014 K X P R E 8 8 I O N pAT~ERMH OF CHLOROPIALST GENES INVOLVED I N I N D E I ~ N D B N T CHLOROPHYLL a l m T ~ E S I S I N LIVERWORT CELLS.

S. Takio & T. Satoh Dept. of Biological Science, eiroshima 739, Bigaahi-Siroshima, Japan.

University,

LIGHT-

P. O. Box

We have isolated suspension cultures of two cell lines from the liverwort, Marchantia paleacea vat. diptera. Both cell lines showed high level of chlorophyll in the light. In the dark, one cell line (yellow line) showed much reduced level of chlorophyll, while another line (green line} showed the same level as observed in the light. To elucidate regulatory mechanism in light-independent chlorophyll synthesis, transcript levels of chloroplast genes in these liverwort cells were determined. Both cell lines accumulated considerable amount of rbcL transcript in the dark and light. Transcripts for psaA and psbB were reduced in the dark in both cell lines. Transcript for chlL involved in light-independent chlorophyll synthesis was detected in green line both in the light and dark, while it was not in yellow line. These facts indicate that high level of chlorophyll in dark-grown cells of green line is due to increased expression of chlL gene.

PHOTOSYNTHESIS AND LIGHT ENERGY DISSIPATION IN SENESCING LEAVES OF GRASS MUTANTS BLOCKED IN CHLOROPHYLL DEGRADATION Nad6ee Souriaula, Barbara Hauck1, Christine Foyer2 and Howard Thomas1. ~Department of Cell Biology and 2Department of Environmental Biology, IGER, PIas Gogerddan, Aberystwyth, Dyfed SY23 3EB, UK. Mutation of the gene sid in grasses of the genera Festuca and Lolium disables chlorophyll degradation at the point of macrocycle opening by the enzyme phaeophorbide a dioxygenase. As a result the senescing leaves stay green. Nevertheless, photosynthetic CO2 assimilation declines in a similar manner in both wild-type and mutant plants and components such as ribulose-1,5bisphosphate carboxylase-oxygenase are lost at comparable rates. Chlorophyll a fluorescence analysis revealed that the photosystem II quantum efficiencies declined in parallel with increasing irradiance in all plants, suggesting the absence of novel alternative electron sinks in the stay-green mutant compared to the wild-type. The stay-green mutant did not appear to he substantially more succeptible to photoinhibition than the wild-type, suggesting that a very effective mechanism of energy dissipation is operating. Non-photochemical quenching was increased in the mutant compared to the wild-type. Analysis of the pigment composition and pigment distribution will be discussed in relation to the requirement for a massive energy sink that protects the excess chlorophyll from photooxidation in the stay-green mutant.

163

Poster P-17-018 PURIFICATION AND CHARACTERIZATION STRUCTURALLY DIFFERENT FORMS OF CARBOXYLASE IN PISUM SATIVUM

OF TWO ACETYL-CoA

C. Albat~, L. Dehaye, C.Job, J. Jullien, D. Job & R. Douce Unit~ Mixte CNRS/RhSne-Poulenc Agrochimie, 14-20 rue Pierre Baizet, 69263 Lyon Codex 09, France Young pea leaves contain two structurally different forms of acetyl-CoA carboxylase (ACC). A minor form was detected in the epidermal tissue. This enzyme was soluble and was purified to homogeneity from young pea leaf extracts and mature dry seeds. It consisted of a dimer of two identical biotinyl subunits of molecular mass 220 kDa and was refered to as ACC220. In this respect, this multifunctional enzyme was comparable to that described in other eucaryotes. A predominant form was present in both the epidermal and mesophyll tissues. In mesophyU protoplasts, ACC was detected exclusively in the soluble phase of chloroplasts. This enzyme was purified from pea chloroplasts and consisted of a complex composed of two functional domains dissociable and reassociable. The biotin carboxylase domain containing a biotinyl polypeptide of 38 kDa and a biotin-frce polypeptide of 32 kDa was biochemicaily characterized. This complex subunit structure was referred to as ACC38. In contrast to ACC38, ACC220 was also found to carboxylate propionyl-CoA. Biochemical parameters and kinetic properties were determined for both ACC forms including proptonyl-CoA carboxylase (PCC) activity catalysed by ACC220. Finally, both ACC exhibited different sensitivities towards the herbicide qnizalofop. This compound had no effect on ACC38, while ACC and PCC activities eatalysed by ACC220 were strongly inhibited.

P-17-021 The effects of Ce on chlorop_hyll-protein of Cucumber

chloroplast in relation to F~ +

Z.X.Chu, M.H.Mu, F.Z.Wang and H.X.Shao (Institute of Botany, Academia Sinica, Beijing 100044, China.) The cucumber seedlings are cultured in Hoagland solution containing 5ppm CeC13. It was proved that Ce can promote the chlorophyll formation in cucumber leaves, and has advantageous influence on the development of PSI Chl-protein complexes, the chlorophyll content are increased by 36%, the relative content of PSI Chl-protein complexes (CPIa 2 and CPI) and their 110KD polypeptide in treated chloroplast is higher than those of control chloroplasts. The results also showed that Ce decrease the relative contents of LHCP and their 27KD polypeotide in chloroplasts. It was observed that promotion of Ce is relative to l~e2+ , if culture solution lacking Fe2÷, Ce not only no advantageous influence but the inhibition was observed, the amout of chlorophyll decrease by 58%, the relative content of PSI Chl-protein complexes is also reducd. When the cucumber seedlings are grown in Hongland solution containing Ce despite of the presence or absence of Fe2+, the chloroplast structure is changed, there are less grana membrane and more stroma membrane in comparison to those of control chloroplast. It was also evidenced that Ce can delay the oxidizingprocess of Fe 2+ to Fe 3+, and enhance Fe content in leaves with 13.85%.

P-17-019 GENETIC REDUCTION

BORYANUM

ANALYSIS OF THE

ON PROTOCHLOROPHYLLIDE CYANOBACTERIUM PLECTONEMA

"Z Fuiita , H. Takagi & T. Hase Institute for Protein Research, O s a k a University, 3-2 Yamadaoka, Snita, Osaka 565, Japan The protochlorophyllide (Pchlide) reduction is a key step in the chlorophyll biosynthetic pathway o f plants. Many plants except for angiosperms have two different Pchlide reduction systems; one is a light-dependent system and the other is a l i g h t - i n d e p e n d e n t system. Previous genetic analysis of the cyanobacterium Plectonema boryanum has shown that this cyanobacterium has both systems for Pchlide reduction, and two genes (chlL and chiN) forming an operon are involved in the light-independent system. Recently we cloned the third gene (chlB) essential for light-independent system using the liverwort chlB gone as the probe. Western blot analysis using the antiserum against the ChlB protein expressed in E. coli indicated that the ChlB protein was localized in the membrane fraction o f this cyanobacterium. In addition, a g e n e for the lightdependent Pchlide reductase o f this cyanobacteriurn was cloned by a PCRamplified fragment using a set o f synthetic primers for conserved regions of the Pchlide reductases from higher plants. The nueleotide sequences and expression o f these genes and the phenotypes o f several mutants lacking these genes will he presented.

P-17-020 CHLOROPLAST SYNTHESIS NAY BE A PHOSPROINOSITIDE-4~)IATED Harvard L~man

EVENT

Synechocystle

OF

THE

sp.

MEMBRANE-BOUND

412

DESATURASE

The phospholipase C-inositol phospholipid(PLC-PiP2)tran s--~-hrane signalling system is seen in plant & alg~l systems.

(Cote & Crain.1994.BioEssays 16,39). An external signal activates PLC generating two second messengers,Inositol-triphosphate(XP 3) & diacyglycerol. IP. opens Calciul(Ca) channels releasing Ca & activating cell~la~ events. Lithitmm ion and Ca channel blockers Lanthanum & Cadmium C~loride inhibit this systel. Chloroplast synthesis in Ett~le_na ~racilis is specifically and reversably inh4hlted by these reagents. Potentiation, wherein dark-grown cells are preilltmainated,returned to darkhess,then exposed to constant light,show no lag in ~hlorophyll synthesis due to transcription/translation induced by I~tentiating light. Lithium,given during the potentiating light and then removed in constant light h~d no inlLi.bJ.tory effect on chlorophyll synthesis and also led to a t-~o-fold acctmmlation of chlorophyll over controls,implying that Lif/liul inhibits a pest-transcription/translation step° EGTA & Ca-free m e d i ~ do not affect chlorophyll synthesis indicating that Lanthan~ & ~ block i n ~ Ca channels. These data indicate that light-mediated chloroplast synthesis in En~lena is regulated at a post-transcripten/translation step by PLC-PIp 2.

IN

P C C 6803

L . M u s t a r d v 1.2, D . L o s t, Z . G o m b o s 2, N . M u r a t a I XNational I n s t i t u t e for B a s i c Biology, Okazaki, 444 Japan; 2Inst. of Plant Biol., BRC, H u n g . A c a d . Sci., H6701 Szeged, H u n g a r y The AI2 d e s a t u r a s e in c y a n o b a c t e r i a is r e s p o n s i b l e for d e s a t u r a t i o n at the 412 p o s i t i o n of membrane lipid fatty'acids a n d p l a y s a r o l e in the l o w - t e m p e r a t u r e a d a p t a t i o n of the organism. In Synechocystis sp. PCC 6803, its a m i n o - a c i d s e q u e n c e s u g g e s t s that it is a m e m b r a n e - b o u n d enzyme. A n i m m u n o c y t o c h e m i c a l a p p r o a c h w a s m a d e to l o c a l i z e the 412 d e S a t u r a s e in the cells of Synechocystis w i t h an a n t i b o d y r a i s e d a g a i n s t a synthetic oligopeptide, w h i c h c o r r e s p o n d s to the Ct e r m i n a l p a r t of t h e enzyme. In the w i l d t y p e c e l l s we f o u n d t h a t l a b e l s w e r e l o c a l i z e d in b o t h p l a s m a a n d t h y l a k o i d m e m b r a n e s . In Fadl2 m u t a n t cells, w h i c h l a c k this e n z y m e a n d c a n n o t adapt to l o w e r t e m p e r a t u r e s , t h e r e w e r e n o s p e c i f i c labeling. T h e s e r e s u l t s s u g g e s t that the 412 d e s a t u r a s e is l o c a t e d in b o t h types of membranes of Synechocystis a s s u r i n g fast a d a p t i v e r e s p o n s e to the c e l l s u n d e r l o w - t e m p e r a t u r e c o n d i t i o n s .

P-17-023 PORPHYRIN DEGRADATION IN A PLANT SOLUBLE FRACTION

Dept. of Biochemistry & Call Biology.

State university of Now York,Stony Brook,Stony Br~k,NoYolI794

164

P-17-022 LOCALIZATION

K. Yoshida & P. B6ger Lehrstuhl fiir Physiologic und Bioehemie der Pflanzen, Universita't Konstunz, D-78434 Konstanz, Germany Diphenyl ether-type herbicides (bleaching herbicides) inhibit protoporphyrinogen oxidase in chloroplasts. Its mode of action is thought to cause accumulation of protoporphyrinogen (protogen) in the chloroplast followed by transportation out of the organelle with subsequent oxidation of protogen to protoporphyrin IX (proto IX). This tetrapyrrole is activated by light and phytotoxic radicals are produced. Following a report of Jaeobs et al. (1994, Pestic. Biochem. PhysioL, 50, 23) we found that a soluble plant protein fraction of herbicide-tolerant plants catalyzes the degradation of protogen and this may closely correlate with the plant protection mechanism against bleaching herbicides. But the details are unclear. Thus we studied the degradation of protogen in cell extracts from mustard and cress. A fraction from mustard seedlings germinated in the light for 7 days showed stronger degradation of protogen than that of seedlings obtained in the dark. Cress seedlings had no such activity. The addition of a safener, naphthalie anhydride, showed no effect on the degradation of protogen. We purified this fraction and its charactristics will be presented.

Poster

P-17-024

P-17-027

CHARACTERIZATION A N D PROPERTIES OF Mg-DECHELATING SUBSTANCE AND PHEOPHORBIDASE WHICH ACT IN BREAKDOWN OF CHLOROPHYLLS

EVIDENCE FOR LIGHT-INDEPENDENT CHLOROPHYLL .4 SYNTHESIS IN A BARLEY MUTANT THAT LACKS CHLOROPHYLL B.

SkioJ,,~Y., W a t a n a b e , K., T o m i t a , N., T s u c h i y a , T. and T a k a m i y a , K. Fac. o f B i o s c i e n c e a n d B i o t e c h n o l o g y , T o k y o I n s t i t u t e o f Technology, Nagatsuta, Midoriku, Yokohama 226, Japan

Jane Walmsley and Heather Adamson, School of Biological Sciences, Macquarie University, North Ryde N.S.W. 2109, Australia.

N e w s u b s t a n c e , M g - d e c h e l a t m g s u b s t a n c e (MDS) a n d e n z y m e , pheophorbidase which act in breakdown of Chls were found in c r u d e e x t r a c t s o f Chenopoclium album. MDS w h i c h c a t a l y z e s a M g - r e l e a s i n g r e a c t i o n f r o m C h l i d e to P h e o p h o r b i d e ( P h e d ) was a low molecular mass, heat-stable substance. Mgdechelation reaction was stimulated by chelators and i n h i b i t e d b y M g 2+ i o n . P h e o p h o r b i d a s e is a n e n z y m e w h i c h catalyzes a decarbomethoxylation reaction from Phed into a pyrophed precursor. The precursor formed was converted n o n - e n z y m a t i c a l l y to p y r o p h e d . O p t M n u m p H o f t h e r e a c t i o n w a s 7.5. K m v a l u e f o r p h e d a w a s 12.5 ~tM a t p H 7.0.

P-17-025 Biogenesis of Chl a apoproteins of higher plant photosystems I and II

LA. Eichacker & M. Helfrich Botanical Institute HI, Menzingerstr. 67, 80638 Munich, Germany The assembly of plastid encoded chlorophyll a (Chl a)-binding apoproteins P700, CP47, CP43, D2 and D1 (Chl-P) with Chl a is highly regulated to protect photosynthetic activity against photooxidative destruction. Mg- and Zn-pheophorbide (Pheide) a and b were used to investigate stabilization of de novo synthesized, against proteolysis. Intact etioplasts were capable of taking up externally offered Pheidederivatives and geranylgeraniolpyrophosphates (GGPP) and were superior over lysed etloplasts with respect to pbeophyfin (Phe)- and protein-synthesis. Intact etioplasts esterified Mg- and Zn-Pheides a and b equally well, but Zn-Phe was highly superior (3-18 fold) with respect to Chl-P stabilizing capacity. Mg-Phe b did not stabilize ChlP, whereas Zn-Phe b could not be distinguished from Zn-Pbe a. Stabilization of ChiP by Mg-Phe a showed a general threshold value of 48 pmole/107 etioplasts, (Pmoles) whereas Zn-Phe a of 3, 7, 11 and 13 Pmoles initiated stabilization of ChlP, respectively. High concentrations > 120 Pmoles of de novo synthesized Mg-Pbe a saturated accumulation of Chl-P, whereas Zn-Phe a >24 Pmoles (P700) or >70 Pmoles (CP47, CP43, D2, D1) destabilized ChI-P and displaced Mg-Pbe a or Chl a bound to Chl-P. HPLC revealed Mg-Phe a-phytyl, whereas Zn-Phe a accumulated only as nonhydrogenated Zn-Phe a-GG indicating an importat role i, for the centralatom of the Phe and ii, for the steric configuration of the esterified diterpen alcohol.

P-17-026 CONTRIBUTION OF LIPIDS TO PSII

Nodhir0 Satol, Kintake Sonoike2, Mikio Tsuzuki 1, and Aldhiko Kawaguchi3 1School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-03, 2Dept of Botany, The University of Tokyo, Bunkyo-ku, Tokyo 113, 3Dept of Biology,The University of Tokyo, Meguro-ku, Tokyo 153, Japan We measured photosynthetic activities of mutants impaired in lipid metabolism of chloroplasts for investigation of the roles of lipids in chloroplasts. A mutant of Chlamydomonas reinhardu'i defective in sulfoquinovosyl diacylglycerol (SQDG) showed 40% reduction in PSII activity with little effect on PSI activity, as compared with the parent. Chlorophyll-protein complexes, i.e., PSI complex, PSII reaction center complex, and LHCII, were isolated from the parent for examination of lipids bound to these complexes. The analysis indicated that SQDG was distributed predominandy to PSH reaction center complex and LHCII, but not to PSI complex. These results suggest that SQDG is responsible for PSII activity by associating with PSH complex.

Dark-grown seedlings of the Chlorina 2 (b-less) mutant of barley

(Hordeum vulgare) were exposed to light for several hours, in the presence of 1-14C glutamic acid supplied via the roots, and then transferred to darkness. Net chlorophyll(ide) content remained constant or increased only slightly during dark treatment. There was however a substantial increase in the radioactivity of the total chlorophyll pool. This finding mirrors previously published evidence of light-independent chlorophyll synthesis and incorporation of label into the tetrapyrrole moiety of chlorophyll a in darkness in the wild-type (Walmsley and Adamson 1995). It indicates that the accumulation of chlorophyll a in darkness in barley cannot be attributed to the formation of chlorophyll a from either chlorophyllide b or chlorophyll b.

P-17-028 HYDROGENATION OF CHLOROPHYLL ALCOHOL SIDE CHAIN AND ITS ROLE ON PHOTOSYNTHESIS

M. Shihata, K. Fujii, M. Tsuyama, Y. Kitagawa, N. Iwata and Y. Kobayushi Faculty of Agriculture, Kyushu University, Fukuoka 812, Japan Hydrogenation of chlorophyll alcohol side chain from geranylge'raniol to phytol was investigated using rice mutants that accumulate Chls a and b with incompletely reduced alcohols. Genetic analysis suggests that the three-step reduction of geranylgaraniol to phytol is catalyzed by the same hydrogenating enzyme for both groups of Chls a and b. Leaf photosynthesis of mutant leaves was sensitive to high light. The photo-sensitivity was due to decomposition of Chls with incompletely reduced alcohol side chains integrated into chlorophyll-protein complexes. Based on the change in lipophilicity of the side ehain~ a possible role of the alcohol moiety is discussed.

P-17-029 BIOSYNTHESIS OF ISOPRENOID CHAINS OF CHLOROPHYLLS AND PLASTOQUINONE IN Scenedesmus BY A NOVEL PATHWAY

J. Schwender 1, H. K. Lichtenthaler1, M. Seemann2, M. Rohmer2 l Botanical Institute II, University of Karlsruha, D-76128 Karlsruhe, Germany; 2 Ecole Nationale Sup6rleure de Chimle de Mulhouse, 3, rue Alfred Werner, F-68093 Mnihouse Cedex, France;

Scenedesmus obliquus was fed with glucose, labelled with 13C at different positions. The phytol side chain of chlorophylls and plastoquinone-9 were analysed by 13C-NMR-spectroscopy. The 13C label pattern of the two isoprenoid chains (C20 and C45 ) were in disagreement with the condensation of three acetate units via mevalonic acid to form isopentenyl pyrophosphate (IPP), In contrast, the 13C-label showed a new pathway whereby a C3-unit is condensed with a C2-unit to form a Cs-precursor, which in a transposition step yields the isoprene carbon skeleton of IPP. The same pathway was found in eubacteria for biosynthesis of the isoprenoid hopanoids and ubiquinone (Rohmer et al., 1994, Biocbem. J., 295, 517-524). Because of the close relationship of green algae to higher plants we suppose that this novel pathway also exists in higher plants, which is presently investigated.

165

Poster P-17-030 CHLOROPHYLL FORMATION IN THE LIP1 PEA MUTANT.

P-17-033 STUDY OF UV-B EFFECT ON PHOTOACTIVE PROTOCHLOROPHYLLIDE COMPLEXES PHOTOCONVERSION.

C. Sundqvist, I M. Seyyedi ~and M. P. Timko 2, lBotanical Institute, Carl Skottsbergs Gata 22, S-413 19 GOte-borg Sweden; 2Dept. of Biology, Univ. of Virginia, Charlottesville, VA 22901, USA.

P. EullaffrQy, P, Juneau, R. Salvetat & R. Popovic D~pt. de Chimie, Universit~ du Quebec ~ Montreal, C.P. 8888, Succ. A, H3C 3P8, Montreal, PQ, Canada

The lipl mutant of pea cv Alaska has a low content of the PHYA polypeptide phytochrome I (Frances et al., The Plant Cell 4 1519, 1992). When grown ill darkness the lipl mutant has expanded leaves mad the internode length is reduced. The plastids lack regular prolamellar bodies but have extended agranal thylakoids. Some areas with membrane associations resembling transfomled prolanaellar bodies are found. The protochlorophyllide concentration is higher in the lipl mutant than in the wild type Alaska pea. Only minor amounts of the long-wavelength form of protochlorophyllide is found in the lip1 mutant whereas the wild type has about equal alnounts of short-wavelength and long-wavelength protochlorophyllide in their leaves. With flash irradiation only the long-wavelength form is converted to chlorophyll(ide) but with continuous light there is a decrease also in the content of short-wavelength protochlorophyllide. In fact the chlorophyll accumulation is more rapid in the mutant than in the wild type.

Absorbance spectroscopy was used to study the effect of ultraviolet-B radiation (UV-B) on photoconversion of photoactive protochlorophyllide (Pchlide) complexes and their regeneration. These light-dependent steps of Chlorophyll biosynthesis are catalysed by Protochlorophyllide OxidoReductase enzyme (PCRI. Dark grown barley leaves {Hordeum vulgare) were illuminated with continuous non-saturated light supplemented or not by UV-B. Under both conditions, we observed a phototransformation of Pchlide into Chlorophyllide (Chlide6~2). We noticed a faster accumulation of Chlide672 under UV-B illumination. This confirms that PCR is a flavoprotein and it appears that flavins are involved in primary processes of Pchlide photoreduction in etiolated leaves. In conditions which produced Chlide6~2 no regeneration of photoactive Pchlide can be observed in the dark. Based on these data, mechanisms of Pchlide photoconversion are discussed.

P-17-031 n~X'UROGmNEITY O F T H E P H O T O I N A C T I V E P R O T O C H L O R O P H Y L IDE POOL AT D I F F e R e N T BEAN LEAF D E V E L O P M E N T STAGES.

B. S c h o e f s I , M. Bertrand 2 & F. F r a n c k I . iLab. Photobiol. (B22), Liege univ., B-4000 Liege; 2 I N T E C H M E R , D i g u e de C o l l i g n o n , F - 5 0 1 1 0 T o u r l a v i l l e Higher plants developing in darkness accumulate protochlorophyllide (Pchlide) in two different functional states termed photoinactive and photoactive Pchlide. Photoinactive Pchlide is c h e m i c a l l y and s p e c t r a l y h e t e r o g e n o u s and a c t u a l l y w o u l d r e s u l t of t h e s u p e r p o s i t i o n of 4 d i s t i n c t s p h o t o i n a c t i v e P c h l i d e s p e c t r a l f o r m s (Cohen & Rebeiz, 1981, P l a n t Phy~io], 67, 98). W e h a v e r e i n v e s t i g a t e d the heterogeneity of the photoinactive pool at d i f f e r e n t b e a n leaf d e v e l o p m e n t s t a g e s b y m e a n s of 77 K e m i s s i o n and e x c i t a t i o n f l u o r e s c e n c e m e a s u r e m e n t s . A t the d i f f e r e n t d e v e l o p m e n t stages, w e f o u n d the same 3 photoinacive Pchlide spectral forms w i t h e m i s s i o n m a x i m a a t 626, 631, 636 n m (± 1.5 nm). The role of e a c h of t h e m w i l l be discussed.

P-17-032 ROLE OF NADPH-~ROPHYLLIDE-REDUCTASE PROTECTION OF NEWLY FORMED CHLOROPHYLLIDE

B. S c h o e f s & F. Franck. Lab. Photobiol. Univ., B - 4 0 0 0 Liege, Belgium.

IN P H O T O (CHLIDE).

(B22),

Liege

C h l i d e p h o t o d e s t r u c t i o n kinetics, u n d e r d i f f e r e n t red l i g h t intensities, h a v e b e e n m e a s u r e d in intact b e a n l e a v e s w h e r e p h o t o a c t i v e P c h l i d e has b e e n t r a n s f o r m e d b y a s a t u r a t i n g flash. T h e k i n e t i c s of in v i v o C h l i d e a b s o r b a n c e d e c r e a s e s h o w e d a lag i n d i c a t i n g t e m p o r a r y r e s i s t a n c e t o p h o t o d a m a g e . The d e c r e a s e p h a s e w h i c h followed, w a s c o r r e l a t e d w i t h an i n h i b i t i o n of the C h l l d e p h o t o c y c l e in w h i c h C h l i d e 6 8 4 and C h l i d e 6 7 8 c o n v e r t t o e a c h o t h e r in the light. S i n c e t h e s e two s t a t e s d i f f e r b y t h e i r b i n d i n g t o N A D P H or NADP+, w e hypothesized that the acceleration of Chlide d e s t r u c t i o n in v i v o w a s c a u s e d by a d e p l e t i o n of the N A D P H s u p p l y in t h e s u r r o u n d i n g s of the complex. S u c h N A D P H c o n s u m p t i o n was o b s e r v e d d u r i n g i r r a d i a t i o n of i s o l a t e d e t i o p l a s t s u s p e n s i o n s . We s u g g e s t t h a t the reductase has a protective role against Chlide p h o t o d e s t r u c t i o n p r o v i d e d N A D P H is available.

166

Poster P-18-003 Cloning by in vivo complementation of the Gap2 gene of Synechocystis sp. PCC 6803supports the amphibolic role of the cyanobacterial NAD(P)dependent glyceraldehyde-3-phosphate dehydrogenase F. Valverde. M. Losada and A. Serrano Instituto de Bioqufmica Vegetal y Fotosfntesis, CSIC y Universidad de Sevilla, Apdo. 1113, E-41080 Sevilla, SPAIN

P o s t e r s e s s i o n 18

Evolution o f P-18-001

Cyanobacteria possess one phosphorylating NAD(P)-depe.ndent glyceraldehyde-pnospnate ¢.~..nyarogenase (GAPDFI) equally active with either coenzyme that on me aasis of bmchemical work has bee.n clatmed to participate in both glycolysis ann tnenRPP pathway. We clonea mis amphibollc enzyme from SynecT~bcysfis sp. rt:t~ o~soa oy iuncnona~ comDlementation oz afap- mutant of E. coli w~m a global genomic library in the p'BS SK+ vector, I~emg the first time that this cloning strategy has been used ftr a.photosynthetic GAPDH..The overexpression ot mis enzyme ant/its purincafion nave oamonstratea not only its double coenzyme usage but also that it can certainly, operate as a glycolytic enzyme. Western blot analysis indicate that the 38 kDa subunit of the cyanobacterial enzyme is cor~cfly expressed by the .r.e.c.o mbinant plas.mid..The pmtein s~luence ob't~ned fro.m me ~ene snows a very n?gn oegree, oIslmuanty tUP to ~sa~) wire ChLOe.plastic ~ A e o r i s rLom ~ ,nign.erplants ag.a me t, apzl~ene o.fAnabaena variaoi!is;ar~a,a ~esser one t20~2 wltn glycolync enzymes, i ne existence ot omy mls NA~.r)dependent t.iAPIJH in ~/necnofystis cells is indicated by both biochemical oat,a ~omy one aqtivit], anna oDserv.ea.m column cttromatogr;aphy during p .m-il.tcationot me enzyme.).an.o immunological .studies kcompi.ete mmmiion ot both NAD ana i~,"tLJi" actlwtles Dy a monospecmc antmoay in Cell-tree extracts}, t-towever~ southern blots using as probes the cloned GAPDH genes of Synecnocystis ann E. coli indicate that two.Ga p genes should be ]~resent in the genome of 3ynecnocystis. taupportea oy grant PB 91-085 of DGICYT)

photosynthesis - P-18-008

P-18-001

P-18-004

SEQUENCE OF a~l; a ~ H GENES OF Prochloron sp. ATPASE GENE CLUSTER: IMPLICATIONS FOR CHLOROPLAST ORIGIN.

C H A R A C T E R I Z A T I O N OF A GENE E N C O D I N G A FUCOXANTHIN-CHLOROPHYLL PROTEIN FROM THE C H R O M O P H Y T I C ALGA, HETEROSIGMA CARTERAE

~g!92.I~f, glg~gg~*, Anthony W.D. Larkum* and Roger G. Hiller + School of Biological Sciences. *University of Sydney, NSW. 2006. Australia+ Macquarie University, NSW. 2109. Australia.

D. G. Durnford & B.IL Green Department of Botany, University of British Columbia, 6270 University Blvd, Vancouver, B.C., Canada, V6T 1ZA.

The phylogenetic position of the Prochlorophyta:prokaryotes that contain chlorophyll a and b as well as a chlorophyll c-like pigment is contentious. Prochlorophytes may be an anciently diverged group widely separated from the Cyanobacteria that are ancestral to the algal and higher plant chloroplast or are an anciently diverged group widely separated from the Cyanobacteria.that have independently aquired chlorophyll b and c and which may or may not be related to chloroplasts. The ATPase operon is a conserved gene cluster that has been well characterised. In the Cyanobacteria the atpl gene product is quite distinctive with a highly charged carboxyl terminus, consisting of 8 (Synchecoccus) to 9 (Anabaena) amino acid residues, where 6 out of 8 and 8 out of 9 of these residues are either glutamic acid or histidine. We have have sequenced the atpl and atpH genes of the ATPase operon from a Prochloron sp to clarify the phylogenetic position of this organism.

Heterosigma carterae is a unicellular marine raphidophyte which, along

P-18-002 PHYLOGENETIC ANALYSES OF PHOTOSYSTEMS I A N D II R E A C T I O N C E N T E R P R O T E I N S E Q U E N C E S

with other chromophytic algae, is characterized by the presence of two additional m e m b r a n e s around the chloroplast and by the possession of both chlorophylls a a n d c. A gene encoding a fucoxanthin-chlorophyll a + c protein (FCP) has been cloned and sequenced. This gene is part of a large nuclear encoded multigene family with over twenty members. Southern blot analysis indicates the presence of Fcp genes with varying degrees of sequence divergence, suggesting a gene complexity comparable to the chlorophyll a + b (CAB) gene family of terrestrial plants. The FCP sequences from different algae form a group apart from the peridinin-chlorophyll protein (I?CP) sequences of the dinoflagellates in a phylogenetic analysis. The FCPs and PCPs are evolutionarily related to the CABs, t h o u g h the relationship is distant.

P-18-005 The Phylogeny of Pheophytin-Quinone Reaction Center Proteins

Steven C. Halls, Stephen R. Downie, & Qovindiee Dept. o f Plant Biology, U n i v e r s i t y o f Illinois, Urbana, U S A

P. J. Lockhart ~& A. W. D. Larkum2 1Dept of Plant Biology and Plant Biotechnology, Massey University, Palmerston North, New Zealand; 2School of Biological Sciences, University of Sydney, NSW, 2006, Australia;

T h e c o m p l e t e a m i n o acid s e q u e n c e s for the reaction center proteins o f PS I a n d II were obtained f r o m v a r i o u s g e n e s e q u e n c e depositories, aligned, a n d each a n a l y z e d p h y l o g e n e t i c a l l y u s i n g m a x i m u m p a r s i m o n y . In total, 39 p.~_b_bA(D1) and 11 p_t.b_bD(D2) s e q u e n c e s , r e p r e s e n t i n g different genera, a n d 14 p_.~A a n d 14 p._~B s e q u e n c e s , r e p r e s e n t i n g 13 g e n e r a each, were a n a l y z e d . The genera chosen included angiosperms, gymnosperms, bryophytes, algae, a n d cyanobacteria. T h e multiple a l i g n m e n t o f each o f the reaction center subunits was facilitated by the introduction o f very few or no gaps. W e present for the first time evolutiona r y trees b a s e d o n PS I reaction center proteins a n d e x p a n d e d trees b a s e d on PS II proteins. In m o s t cases, the e v o l u t i o n a r y relationships inferred on the basis o f m a x i m u m p a r s i m o n y were c o n c o r d a n t with traditional classification s c h e m e s .

Pheophytin-Quinone photosynthetic reaction center proteins in anoxygenic and oxygenic photosynthesis have arisen by gene dupl;,~ation. Based on functionally similar roles of L and D1 proteins and of M and D2 proteins a strong expectation has been that comparative studies of their sequences would find that D1 is phylogenetically most closely related to L and that M is most closely related to D2. Surprisingly, a number of sequence studies have now led to a conclusion that this is not so. Inference from these studies has been that two independent gene duplications have given rise to reaction center proteins of analogous function. In this contribution we demonstrate the problem of making phylogenetic inferences from primary sequence comparisons of PheophytinQuinone proteins. We show that little confidence can be had in the conclusions drawn from recent published sequence studies.

167

Poster i

P-18-006 A STRUCTURAL COMPARISON OF THE PHOTOACTIVE YELLOW PROTEINS (PYP) FROM THREE HALOPHILIC PURPLE PHOTOTROPHIC BACTERIA G. Van Driessche I , B. Samyn 1 , M. Koh 1 , G. Van den Broeck I , T.E. Meyer2 & J. Van Beeumeq 1 1 Dept. of Biochemistry, University of Gent, 9000 Gent, Belgium; 2 Dept. of Biochemistry, University of Arizona, Tucson, AZ, 85721 PYP has photochemical activity remarkably similar to the bacterial rhodopsins, but is a small water-soluble protein unrelated to the rhodopsins. The sequence and three-dimensional structure of the prototypic protein from Ectothiorhodospira halophi/a have been determined previously. We now report the complete amino acid sequences of Rhodospirillum salexigens and Chromatium salexigens PYP. Although isolated from three different families of bacteria, the PYP sequences (125 amino acids) are 70-76% identical and no insertions or deletions are necessary for alignment. This suggests that PYP has the same important function in all purple bacteria from which they were isolated and that most of the amino acid residues are necessary to maintain structure and function. By most standards, PYP would be called a "slowly evolving protein".

P-18-007 Origins of plant and algae organelles as inferred from comparisons of HSP60 and HSP70 stress proteins. Dept. of Microbiology. Faeultad de Ciencias Bioquimicas y Farmac6uticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina. Although a xenogenous, bacteria/ origin for the organdies is now accepted, much less agreement exists on the number of primary endosymbiotie events and specific affiliation of the bacteria that provided for plastids and mitochondria in the distinct eukaryotic lineages [Viale, A., and Arakaki, A. (1994) FEBS Lett. 341, 146-151]. We have constructed evolutionary trees based on highly conserved maeromolecules located on the organdies (including the molecular chaperones HSP60 and HSP70). A monophyletic origin for plant and non-plant mitochondria was inferred from these analyses, being their closest ancestors among the intracellular parasites of the aproteobacterial Ehrlichia/Rickettsia group. HSPs present in plastids from plants, green and non-green eukaryotic algae grouped exclusively with their cyanobaeteria homologs. A model assuming a monophyletic origin for plastids in all photosynthetic eukaryotes is proposed to reconcile conflicting trees obtained from different molecules.

P-18-008 PHYLOGENETIC ANALYSIS OF PHOTOSYNTHETIC REACTION CENTERS OF PURPLE BACTERIA AND GREEN FILAMENTOUS BACTERIA

K.V.P.Nagashima, S.Hanada, A.Hiraishi, K.Shimada & K.Matsuura Dept. of Biology, Tokyo Metropolitan University, Hachioji, Tokyo 192-03, Japan Nucleotide sequences of genes coding for photosynthetic reaction centers (RC) were determined in more than I0 purple bacteria and a new green filamentous bacterium. About 90 per cent of pull and M genes were amplified by polymerase chain reaction with primers designed based on the consensus sequences of these genes, and then cloned and sequenced. Phylogenetic trees based on deduced amino acid sequences of these genes were compared with that of 16S rRNA sequence, and the followings were suggested. (1) RC trees are primarily consistent with the tree of 16S rRNA, meaning RC genes generally came from the line of decent. No implication of a horizontal RC gene transfer was apparent from purple bacteria to green filamentous bacteria. (2) RC genes of beta and gamma subclasses of purple bacteria originated from a horizontal gene transfer from an ancestral Rhodoprillum species in alpha subclass of purple bacteria. (3) RC's without the bound cytochrome subuni% are not a single origin, in terms of the evolutionary deletion of the cytochrome subunit.

168

Poster

P-19-20-003 DIURNAL REGULATION OF SUCROSE PHOSPHATE SYNTHASE (SPS) IN TREES Udav Pathre, A.K. Sinha, P. A. Shirke, and P.V. Sane National Botanical Research Institute, Lucknow 226 001, INDIA.

Poster session 19-20

Enzymology of the photosynthetic metabolism P-19-20-001

-

P-19-20-074

P-19-20-001

The regulation of SPS in relation to diurnal changes in CO2 assimilation rate and sucrose synthesis was examined in P. juliflora. During a diurnal rhythm SPS activity revealed three distinct peaks when analysed for 24 hr period. The first peak appeared immediately after illumination at 8 AM and declined at 10 AM. The second peak was at 12 noon and overlapped with photosynthesis peak. It decreased gradually with decrease in PPFD along with photosynthesis. The SPS activity recovered in dark and showed a third peak at 12 night. These changes involved both alteration in maximal activity as well as changes in activation state of the enzyme as revealed by a change in sensitivity to inhibition by inorganic phosphate (Pi). The relationship between PPFD, assimilation rate and SPS indicated that light activation of the enzyme is determined by a product of carbon assimilation, rather than directly by light. Our results provide direct evidence to the proposal, that SPS exists in kinetically distinct forms in vt'vo, regulated by Pi availabilty and accumulation of sucrose.. Kinetic analysis of the two forms revealed small changes in the Km for fructose 6-phosphate and UDP-glucose, but large changes in the sensitivity to allosteric activator Glucose 6-phosphate and inhibitor, Pi,

P-19-20-004

GAS-EXCHANGE AND U L T R A S T R U C T U R A L ANALYSIS O F TRANSGENIC POTATO PLANTS EXPRESSING A MRNA. A N T I S E N S E C O N S T R U C T T A R G E T E D T O T H E CP-FBPASE

A D P - G L U C O S E L I M I T E D CHLAMYDOMONAS C E L L S PHENOCOPIES OF STARCH SYNTHESIZED PHOTOSYNTHESIS.

Michael Muschak, Susanne Hoffraann-Benning,Heidi Fuss, Jens Kol3mann,Lothar Willmitzer and Joachim Fisahn; Institut fiir Genbiologische Forschung, lhnestr. 63, 14195 Berlin, Germany Gas-exchange measurementS and transmission electron microscopy were performed on transgenie and wild type potato plants. Transgenic potato plantS expressing different levels of the ehluroplastic isoform of fructose-l,6-bisphosphatephosphatase(FBPase) were created via the mR/qA antisense technique. Assimilation rates (A), transpiration rates (E), leaf conductances (g) and intercellular CO2 concentrations (Ci) were calculated from the measured gas exchange parameters. Plants containing a cp-FBPase activity of 40% of the wildtype activity showed no reduction in the assimilation rate at photon flux densities of 500/zrnol m-2 see-1 while at 1000 panol m-2 sec"1 small differences in the assimilation rate were found. Progressive attenuation in the FBPase activity induced a further reduction in the assimilation rates at both light intensifies.Measurementsof the CO2 compensationpointS revealed identical values for the transganic and wildtype plants. Under anoxic conditions a 30% increase in the assimilation rate occurred in all plantS. Respiration rates collected during the dark period were similar among the wildtype and the FBPase-antisense plantS. Using transmission electron microscopy we demonstrated that the size and the number of starch grains were significantly lower in plants with FBPas¢ activities below 40%. A shift to a higher amount of small grana stacks occurred within the chloroplasts of the transgenic potato plants. "This work was supported, in part, by a DFG grant to Joachim Fisahn #Fi 571 1-1."

N. Van den Koornhuyse 1, N. Libessart 1, j. Preiss2 & S. Ball1 Dept of Biological Chemistry, UMR 11,1 C.N.R.S., Univ des Sciences et Technologies de Lille, 59655 France; z Dept of Biochemistry, Michigan State University, East Lansing, MI 48824 USA

P-19-20-002

DISPLAY DURING

A full-length cDNA encoding to the Chlamydomonas large subunit of ADPglucose pyrophosphorylase has been cloned and sequenced. Disruptions in the heterototramer structural genes have been obtained by random integration of the ARG7 gene. The sta6-1::/IRG7 gene disruption leads to a couapse m all conditions tested of both starch (0.05% of wild-type amounts) and enzyme activity. This defect complemented the previously characterized stal-1 mutation that rendered the enzyme less responsive to the allosteric activator 3-PGA. The residual starch in all these mutants is shown to be devoid of amylose despite the presence of GBSS, ~ e amyiose biosynthetic enzyme. The overall mutant amylopectin structure displayed during storage an exact phenocopy of the polysaccharide extracted from wild-type photosynthesizing cells.

P-19-20-005

REGULATORY PROPERTIES OF NADP-MALIC ENZYME FROM WHEAT CHLOROPLASTS

S T O R A G E AND P I t O T O S Y N T H F ~ I S : T H E DUAL N A T U R E O F S T A R C H S T R U C T U R E AND FUNCTION IN CHLAMYDOMONAS.

L.Achnine & A.T.Eprintsev Dept of Plant Physiology and Biochemistry, Voronezh State University, Voronezh 394693, Russia

N. Libessart, N. Van den Kooruhuyse, A. Decq & S. Ball Dept of Biological Chemistry, UMR 111 C.N.R.S., Univ des Sciences et Technologies de Lille, 59655 France

Highly purified NADP-dependent malic enzyme (NADP-ME; EC 1.1.1.40) was obtained from chloroplasts of wheat leaves. It was able to catalyze oxidative decarboxylation of malate (in the presence of Mg 2.) and decarboxylation of oxaloacetate (using Mn2+). Fumarate, 2-oxoglutarate, succinate, oxaloacetate and potassium ions are activators whereas EDTA, tdphosphoglycerate, citrate, AMP, ADP, ATP and NADH are inhibitors of the enzyme. Wheat NADP-ME is localized only in chloroplasts being present as sole molecular form. It is proposed that in wheat which is C3-plant NADP-ME in conditions of NADPH excess in intensive light enrichs chloroplast stroma by endogenous CO2 via reduction of oxaloacetate by NADPmalate dehydrogenase (MDH) and consequent decarboxylation of malate. Kinetic properties of ME and MDH favour this consequence of reactions.

Growth arrested Chlamydomonas cells accumulate a storage pol~csaccharide that bares strong structural and functional ressemblance to higher plant storage starch. We find that log phase photosynthetically active algae accumulate granular glucans whose shape cellular location and structure differs markedly from that of storage starch. That synthesis of th .e~.. two types of polysaccharides are controlled by a.commonb.ut also a specific of genes as evidenced by the identification ot a new cntamyaomonas tai/J*l locus specifically involved in the biosynthesis of storage starch. Mutants defective in STA4 accumulate a new type. of hi .gh..aynylo~ storage starch displaying an altered amylopectin chain size dismbutmn.It ts expected that the dual nature and functions (storage or photosynthesis) of starch synthesis in unicellular green algae will yield new insights as to the biological reasons for the emergence of starch in the eukaryotic plant cell.

169

Poster

P-19-20-006

P-19-20-009

IDENTIFICATION OF A POTENTIAL REDOX-SENSITIVE INTER-DOMAIN DISULFIDE IN THE SEDOHEPTULOSE BISPHOSPHATASE OF CHLAMYDOMONASREINHARDTII

IDENTIFICATION OF A THIOL-REAGENT SENSITIVE CYSTEINE IN THE V I C I N I T Y OF THE ACTIVE SITE OF SORGHUM NADP-MALATE D E H Y D R O G E N A S E

L.E. Anderson 1, H.C. Huppe2, D. Li 1 & F.J. Stevens3 1Biology, University of Illinois at Chicago, Chicago, IL 60607 USA; 2Biology, Queen's University, Kingston, ON, Canada K7L 3N6; 3Mechanistic Biology and Biotechnology, Argonne National Laboratory, Argonne, IL 60439, USA

E. Issakidis. M. Lemaire, P. Decottignies, E. Ruelland, J-P. Jacquot & M. Miginiac-Maslow, URA CNRS 1128. Institut de Biotechnologie des Plantes. B,~t. 630. Universit6 de Paris-Sud 91405 Orsay Cedex France

There are two Cys residues which appear to be close enough to one another to form a disuffide bond in the simulated tertiary structure of the Ch/amydomonasreinhardtiisedoheptulose bisphosphatase (SBPase). These two cysteines are juxtaposed on the nucleotide and carbon substrate domains. Examination of the redox modulation of the algal SBPase confirms that it resembles the higher plant enzyme in being activated by reduction. In the wheat and Arabidopsisenzymes both redox-sensitive Cys residues appear to be located on the regulatory nucleotide-binding domain. Apparently different Cys residues are involved in modulation in the algal and higher plant SBPases. Supported by US NSF, NSERC Canada, US DOE, UIC Research Board, and Argonne National Laboratory.

P-19-20-007 L E V E L E X P R E S S I O N OF R E C O M B I N A N T CHLOROPLASTIC FRUCTOSE-1,6-BISPHOSPHATASE MUTAGENESIS OF ITS REGULATORY SITE HIGH

PEA AND

J.P. Jacouot. J Lopez-Jaramillo °, A. Chueca °, J. Cherfils, S. Lemaire, B. Chedozeau, M. Miginiac-Maslow, P. Decottignies, R. Wolosiuk and J. LopezGorge° URA CNRS 1128. Institut de Bintechnologie des Plantes. Bat. 630. Universit6 de Paris-Sud 91405 Orsay Cedex France, ° CSIC, Granada, Spain The cDNA fragment coding for mature chloroplastic pea fructose-l,6bisphosphatase was introduced by PCR in the expression vector pET-3d, resulting in the construction pET-FBP. The recombinant enzyme could be purified from E. coli BL21 (DE3) cells with a final yield of 20 rag/liter culture. In every respect, recombinant FBPase was similar to the chloroplastic protein and in particular its redox regulatory properties were conserved. In a second series of experiments based on three-dimensional modeling of the chloroplastic protein and sequence alignments, two Cys residues of the recombinant enzyme (Cys173 and Cys178) have been mutated into Ser residues. An always active enzyme which did not respond to thiol reagents as well as to light activation was obtained, confirming the putative regulatory role of the insertional sequence characteristic of the chloroplastic enzyme.

P-19-20-008 THE ACTIVE SITE OF SORGHUM L E A F N A D P - DEPENDENT MALATE DEHYDROGENASE ~ 1 , J.-M. Schmitter 2, M. Miginiac Maslow 1 and P. Decottignies 1 II.B.P., U.A. CNRS 1128, Universit6 Paris-Sud, Bat 630, 91405 Orsay Cedex, France; 2Lab. de Biochimie, Ecole Polytechnique, Palaiseau, France In sorghum leaf, the NADP-dependent malate dehydrogenase (NADP-MDH) is localized in the chloroplast of mesophyll cells, where it takes part in the photosynthetic CO2 fixation pathway. In NAD-MDHs, a His residue is involved in catalysis, in interaction with an Asp, Both amino acids being conserved in all MDHs, we studied their role in sorghum NADP-MDH (His229 and ASp201). We demonstrated that diethylpyrocarbonate, a histidine specific reagent, inactivated NADP-MDH by derivatization at the active site. DEPC-treated NADP-MDH was cleaved with trypsin and the peptides analyzed by sequencing and mass spectrometry: the single amino acid derivatized was His229. This result was confirmed by site-directed mutagenesis. When His229 was replaced by Ala, Asn or Gin, the mutated proteins were totally inactive. All these results demonstrate that His229 is essential for catalytic activity, acting as an acid-base catalyst. Asp201 was changed to Ala or Asn, both mutated proteins exhibited a low residual activity (4%). Their kcat were 6-fold reduced and their apparent Km for oxaloacetate were dramatically increased (50-80 fold). Thus, Asp201 acts in interaction with His229, at the active site of sorghum leaf NADP-MDH.

170

The light-dependent chloroplastic NADP-malate dehydrogenase can be changed into a permanently active enzyme by site-directed mutagenesis consisting in the replacement of four regulatory cysleines (two at the N-terminus and two at the C-terminus of each subunit) by serines or alanines. This permanently active mutant is still sensitive to thiol reagents, suggesting the presence of one or several cysteine(s) at or close to the active site. In order to identify these cysteines, site-directed mutagenesis was performed on the cDNA encoding wildtype sorghum leaf NADP-malate dehydrogenase. The replacement of at least one of the internal cysteines by an alanine yields an enzyme whose activity is much less sensitive to thiol reagents, and which is much more stable in its activated form. This finding suggests that the mutated cysteine has no catalytic function, but is located close to the active site.

P-19-20-010 PHOTOSYNTHESIS, GROWTH AND ALLOCATION AT LOW NITROGEN TRANSGENIC TOBACCO wITH REDUCED AMOONTS OF PHOSPHORIBULOKINASE

FM B a n k s l, D H a b a s h I, M A J P a r r y I, JS K n i g h t 2, JC G r a y 2, M J ' IACR-Rothamsted, UK; 2 C a m b r i d g e U n i v e r s i t y , U K

IN

Paul I

T h e C a l v i n c y c l e a c c o u n t s for m o r e t h a n a t h i r d of t h e n i t r o g e n (N) c o n t e n t of C3 leaves. To test w h e t h e r this i n v e s t m e n t c o u l d be reduced without limiting p l a n t growth, transgenic plants with a l t e r e d a m o u n t s a n d p r o p e r t i e s of d i f f e r e n t p h o t o s y n t h e t i c e n z y m e s are being analysed under different conditions. Transgenic tobacco expressing an i n v e r t e d c D N A c l o n e e n c o d i n g p h o s p h o r i b u l o k i n a s e (PRK) h a v e b e e n g r o w n u n d e r d i f f e r e n t l e v e l s of N n u t r i t i o n to e x a m i n e w h e t h e r i n v e s t m e n t of N in P R K is o p t i m i s e d at l o w N, a n d h o w the b a l a n c e b e t w e e n p h o t o s y n t h e t i c components and growth and a l l o c a t i o n w e r e a f f e c t e d . D a t a s h o w t h a t at l o w N (0.4 mM) P R K is s t i l l in l a r g e e x c e s s u n d e r c o n s t a n t l o w i r r a d i a n c e a n d t h a t a r e d u c t i o n in P R K a c t i v i t y in t r a n s f o r m a n t s limits photosynthesis w h e n i r r a d i a n c e is i n c r e a s e d in t h e s h o r t term. In p l a n t s g r o w n at l o w N at c o n s t a n t low light expressing 5% of w i l d - t y p e PRK a c t i v i t y , p h o t o s y n t h e s i s a n d p l a n t g r o w t h w e r e r e d u c e d c o m p a r e d to w i l d type. T h e d a t a s u g g e s t t h a t e v e n u n d e r l o w N, w h e n a s s i m i l a t e s u p p l y is u s u a l l y in excess, a small reduction in rate of CO 2 a s s i m i l a t i o n d u e to l o w P R K a c t i v i t y c a n l i m i t g r o w t h .

P-19-20-011 STUDIES ON GENETIC ENGINEERING OF RICE Rubiseo* Gan-An Wang, Hai-Bao Chert & Yu Wang Shanghai Institute of Organic Chemistry, Chinese Academy of Sciances 354 Feng Lin Lu, Shanghai 200032, FAX: 86-21-4166128, CHINA To facilitate systematic studies on the structure-functional relationship of Rubisce, a designed structural gene for the large subonit ( rboL ) of rice Kuhisco has bean synthesized by mainly using an ssDNA method [ I-L-B. Chen, et aL, NucL Acids Res. (1990) 18, 871-878 ]. High expression of the synthetic gene in E. coli resulted in producing a genetic engineering rice rbcL in high yield ( 120 nag of pure protein per liter cu~tre ) and opening a way to study the assembly of the rbcL and small subun~ ( rbeS ) to form rice Rubisco in vitro. In the preumce of chemical reagents, reconstitution of the engineering rbcL and the rice rbcS, which was isolated from high expression in E. coli of a designed synthetic gene for rice rbcS [ I~-B. Chen & G.-A. Wang, Curt. Plant Sci. Biotechnol. Attic. (1993) 15, 160-164 ], ledto the formation of a product, which was detected by Western blotting f~om a native polyacrylamide gel to show an electrophoretic mobility same as that of natural rice Rubisco. * Project supported by grant from the High Technology Development Program of China

Poster

P-19-20-012 2-CARBOXYARABINITOL 1-PHOSPHATE (CA1P) DOES NOT R E G U L A T E RUBISCO IN PHASEOLUS VULGARIS IN THE NIGHT Anwaruzzaman, Y. Nakano 1 & A. Yokota Plant Mol. Physiol. Lab., Res. Inst. Innov. Tech. Earth (RITE), Kizu, Kyoto 619-02, Japan; 1Dept. Appl. Biochem., Univ. Osaka Pref., Osaka 593, Japan RuBisCO requires CO 2 and Mg 2÷ for activation before catalysis. Complete activation occurs at pH 8 with more than 100/,aM CO 2 and 10 mM Mg 2+. It has been reported that CA1P, the nocturnal inhibitor of RuBisCO, is synthesized in the night stroma to bind to activated RuBisCO and thereby keep it inhibited in P. vulgaris and many other plants. However, all of these results were collected by assaying RuBisCO after extraction of leaves at pH 8 which is not the night stromal pH and the extraction might have produced artifacts in the results. In the night, stromal pH is 7 and Mg '÷ is present at less than 5 mM. In this study, we examined the interaction of RuBisCO and CA1P /n vivo without extraction of P. vulgaris leaves. The leaves at the predawn was promptly freezed and ground in liq. N 2 to fine powder. After evaporation of N2, the frozen leaf powder was directly transferred to the assay mixture of RuBisCO. RuBisCO was not in the complex with CA1P, but 70% of the total potential activity was detected in this direct assay. T h e / n vi~o analysis of RuBisCO purified from spinach leaves showed that this high activation state even at pH 7.0 was due to the stimulation of activation by inorganic phosphate. These results indicate that CA1P is not the nocturnal inhibitor of RuBisCO in P. vulgar~.

P-19-20-015 CARBOXY-TERMIANL TRUNCATION OF PHOSPHOENOLPYRUVATE CARBOXYLASE BY LIMITED PROTEOLYSIS INFLUENCES CATALYSIS A.S.Bhagwat & G.B.Maralihalli Molecular Biology & Agriculture Division, Bhabha Atomic Research Centre, Bombay-400 085 (India) Maize phosphoenolpyruvate carboxylase (PEPC) was rapidly inactivated by limited proteolysis by trypsin at 37°C. The inactivation which followed biphasic kinetics was completely protected by PEP + Mg 2+. The inactivation results from a fairly specific cleavage of 20 kDa peptide from the enzyme subunit. Limited proteolysis under catalytic condition , although yielded a truncated enzyme of 90 kDa did not affect the catalytic activity. The proteolysed enzyme under these conditions was , however, desensitized to glucose-6-phosphate and malate. No change in the total sulfhydryl group and drastic reduction in the intrinsic fluorescence on limited proteolysis together with the phosphorylation data strongly suggest that trypsin specifically cleaves highly conserved C-terminal portion of the subunit affecting the catalysis and the native enzyme used was a full length protein. A possible role for the C-terminal domain in catalysis and /or structural integrity of the enzyme molecule will be discussed.

P-19-20-013 Herbicidal Effects of a N e w Class of Imidazoleglycerol Phosphate Dehydratase

Inhibitors

of

Plant

Sachiyo Tada, Mika Hatano, Yumiko Nakayama, 'Raymonde Fonnd-Pfister & Daisaku Ohm International Research Laboratories, Ciba-Geigy Japan Ltd., Takarazuka 665 Japan, JCiba-Geigy Ltd., Agricultural Division, CH-4002 Basle, Switzerland cDNAs encoding imidazoleglycerol phosphate dehydratase (IGPD), involved in the His biosynthesis, from Arabidopsis and wheat were expressed in the insect cells by using the baculovirus expression system. The wheat recombinant IGPD was inhibited in a competitive manner by both IRL 1803 and IRL 1856, newly synthesized Iriazole phosphonates, with the Ki values of 10 nM and 23 nM, respectively. IRL 1803 and IRL 1856 inhibited the growth of cultured basil cells with IC50 of 2.9 pM and 41 v-M, respectively. These cytotoxic effects were completely reversed by the addition of His. Plant growth was controlled within 10 days of the treatment (4 kg/ha). We determined IGPD contents and extractable IGPD activity levels of these inhibitor-treated plants. Western blotting showed that the IRL 1803- and IRL 1856-treated plants contained comparable levels of IGPD protein, while no extractable activity was detected in the IRL 1803-treated plants. These results indicated that the loss of the extractable activity in the IRL 1803-treated plants was not due to the decrease in the IGPD protein content but caused by the formation of a stable enzymeinhibitor complex, implicating that IRL 1803 is a slow,binding inhibitor.

P-19-20-014 LICHT ACTIVATION OF SEDOIIEPTULOSE - 1 , 7 - BISPHOPHATASE BY A RECONSYIlux'~ THYLAKOID SYSTEN PROYEIN-~azx~ INTERACTIOIlS

t h y l a k o i d system i n c l u d i n g i s o l a t e d

J.

ON S O M E

Kv~to5

Department Molecular

Cesk6

of Photosynthesis, Institute Biology A S CR, Branigovsk6 31, Czech Republic

of Plant C Z - 3 7 0 12

Bud~jovice,

Activities of both ribulose-l,5-bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxylase (PEPC), oxygen evolution by leaf pieces, and chlorophyll contents o£ t o b a c c o t r a n s f o r m e d with Ri plasmid A4b w e r e studied. Three investigated transgenic regenerant clones cultivated i n v i t r o on a solid agar medium neither showed pronounced morphological diversity nor significantly differed in chlorophyll contents from control plants. However, the transformation caused 27 t o 83 % d e c r e a s e in the leaf oxygen evolution and in both RuBPC a n d PEPC a c t i v i t i e s . Therefore, the transformation brought about a reduction of active PEPC a s well as activable RuBPC amounts in plant tissues. Individual clones of transformants did not differ mutually.

P-19-20-017 C O V A L E N T M O D U L A T I O N O F 6-PHOSPHOFRUCTO-2-KINASE/ F R U C T O S E - 2 , 6 - B I S P H O S P H A T A S E IN SPINACH L E A V E S Ellen G. Rowntree & Nicholas J. Kruger Dept of Plant Sciences, University of Oxford, Oxford, OXl 3RB, UK

C. QUEIROZ-CLARET & J.C. HEUNIER Chimie b i o l o g i q u e , I n s t t t u t N a t i o n a l Agronomique, F-78850 T h i v e r v a l - G r i g n o n , France A reconstituted

P-19-20-016 E F F E C T O F R i P L A S N I D T-DNA T R A N S F O R M A T I O N PHOTOSYNTHETIC CHARACTERISTICS OF TOBACCO

spinach

thylakoids, ferredoxin (Fd), thioredoxin (Td) and ferredoxln-thioredoxin reductaee (FTR) was shown to provide sufficient electron transfer flow to mediate photoactivation of sedoheptulose-l,7-bisphosphataee (SBPase). Progress curves were linear under these conditions, in contrast to the lags observed after reductive activation of SBPase by DTT. In this case the lag was suppressed in the presence of Fd + Td, and addition of FTR to this incubation medium resulted in substantial increase of SBPase activity. Evidence obtained by gel filtration suggests that during the activation process, incubation of SBPase with Fd, Td and FTR, resulted in enzyme-protein or enzyme-enzyme associations and/or conformational changes.

Activities of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase, responsible for the synthesis and degradation of the signal metabolite fructose 2,6-bisphosphate, vary diurnally in spinach leaves. A rapid decrease in the ratio of kinase/phosphatase activity (K/P) following transfer of leaf disks from light to dark at the end of the photoperiod is prevented by treating with okadaic acid. A similar decrease in K/P ratio occurs in desalted extracts obtained from leaves after 9 h illumination. This decrease is unaffected by a combination of protease inhibit.ors, but is prevented by similar concentrations of okadaic acid and calyculin A (Io.5 ~ 15 riM). These results suggest the involvement of a type 2A protein phosphatase. Incubation of darkened leaf disks on sucrose, but not sorbitol, alters the K/P ratio by a process that is inhibited by okadaic acid. We propose that reversible (de)phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, in response to changes in leaf metabolite status, contributes to the control of fructose 2,6-bisphosphate metabolism and thus regulation of carbon partitioning in leaves.

171

Poster

P-19-20-021

P-19-20-018 EXPRESSION OF NADP-MALIC ENZYME ISOFORMS GREENING OF ETIOLATED MAIZE SEEDLINGS,

DURING

THE

(~ados $. Andreo, Maria. F. Ddncovich & Verbnica G. Maunno. Centro de Estudios Fotosint~ticos y Bioquimicos (CEFOBI). Suipacha 531. 2000 Rosario, Argentina.

Two isoforms of NADP-malic enzyme, which are expressed in a differential way during the greenning of etiolated seedlings, were detected in maize leaves. One of the forms, present mainly in etiolated maize leaves, has a molecular mass of 72 kDa and low specific activity in comparison with the form found in green leaves, which has a molecular mass of 62 kDa. The larger form of the enzyme has higher K m values for NADP and malate and lower pH optimum. The antibodies raised against the "green" form react with the "dark" form, suggesting a high degree of homology between both forms. The "green" form of the enzyme increases several-fold during greening of dark-grown seedlings, while the "dark" form remains practically constant after a transient increase. Northern blot analysis using a specific probe reveals the increase of a 2.2 kb mRNA. This study was also made in separated mesophyll and bundle sheath calls. Southern hybridization analysis with genomic DNA suggests the presence of more than one gene encoding NADP-malic enzyme in maize.

THE LIGHT-TRANSDUCTION PATHWAY LEADING TO PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE (PEPCK) ACTIVATION IN C4 MESOPHYLL PROTOPLASTS Giglioli N. 1, Pierre J-N. 1, Brown S. 2, Vidal j.1 llnstitut de Biotechnologie des Plantes, Universit6 Paris XI, Orsay, France; 2 Institut des Sciences V6g6tales, avenue de la Terasse, Gif-sur-Yvette, France. In leaves of C4 plants, PEPC is regulated by a light-dependent phosphorylation, which increases its catalytic activity (2 fold at pH 7.3) and decreases its sensitivity to the retro-inhibitor, L-malate (2.7 fold at pH 7.3). In Di~itafia sana'uinalis or Sorghum mesophyll cell protoplasts we have now identified some components o f the cascade leading to the stimulation of the PEPCK, namely 1) an alkalinization of cytosolic pH Coy ammonium chloride or by the photosynthetic metabolite 3-phosphoglyceric acid), 2) the activation of a phospholipase C that produces the second messenger inositol-l,4,5 triphosphate (IP3), 3) an increase of cytosolic calcium via the opening of a IP3gated tonoplast calcium channel, 4) activation by this calcium flow of a putative Ca2+/calmodulin-dependent protein kinase, as indicated by the strong inhibition of PEPC phosphorylation by W7 and other calmodulin antagonists, 5) finally, a change in the turnover of the calcium-independent PEPCK (or an activation factor), as suggested by the use of the protein synthesis inhibitor cycloheximide, which severely blocked PEPC phosphorylation in situ.

P-19-20-022

P-19-20-019 I S O L A T I O N OF c D N A S FOR N A D P - M A L I C ENZYME I S O F O R M S F R O M A CAM PLANT, A l o e a r b o r e s c e n s * Hidao Honda and Hiroaki Shimeda life Science Instititue, Mitsui Toatsu Chemicals, Inc., Togo 1144, Mobara 297, Japan Crassulacean acid metabolism (CAM), a specialized mode of photosynthetic carbon assimilation, is a functional adaptation to arid environments and a CO2concentrating mechanism. Atmospheric CO2 is fixed at night and subsequently stored as malate il the plant vacuole, then in many CAM plants it is utilized by NADP-malic enzyme (NADP-ME) activity during the daytime. In order to isolate NADP-ME cDNAs from a monocot CAM plant, A/oe arborescens, we carried out RT-PCR using primers synthesized based on the conserved regions of other known NADP-ME genes. An amplified cDNA fragment (0.5 kb) was sequenced and indicated a homology to the rice NADP-ME. Using this fragment as a probe, cDNA clones were isolated from a library constructed from aloe green leaves. Since these clones were classified into two groups depending on their physical maps, nucieotide sequences of the representative clones from each group were analyzed. ORFs in these clones consisted of 586 (AME1), 592 (AME2) amino acid residues, respectively, which showed high similarity with each other (90%). They also showed high level of identity ranging over 7684% (AME1) and 77-84% (AME2) to other known plant NADP-ME's. These results suggests that the aloe genome contains mere than two copies of NADPME genes. Southern blot analysis of the aloe genome confirmed this result. •This work was supported by PEC (Petoleum Energy Center of Japan).

P-19-20-020 THE EFFECT OF pH ON THE COVALENT AND METABOLIC CONTROL OF C4 P H O S P H O E N O L P Y R U V A T E CARBOXYLASE FROM SORGHUM LEAF. Echevarria C., Pacouit V*., Bakrim N.*, Osuna L., Delgado B., Arrio-Dupont M.*, Vidal J.* Lal~oratodo de Fisiologia Vegetal, Sevilla, Spain. *institut de Biotechnologie des Plantes, Universit~ Paris XI, Orsay, France. The influence of pH on the in vitro activity and regulatory properties of Sorghum leaf C4 phosphoeno/pyruvate carboxylase (PEPC) was investigated with respect to the pbospborylation status of the enzyme. Pbosphorylation induces an increase in the catalytic activity (4-5 fold and 2-fold at pH 7.1 and 7.3, respectively) and in the IC50 for L-malate (1.1 and 2.7-fold at pH 7.1 and 7.3, respectively) of PEPC. The positive effector glucose 6-P acted similarly to phosphorylation, however, a combination of both factors led to a much larger increase in the IC50 for L-malate (15 mM) than that observed by a single factor. In reconstituted assays performed at pH 7.1 or 7.3, in the presence of a protein-kinase, L-malate decreased the phosphorylation rate of PEPC while glucose-6P (5 rnM) plus PEP (3raM) relieved the inhibition. Together with previous findings indicating that the photoinduction of PEPC protein kinase was strictly dependent on cytosolic alkalizafion in mesophyll cell protoplasts, the present data support the view that cytosolic pH is an important component of the overall regulation of PEPC in C4-photosynthesis. Finally, the role of phosphorylation appears to be a mechanism to amplify the regulatory influences of pHc and glucose-6P on PEPC activity.

172

REGULATION OF RUBISCO BY INHIBITORS DIFFERS IN WILD-TYPE AND TRANSGENIC TOBACCO EXPRESSING LIMITING AMOUNTS OF PHOSPHORIBULOKINASE MS Paul I, pJ AndraloJc I, F M B a n k s I, D Habash 1, J Ryan 1, M A J P a r r y I, JS Knight 2, JC Gray2, D W L a w l o r I, AJ Keys I 11ACR-Rothamsted, UK; 2University of Cambridge, UK Rublsco activites were measured in tobacco transformed with an inverted phosphorlbulokinase (PRK) cDNA which reduced PRK activity 20-fold and assimilation rate by twofold. Specific Rubisco activities were higher in transformed plants than in wild type. This difference in activity could be accounted for by differences in amounts of a tight-blndlng inhibitor of Rublsco present in leaves during the light. The retention time of the inhibitory component on HPLC anion exchange columns was distinct from CAIP and suggested the compound was a sugar blsphosphate. Treatment of the inhibitory fraction with alkaline phosphatase abolished inhibitory activity; treatment with CAiP-phosphatase partially removed inhibitory activity. Both wild type and transformants contained CAIP at night and in transformants a further inhibitory compound was also present during the night. The work demonstrates the power of transgenic plants in exposing novel aspects of plant metabolism.

P-19-20-023 MOLECULAR CHARACTERIZATION OF H=O2-RESISTANCE SYSTEM OF FRUCTOSE-1,6-BISPHOSPHATASE AND NADPGLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE IN S y n e c h o c o c c u s PCC7942 T. Takeda. M. Tamoi, T. Ishikawa and S. Shigeoka, Dept. of Food and Nutrition, Kinki Univ., Nara 631, Japan The photosynthetic CO2 fixations of Euglena, Chlamydomonas and cyanobaeteria are resistant to H202, which is due to the insusceptibility of the algal thiol enzymes to H202 in the Calvin cycle. Here, we isolated and characterized genes encoding fructose-l,6-bisphosphatase (FBPase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Synechococcus PCC7942 (S.7942). The 5.5-kbp Bam HI fragment contained a 1017-bp of ORF encoding the FBPase. The deduced amino acid sequence shared about 40% identity with FBPases from various sources, and was similar to those of cytosol-form and E.coli rather than that of chloroplast-form. The FBPase lacked the extra amino acid sequence present in chloroplast FBPase, including cysteine residue (Cvs-174 to Cvs-179) responsible for light-dependent regulation of the chloroplast enzyme. The recombinant enzyme isolated from E. coil was also resistant to 1 mM H202. The 6.6-kbp Barn HI fragment contained the GAPDH gene, which showed approximately 60% identity with NADP-GAPDHs of higher plants. The two specific cysteine residues, shown to be involved in the redoxregulation of chloroplast GAPDH were lacked in the S. 7942 GAPDH.

Poster

P-19-20-027

P-19-20-024 MOLECULAR CHARACTERIZATION FROM EUGLENA GRACILIS Z

OF ASCORBATE

PEROXIDASE

CIRCADIAN RHYTHM OF MALATE CRASSULACEAN ACID METABOLISM

REMOBILIZATION

IN

Dept. Food and Nutrition, Kinki Univ., Nara 631

H. Arata, Y. Furutani & M. Nishimura Dept. Biol., Fac. Sci., Kyushu Univ., Fukuoka 812-81, Japan

Ascorbate peroxidese (AsAP) plays a key role in the removal of H202 in higher plants and algae. In higher plants, the enzyme exists as two isozymes, chloroplastic and eytosolic forms. We have previously demonstrated that in Euglena, which lacks catalese, AsAP is only located in cytosl, but not in chloroplasts. Euglena AsAP shows the similar enzymlogical properties as cytosolie form of plants and has its own properties (e.g. elimination of lipid hydroperoxide like glutathione peroxidese). In order to elucidate molecular characterization of Euglena AsAP, we raised monoclonal antibodies against purified AsAP. All monoclonai antibodies (EAP 1-8) showed high homology to cytosolic AsAP from plants, as judged by western blot analysis. EAP 1 was also specific for the ehloroplastic AsAP from spinach leaves. The amino acid sequences of the N-terminai region of Euglena AsAP showed little homology with those of cytosolic and chloroplastic AsAP from plants. However, the sequences of some endogenous peptides digested by peptidase showed high degrees of homology with those of cytosolic AsAP. The analysis of the isolated cDNA clone probed with monoclonal antibodies and deduced nucieotide from peptide is in progress.

CAM p l a n t s show p e r s i s t e n t circadian r h y t h m s of c a r b o n metabolism under constant environmental conditions. Periodical phosphorylation of phosphoenolpyruvate carboxylase is one of the m e c h a n i s m s that m a i n t a i n the e n d o g e n o u s r h y t h m . We investigated if any other process was regulated by the circadian clock, focusing on the remobilization of malate in the vacuole. A CAM plant, Kalancho~ daigremontiana, evolves oxygen in the absence of external CO2 as a result of photosynthesis with malate as the carbon source. Under the 12-hr light/12-hr dark regime, the rate of oxygen evolution in the CO2-free condition is low during the dark period and increased after the start of the light period. This rhythm persisted under constant light conditions. The peak of the malate-dependent oxygen evolution appeared after the vacuolar malate concentration started to decrease. These results indicate that the processes involved in the malate remobilization (translocation of malate across the vacuolar m e m b r a n e or decarboxylation of malate, or both) are regulated by the endogenous circadian clock.

T. ISHIKAWA. T. TAKEDA, and S.SHIGEOKA,

P-19-20-025 ENZYMATIC AND MOLECULAR CHARACTERIZATION OF CATALASEPEROXlDASE IN Synechococcus PCC 7942 S. Shineoka, T. Takeda, T. Ishikawa and M. Mutsuda Dept. of Food and Nutrition, Kinki Univ., Nara 631, Japan The H202-seavenging system of cyanobacteria is divided into two groups, the first group contains ascerbate peroxidase anti catalase, and the second group only cataiese. Here, we studied the enzymatic and molecular characterization of catalaseperoxidase from Synechococcus PCC 7942. Catalase-peroxidase has been purified to electrophoretic homogeneity by ion-exchange chromatography, hydrophobic chromatography and gel filtration. It had a Mr ot 150 kDa and consisted of two identical subunits of Mr of 75 kDa. The/d'n value lor H202 was 5.1 mM. The inhibition of the enzyme activity by NaN3 and KCN and the absorption spectrum with a peak at 403 nm showed that the enzyme was a hemoprolain. 3-Amino-l,2,4-triazole, a specific inhibitor of typical catalase, had no effect on the enzyme activity. A DNA fragment was amplified by PCR, using primers synthesized from consensus sequences of cataiase-peroxidase genes from prokaryotie cells and used as a probe to screen a genomie library. The gone for cataiase-peroxidase contained a 2160-bp of an ORF, encoding a protein comprising 720 amino acid residues with a mol wt of 79 kDa. The deduced amino acid sequence was 50-65% identical with those ot catalese-peroxidase from prokaryotic cells.

P-19-20-026

P-19-20-028 BINDING OF LIGANDS TO THE GLUCOSE-6-PHOSPHATE ALLOSTERIC SITE IN MAIZE-LEAF PHOSPHOENOLPYRUVATE CARBOXYLASE A. Tovar-M6ndez, H. Yampara-Iqulse, C. M6jica-Jim6nez & R. A. MufiozClares. Dept of Biochemistry, Faculty of Chemistry, UNAM. 04510 M6xico City, M6xico. Phosphoenolpyruvate carboxylase [PEPC] of leaves of C4-plants is a regulatory enzyme subjected to allosteric control. Its activity is increased by glucose-6-phosphate [glue-6-P] and some phosphoenolpyruvate [PEP] analogues, which are supposed to bind to the same allosteric site than gluc-6-P. At present, little is known about the full relationship between binding and observed steady-state kinetics. We desensitized the maize-leaf enzyme to gluc6-P by incubating it with pyridoxal 5'-phosphate, and we found that the rate and equilibrium point of desensitization are dependent on the concentration of gluc-6-P in the incubation medium. Taking advantage of this, we measured the binding of ligands to the glu-6-P-allosteric site and studied the factors which modulate this binding, such as pH, Mg2+, PEP, malate, glycine, glycerol and polyethyleneglycol, in an attempt to further extend our understanding of the allosteric mechanism of this enzyme.

P-19-20-029

REDUCTION OF PHOSPHOGLYCERATE KINASE IN TRANSFORMANTS OF C H L A M Y D O M O N A S R E I N H A R D T I I

THE NATURE AND ALTERNATE FATES OF THE RIBULOSE 1,5-BISPHOSPHATE (RuBP) C A R B O X Y L A S E / O X Y G E N A S E (RUBISCO) OXYGENATION INTERMEDIATE.

h ~ Jon Y. Suzuki, Kaom Kitayama & Robert K. Togasaki Dept. of Biology, Indiana University, Bloomington. IN 47405. USA

Mark R. Haroel, Yuh-Ru Chen, and Fred C. Hartman. Protein Engineering Program, Biology Div., Oak Ridge Natl. Lab., Oak Ridge, TN 37831, USA.

Transformants for an antisense gene of the chloroplastic phosphoglycerate kinase (PGK) of Chlamydomonas reinhardtii (C. reinhardtiO were isolated by co-transformation of an arginine auxotroph (CC-425) with the transformation selection vector pArg (argiainosuccinate lyase) and an antisense POK construct using the arylsulfatase promoter of C. reinhardtii One transformaat (strain AS107) was found to have a 30% reduction of PGK specific activity due in part to an observed 85% reduction of PGK protein level compared to the parent strain. Although the transcripts from the integrated antisense PGK construct could not be detected, the level of the endogenous PGK transcript was decreased by 50%.

Rubisco's carboxylation efficiency is limited by the enzyme's competing oxygenase activity. Two interacting, intersubunit active-site side chains (Lys329 and Glu48) affect these two activities: both facilitate gaseous substrate addition to the RuBP-enediol, stabilize reaction intermediates, and mitigate improper processing of intermediates. Mutagenesis at either position in R. rubrum Rubisco debilitates carboxylation efficiency and induces formation of novel side products. Both E48Q and K329A produce D-glycero-2,3-pentodiulose 1,5-bisphosphate by elimination of H202 from a peroxy adduct of RuBP. Whereas K329A rearranges the dicarbonyl to 2-carboxytetritol 1,4-bisphosphate (CTBP), E48Q degrades it via C-C scission. These results provide proof for the putative peroxyketone oxygenation intermediate and show that this intermediate, as well as the diearbonyl, can suffer multiple fates. Although wild-type Rubisco also converts the isolated dicarbonyl to CTBP, neither compound is found following wild-type turnover of RuBP. Thus, the peroxy intermediate is stabilized for normal processing, despite its negative impact on carboxylation. Lys329 and Glu48 facilitate forward processing of the intermediates in both pathways. (Supported by USDOE under contract DE-AC0584OR21400 with Martin Marietta Energy Systems, Inc.)

173

Poster

P-19-20-030 ENZYMOLOGICAL EVIDENCE FOR INVOLVEMENT OF A CALCIUM-DEPENDENT PROTEIN KINASE IN REGULATORY PHOSPHORYLATION OF PEP CARBOXYLASE IN MAIZE

K. Izuil~_N, Yabuta1, N. Ogawa1, Y. Ueno 1, T. Furumoto 1, Y. Saljo 1, S. Hata 1, & J. Sheen2 1Dept of Agricultural Biology, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan; 2Dept of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA The activity of maize PEPC for C4 photosynthesis is known to be lightregulated through reversible phosphorylation of Set-15. Previously we suggested the participation of a calcium-dependent protein kinase (CDPK) in this phosphorylation (Ogawa & Izui, 1992, FEBS Lett. 302, 86-88). It was revealed by gel-fdtration chromatography that both CDPK and calcium-independent PK with molecular masses of 100 kDa and 30 kDa, respectively, are capable of phosphorylating PEPC. We purified CDPK near homogeneity and characterizxxt. The molecular size of its subunit detected through autophosphorylating activity was about 50 kDa as estimated from SDS-PAGE. The HPLC analysis of phosphorylated peptide liberated from PEPC by lysylendopeptidase, and the experiments using a synthetic peptide (Metl-Gln22 of maize PEPC) and several mutagenized PEPCs of maize (S15D, K12G and K12N) as substrates showed that this CDPK phosphorylates Ser-15. To identify this PK, we cloned several cDNAs encoding CDPKs.

P-19-20-033 TRANSFORMATION

OF

THE

Arabidopsis

rca

MUTANT

WITH

ALTERED

Arabidopsis RUBISCO ACTIVASE cDNA BY V A C U U M I N F I L T R A T I O N Russ Kallis t & Archie R. Portis, Jr. 2 tSchool of Life Sciences, University of Illinois and 2USDA/ARS Photosynthesis Research Unit, Urbana, IL 61801 Vacuum infiltration of the Arabidopsis rca mutant has resulted in 36 putative transformants. Binary vector pCGN1547 carrying an altered Arabidopsis rubisco activase cDNA coding for 42kD rubisco activase (QlllD), and the NPTII gene coding for kanamycin resistance was transformed into Agrcbacteriumtumefaciens EHA101. Agrobacteria with this construct were introduced into bolting Arabidopsis rca plants by vacuum infiltration (Bent et al. Science 265:1856-1860, 1994). Seeds from infiltrated plants were plated on selection media containing 50mg/L kanamycin and germinated at 100-150 ~E m "2 s "~ light and 1% CO 2. Seedlings that grew secondary leaves were transferred to a second selection plate and following the emergence and expansion of several secondary leaves, putative transformants were transferred to ambient CO 2. After several more days on antibiotic selection plates, putative transformants were transferred to soil. The 42kD rubisco activase polypeptide was detected in soluble leaf extracts from 33 of 36 putative transformants by immunoblotting using spinach rubisco activase polyclonal antibodies.

P-19-20-034

P-19-20-031 CARACTERIZATION OF RECOMBINANT AND NATIVE RAPESEED CHLOROPLAST FRUCTOSE-1,6-BISPHOSPHATASE

ON THE SEARCH FOR C4-SPECIFIC STRUCTURES IN PLANT PHOSPHOENOLPYRUVATE CARBOXYLASES

Roberto Rodri~uez-Suarez and Ricardo A. Wolosiuk Instituto de Investigaciones Bioquimicas (Fundaci6n Campomar, FCEN-UBA, IIBBA-CONICET), Antonio Machado 151, (1405) Buenos Aires, Argentina.

Per Svens$on.1 Oliver BlUing2 and Peter Westhoff2 1. Dept. of Biochemistry, Arrhenius Laboratories for Natural Sciences Stockholm University, S-106 91 Stockholm, Sweden 2. Institut fiir Entwickiungs- und Molekularbiologie der Pflanzen, HeinrichHeine-Universit~it DUsseldorf, D-40225 Dtisseldorf, Germany

Escherichia coli was transformed with a plasrnid containing the DNA sequence that coded for the mature form of rapeseed chloroplast fructose-l,6-bisphosphatase (CFBPase). Following gene expression, the recombinant enzyme was purifed to apparent homogeneity. In parallel, the modification of the purification procedure provided an homogeneous preparation of CFBPase from rapeseed leaves. Gel filtration experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the recombinant and the native rapeseed CFBPase were tetramers [160 kDa] comprised of four identical subunits. Moreover, the Edman degradation evinced an unique N-terminal amino acid sequence [A-V-A-A-D-A-T-A-E-T-KP-] in both enzymes. The expression of the gene that encodes rapeseed CFBPase in Escherichia coli produces high yields of a polypeptide that assemblies into a functional enzyme similar to the counterpart isolated from leaves.

The evolution of C4 photosynthesis are thought to have occurred several times independently in angiosperms. The enzyme phospboenolpyruvate carboxylase (PEPCase) is of special interest for the elucidation of the molecular event in the evolutionary process since it is a key enzyme of the C4 cycle, but also plays a central role in basic metabolism of C4 as well as C3 plants. The isoform of PEPCase that are involved in the C4-cycle show distinct kinetic and regulatory properties when compared with the C3-isoform. However, it is not known which changes in the primary structure of the C3 ancestor gene that were necessary to bring about a functioning PEPCase in the C4 photosynthetic pathway. In the search for the domains that are responsible for the C4 specific properties of PEPCases we have cloned two closely related ppc cDNAs into a bacterial system. One eDNA stems from the C3 plant Flaveria pringlei and the other comes from the C4 plant F. trinervia. We will present results concerning the properties of the bacterially expressed plant enzymes and of chimeric enzymes obtained by ligating certain parts of the two cDNAs with each other.

P-19-20-035

P-19-20-032 cOMPENSATION FOR REDUCED A N T I S E N S E ARABI~PSlN.

ACTIVABE

CORTENT

IS

RUBISCO

~CTIVASE

Nancy A. Eckardt ~ and Archie R. Portis, Jr. ~ *Department of Plant Biology, University of Illinois and 2USDA/ARS Photosynthesis Research Unit, Urhana, IL 61801, USA. Rubisco activase (RA) catalyzes the light activation of rubisco in vivo. We are investigating the role of R A i n light regulation of photosynthesis using Arabidopsis transformed w i t h R A a n t i s e n s e cDNA. Plants with 30% and <10% wild-type (wt) RA content were grown under high (600 ~mol photons m -z s -t) and low (200 ~mo! m "2 s -z) light. Plants with < 1 0 % w t R A s h o w e d r e d u c t i o n s in growth and net photosynthesis (A) at all light levels. Plants with 30% w t R A showed significant reductions in growth and light-saturated A only when grown under high light. Light-saturatedA as a function of RA content for high-light grown plants described a sigmoidal as opposed to a linear response, suggesting that plants with 30% wt RA compensated in some way for the effect of reduced RA on photosynthesis. Leaf extracts of high-light grown plants showed an increased ATP/ADP ratio in 30% wt RA plants compared to wt. Since RA activity is dependent on the ATP/ADP ratio, the 30% wt RA plants may have increased RA specific activity. We are continuing to test this hypothesis in further experiments with both high- and low-light grown plants.

174

Identification and Phosphate Exchange

Characterlsation Activity in Leaf

of a CA-CAIP Extracts from

French Bean (Phaseolus vulaarls L.) PJ Andralojc, JE Ryan, MAJ Parry and AJ Keys IACR-Rothamsted, Harpenden, Herts, AL5 2JQ, UK. 2-Carboxyarabinitol-l-phosphate ( C A I P ) is a naturally occurring, tight-binding inhibitor of R u b i s c o , a n d is p r o d u c e d a t n i g h t . T h e r e a p p e a r t o b e t w o r o u t e s of C A I P s y n t h e s i s in v l v o . T h e f i r s t is t h e c o n v e r s i o n of recently assimilated c a r b o n i n t o C A I P , in r e s p o n s e t o a f a l l in l i g h t intensity. The second involves the phosphorylation of a p r e - e x i s t i n g p o o l o f 2carboxyarabinitol (CA), in t h e d a r k . D u r i n g attempts to identify the enzyme responsible for phosphorylating CA, a C A - C A I P p h o s p h a t e exchange activity was identified, by which the phosphate moiety of [12C]CAIP was transferred to [x4C]CA. The properties and implications of this activity will be presented and the associated enzyme identified.

Poster

P-19-20-036 CO2/O2 SPECIFItLt'Aq6 OF SITE-DIRE~-A'J~u M U T A N T S OF RUBISCO

P Madgwick, S Parmar, AJ Keys and M~I_P~q~DC~ Biochemistry and Physiology Department, IACRRothamsted, Harpenden, Herts. AL5 2JQ,r UK. Mutagenesis /n vitro of the gene encoding the large subunit of Rubisco (EC 4.1.1.39) from Anacystis n/du/ans has been used to modify catalytic properties. Residues at the C-terminal end of loop 6 of the ~/a barrel structure of the large subunit influence specificity towards the gaseous substrates, CO2 and Ch. The introduction of a bulky, negatively charged side chain, by site-directed mutagenesis of alanine 340 decreased specificity by 17% but the introduction of a bulky, basic sidechain increased the enzyme's specificity for CC5 by up to 18%. Alanine 340 does not interact directly with the transition state analogue CABP and effects on the reaction of the enediolate intermediate with the gaseous substrates must be indirect.

P-19-20-037 MOLECULAR CHARACTERIZATION OF RECOMBINANT ASCORBATE PEROXIDASE OF CUCUMBER K. Amako & K.Asada Res. Inst. Food Sci., Kyoto University, Uji., Kyoto 611, Japan. Ascorbate peroxidase (APX) plays a role in scavenging of hydrogen peroxide in plant tissues. We isolated cytosolic APX gene from cucumbe% e,x. p ~ r~mbinant ~ (rcAPX) in Kco/i, and analyzed of vvAPX. Purifim rcAex saowee me same characteristics as native cAPXs from tea, pea and spinach leaves. Inte~estingly, rcAPX showed catalase activity as well as thyroid peroxidase. Compound I of rcAPX showed the a-peak at 413 nm, which suggested that one oxidation equivalent belong to an amino acid residue, not to the porphyrin ring, as in the case of yeast Cyt c peroxidase (CCP). Excess hydrogen peroxide caused rapid disappearance of the or-peak, concomitantly decrease of APX and catalase activities. The conse~eed Cys and Trp residues were replaced by site-directed mulagenesis to Ser (C32S) and Phe (W41F, W180F) or Tyr (W180Y), respectively. C32S was active, and the Cys was supposed not to participate in the catalytic cycle of APX. W41F was active but W180F and W180Y were not, which were agreed with the case in CCP [Fishel et al. (1987) Biochemistry, 26, 351; Manro et al. (1988) Biochemistry, 27, 6243], and W180 was expected to the amino acid radical in Compound I of APX.

P-19-20-038 NON-DESTRUCTIVE MONITORING OF NAD(P)H IN INTACT ISOLATED CHLOROPLASTS AND LEAVES ~ , F. Morales, E. Langrand, M. Bergher & I. Moya. LURE - CNRS, Bat 209 D, 91405 Orsay, France We wanted to estimate the redox state of NAD(P)H in chloroplasts and leaves by using UV-excited blue-green fluorescence (BGF). Therefore, we investigated the spectral and lifetime characteristics of BGF of leaves and chloroplasts and used a fluorimeter insensitive to actinic illumination to follow simultaneously the changes in BGF and red (chlorophyll) fluorescence. We found that in intact isolated chloroplasts, BGF originated indeed from NAD(P)H. This fluorescence depended on a soluble stromal component, and the excitation and emission spectra, when corrected for reabsorption, corresponded to that of NAD(P)H. Variable BGF was increased by Photosystem I light, and followed the changes of the photochemical quenching coefficient of chlorophyll fluorescence under a variety of light regimes and metabolic states. The variable BGF was also present in mesophyll but was hardly seen in intact leaves. Time resolved fluorescence spectra at different level of leaf organization showed that the absence of variable BGF in outdoor grown leaves was due to the presence of epidermis, it's strong contribution to the detected fluorescence, and the effect of shading. Still, NAD(P)H fluorescence could be detected even in intact leaves in the long-lived component of the nanosecond fluorescence decay.

P-19-20-039 SPECTRAL AND TIME-RESOLVED SIGNATURES OF S U G A R B E E T (BETA V U L G A R I S L.) L E A V E S F O R T H E C H A R A C T E R I Z A T I O N OF T H E I R P H Y S I O L O G I C A L S T A T E F. Morales 1, Z.G. Cerovic 1, Y. Goulas 1, R. Belkhodja 2, A. Abadfa 2, J. Abad/a 2 & I. Moya 1 1LURE - CNRS, BAt 209 D, 91405 Orsay, France; 2CSIC, Aula Dei Experimental Station, Apdo 202, 50080 Zaragoza, Spain When excited with UV light s u g a r beet (Beta vulgaris L.) leaves emit red (chlorophyll) fluorescence and blue-green fluorescence (BGF). Iron deficiency was used to change the physiological state of the leaves. Red and BGF were affected by iron deficiency. This nutritional stress impaired the photochemical efficiency of photosynthesis. Evidence comes from a decrease of the (F'm - Fs) / F'm red fluorescence p a r a m e t e r , which was well correlated with the q u a n t u m yield of oxygen evolution and with the m e a n fluorescence lifetime, calculated fi-om the decay after single shots of a picosecond laser. Spectral and time-resolved m e a s u r e m e n t s of BGF indicated t h a t ferulic acid, flavins, and nicotinamide nucleotides contribute to this fluorescence. Flavins accumulate in response to iron deficiency, changing the BGF. Its use for an early detection of iron deficiency deserves investigation.

P-19-20-040 ISOLATION OF MULTIPLE FORMS OF PHOSPHORIBULOKINASE AND GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM A MUTANT OF CHLAMYDOMONAS REINHARDTII. L~Axilaa, B.Gontero, S. Lebreton and J. Ricard. Univ Paris VII. CNRS, Tour 43. Institut J.Monod. 2, place Jussieu. 75 251 Paris Cedex 05. France. A mutant strain of Chlamydomonas reinhardtii, has already been characterised in which the arginine 64 of phosphoribulokinase (PRK) has been replaced by a cysteine residue. In this strain, we have isolated dimeric PRK and tetrameric glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Beside these two forms, two other forms are eluted in the washing step of an anion exchanger column. The PRK and the GAPDH not retained on the DEAE column have a higher molecular mass than the dimeric PRK and the tetrameric GAPDH. This result is therefore suggestive of a different degree of polymerisation for these two enzymes. At this moment, it is not clear if there is a complex between these enzymes as it is described for the wild type (WT). Contrary to the bi-enzyme complex purified in the WT, these high molecular forms of PRK and GAPDH are not depolymerised upon very high concentrations of dithiothrei'tol (up to 20 mM). Nonetheless, the high molecular form of PRK has a latent activity and is activated by thioredoxin. The existence of a possible complex between the mutated PRK and GAPDH is discussed.

P-19-20-041 PHOSPHORIBULOKINASE AND GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM CHLAMYDOMONASREINHARDTII FORM A BI-ENZYME COMPLEX. B.Gontero, S. Lebreton, L. Avilan and J. Ricard. Univ Paris VII. CNRS, Tour 43. Institut J.Monod. 2place Jnssieu. 75 251 Paris Cedex 05. France. We have isolated and purified a chloroplast enzyme complex from the green alga, Chlamydomonas reinhardtii. This complex is made up of two enzymes, the phosphoribulokinase (PRK) and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH). By gel filtration, the molecular mass of this structure is found to be around 320 kDa. Under native electrophoresis, using silver nitrate staining, this complex seems to be homogeneous. The same band is recognised both by spinach PRK and GAPDH antibodies. Under denaturing conditions, in SDS electrophoresis, two very closed bands corresponding to molecular masses of 38 and 40 kDa, are detected corresponding to PRK (R) and GAPDH (G) subunits. The activity of PRK is latent in the complex and increases with reducing conditions (in presence of dithiothreRol). Nonetheless, too high concentrations of reducing agent lead to complete dissociation of this complex. The NADPH dependent activity of GAPDH also increases upon reduction while the NADH dependent activity is not affected. Possible structures suggested for this bi-enzyme complex are 6R2G, 6G2R, 4R4G.

175

Poster

P-19-20-042 )i%LATE D ~ 4 Y D ~ E

FROM GREEN

B. Synstad, K. Naterstad, O. Emmerhoff, R. Sirev~q Dept. Biology, University of Oslo, Box 1066 Blindern, Norway

0316 Oslo,

M~late dehydrogerksse (~E]H) from the thezmophilic green gliding bacterit~ Chlorof]exus ailrantiacus, the mesophilic green sulfur bacteritml ..ChlorobitEa vibrioforme, and the moderate thezmophilic .Cb. tepidum have been purified and characterized, and the corresponding genes isolated, s e ~ and characterized. The primary structure of these enzymes are more similar to ~ t e dehydrogenase (IZX4) from other eubactezJ.al sources than to from such .sources, hilt do have the shine acid residues ~eristic of the ~ catalytic site. The m~% genes were cloned in the expression vector pErlla and also in pUC19, and in both cases large ~ t s of the protein were produced from the foreign genes in Escherichia coli. The expression systems have been effectively used as a tool to investigate the role of specific DNA s ~ c e s and amino acid residues in the regulation, structure and fonction of the various ~ffX~s, and in the thezxnophilic properties of ~ from C,, aurantiacus and

P-19-20-045 Carboxylation and oxygenation of ribulose-l,5bisphosphate ( R u B P ) : a m o d e l on t h e l e v e l o f t h e partial reactions. Juta Vii1 Institute of Experimental Biology,Estonian Academy of Sciences,Harku, Harjumaa, EE3051 Estonia A mathematical carboxylation and

model of the kinetics of oxygenation o f RuBP i s p r o p o s e d

for studying the regulation of the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The model is b a s e d o n r a t e c o n s t a n t s o f the p a r t i a l reactions of the processes and on concentrations of substrates, intermediates, and products. The aim of the model is to e l u c i d a t e the contribution of changes in t h e s e p a r a m e t e r s to the integrate response of carboxylation and oxygenation and to e v a l u a t e the parameters inaccessible for direct measurements. This allows to d i s t i n g u i s h between changes in t h e a c t i v i t y of R u b i s c o due to s h i f t s in a f f i n i t i e s to s u b s t r a t e s o r p r o d u c t s or in the c o n c e n t r a t i o n of competent reaction centres.

C. tepidmn in p a r t i c u l a r .

P-19-20-043 DETERMINATION OF REACTION MECHANISM OF PHOSPHORIBULOKINASE FROM A CYANOBACTERIUM S Y N E C H O C O C C U $ PCC 7942 AND THE MODEL OF CALVIN CYCLE WITH THE MECHANISM A. Wadano, R. Sato, K. Haranou and K. Nishikawa La.bo~tory of Applied Molecular Biology, Department of Applied Biochemist[y College of Agriculture, University of Osaka Prefecture. Phosphori'bulekinase (PRK) catalyzes the ATP-dependent pnospnorvlation of D-nbulose 5-pliosphate(Ru5P) ~o form D-ribulose 1 5bispliospfiale (RuBP). RuBP is a substrate for RuBP ~ea~nOXy'las~/ox~enase (RuBisCO), which is the first enzyme of Calvine n ~ n cycle. I nere are a tot of knowledge about the kinetics of R~BisCOr, but a litt!e is .known about the Kinetics of PRK. The reaction of from spinach is said to follow a random Bi Bi mechanism. On the other hand, the[ea[e almost no experimental evidences about the reaction mecnan*sm or ri,,h from cyanonactefia, although it has a different mgiecmar stru.%mIe " n20mt~at of.the enzyme fLoWn ~ ~ e higher plant. ~ m y m u e s o r rKr~ from ~ynecnococcus *-t~c/v4z are 0.13- and 0.047 mM for Air" and Ru5P, respectively. 6-Phosphogluconale inhibits the reaction non competitively against ATP, but competitively a~ainst RuSP, s .bowing that A TP can birid tothe enzyme either 5efore or after the omamg or t~u3r', t ne tree o* inhibition of RuBP is noncompetive, which !s not ~ntradi.c!qry to tfi6 .conclusion that the type of the enzyme reaction is a ranaom t~l r~l mecnamsm. We also constructed a model of Calvin cycle of a cyanobacteria with the mechanism.

P-19-20-044 STRUCTURAL STUDIES PHOSPHORIBULOKINASE

ON

RHODOBACTER

SPHAEROIDES

H.M. Miziorko, H. Koteiche +, D. Roberts, J.A. Runquist, C. Narasimhan, & J.J. Kim. Dept. of Biochemistry & +Biophysics Res. Inst., Medical College of Wisconsin, Milwaukee, WI 93226 USA Spectroscopic and X-ray diffraction approaches are being applied to study the structure of phosphoribulokinase (PRK), a regulated Calvin cycle enzyme which catalyzes the ATP-dependent phosphorylation of ribulose 5-phosphate. R. sphaeroides PRK, an octamer of 32 kDa sttbunits, is allosterically activated byNADH. For spectroscopic studies, a proxyl spin label (SL) has been coupled to the phosphoryl chain of adenine nucleotides. ATP-SL can occupy both ATP substrate and NADH activator sites on recombinant PRK. Methodology to selectively direct the probe to either site has been developed. ATP-SL mimics ATP by binding cooperatively to the substrate site and also mimics NADH by displaying hyperbolic saturation of the activator site. The spin-probe approach facilitates evaluation of the structural integrity of recombinant wild-type and mutant PRK enzymes. Diffraction quality (2.6 ~ resolution) crystals have been produced from recombinant PRK. Preliminary data indicate a cubic space group P432 with unit cell parameters a=b=c=129.55 A. Solution state methodology results suggests strategies for crystallization of PRK containing bound substrate or activator. Supported by USDA NRI-CRGO Photosynthesis (93-373069181).

176

P-19-20-046 EFFECT OF STRONAL pH IN FRUCTOSE-I,6-BISPHOSPHATASE THIOREDOXIN f (Td f) INTERACTION A.Reche, J.J.L~zare, R.Hermose and A.Chueca Plant Biochemistry Department, Estaci6n Experimental (CSIC), Prof. Albareda i, 18008-Granada, Spain

(FBPase}/

del Zaid~n

Chloroplast FBPase is modulated by Td f, which links photosynthetic electron transport to a reductive activation of the enzyme. Since dark-light transition induces a rise in the stromal pH (7.0 ÷ 7.9), we have studied if this change has any effect in the FBPase/Td f interaction, earlier found as a putative previous step to the enzyme activation. Both purified pea leaf proteins exhibit an interaction at pH 7.9 in the presence of DTT, as shown by filtration through a FPLC-fitted Superose 12 column, from which they elute together as a 225 kDa complex. This interaction does not exist at pH 7.1 and at pH 7.9 without DTT. When increasing concentrations of Td f are incubated with a constant amount of enzyme, a rise in the FBPase/Td f complex is observed up to a 1:4 stoichiometry. The enzyme-Td binding can be also detected in chloroplast lysates when the lysis is carried out at pH 7.9 in the presence of DTT. These results seem to show an "in vivo" FBPase/Td f interaction in illuminated leaves, conditions under which the pH and the redox state of the stroma is similar to those mimiced in "in vitro" assays.

P-19-20-047

INFLUENCE OF EXTERNAL FACTORS ON THE CATABOLISM OF STROMAL PROTEINS IN ISOLATED PEA CHLOROPLASTS Pia A. Stie,qer and Urs Feller, Institute of Plant Physiology, University of Bern, Altenbergrain 21, CH-3013 Bern, Switzerland Isolated chloroplasts are a suitable system to investigate the catabolism of nuclear encoded stromal enzymes. Proteins are no longer imported after chloroplast isolation and only the proteolytic systems present inside the organelles may contribute to degradation. Intact pea chloroplasts were incubated under various conditions, reisolated to separate intact from lysed plastids, and analysed by SDS-PAGE and immunoblotting. Stromal proteins were degraded at different rates in intact chloroplasts incubated under low light (45 ~mol m -2 sl); proteins were quite stable in the dark. Including CuSO4 in the incubation medium caused precipitation of stromal proteins. However, methyl viologen treatment accelerated protein degradation and did not cause protein precipitation. Thus, isolated chloroplasts contain proteolytic systems capable of degrading stromal proteins and the degradation of proteins is stimulated by low light and methyl viologen.

Poster P-19-20-048 FBPase BINDING SITE INVOLVED IN T~E ENZYMK-Td INTERACTION R.Hermoso, R.Csstillo, M.Sahrawy and J.L6pez Gorg~ Plant Biochemistry Department, Estaci6n Experimental (CSIC), Prof. A l b a r e d a i, 1 8 0 0 8 - G r a n a d a , S p a i n

del Z a i d ~ n

L o o k i n g for s t r u c t u r a l d i f f e r e n c e s w h i c h e x p l a i n the d i f f e r e n t b i n d i n g a b i l i t y for t h i o r e d o x i n s (Td) s h o w e d by s p i n a c h a n d p e a c h l o r o p l a s t fructose-l,6-bisphosphatases (FBPase), we a n a l y z e a 19 a m i n o a c i d s f r a g m e n t ( 1 4 9 p r o - 1 6 7 G l y in pea) w h i c h p r e c e d e s the - S - S - r e g u l a t o r y site. T h i s f r a g m e n t e x h i b i t s low h o m o l o g y among c h l o r o p l a s t F B P a s e s , a n d s h o w s in the p e a e n z y m e a d i s o r d e r e d s e c o n d a r y s t r u c t u r e . The c D N A s e q u e n c e for this f r a g m e n t w a s a m p l i f i e d by P C R f r o m a c l o n e c o d i n g for p e a F B P a s e , s u b c l o n e d in p G E M E X - I , a n d e x p r e s s e d in E. d o ~ K 3 8 as a f u s i o n p r o t e i n . T h i s a p p e a r s as i n c l u s s i o n b o d i e s , from w h i c h was r e c o v e r e d by u r e a e x t r a c t i o n . The f u s i o n p r o t e i n s t r o n g l y i n t e r a c t s w i t h p e a Td m, as s h o w e d by E L I S A a n d S u p e r o s e 12 f i l t r a tion, but s u r p r i s i n g l y e n h a n c e s F B P a s e a c t i v i t y in i n h i b i t i o n e x p e r i m e n t s w i t h F B P a s e and Td m. P r e l i m i n a r y a s s a y s h a v e s h o w n i n h i b i t i o n of F B P a s e a c t i v i t y in the p r e s e n c e of s p e c i f i c IgG a g a i n s t the 19 a m i n o a c i d s insert. T h e s e r e s u l t s s e e m to s h o w s t r u c t u r a l m o d i f i c a t i o n s of Td by F B P a s e b i n d i n g . A s s a y s w i t h p e a F B P a s e d e l e t e d in the 19 a m i n o a c i d s f r a g m e n t , a n d s u b s t i t u t e d for t h a t of the w h e a t enzyme, are n o w in p r o g r e s s .

P-19-20-049 EFFECT OF TEMPERATURE AND IRRADIANCE ON C4 PHOSPHOENOLPYRUVATE CARBOXYLASE (PEPC) FROM PASPALUM DILA TA TUM LEAF,

A. B. Silva and M. C. Arraba~a. Dep. de Biologia Vegetal, Faculdade de Ci~ncias da Universidade de Lisboa, 1700 Lisboa, Portugal. In Paspalum dilatatum leaves the content of PEPC-protein and the sensitivity to effectors were higher under cold environmental conditions. This is m a y be due to light and/or temperature. The influence of these parameters on the in vitro activity and L-malate sensitivity of P. dilatatum leaf C4 PEPC was studied. The plants were grown under controlled conditions at 25/18°C and 10/8oc. Before each experiment the first expanded leaf was illuminated at 900 ~tmol.m-2.s-1 or maintained in darkness for two hours and assays always at 30 °C. Vmax did not change significantly with irradiance but increased 2.5 fold in plants grown at 10 o c . This is due to a h i g h e r level of P E P C - p r o t e i n as quantified b y immunoprecipitation. The IC50 for L-malate is higher in illuminated plants but the difference is more evident in plants g r o w n at 25 o c (2.3 to 3.5-fold increase). At 10 o c the IC50 ratio was smaller due mainly to a higher value in dark-plants, suggesting that the enzyme dephosphorylation is not complete. Experiments are being done in order to confirm this fact.

P-19-20-050 R E G U L A T I O N OF C4 P H O S P H O E N O L P Y R U V A T E C A R B O X Y L A S E OF PASPALUM DILATATUM LEAF UNDER ENVIRONMENTAL CONDITIONS.

A. B. Silva*, J. Vidal # and M. C. ArrabaTa* *Dep. de Biologia Vegetal, Fac. de Ci~ncias de Lisboa, 1700 Lisboa, Portugal. #Institut de Biotechnologie des Plantes, U R A CNRS D 1128, Universit6 ParisSud, 19 405 Orsay, France

Paspalum dilatatum cv Raki is, as far as we know, the only C4 plant that grows during all the year in Portugal. In order to understand the possible involvement of PEPC in the mechanisms responsible for the capacity to grow under winter conditions, we have determined the activity and regulatory properties of C4PEPC. The enzyme was assayed in the first leaf, a month after cutting. A twofold increase in PEPC activity was observed in autumn and at the end of winter as compared with spring and summer levels. This fact suggests that the content in PEPC-protein is higher in cold weather, which was confirmed by western blotting. Both lower malate sensitivity and enhanced activation by the allosteric effector glucose-6-phosphate o f PEPC, as observed during the day, suggested that its phosphorylation status is higher in summer. These results point to the contribution o f P E P C to the survival of P. dilatatum u n d e r adverse environmental conditions.

P-19-20-051 CIRCADIAN ACTIVITY OF SUCROSE PHOSPHATE SYNTHASE IS A RESULT OF CIRCADIAN REGULATION OF SPS-PHOSPHATASE

~ 1 and Donald R. Ortl.2; 1Dept. of Plant Biology, University of Illinois, Urbana, IL 61801 & 2photosyn Research Unit, USDA/ARS Sucrose phosphate synthase (SPS), a key enzyme in sucrose biosynthesis, is regulated by protein phosphorylation and shows a circadian pattern of activity in tomato. SPS is most active in its dephosphorylated state which n o r m a l l y coincides with daytime. Applying okadaic acid, a potent phosphatase inhibitor, prevents SPS activation. More interesting is t h a t a brief treatment with cycloheximide, a cytoplasmic translation inhibitor, also prevents the light activation of SPS, without any effect on the amount of SPS protein. Cordycepin, a transcription inhibiter, has the same effect. Both of these inhibiters also inhibit the activation phase of the circadian rhythm in SPS activity. Conversely, cycloheximide and cordycepin do not prevent the decline in circadian SPS activity. T h e s e observations indicate t h a t SPSphasphatase activity, but not SPS-kinase activity, is controlled at the level of gene expression. Taken together, it seems clear t h a t there is a circadian rhythm controlling the transcription of SPS-phosphatase which subsequently dictates the c i r c a d i a n r h y t h m in SPS activity via effects on its phosphorylation state.

P-19-20-052 NOLECUT.d~ BZOLOGZC3tL APPRO&CH TO ~ N . T J ~ 9 ~1~ ROLE OF ~ P M3~L~TE IIRYDRCKgF.N~SE IN THZ I~.GUT_.~TZON OF C4 P H O T O ~ B Z ~ S.J.Trevanion I, R.T.Furbank 2, & A.R.Ashton 2 iCooperative Centre for Plant Science, P.O.Box 475, c/o RSBS ANU, Canberra. ACT 2601. AuStralia. 2CSIRO Division of Plant Industry, PO Box 1600, Canberra. ACT 2601. Australia. NADP-malate dehydrogenase catalyses the reduction of oxaloacetic acid to malate, an important step in the fixation of CO 2 by C4 plants. Although activity of the enzyme is regulated by covalent modification, its significance in regulating C4 photosynthesis is poorly understood. To address this problem we have generated transgenic Flaveria bSdentis plants with reduced activity of the enzyme. Activity was reduced to 5% of wild type (wt) with no apparent effect on growth or development. In one individual transformant with 40% activity of the wt, the ~aximum rate of photosynthesis was reduced by about 40%. However, there was no effect on quantum yield, or on the response of photosynthesis to changes in [CO2]. ~nalysis of the T1 generation from a n u ~ r of independent transformants is currently under progress. We will also discuss initial attempts to overexpress forms of the enzyme modified to alter their kinetic properties.

P-19-20-053 EFFECT OF PHOTORESPIRATION INHIBITORS ON PHOTOSYNTHESIS IN MAIZE LEAVES Be~olia Gonzalez-Moro. Maite Lacuesta, Alberto Muiioz-Rueda & Carmen Goiazaiez-Murua. Dept. Biol. Veg. & Ecol., Univ. Basque Country, Apto 644 E-48080 Spain C4 plants contain the enzymes of the photorespiratory pathway and the photorespiratory nitrogen cycle but CO2 liberated is not released into the atmosphere. However, in spite of the lower rate of photorespiration in C4 plants than in C3 plants, it became evident when photorespiratory cycle is interrupted by phosphinothricin (PPT) a glutamine synthetase inhibitor or aminooxiacefic acid (AOA) an aminotransferase inhibitor. We have examined the inhibition caused by both of them on PEPcarboxylase, Rubisco and NADP-ME activities and on the levels of PGA and RuBP of a C4 plant like maize. Neither AOA nor PPT seemed to affect PEPCasa, whereas a slight increase of NADP-ME were produced. PPT did not produce significant changes in Rubisco activity but about 35% was reduced with AOA. A close correlation became apparent between glycolate accumulation and the inhibition of both Rubisco activity and photosynthesis. The inhibition of photosynthesis produced by PPT was accompanied by a decrease of PGA, whereas a high RuBP accumulation was observed in plant treated with AOA. This research was supported, in part, by Direcci6n General de Investigaci6n Cientfllca y T6cnica (PB92-0468) of Spain.

177

Poster

P-19-20-054 Reconstruction

depends

of L8S 8 RubisCO

on 188KD p r o t e i n

from

denatured

subunits

and Mg-ATP

M I A O You-Gang & LI Li-Ren (Shanghai institute of Plant Shanghai 200032, China)

Physiology,

Academia

Sinica,

It was d e t e c t e d by w e s t e r n blot with antibody against RubisCO that there was a m o l e c u l a r weight of 188KD protein which p r o d u c e d a positive reaction with RubisCO antibody besides RubisCO in crude extract. It was estimated by twod i m e n s t r a t e d PAGE and w e s t e r n blot that 188KD protein contains one RubisCO large subunit and 4-5 subunits of 26KD. In addition, b y w e s t e r n blot of crude extract from rice with the antibody against (cpn60)14 complex from rice it was found that there were lots of cpn60 monomers in the crude extract. A f t e r removing RubisCO and (cpn60)14 complex from crude extract by gel filtration, the d e n a t u r e d RubisCO by 8 mol/L urea, Mg-ATP and K + were added to the p a r t l y p u r i f i e d extract, it was seen that a b a n d of native L8S 8 RubisCO appeared on the gel of n o n - d e n a t u r l n g PAGE. Further experiment showed that the assembly of the d e n a t u r e d RubisCO in vitro was d e p e n d e d on the 188KD protein complex besides cpn60 monomers and Mg-ATP. It was suggested that the 26KD p r o t e i n may play a role of ch-cpnl0.

P-19-20-057 OCCURRENCE OF RIBOSE 1,5-BISPHOSPHATE ISOMERASE IN SPINACH CHLOROPLASTS AND ITS PROPERTIES H. Shibata 1, H. Yamashita 1, J. AkiyoshP, S. Sano 2 & A. Yokota 2 TD-ept. Biol. Sci., Fac. Agric., Shimane Univ., Matsue, Shimane 690, Japan; ~'Plant Mol. Physiol, Lab., Res. Inst. Innovative Tech. Earth (RITE), I~zu, Kyoto 619-02, Japan We have found a new pathway to by-pass the step catalyzed by ribose 5-phosphate isomerase. The new RuBP-regenearting pathway in the Calvin-Benson cycle is comprised of phosphopentokinase and ribose 1,5-bisphosphate (RBP) isomerase. The enzymatic properties of the former enzyme are reported in the accompanying paper. We have purified RBP isomerase from spinach leaves and its properties are reported in this paper. The activity of RBP iomerase in the 6troma was 350/Jmol/mg Chl/hr. RBP isomerase purified from spinach leaves was atetramer (200 kDa) composed of the subunits of 50 kDa. The optimum pH was 7.2, but the enzyme showed significantly high activities at stromal PH's. The apparent K mfor RBP was 1.0 mM. [Partly supported by PEC/MITI].

P-19-20-055 VO2÷ AS A PARAMAGNETIC PROBE OF RUBISCO ACTIVASE ~ , Miel Vallejo, And Wayne D. Frasch Department of Botany, Arizona State University, Tempe, Arizona 852871601, USA. Ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco) activase requires ATP and Mg 2+ to perform its catalytic task of activating Rubisco. This function is inhibited by ADP. The stoichiometry of metal binding and the structure of the metal-binding site in the enzyme were investigated using the paramagnetic metal ion vanadyl (VO2+) an electron paramagnetic resonance (EPR) probe. The enzyme was treated to remove bound Mg2+, then titrated with VO2+, alone and in conjunction with ADP or the ATP analog, AMPPNP. The 51V nucleus has a spin I = 7/2; the magnitude of the 51V hyperfine coupling is sensitive to the nature of the ligands in the cation's equatorial plane. The titration curves, along with room-temperature and 125 K EPR spectra, suggest that while VO2+ alone binds non-specifically to the protein, and VO2+ in concert with AMPPNP forms a complex not associated with the protein, VO2+ and ADP form a stable, unique complex with Rubiseo activase. The parallel component of the 51V hyperfine coupling in this complex is most consistent with an equatorial ligand set composed of one water plus some combination of three carboxylates and/or phosphates.

P-19-20-056 PEPCK: THE INITIAL CARBOXYLASE IN A C4-LIKE ALGA Julia B. Reiskind & George Bowes Dept of Botany, Univ of Florida, Gainesville FL 32611-8526, USA

Udotea flabeUum, a marine macroalga (Chlorophyta), has a CO2 concentrating mechanism based on a C4-type of photosynthesis with phosphoenolpyruvate carboxykinase (PEPCK) as the initial carboxylase. Inhibition of PEPCK resulted in C3-1ike photosynthetic physiology and biochemistry. Kranz anatomy was not present in this alga, but percoll gradient and immunogold localization studies indicated PEPCK was cytosolic, associated with non-membranous spherical bodies, while Rubisco was chloroplastic. Sufficient activity of the decarboxylase, NAD-malic enzyme, to support the photosynthetic rate was found; however, the location of this enzyme has yet to be defined. PEPCK was purified and kinetically characterized; it was a tetramer with a 70 kD subunit. Amino acid sequencing indicated the Nterminus was blocked; pilot studies of cyanogen bromide lysis showed eight bands. Studies are underway to determine the sequence. This project was funded by the USDA\CSRS\NRICG Photosynthesis and Respiration Program, grant no. 91-37306-6000.

178

P-19-20-058 DIRECT PHOSPHORYLATION RIBULOKINASE

OF

RIBOSE

5-PHOSPHATE

BY PHOSPHO-

S~.Sano1, A. Yokota1, J. Akiyoshi2 & H. Shibata= tPlant. Mot Physiol. Lab., Res. Inst. Innovative Tech. Earth (RITE), Kizu, Kyoto 619-02, Japan; ZDept. Biol. Sci., Fac. Agric. Shimane Univ., Matsue, Shimane 690, Japan It has been believed that ribulose 1,5-bisphosphate (RuBP) in the (Salvin-Benson cycle is generated by phosphoribulokinase (PRuK) catalyzing the phosphorylation of ribulose 5-phosphate (Ru5P), one-third of which is from ribose S-phosphate (R5P). However, the rate of the isomerizetion from RSP 1o Ru5P catalyzed by R5P isomerase in the stroma, which is retarded by high concentration of a inhibitor 3-phosphoglycerate and the low concentration of the substrete R5P, cannot satisfy that of the turnover of the Calvin-Benson cycle. Here, we propose a pathway to bypass the R5P isomerase step in the cycle. Phosphoribekinase (PRK), which catalyzes the direct phosphorylation of FI5P with ATP, was found in the stroma of spinach chloroplasts and was purified from spinach leaves. PRuK of the ice plant produced in E. coil was also purified. These two kinases could catalyze the phosphoryletion of the both of RSP and Ru6P and had the same ratio of the activity with Ru5P to that with R5P, about 1.2 to 1. Therefore, the PRK activity in the stroma is due to low specificity of PRuK. We conclude that PRuK functions as phosphopentokinase in the stroma to by-pess the R5P isomerase step with aid of ribose 1,5-bisphosphate isomerase. [Partly supported by NEDO]

P-19-20-059 THE ISOLATION OF MUTANTS OFAMARANTHUS EDULIS WITH ALTERATIONS IN C4 PHOTOSYNTHESIS Lea, P.J.*, Leegood, R.C. 2, Oltek, L.A. 1, Lacuesta, M. s & Dever, L.V.1 1Dh,ision of Biological Sciences, University of Lancaster, Lancaster, LA1 4YQ, U.K.; 2 Robert Hill Institute, University of Sheffield, Sheffield, Sl0 2YQ, U.K.; 3 Biologic Vegetal, Facukad de Farmacia, Universidad del Pals Vasco, O-1007 Vitoria, Spain. Two mutants ofAmaranthus edulis have been isolated. LaC4 73 has been shown to contain less than 10% of the wild type leafNAD-malic e~yme activity. Western Blot analysis indicated that the mutant contained normal levels of the two subunits of the enzyme protein of 60 and 65 kDa. LaC473 exhibited very low rates of CO2 as~milation in air, which responded in a linear manner tO increased internal CO2 concentrations. Four different mutant lines have been isolated that accumulate glycine, up to 90% of the total amino acid pool in the leaves. The rate of ammonia evolution following the addition of the glutamine synthetase inhibitor, phosphinothricin was reduced in all of the glycine accumulating mutant lines. Western Blot analysis of the four glycine decarboxylase proteins indicated that in LaC4 2.11, there was evidence of a reduction in the level of the P protein.

Poster

P-19-20-060

P-19-20-063

THE CHARACTERISATION OF A MUTANT OF A M A R A N T H U S E D U L I S LACKING P H O S P H O E N O L P Y R U V A T E CARBOXYLASE

STUDY OF THE p - H Y D R O X Y P H E N Y L P Y R U V A T E DIOXYGENASE FROM CULTURED CARROT CELLS.

Dever~ LV. 1, Leegood, R.C. 2 and Lea, P.J) t Division of Biological Sciences, University of Lancaster, Lancaster LA1 4YQ, U.IC, 2 Robert Hill Institute, University of Sheffield, Sheffield Sl0 2UQ, U.IC

M. Matrin~e t C. Lenne 1, K. E. Pallett 2 & R. Douce 1. ILabo. Mixte CNRS UMR 41, Rh6ne-Poulenc Agrochimie BP 9163, 69263 Lyon Cedex 09, France. 2 Ddpartement Biotechnologie,Rh6ne-Poulenc Agrochimie BP 9163, 69263 Lyon Cedex 09, France

A mutant ofAmaranthus edulis (designated LaC4 2.16) has been isolated, that contained less than 5% of the normal level of PEP carboxylase activity in the leaves, Western blot analysis has shown that the mutant was also lacking the major PEP carboxylase protein. The mutant plant was unable to grow in normal air but grew slowly at elevated levels of CO2 (0.7%). The photosynthetic rate of the mutant was very low in air, but responded in a linear manner to increased internal CO2 concentrations. Heterozygous F~ plants containing approximately 50% of the wild type PEP carboxylase activity and protein have also been obtained. The F1 plants show a small reduction in growth rate and CO2 assimilation rate at high light intensities. Analysis of the F2 seedlings has indicated that the mutation segregates in a normal Mendelian fashion as a single gone.

In plants, p-hydroxyphenylpyruvate dioxygenase activity is involved in the biosynthetic pathway leading to plastoquinones and tocopherols. It is also involved, as in other organisms, in the degradation of tyrosine. This enzyme catalyzes the sequential decarboxylation and hydroxylation of phydroxyphenylpyruvate into homogentisate. Recently, it has been shown that this enzymatic activity is inhibited by a new family of bleaching herbicides. A tentative explanation for this bleaching effect, which is associated with an accumulation of the carotenoid precursor phytoene, would be an indirect inhibition of the phytoene desaturase activity, as a consequence of a plastoquinone-cofactor pool depletion, p-Hydroxyphenylpyruvate dioxygenase thus appears to be a new and very promising herbicide target enzyme. In order to improve our knowledge of the phmt enzyme and to better understand the mechanism of its inhibition by these herbicides, we have initiated a thorough characterization of the p-hydroxyphenylpyruvate dioxygenase enzyme from cultured carrot cells.

P-19-20-061 CP12 - A NEW LIGHT REGULATOR FOR THE CALVIN CYCLE ? N, Wedel, J. Soil & K. Pohlmeyer Botanisehes Institut der Christian Albrechts-Universit~t, Am Botaniseben Garten 1-9, 24098 Kiel, FRG The main difference in the primary structures of subunits GAPA and GAPB of the higher plants clfloroplast GAPDH (EC 1.2.1.13) is the C-terminal extension of about 30 amino acid residues of GAPB [Bfinkraann etal., Plant Mol. Biol. 13 (1989) 81-94]. This extension contains two cysteine residues, spaced by eight other amino acids which probably can form a peptide loop depending on the redox state of the chloroplast. Recently we have isolated cDNA-elones for a new nuclear encoded chloroplast protein from spinach and tobacco eDNA libraries which we have called CP12. The mature protein consists of 75 or 76 amino acid residues respectively and shares striking homology to the 'cysteine motif of the C-terminal extension of GAPB in having that very conserved motif two times spaced by an ohelix of 39 and 40 amino acid residues respectively. Specific binding of the stromal GAPDH to overexpressed and immobilized CP12 and other biochemical in vitro studies with isolated stromal protein complexes from spinaeh as well as the analysis of sense and antisense constructs for CP12 in transgenic tobacco plants suggest that CP12 is a member of the redox regulation machinery for photosynthetic dark reactions in the higher plants chloroplasts.

P-19-20-062 PURIFICATION AND SOME PROPERTIES OF AN SDS-ACTIVATED PROTEASE IN ZEA MA YS

Takafumi YAMADA, Arata OZAWA, Hiroyuki OHTA, Tatsuru MASUDA, Yuzo SHIOI, Ken-ichiro TAKAMIYA, Dept. Biol. Sci., Tokyo Inst. Technol., Nagatsuta, Midori-ku, Yokohama 226 We have reported the properties of SDS-activated protease (39 kDa) from leaves of Z may$ (1). Recently, we found another type of SDSactivated protease with different properties from that previously reported. The protease was purified using several chromatographies (ionexchange, gel filtration etc.) and preparative electrophoresis. Polyacrylamide gel electrophoresis (PAGE) gave a single band of 183kDa under nondenaturing condition. The active subunit was approximately 50 kDa in SDS-PAGE. The properties of the purified enzyme were as follows;1 ) 0.5% SDS was optimal for activation. 2) I~mercaptoethanol (B-ME) stimulated the activity, and the optimum was 1% in the presence of 0.5% SDS. However, without SDS, I~-ME did not activate the enzyme. 3) Nonionic and ionic detergents tested except for SDS had little effect on the activity.4) The optimal pH was around 5. Inhibitor experiments suggested the protease to be an SHprotease. (1) Takamiya,K. et al. (1992) Plant Physiol.,99,138

P-19-20-064 THE EFFECT OF REDUCED SBPASE LEVELS ON LEAF CARBON METABOLISM E.P. Harrison. J.C. Lloyd and C.A. Ralnes. University of Essex, Department of Biology, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK. Sedoheptulose-l,7-bisphosphatase (SBPase) is located in the chloroplast stroma where it functions in the photosynthetic carbon reduction (Calvin) cycle. The role of SBPase in the regenerative part of this cycle means that this enzyme competes with the sucrose and starch biosynthetic pathways for triose phosphates. Our goal is to determine the relative importance of SBPase in regulating carbon flux through the cycle and the export of intermediates to starch and sucrose. To address this question we have produced a number of independent transgenic tobacco lines expressing an SBPase antisense construct. These plants display a characteristic phenotype, of chlorosis in cells adjacent to the veins, which varies in severity and appears in a developmentally related manner. A range of SBPase protein levels has been found in these plants, measured by western blot analysis, and correlates with the reduced growth rates and altered carbohydrate levels. Photosynthesis (measured as light dosage response curves) was also found to be reduced.

P-19-20-065 LIMITATION OF NON-STEADY-STATEPHOTOSYNTHESIS BY RUBISCO ACTIVASE Edward T. Hammondi, T. JolmAIKIrews2 and Ion E. Woodrow! ! Schoolof Botany, The Universityof Melbourue, Parkville, VIC 3052, Australia 2Research Schoolof Biological Scieaces, AustralianNational University,PO Box 475, Canberra, ACT 2601, Australia Light-mediated activation of the enzyme ribulose bisphosphate carboxylase (Rubisco) involvesat least two slow (ie. characterised by a relaxation time of one or more minutes) processes. The first involves the addition of an activator CO2 to a lysine residue of the large subunit of Rubison (carbamylation); and the second process involvesthe removalof ribulose 1,5-bisphosphate(and possiblyother tightly bound compounds) from an inactive fi3rm of Rubisco by the enzyme Rubisco activase. Studies using tobacco (Nicotiana tabacum) - both wildtype and "anfisense mutants" with reduced amounts Rubiseo activase - show that the relative contribution of these two slow processesto the overall kinetics of Rubiseo activation depends on the proportion of the total Rubiseo pool activated. Activase activity largely limits Rubisco activation following relatively large increases in PPFD; the initial rate and the relaxation time of activation are directly proportionalto activase concentration. Carbamylation, however, is only limiting following relatively small changes in PPFD.

179

Poster

P-19-20-066 PURIFICATION

OF C A I P - P H O S P H A T A S E IMMUNOLOGICAL A N A L Y S I S

AND

A PRELIMINARY

JE Rya~, A J Key~ & MAJ Parry, Biochemistry and Physiology Department,IACRRothamsted, Harpenden, Herts. AL5 2JQ, UK. In m a n y species of plants the tight binding inhibitor, 2-carboxy-D-arabinitol 1-phosphate (CAIP) is a major cause of the diurnal variation in Rubisco activity. The enzyme which breaks down CAIP in the light is CAIP-phosphatase. It has been purified to h o m o g e n e i t y from Phaseolus vulgaris cv. Tendergreen using a modified procedure. Several peptides from tryptic digests have been sequenced. Polyclonal antisera to CAiP-phosphatase has been raised in rabbits. The antisera recognised purified CAIPphosphatase from P.vulgaris in Western blots as well as the enzyme in leaf extracts. The cross reactivity of the polyclonal antibodies was tested against leaf extracts from several other species. The antisera is being used to develop an affinity purification procedure to produce larger quantities of pure CAIPphosphatase.

P-19-20-069 Evidence for an enzymatic complex PEPc/NADP-MDH in C4 plants (sugar cane) P. BALLETI , F. CADET1, C. ROUCHl and J-C MEUNIER2. 1Lab. Biochimie, Universit6 de la R6union,2Lab. Biochimie, CBAI, INA-PG A multienzymatic complexe (five enzymes) has been found by Gontero et al. (1) in the Calvin cycle of C3 plants. After optimisation of extraction conditions, by a combination of different chromatography techniques, we have obtained a stable~ highly pudfiexi, enzymatic complex Phosphoenol Pyruvate Carboxylase (PEPc)/NADP-Malate dehydrogenase (NADP-MDH) from C4 plants. In the mesophylI of C4 plants (such as sugar cane) takes place the Hatch and Slack cycle where the first reaction is a C02 fixation catalysed by PEPc. The product of this reaction, OAA is, in a second step, reduced in malate by a NADP-MDH. The system ferredoxin/thioredoxin also appears to regulate this second enzyme (NADP-MDH) not directly associated with the Calvin cycle. The purification procedure of PEPc/NADP-MDH complex involves: DEAEcellulose, hydroxyapatite column, affinity chromatography on chelating gel and molecular sieving. Properties of this complex are under investigation. Its molecular weight is higher than 200 kDa. This complex is functional as NADP + (with malate) is formed from PEP. 1- B. Gontero, M.L Cardenas and J. Ricard, (1988) Eur. J. Biochem, 173, 437-443.

P-19-20-067 MODIFICATION OF CARBON METABOLISM IN TOMATO PLANTS OVEREXPRESSING SPS IN THE LEAVES. E. Murchle I , N. GaitierI , T. Betsche 2, C. Th~penier 2, G. Lasceve2, P, Quick3, C.H. FoyerI . 1: Lab du M(~tabolisme. INRA. 78026 VERSAILLES Codex. France 2: eept de Physlologie V6g6tale. CEA. 13108 St PAUL/DURANCE. France 3: Dept of Animal and Plant Sciences. SHEFFIELD $I0 2UQ. England Photosynthetic carbon assimilation, carbon partitioning and foliar carbon budgets were measured in air and in high CO2 in the leaves of transformed tomato plants expressing a maize sucrose-phosphate synthase (8PS) gene in addition to the native anzyme, and in untransformed controls. Net photosymheals was measured in air and at elevated CO2 (800-1500 ppm) on whole plants grown in air. CO2 assimilation was significantly increased in the high SPS exprossors compared to the untransformed controls and was highest where SPS activity was greatest. The rate of sucrose synthesis was increased relative to that of starch. The sucrose/starch ratios were higher throughout the photoperiod in the leaves of all plants expressing high SPS activity, both in air and at elevated CO2. This resulted in a greatly decreased level of carbohydrate accumulation in the leaves of plants grown at high CO2. We conclude that SPS activity is a major point of control of photosynthesis and is particularly important in conditions of saturating light and C02.

P-19-20-070 CLONING AND ANTI-SENSE RNA CONSTRUCTS OF A STARCH BRANCHING ENZYME GENE FROM BARLEY ENDOSPERM

P.~.Sathish. Chuanxln Sun, Anna Deiber & Christer Jansson Dept. of Biochemistry, The Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden Starch is the major reserve carbohydrate found in the endosperm of barley. Like most other starches, barley starch is composed of the essentially linear a-1,4 polyglucan amylose and the heavily branched a-1,4; a-1,6 polyglucan amylopectin. Starch branching enzyme (SBE) introduces the a-1,6 branches in the growing amylopectin molecule. We have cloned a gene for SBE in barley endosperm using the Rapid Amplification of cDNA Ends (RACE) technique. The cloned cDNA shows a high identity score with the sbel cDNA from maize and rice and is, consequently, referred to as the barley sbel eDNA. Comparative analyses of the barley sbel cDNA and experiments with sense and anti-sense constructs will be dsicussed.

P-19-20-068

P-19-20-071

SUBSTRATE AND INHIBITOR BINDING TO RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (RUBISCO)

ISOLATION OF A STARCH BRANCHING ENZYME FROM

J. Frank'. J. Vater= and J.F Holzwarth'

lChuanxin Sun. 'P. Sathlsh, =Bo Ek, 'Anna Deiber & 'Christer Jsnsson 1Dept. of Biochemistry, The Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden and 2Dept. of Cell Research, Swedish University of Agricultural Sciences, P. O. Box

'FHtz-Haber-lnatitute of firs Max-Planck-Socie~/, Faredayweg 4-6, D-14195 Berlin, Germany; ~lnst/tute of Biochemistry and Molecular BiD/of/y, Technlsche Un/versitat BaHin Frenkl/nstr. 28-29, D- 10587 Berlin, Germany The binding events at the active site of the carbondloxld fixing enzyme RUBISCO from spinach were detected with the fluorescent dye toluldinonaphthaiinosulfonete (2,6-TNS). The changes In fluorescence Intensity of the enzyme bound dye upon binding of the sul0strete dbulose t , 5 - b l ~ e (RUBP) and related compounds were recorded by a stopped-flow apparatus, The iodine laser temperature jump technique (ILTJ) was applied to equilibrium mixtures of enzyme (RUBISCO), dye (I"NS), sub=rate (RUBP) or the Inhibitor 6-phosphogluconate (6-PG). The concentration dependence of the reciprocal relaxation time Indicates a simple blmolecular binding step for the subetrate and substrate analogues in the ai0eence of magnesium ions. In the presence of magnesium Ions the concentration dependence of the reclprocat relaxation time shows saturation cheractedstlcs. The binding kinetics of the substrate (RUBP) and the transition state analogues cerboxyarablnitol 1,5-blsphosphate (CABP) and carboxydbitol 1,5-blsphosphate (CRBP) to different states of the enzyme and their consequences for the mode of binding will be discussed.

180

BARLEY

ENDOSPERM

7055, S-75007 Uppsala, S w e d e n

Starch branching enzyme activity was followed in developing barley (Hordeum vulgate L. cv Golf) caryopses during a period of one month after anthesis. Four branching enzyme activity fractions were resolved. From one of these fractions a branching enzyme preparation containing two related polypeptides of 51 and 50 kDa was obtained. Native polyacrylamide gel electrophoresis and gel filtration showed that the 51/50 kDa polypeptide is monomeric. A combination of phosphorylation stimulation and amylose branching gel assays, SDS-PAGE, and TLC was used to demonstrate the branching activity of the 51150 kDa polypeptide.

Poster

P-19-20-072 ISOPRENE SYNTHASE:

A THYLAKOID-BOUND PROTEIN

MarY_ Wildermuth and Ray Fall, University of Colorado. Boulder, CO. Isoprene synthase is the enzyme responsible for the production of the volatile C5 hydrocarbon, isoprene, by plants. Isoprene formation in numerous C3 plants is interesting in that 1) the function of plant isoprene production is unknown, 2) large quantities of isoprene are emitted -,350 teragrams annually, and 3) a plant may release 2-10% of its fixed ca:ben dioxide as isoprene, It has been postulated that isoprene synthase is located in the chloroplast due to its liglltaepenclent response and its enzymatic activation under conditions occurring in the chloroplast strorna during illumination. Subcellular localization o~ isoprene synthase using Salix spp. and Mucuna spp. indic,ate that isoprene synthase is indeed located in the chloroplast. Additional experiments with Salix further localize isoprene synthase to the thylakoid membrane. Isoprene synthase appears to be bound tightly to the thylakoid membrane as washes, mild detergent treatments, and sonication do not release activity. Its sensitivity to proteolytic treatment suggests that the enzyme has a large external domain containing the active site. We propose that isoprene synthase is anchored to the thylakoid membrane with its catalytic domain exposed to the chloroplast stroma.

P-19-20-073 TEE IDENTIFICATION CHLOROPLAST ACID LABILE

AND CHARACTERISATION PHOSPHOPROTEINS

OF

Paul E~vies, Anna T u l l ~ & John F. Allen. Plant Cell Biology Department, Lund University, Box 7007, S220 07 LL~q3 Sweden. The involvement of chloroplast phosphoproteins in the regulation of photosynthesis has been kno~q for many years. The study of such chloroplasts p h o s ~ o t e i n s radiolabelled in the presence of either [~2p]-ATP or 3 2 p - o r t ~ t e , under controlled pH ccrditions, has revealed the presence of several pbosphcproteins where l~hosphorylatien is acid labile. Acid labile phosphoproteins are cummcnly involved in two cc~ocnent regulatory mechani~s in bacteria. %~lese studies are therefore directed towards the ic%~ntificatien and characterisation of the acid labile p~sphoproteins found in chloroplasts. The detenni/~tion of the role of such two ccnloonent regulatory systems in the photosynthetic machinery within the chloroplasts of higher plants has also been studied.

P-19-20-074 A 2,4-D I N D U C I B L E G L U T A T H I O N E S-TRANSFERASE F R O M S O Y B E A N ( G L Y C I N E M A X ) . I N D U C T I O N AND EXTRACELLULAR LOCALISATION T h o m a s Flurv I & Klaus Kreuz 2 IBiologisches I n s t i t u t II, Albert Ludwigs-Universit~it Freiburg, Seh~inzlestrasse 1, D-79104 Freiburg, Germany; 2Ciba Crop Protection, Plant B i o c h e m i s t r y Ciba-Geigy Ltd, CH-4002 Basle, Switzerland Protein a c c u m u l a t i o n of a Glutathione S - t r a n s f e r a s e (GST, EC 2.5.1.18) isozyme from soybean (Glycine max. [L.] Merr. cv. Williams) was found to be induced by a large diversity of m o l e c u l e s , including synthetic auxins, synthetic and naturally occurring cytokinins, arachidonic acid, oxidants and some auxin t r a n s p o r t inhibitors. It is concluded that the induction is not related to auxin activity or to changes in the e n d o g e n o u s auxin levels. Inducible GST protein and activity was found in the extracellular w a s h i n g fluid o f h y p o c o t y l s . This s u g g e s t s the existence of an extracellular GST in s o y b e a n hypoeotyl.

181

Poster P-21-003 ROLE OF PHOSPHORYLAT1ON/DEPHOSPHORYLATION IN PHYTOCHROME MEDIATED STIMULATION OF NITRATE REDUCTASE CHANDOK, M.R. and SOPORY, S.K. M~lecular Physiology Laboratory, School of J a w a h a r l a l Nehru University, New Delhi-l10067.

Poster session 21

Integration

of the C,N,P,S metabolisms

P-21-O01 - P-21-021

A.U.leamberdiev & N.V.Bykova Dept of Plant Physiology and Biochemistry, Voronezh State University, Voronezh 394693, Russia Pathways of glycine photosynthetic metabolism were investigated in leaves of wheat and maize. Oxidation of glycine during greening reveals strong increase of cyanide resistance, and its preference to other respiratory substrates is formed only after transition of plants to light. Glycine and serine conversion during photosynthesis takes place not only via glycine decarboxylase and serine hydroxymethyltransferase, but also via reactions catalyzed by glycine oxidase and serine dehydratase. These enzymes were partially purified and characterized. They are localized in chloroplasts and the cytosol. Their activity are about 3 times higher in C 3plant (wheat) than in C4-plant (maize). It is proposed that they catalyze reactions of nitrogen and carbon outflow from photorespiratory glycolate cycle via conversion of amino acids after their transport from peroxisomes and mitochondria into chloroplast and cytosol.

P-21-002 LIGHT DEPENDENT REGULATION SYNTHETASE IN PURPLE SULFUR

OF GLUTAMINE BACTERIA.

E. K h a t i n o v D e p t . of M i c r o b i o l o g y , M o s c o w S t a t e U n i v e r s i t y , M o s c o w 119899, Russia. R e g u l a t i o n of g l u t a m i n e s y n t h e t a s e (GS) a c t i v i t y of p u r p l e s u l f u r b a c t e r i a Thioeapsa roseopersicina B B S a n d Eetothiorhoclospira shaposhrdkovii D S M 243 by ammonium and light was c o m p a r a t i v e l y s t u d i e d . I n b o t h b a c t e r i a G S is r e g u l a t e d b y a d e n y l y l a t i o n in r e s p o n s e to t h e i n c r e a s i n g c o n c e n t r a t i o n of a m m o n i u m . T h e p r o c e s s of a d e n y l y l a t i o n in T roseopersicina, b u t n o t in E. shaposhnikovi~ r e q u i r e s i l l u m i n a t i o n of c e l l s T h e d e t e r m i n a t i o n of A T P c o n c e n t r a t i o n in cells of t h e s e b a c t e r i a i n c u b a t e d u n d e r d i f f e r e n t c o n d i t i o n s a l l o w e d to c o n c l u d e t h a t t h e energy charge is n o t i n v o l v e d in r e g u l a t i o n of G S in T. roseopersicina d u e to t h e c a p a c i t y of t h i s b a c t e r i u m f o r s u b s t r a t e p h o s p h o r y l a t i o n in t h e d a r k , w h i l e in E. shaposhnikovii t h e regulation of t h e e n z y m e is a p p a r e n t l y c o u p l e d w i t h cell e n e r g y c h a r g e . T h e r e g u l a t i o n of a m m o n i u m t r a n s p o r t is also s t u d i e d a n d t h e p o s s i b i l i t y of c o u p l i n g of t h i s p r o c e s s w i t h r e g u l a t i o n of G S a c t i v i t y is d i s c u s s e d .

182

Sciences,

N i t r a t e r e d u c t a s e (NRI is one of the key e n z y m e of nitrogen metabolism and has been f o u n d to be r e g u l a t e d by p h y t o c h r o m e f°frl. C u r r e n t l y we are investigating the role of second messengers g e n e r a t e d as a c o n s e q u e n c e of a c t i v a t i o n of p h y t o c h r o m e by light f o r the s t i m u l a t i o n of NR. Involvement of polyphosphoinositide c y c l e and protein kinase C type e n z y m e in Pfr m e d i a t e d s t i m u l a t i o n of NR gene expression has been shown. We have also c h a r a c t e r i z e d a protein kinase which was found to use purified NR as a substrate. Interestingly H-7, an inhibitor of protein kinase C inhibited light s t i m u l a t e d NR t r a n s c r i p t level while it increased the NR a c t i v i t y . O k a d a i c acid, an inhibitor of phosphatase i n c r e a s e d the level of NR t r a n s c r i p t but d e c r e a s e d the NR activity. This indicated t h a t phosphorylation m a y be involved in P f r m e d i a t e d s t i m u l a t i o n of NR t r a n s c r i p t level w h e r e a s dephosphorylation may be involved in increasing the NR a c t i v i t y . Role of phosphorylation/ d e p h o s p h o r y l a t i o n in p h y t o c h r o m e m e d i a t e d s t i m u l a t i o n of n i t r a t e r e d u c t a s e will be discussed.

P-21-004

P-21-O01 METABOLIC PATHWAYS OF GLYCINE AND SERINE CONVERSION IN P H O T O S Y N T H E T I C P L A N T CELL

Life

INTERACTIONS BETWEEN P H O S P H A T E UPTAKE, RESPIRATION AND PHOTOSYNTHESIS David A. Gauthier and David H. Turpin. Department of Biology, Queen's University, Kingston, Ontario, Canada. K 7 L 3N6 We have employed phosphate (Pi)-limited chemostat cultures of the green alga Selenastrum minutum to develop a model depicting possible interactions between Pi uptake and assimilation with respiration and photosynthetic processes. Previously, we have proposed that increased respiration accompanying H+/Pi cotransport in the dark is controlled by the ATP requirement of a plasma]emma H+-ATPase (Gauthier and Turpin (1994) Plant Physiol 1114: 629-637). Metabolite changes indicated that the respiration of starch contributed less to the production of ATP during Pi assimilation under satm-ating light than in the dark. Mass spectrometry also indicated little change in mitochondrial respiration during Pi assimilation in the light, however, decreases in both 0 2 evolution and C O 2 fixation were observed. This suppression of photosynthesis m a y result from rapid triose-phosphate export from the chloroplast. Room temperature and 77K fluorescence indicated a transition from state I to state II during Pi assimilation in the light. Increased cyclic photophosphorylation accompanying the state transition could increase the ATP:NADPH production ratio and satisfy the energy requirements for Pi uptake in the light.

P-21-005 CHARACTERIZATION of GLUTAMATE SYNTHASE Chloroblum Ilmicola f. thlosuffMophilum NCIB 8327

PURIFIED

from

.Tong-Uk Na 1, H. Youn, S.-H. Cho, C.-S. Hwang Y.-C. Hah and S.-O. K a n t Department of Microbiology, College of Natural Sciences, and Research Center for Molecular Microbiology, SeouI National University 1Environment Research Center, Korea Institute of Science and Technology, P.O.Box 131, Cheongrayng, Seoul 151-650 Korea A green sulfur bacterium, Chlorobium limicola f. ttu'osulfatophilum NCIB 8327, was grown in modified Pfennig's medium including glutamate as a nitrogen source. Glutamate synthase was isolated through a series of ultrafiltration, Sephacryl S-300 gel permeation chromatography, DEAE-Sepharose CL-6B ion exchange chromatography, and preparative HPLC. The recovery and purification fold of the enzyme were 5% and 90.5, respectively. The homogeneity of the isolated enzyme was confirmed by polyacrylamide gel electrophoresis. The relative molecular mass of the native enzyme was estimated to be 137,000 by gel permeation chromatography. The enzyme consists of nonidentical two subunits with relative molecular mass of 89,000 and 46,000, which was estimated by SDS-polyacrylamide gel electrophoresis. The optimal temperature and pH of the enzyme are 35 ~ and 8.0, respectively. The enzyme is stable below 45 "C and around pH 7.0. K= values are 0.21 mM for L-glutamine and 0.13 rnM for a-ketoglutarate. The enzyme activity was almost not affected by amino acids used in this experiment, but by ADP, ATP and NADP*.

Poster P-21-006 FREE AMINO ACIDS IN MICROALGAE

P-21-009 IDENTIFICATION OF A cDNA ENCODING A CYTOSOLIC NADP-DEPENDENT ISOCITRATE DEHYDROGENASE

V.Martin-J6z6quel & A.Cueff Equipe Phytoplancton, Observatoire Oc6anologique de Roscoff, CNRS, Station Biologique, Place Georges Teissier, F-29680, Roscoff C6dex,Francc. The individual pool of free amino acids were studied in marine microalgae, as indicators of both carbon and nitrogen metabolisms. Experiments were focused either on light regulation on the autotrophic carbon incorporation, either on mineral nitrogen assimilation. The light/dark cycle was studied in T h a l a s s i o s i r a weissflogii, related with exponential or stationnary phase of growth. The daily light cycle in three dynamic regimes was studied in Chorella ovrenoidosa. The light intensity was studied in Prochlorococcus. Results were essentially related to the path of the carbon between the major families of amino acids, determined by there heads (ASP,GLU,ALA,GLY). The nitrogen regulation was studied under deficiency of nitrate, and related with light intensity, in Dunaliella tertiolecta. Results showed closed relationships between photosynthetic and photorespiratory metabolisms, (GLU,ASP,SER)

P-21-007 Nitrogenese Activities And H= Production In Continuous Cultures Of Nostoc Flagelliforme Rixs R. Lichtl, David O. Hall, Michael J. Bazin King's College London, Division of Life Sciences, London W8 7AH, UK

Nostoc flagelliforme is the subject of a study on H2 metabolism and optimisation of H 2 production. The role of the key enzyme nitrogenase and the influence of hydrogonasas on the production and consumption of H2 was established. A ohemostat was used to study the effect of growth rate, temperature and gas phase on nitrogenase activites and H2 production. Continuous growth ensured constantly high nitrogenase activities and H 2 production rates over unlimited periods of time. Increasing the dilution rate D from 0.014 h°1 to 0.022 h-t caused a doubling of the nitrogenase activity. H2 production increased 10fold with maximal rates of 45 p.mol mgChla-lh-1. Within s temperature range of 25 to 38°C, the optimum was found to be 34°C for H 2 production and 38°C for nitrogenase activity. Decreasing atmospheric nitrogen from 70% to 35% and 10% did not effect nitrogenase activities and H 2 production rates significantly. At 5% N2 a marked increase in heterooyst frequency, H 2 production, and nitrogenase activity could be observed.The activity of reversible hydrogenase can be induced 40fold in anaerobic batch culture. In continuous culture, a significant induction of that enzyme did not occur, but a slow increase in its activities could be noted. The expression of those enzymes under different gas phases is the subject of further investigation.

P-21-008 L O C A L I Z A T I O N A N D C H A R A C T E R I Z A T I O N OF THE GENE E N C O D I N G THE PERIPLASMIC NITRATE REDUCTASE FROM Rhodobacter sphaeroides forma sp. denitrificans Carole Schwintner1, Sandrine Cahors1, Monique Sabaty 2 and Pierre Richaud l I CEA-DPVE/SBC- C.E. Cadarache, 13108 St. Paul lez Durance, France 2 Dept. Microbiology and Molecular Genetics - Univ. Texas - Medical School Houston, Houston 77225 Texas, U.S.A.

Rb. sphaeroides forma sp. denitrificans reduces NO3" to N2 by a dissimilatory process including four successive reduction steps. A periplasmic nitrate reductase catalyzes the first step. We have obtained trypsic endopeptides for this enzyme. Their sequences perfectly align with the periplasmic nitrate reductase from Alcaligenes eutrophus HI6. We have defined 3 different degenerated oligonucleotides on both strands and used them in hybridization and PCR amplification experiments. All 3 radiolabelled primers hybridize with one of the four large size endogenous plasmids. By PCR we have obtained two overlapping fragments (550 and 1950 base pairs as expected). The cloning and sequencing of both fragments are in progress. The first one will be also used in hybridization experiments on the chromosome and plasmid DNA to confirm the results obtained with the different oligonucleotides.

S. Gfilvezt, E. BismuthI, P. Decottignies 1, F. Dubois2, M. Lancien t, R.S. Sangwan2, C. Cr6tin l, P. Gadal 1 and M. Hodges I ~institut de Biotechnologie des Plantes, (URAl128 CNRS), B~it. 630, Uulversit6 de Paris Sud, 91405 Orsay and 2Laboratoire d'Androgen6se et Biotechnologie, Universit6 de Picardie, 80039 Amiens c6dex, France In the light, ammonia produced by photorespiration in leaves of C3-plants can be 10-20 times higher than that formed by primary ammonia assimilation. This nutrient is incorporated into an organic compound by the glutamine synthetase/glutamate synthase pathway. To be active, this cycle requires a continuous supply of 2-oxoglutarate, but at the present time the knowledge of its production site is poorly understood. A eDNA which encodes an NADPdependent isocitrate dehydrogenase (ICDH) has been isolated from a tobacco cell culture library. To assign this cDNA to a specific ICDH isoenzyme, firstly we have used an immunocytochemical approach to demonstrate that the major ICDH isoenzyme of tobacco leaves is located in the cytosolic compartment of tobacco cells. Secondly, an amino acid sequence comparison of the N-terminus and several tryptic peptides of the purified eytosolic ICDH with that deduced from the eDNA shows that this clone encodes the eytosolic isoenzyme. The eDNA has been used to investigate the expression pattern of cytosolic ICDH transcripts in various plant tissues using a specific T-non-coding region probe.

P-21-010 IDENTIFICATION OF A cDNA ENCODING A NON-CYTOSOLIC NADP-DEPENDENT ISOCITRATE DEHYDROGENASE M. Hodge~ l, S. Gfdvezl, P. Decottigniest, E. Bismuth l, F. Dubois2, M. Lancien t, R.S. Sangwan2, C, Cr6tin 1 and P. Gadal 1 ]Institut de Biotechnologie des Plantes, (URAl128 CNRS), Bat. 630, Universit6 de Paris Sud, 91405 Orsay and 2Laboratoire d'Androgen~se et Biotechnologie, UniversitE de Picardie, 80039 Amiens c6dex, France It is widely accepted that 2-oxoglutarate represents the net source of carbon for reduced nitrogen passing through the glutamine synthetasc/glutamate synthase pathway. However, the subceUular localization of the isocitrate dehydrogenase (ICDH) implicated in its synthesis is not known, A eDNA has been isolated from a tobacco cell culture library which encodes an NADP-dependent ICDH. An amino acid sequence comparison of the N-terminus and several tryptic peptides of the pmified chloroplastic ICDH (ICDH2) with that deduced from the eDNA infers that this clone could encode the chloroplastic isoenzyme. Indeed, the amino acid sequence deduced from the 5'-end of the eDNA has the characteristics of a pepfide signal, The immunolocalization of ICDH2 shows that antigens are localized in chloroplasts, mitochondria and peroxisomes but not in the cytosol. The eDNA has been used to investigate the expression pattern of non-cytosolic ICDH transcripts in various plant tissues using a specific 3'-noncoding region probe. We suggest that a single ICDH gene might encode at least two different ICDH isoenzyrnes depending on the site of translation initiation,

P-21-011 EFFECTS OF NITROGEN NUTRITION ON ACTNITIES OF THE PHOSPHOENOLPYRUVATE CARBOXYLASE AND SUCROSE-PHOSPHATE SYNTHASE, AND THE LEVELS OF SUCROSE IN SUGARCANE LEAF. B. Suoiharto 1, Sumadi t, G.F. Hutasoit 2, and M. JusuP ~Dept of Biology, Jember Univ., Jember 68101 Indonesia; 2indonesian Sugar Research Institute, Pasuruan 67126 Indonesia; adept of Botany, Bogor Institute of Agdc., Bogor 16132 Indonesia. The activities of phosphoenolpyruvatecarboxylase (PEPC) and sucrose-phosphate synthase (SPS), and levels of sucrose were determined in response of the differences of nitrogen concentration. Nitrogen nutrition had dramatic effects on PEPC and SPS activities, and sucrose levels. The increase of nitrate or ammonium concentrations resulted in greater activities of PEPC and SPS in parallel with the increase of sucrose levels. Western blot analysis using anti-maize PEPC shown that the increase of PEPC activities was due to increasing of PEPC-protein contens, suggesting of transcriptional:regUlation of PEPC content by nitrogen in sugarcane leaf. However, nitrogen nutrition had a little effect on dbulose-l,5-bisphosphate carboxylase/oxygenase (Rabisco)-protein content. The results indicate that the increase of sucrose levels in sugarcane mature leaf at optimal nitrogen nutrition is related to greater activities of SPS and PEPC rather than Rubisco or others sucrose metabolizing enzymes.

183

Poster P-21-012

P-21-015

PHOTOSYNTHESIS, GROWTH AND CARBON PARTITIONING OF BETA VULGARIS L. AS INFLUENCED BY NITROGEN SOURCE, NO3" VERSUS NH4 +

M E T H I O N I N E B I O S Y N T H E S I S IN H I G H E R PLANTS: PURIFICATION AND MOLECULAR CHARACTERIZATION OF CYSTATHIONINE B-LYASE

N. Terry 1 & T. K. Raab 2 1Dept. Plant Biology, University of California, Berkeley, CA 94720, USA; 2Dept. Environmental, Population and Organismic Biology, University of Colorado, Boulder, CO 80309, USA

S. Ravanel. M. Droux & R. Douce Unit6 Mixte CNRS/Rh6ne-Poulenc Agrochimie, 14-20 rue Pierre Baizet, 69263 Lyon Cedex 09, France

Sugar beet plants cultured in growth chambers were transferred from nitrate to ammonium (each supplied in half-Hoagland's solution). Ammonium nutrition decreased growth and the expansion of individual leaves; however, photosynthesis per leaf area was increased. The increase in photosynthesis was associated with a doubling of chloroplast volume, a 62% rise in chlorophyll content, a 4.3-fold increase in soluble leaf protein and increases in the activities of some stromal photosynthetic enzymes. There was no effect on stomatal conductance. Sugar beets acclimated to ammonium with a 2.5fold increase in ghitamine synthase in fibrous roots and a 1.7-fold increase in leaf blades. Rapid ammonium assimilation into glutamine consumed carbon skeletons, leading to a depletion of foliar starch, sucrose, and maltose. Nitrate-fed plants contained substantially larger concentrations of osmolytes (nitrate, cations, and sucrose), which may have contributed to the faster rates of leaf expansion in nitrate-fed compared to ammonium-fed plants.

Cystathionine 13-1yase, the second enzyme involved in the methionine biosynthetic pathway in plants, was shown to be associated with both the cytosolic and the chloroplastic compartments. The enzyme was purified over 16,000-fold from spinach (Spinacia oleracea L.) leaf chloroplasts. Cystathionine 13-1yase is a typical pyridoxal-phosphate-dependent enzyme which consists of four identical subunits of Mr 44,000. A cDNA encoding cystathionine B-lyase was cloned from an Arabidopsis thaliana expression library by functional complementation of an Escherichia coli mutant deficient in this enzyme. The full-length nncleotide sequence (1.7 kb) translates into a 464 amino-acid protein with a predicted Mr of 50,372. A. thaliana cystathionine 13lyase exhibits 22% sequence identity with the E. coli corresponding enzyme and contains an additional 70 amino-acid N-temainal sequence compared with the bacterial protein. Since the general femures of chloroplast transit peptides could be observed in this amino-terminal extension, we predict that this cDNA encodes the chloroplastic isofom~ of cystathionine f3-1yase.

P-21-013 EFFECTS OF PHOSPHATE LIMITATION ON PIGMENTATION AND PROTEIN COMPOSITION IN SYNECHOCOCCU$ VULCANU$ IC Ishikawa, K. Kashinu, H. Koike & K. Satoh Dept. Life Science, Fac. Sci., Himeji Inst. Tech., Harima Science Garden City, Hyogo 678-12, Japan Phosphate is one of the most important nutrients for plants and algae. Detailed molecular mechanisms of their response to phosphate limitation are not much elucidated. We have investigated the effect of phosphate limitation on growth, pigmentation and protein composition of membranes in a thermophillc cyanobacterium, Syneehococcus vzdcanus. When cells are Wansferred to a phosphate-limited medium, they continue to grow with the same rote as that in a normal medium, and contents of phycobiliproteins and chlorophyll were not much affected in three days. Longer exposure to phosphate limitation, however, caused a decrease in biliproteins and chlorophyll, while a dramatic increase in carotenoid content was observed. Analaysis of protein composition by SDS-PAGE revaled that some protion were new by synthesized membrane fractions.

P-21-016 EFFECT OF NITROGEN AND ERBICIDE ON CROP PHOTOSYNTHESIS AND GRAIN YIELD OF BARLEY AND WHEAT. E. Pea, F. Pierandrei and G. Di Monte Ist. Sperimentale per la Nutrizione delle Piante Via della Naviceila, 2 00184 Roma. Italy. Efficient N uptake and assimilation are essential for barley and wheat growth and yield. Assuming sufficient soil water is available, nitrogen is often the most important factor limiting plant growth and crop yield. Insufficient N can reduce grain yield and quality belowe acceptable levels, while excessive N usually produces indesiderable high protein levels. On the other hand, cereal fields are often infested by weed wich compete with the crops for the N available in the soil as well as for the N fertilizer. Soil water was not limiting during the period of photosynthesis measurements: leaf CER and Ci/Ca ratio for all N treatments increased in response to the use of erbicide. On the other hand a decrease was found in WOE. Significant effects on "ct"(the slope of photosynthesis-light curves) and Pnmax were found for all treatments; "ct" was higher for untreated plants. Furthmore the use of herbicide have much more effect on untreated plants (Pnmax > 26%) with respect to the treted plants (Pnmax> 13 %). The protein content incresed with incrising N fertilizer, the use of herbicide decresed the protein content in both fertilized and unfertilized plants.

P-21-017

P-21-014 BIOLOGICAL ROLE OF POLYHYDROXYBUTYRATEIN CYANOBACTERIA M. Mivake & Y. Asada National Institute of Bioscience and Human-Technology, Higashi 1-1, Tsukuba-shi, Ibaraki-ken, Japan 305 Some cyanobacteria accumulate polyhydroxybutyrate (PHB) which is a common energy storage in bacteria. However, there have been some results suggesting that the PHB in cyanohacteria plays other roles, for example, quenching excess NADPH (Philippis er aL, 1992, FEMS Microblol. Rev. 103 187-194) or a response to a certain nutritional starvation (Stal, 1992, FEMS Microbiol. Rev. 103 169-180). These suggestions have been supported by the following facts: 1) The cyanobacteria accumulate glycogen as an energy storage. 2) The content of PHB was very low compared with glycogen in cyanobacteria. 3) The cyanoabcterla do not possess complete TCA cycle. We have isolated a thermophilic cyanobactertum which accumulates PHB (> 20 96 of total dry weight) under nitrogen limited conditions. The content of PHB was similar to that of glycogen. However, the behavior of the accumulation and decomposition of the compounds were different each other. PHB would play as an acetylCoA storage for amino acid synthesis whereas the glycogen plays as a substrate for the endogeneous respiration.

184

A M I N O ACIDS A S S I M I L A T I O N IN TOMATO Estela M. Valle, Javier P a l a t n i k and Silvana B.Boggio Area B i o l o g l a Molecular, Fac. Cs. Bioquimicas, U.N.R. S u i p a c h a 531, 2000 Rosario, A r g e n t i n a The transport of leaf assimilates to developing fruits does not limits its movement but the metabolic activity of a particular fruit.We have explored the transport of amino acids and the assimilation capacities of leaves and fruits of tomato. Analysis of the composition of the phloem sap, fruit and leaf extracts during photosynthesis revealed that while glutamate was shown to be one of the most abundant amino acid in the phloem sap, glutamine seems to be the major free amino acid present in the fruit before ripening. The study of enzyme activities showed that glutamine synthetase and glutamate dehydrogenase (GDH) where active enough to sustain glutamate and glutamine assimilation in the fruits. By Western blot analysis we observed that GDH accumulated during fruit ripening (GDH antibodies were kindly provided by Dr. K.A. Roubelakis-Angelakis) suggesting that it contributes to maintain the low ammonia levels during fruit ripening. This work was supported by the Volkswagen Foundation

Poster II

P-21-018 THE R O L E OF S H O R T A N D LONG-TERM REGULATION OF GLUCOSE 6-PHOSPHATE D E H Y D R O G E N A S E IN THE ASSIMILATION OF NITROGEN Heather C. Huppe, Jennifer Moustgaard and David H. Turpin, Department of Biology, Queen's University, Kingston Ontario, K 7 L 3N6 C A N A D A Photosynthetic electron transport supports nitrogen and carbon assimilation simultaneously in green algae grown with sufficient nutrients. When cells are limited for nitrate, however, the assimilation of nitrate is supported in part by activation of carbon respiration via the oxidative pentose phosphate pathway (OPPP) and the reduction of carbon is decreased. We have proposed that the switch from assimilatory to respiratory carbon metabolism is controlled by redox regulation of the chloroplastic glucose 6-phosphate dehydrogenase (G6PDH) (Huppe et al (1994) Plant Physiol. 105: 1043-1048). W e now report the characterzadon of Chlamydomonas G6PDH. Nitrogen limitation causes a decrease in the specific activity of several enzymes, including nitrate reduetase, however there is a marked increase in G 6 P D H activity. Characterization of G 6 P D H will be discussed in terms of localization and the short and long-term regulation of the enzymatic activity as related to the assimilation of ammonia and nitrate. Wock supported by the National Science and Engineering Council of Canada

P-21-021 P H O T O S Y N T H E S I S IN N A T U R ~ - H I C R O A L G A L G R O W T H LIGHT DILUTE CULTURE (PHOTOASSIMILATION) Nomita Sen in Botanyp

IN D I M

(nee C h o w d h u r i ) , R e t i r e d P r o f e s s o r & H e e d 236 G u p L e s h w a r P r e m n a g a r ~ J a b a l p u r t I N D I A

Interactions between photosynth~sis and nitrogen assimilation have recently been recognised by tracer a n d o t h e r s o p h i s t i c a t e d s t u d i e s . T o p r o v e this in the l i v i n g state s t u d i e s h a v e b e e n u n d e r t a k e n . S t u d l e s have been made with procaryotic and eucaryotic micro -alga~ssumin~ that if t h e r e e x i s t s a n y s u c h i n t e r a c t i o n t h e n the y i e l d w i l l be a f f e c t e d e i t h e r p o s i t i v e l y or n e g a t i v e l y i f g r o w n u n d e r l i g h t l i m i t e d c o n d i t i o n s f o r p h o t o s y n t h e s i s in d i l u t e c u l t u r e in p r e s e n c e of s u i t a b l e o a r b o n a n d n i t r o g e n s o u r c e , T h e e f f e c t s of v a r i o u s c a r b o n s o u r c e s on the g r o w t h of micro--algae have already been reported.This paper r e p o r t s the e f f e c t s o f v a r i o u s n i t r o g e n s o u r c e s on t h e g r o w t h of m i u r o - a l g a e . P r o e a r y o t ~ ¢ algae studied

exhibited Chloroll~ nitrate

6rowth in the medium without nitrate but did not grow in this mediumoMedium with showed

good

growth

in all

the a l g a e

studied,

P-21-019

UPTAKE OF SULFITE AND PHOTOINHIBITION IN A L G A L CELLS H. Sakurai & S. Pomprasirt Dept. of Biology, Sch. of Education, Waseda University, Shinjuku, Tokyo 169-50, Japan The mechanism of sulfite uptake is not elucidated in plants as well as in bacteria. The [35S]-sulfite uptake by Chlorellaand Chlamydomonaswas studied using a centrifugation-filtration method through a silicone layer. At fixed concentrations of sulfite between pH 4-7, the uptake was the higher the lower the pH. When the pH was raised, the uptake rate was instantly decreased. Analyses of intracellular [35S] compounds by HPLC revealed that the metabolic rate of sulfite was considerably slower in the dark than in the light. Cells were preincubated with sulfite in acidic solutions, washed and suspended in a neutral medium, and then illuminated with strong light. Subsequent measurements of photosynthetic activity revealed that photoinactivation was strongly correlated with the amounts of sulfite taken up.

P-21-020 REDUCTION OF OXYANIONS BY PHOTOSYNTHETIC BACTERIA : ROLE OF THE NITRATE REDUCTASE IN THE REDUCTION OF TELLURITE. C~cile Avaz~ri 1, Janine Pommier2, Francis Blasco2, G~rard Giordano2 and Andr~ Verm~glio 1. 1 : LBC/DPVE/CEA Cadarache 13108 Saint Paul-lez-Durance Cedex. 2 : LCB/CNRS Chemin Joseph Aiguier 13009 Marseine. Some species of photosynthetic bacteria reduce several oxyanions. In our study, we have observed that Rhodobacter sphaeroides forma sp. denitrificans reduced tellurite in tellurium. We have shown by differents biochemical approaches that the periplasmic nitrate reductase is involved in the reduction of tellurite. We have found no other evidence for a tellurite reductase. The reduction of tellurite has been observed for both periplasmic and membrane bound nitrate reductases of several others non photosynthetic bacteria. In particular, we have shown in Escherichia ¢oli, by differents approaches (enzymatic activities, immonoelectrophoresis) and with hel F of mutants that the nitrate reductase is the main enzyme responsible for reduction of tellurite in this species. We have also tested one strain : ECOR17, which does not contain tehA and tehB, genes encoding for tellurite resistance. For this strain, we have found that nitrate reductase was not synthetised. Others experiences indicate that tehA and tehB are implicated in the regulation of nitrate reductase. We conclude that the nitrate reductase plays an important role in the metabolisation of oxyanions. Nevertheless, we cannot exclude that a specific reductases are also involved in the oxyanions reduction.

185

Poster

P-22-003 SHORT TERM EXPOSURE OF ~ TO 13 kJ m': d"t WEIGHTED UV-B IRRADIANCE AND DIFFERENT PAR INDUCES A SHADE TYPE RESPONSE IN PALISADE CELLS. Poster

session

22

W.R. Fagerbergt and J.F. Bornraan2. tDept of Plant Biology, Universityof New Hampshire,Durham,New Hampshire,03824, USA; 2Dept of Plant Physiology,Lund University,Box I 17, 22100 Lurid, Sweden.

P h o t o i n h i b i t i o n a n d o x i d a t i v e stress

P-22-001

-

P-22-081

P-22-001 XNHIBITION OF PHOTOSYSTEM I ELECTRON TRANSPORT BY H I G H LIGHT. M. Sharma, U. D w i v e d i a n d R. B h a r d w a j , S c h o o l of Biochemistry, D.A. University, Khandwa Road, Indore-452 001, I n d i a . T h e e f f e c t of h i g h l i g h t s t r e s s on t h e k i n e t i c s o f P 7 0 0 r e d u c t i o n a n d t h e c h a n g e s in P 4 3 0 w h i c h is a m e a s u r e of i r o n - s u l f u r c e n t e r s w a s c h a r a c t e r i s e d in isolated PS I particles. Illumination of PS I particle at high photon flux density (2200 W m "2) under aerobic conditions induced photoinhibition of PSI m e a s u r e d as l i g h t i n d u c e d a b s o r b a n c e c h a n g e a t 700 n m a n d 430 nm. U n d e r a n a e r o b i c c o n d i t i o n , P700 determined as l i g h t induced absorbance change was f o u n d t o d e c r e a s e d . W h e n 400 m24 D i t h i o n i t e o r I00 JaM Anthraquinone-2-sulphonic a c i d w a s included in the mixture, P700 content was not significantly decreased. However, 100 nlEI V i t a m i n E or 5m/H Glutathione were found to protect t h e d e c r e a s e in P700 content. These results suggest that Photosystem I r e a c t i o n c e n t r e P 7 0 0 is a l s o s u b j e c t t o damage by high light.

P-22-002 P H O T O S Y N T H E T I C A D A P T I V E M O D U L A T I O N OF RICE TO LIGHT-STRESS AND ITS GENETIC IMPROVEMENT APPROACH Demao Jiao, Bin Yan, Benhua Ji Inst of Genet & Physiol, Jiangsu Acad Agric Sci, Nanjing 210014, China PSI activity was induced to increase while PSII activity started to decrease in the tolerant-photooxidant cv. under photoinhibition conditions, indicating there was a compensation function in PSI to PSII. Following the increase in free radical generation, RuBPc activity was inhibited apparently but carbon anhydrase (CA) and PEPc activities were induced to enhance significantly. The result was demonstrated as an increase in molecular expression by western blot, signifying that there is a partial compensation function in CA and PEPc to RuBPe. Application of DCDP (PEPc inhibitor) and Diamox (CA inhibitor) showed an alleviative effect of CA and PEPc on the photoinhibition of PSII and photosynthesis. Significant inducible activity of SOD in the tolerant cv. was of higher and longer duration. Application of DDTC (SOD inhibitor) indicated that SOD decreased the photoinhibition of photosynthesis. Thus, there are several endogenous adaptive systems to protect sensitive sites from lightstress damage. For genetic improvement, we developed a simple and effective technique for massive screening tolerant-photooxidative germplasm and suggested that the photoinhibition traits in F 1 is much biased toward their material genotypes.

186

Plants were sampled after exposure to ultraviolet-B(UV-B) and photosyntheticallyactive radiation (PAR) for 30 min, 1 day and 1 week. Quantitativecytologicalmeasurements (stereology)showedthat UV-B with either 600 or 200/.tmol m-2s"t PAR induced shade effect changes in cell structure including decreases in volume and volume density (Vv) of: chloroplasts, starch, mitochondria,vacuoles and raicrobodies. Organelleresponseswere often exaggeratedin the ffrst 30 rain of UV-B exposure. The surface density (Sv) and surface area of the appressed, non-appressedand raargin thylakoids increasedwith UV-B exposure in both high and low PAR co-treatments.Low and high PAR plus UV-B promoted a more rapid increase in the surface area of appressed vs nonappressedthylakoids on the ftrst day of UV-B exposure. This trend was reversed after one week acclimationto UVB in the high PAR leaves but not in the low PAR plus UV-B samples.These results support the possibility that the two thylakoid systemsmay be regulatedseparately. One week acclimation to UV-B and the blocking effects of UV-B absorbingpigmentsdid not change cellular responses. Thylakoid packing ratios (mrs2thylakoid/rata2 leaf surface) increased under UVB exposure thereby increasingthe statistical probability of photon interception and potential damage from UV-B. We also felt there was support for the notion that UV-B may have a regulatoryrole outside of damage commonlyascribedto it.

P-22-004 PHOTOINHIBITION OF PHOTOSYIfI'HESIS IN ANDEANPOTATO (~olantm spp.) SPECIES DIFFERING IN FROST RESISTANCE M a r t i n e z & M. M a e s t r i Dept. of Plant Biology, Federal University of Vigosa, 36570000 Vigosa, Minas Gerais, Brazil. Photoinhibition of photosynthesis and its recovery were studied in two frost resistant (Solanlm curtllobum and Solanum JuzeDczuckil) and two frost sensitive (Solanum tuberosum and Solanum andizena) potato species exposed to 1500 and 400 pmol photons m-2 s-l. Chlorophyll a fluorescence, photosynthetic rate and superoxide dismutase (SOD, EC l. I5.I.l) activity were measured. Photoinhibltion had marked effects on the kinetics of chlorophyll fluorescence. Maximum fluorescence(Fm), variable fluorescence (Fv), and the ratio Fv/Fm were reduced, while initial fluorescence (Fo) was increased. Photosynthetic rate and photochemical activity of photosysthem II were unable to recover in the presence of streptomycin. Resistance t o photoinhibition was much higher in the Andean frost resistant potatoes. A three fold increment in FeSOD activity was found when leaves of S. Durtilobum and S. ~uzeDczuckii were exposed to high irradiance. Our data suggest that SOD activity play a significant role in protection of Andean potatoes against light stress.

P-22-005 DECLINE IN PHOTOSYNTHETIC ELECTRON TRANSPORT ACTIVITY AND CHANGES IN THYLAKOID PROTEIN PATTERN IN SUNFLOWER PLANTS EXPOSED TO OZONE. A. Ranieri, A. Castagna, M. Tognini, C. Tozzi & G.F. Soldatini I s t i t u t o di Chimica Agraria, UniversitA degli studi di Pisa, Via S. Michele degli Scalzi 2, Pisa, 56124, I t a l y The dangerous action of ozone (03), one of the most phytotoxic a i r pollutant, is due to its very high redox potential (+2.07 V). Ozone damages photosynthetic metabolism and this predisposes the apparatus to photoinhibition. To better understand the alterations in photosynthetic components of plants exposed to 03, we examined the a c t i v i t y of PSI and PSII dependent electron transport in sunflower plants subjected to a treatment with ozone (150 ppb f o r 4 hs, repeated f o r 4 days). Results obtained showed a reduction of the whole electron transport chain (-40%) and both PSI (-56%) and PSll (-38%) a c t i v i t i e s in 03 fumigated plants. The analysis of thylakoid polypeptides by two dimensional electrophoresis showed a decrease in optical density of some spots in the pattern of treated plants compared to the untreated ones.

Poster

P-22-006

P-22-009

Post-illumination Resurgence of Non-photochemical Quenching in Dunaliella tertiolecta.

TURNOVER OF THE RCII-DI PROTEIN IN THE DARK INDUCED BY PHOTOINACTIVATION OF PHOTOSYSTEM II

C. Casper-Lindley & O. BjcSrkman Carnegie Institution of Washington, 129 Panama Street, Stanford, CA 94305, U.S.A.

]h~ashi Gon~ 1 & Itzhak Ohad 2 IDept. of Biology, Box 1045, University of Oslo, Blindern 0316 Oslo, N e r w a y ; 2Dept. of Biological Chemistry, The Hebrew University, Jerusalem, 91904, Israel

Non-photochemical quenching (NPQ) in the green alga Dunaliella tertiolecta induced by exposure to bright light relaxes completely within the first minute after darkening. During the subsequent 30 minutes NPQ of Fm and Fo is re-established and then slowly relaxes. Levels of the dark quenching can reach values as high as the NPQ during the light treatment. The extent of the dark quenching depends on the duration and intensity of the previous light treatment. Uncouplers of the thylakoid membranes do not affect the dark quenching whereas inhibitors of mitochondrial respiration extend the dark quenching significantly. Far red light (730 nm) and PS I stimulating light (430 am) suppress the dark quenching. 77K fluorescence spectra show an increase of PS I fluorescence relative to PS II fluorescence during dark quenching. This suggests that state transitions take place. Such state transitions seem to be characteristic of the time following a light treatment and they can not be generated in bright white light or in response to red/far red light. The possible influences of the adenylate energy charge on the reduction state of the plastoquinone pool in darkness will bc discussed.

P-22-007

In Scenedesmus obliquus LF-I mutant " containing an unprocessed precursor DI protein (pDl) and defective in donor side activity of PSII, photoinactivation of PSII was considerably faster than that in wt. The R C I I - D I ( p D I ) protein in p h o t o i n a c t i v a t e d cells was irreversibly modified and could be degraded and re-synth,'sised (turnover) in the dark in both wt and LF-I. Turnover of the D! protein was proportional to the initial degree of photoinact!vation. Diuron retarded DI degradation in the photoinactivated cells. F,emoval of diuron allowed rapid turnover of photoinactivated RCII-DI(pDI) in the dark. The protective effect of diuron was gradually lost in photoinactivated cells even when incubated in the dark. We conclude that the irreversible changes induced by photoinactivation of RCIIDI(pDI) protein in vivo are sufficient to allow complete DI protein lurnover in the dark. Redox control of the DI protein degradation in ,ivo is thus related to availability of oxidised plastoquinone which ~:ffects the access of the modified protein to the cleavage system.

P-22-010

INCREASED PHOTOSENSITMTY OF THE P H O T O S Y N T H E T I C A P P A R A T U S IN A T R A Z I N E - R E S I S T A N T B I O T Y P E F R O M Conyza Canadensis (L.) Cronq.

DISASSEMBLY OF CYANOBACTERIAL PltOTOSYSTEM H COMPLEX INDUCED BY HIGH IRRADIANCE IN VIVO: AN IMPLICATION FOR TIIE MECHANISM OF THE D1 DEGRADATION

l~va Dark6. Gyula Vhradi 2, Endre Lehoczki 1

J. Komenda and J. Masojidek Department of Autotrophic Microorganisms, Institute of Microbiology, Tfebofl, Czech Republic

I Research Group of the Hungarian Academy of Sciences, Dept. of Botany, JATE University, H-6701 Szeged; 2 Research Institute for Viniculture and Enology, H-6000, Kecskem~t, Hungary The photosensitivities of atrazine-rezistant (AR) and susceptible (S) biotypes of Conyza canadensis grown in high light were investigated. Although the efficiency of the primary charge separation (Fv/Fm) did not differ in the biotypes, the effective quantum yield of photochemistry (AF/Fm') was lower in AR plants at any light intensity because of the higher proportion of reduced QA (lower qP) and the lower capacity of nonphotochemical quenching of the excitation energy (lower qN). The higher fraction of QA" is due to the lower electron transport rate because of the lower reoxidation of QA-. The lower qN results from the lower activity of the xanthophyll cycle, which appears in the qE component of qN. These results suggest that the higher reduction state of QA and the lower activity of the xanthophyll cycle may play an important role in the higher susceptibility to photoinhibition of A R plants.

P-22-008 THE RELATION BETWEEN PHOTORESPIRATION PHOTOINHIBITION IN COTTON LEAVES

AND

L-W Gut. D-Q Xu and Y-K Shen Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, PR China The photochemical efficiency of PSII (Fv/Fm) and apparent quantum yield (AQY) were compared in cotton (Gossypium hirsutum) leaves after exposure to strong light (1500-1700 mol photons m-2s"l) for 3 hours in normal air and in 2.1% 02 air It was found that the leaves in 2 1% 02 air showed larger declines in Fv/Fm and AQY than those in normal air, though the photosynthetic rate of cotton leaves in 2.1% 02 air was higher at the beginning of exposure. It indicated that the leaves in 2.1% O 5 air were more liable to be photoinhibited After exposure to strong light the inorganic phosphate(Pi) content of leaves in 2 1°,4 02 air was much lower than that in normal air. Feeding Pi to the leaves through the petioles could make the difference in photoinhibition between the two treatments no longer significant. It is suggested that photorespiralion plays a role in protecting photosynthetic apparatus against photoinhibition under strong light in normal air by enhancing the recycling of Pi, besides its effect through the dissipation of excess light energy.This work was supported by NSFC (39230050).

A gradual disintegration of the PSH core complex initiated by a release of the chlorophyll-protein CP43 was identified during illumination of the Synechococcu~ cells at low temperature which blocked protein syntls~is. This process was slower compared with inactivation of Hill reaction activity and PSH primary charge separation activity. The electrophoretic analysis showed that the release of CP43 preceded the degradation of the D1 protein. The recovery experiments revealed that no disassembly of PSII was needed for the parallel degradation and replacement of the D1 protein at growth temperature. We propose the existence of two fundamentally different mechanisms of the D1 degradation in vivo. The one, which requires ongoing protein synthesis, occurs without an apparent disassembly of PSH and immediately follows inactivation of oxygen evolution. It can be based on the action of an external protease(s) recognizing a conformation change of the QB binding site. The second mechani.~Tn occurs when protein synthesis is inhibited and consists in the action of reactive radicals and oxygen species after the extensive f~notional damage and disassembly of PSH.

P-22-011 T U R N O V E R O F THE D1 P R O T E I N O F P H O T O S Y S T E M II IN FATI'Y ACID MUTANTS O F SYNECHOCYSTIS SP. PCC 6803 Eira Kanervo l, Eva-Marl A r t 1 and Norio Murata 2 1Department of Biology, University of Turku, SF-20500 Turku, Finland; 2Department of Regulation Biology, National Institute for Basic Biology, Okazaki 444, Japan. The effect of thylakoid lipid unsaturation on the turnover of the D1 protein of PSII was studied using cyanobacterial mutants differentially defective in fatty acid unsaturation. The Fad6/desA::Km r mutant, containing only monounsaturated fatty acids, is extremely sensitive to high light. We found that the DI protein content of that mutant decreased drastically during illumination, compared to the wild type. Degradation of the D1 protein proceeded more or less at the same rate in all three strains in the presence of lincomycin. Thus, the reasons for the increased susceptibility of the Fad6/desA::Km r mutant to photoinhibition and the reduction of its D1 protein content should be attributed to incomplete function of the synthesis side of the repair cycle of PSII. Indeed, pulselabeling of the cells with [35S]-methionine revealed inefficient synthesis of the DI protein in the Fad6/desA::Km r mutant under high light. Expression of the psbA gene at the level of transcription was not affected. 187

Poster

P-22-012 PIGMENT AND HISTIDINE RESIDUES DESTRUCTION OF PSil REACTION CENTER COMPLEX INDUCED BY CHEMICALLY GENERATED SINGLET OXYGEN

Ktm-Yun Yang. I.m Xiong, Chong-Qm Tang, Ting-Yma Kuang & Pei-SongTang Lab. of Photnsynthesis,Instituteof Botany,Aedem/a Sin/ca,Beijing100044, P,R. China Exposme of PSH reaction center DI/D2/Cyt b559 complex to chemically generated singlet oxygen in the dark resulted in rapid decreases of CM a aad histidine residues level that was observed upon the photoinhibitoryillumination of PSII rcaetinn center complex in the absence of election acceptor (Tang, C.-Q. et al 1992, In: Research in photosysthesis. Voi. HI, 439-442.). Both strong illnmin=tion aad singlet oxygen induced destruction of Chl a and histidine residues can be alleviated significantly by the addition of free histidine. Furthermore, the presence of singlet oxygen caused the dama£e of 13-Carotene that was not observed in the photoinlu'bition, and the accessory Cld a showed a same vulnerability as P680 to chemically generated singlet oxygen. _Therefore, we propose that the singlet oxygen,generated in the neighborhoodof P680 during aerobic ilhwnlnation, i~ responsible for the photodamage of Chl a and histidine residues in PSII reaction center.

P-22-015 THE 02 DEPENDENT I N m m m o N TOBACCO PLANTS

OF PHOTOSYNTItJ~SIS IN

F. V~cha & M. Din-chart Inst. Plant MoL BioL, Brani~ovsk~i 31, ~esk6 Bud6jovice 370 05 Czech Repubfie The tobacco plants grown hydroponicaly in defined conditions were transferred into the growing chambers with reduced CO2 atmosphere. The concentration of oxygen was adjusted to 20% and 10%, respectively. The activity of photosynthesis, measured as the electron Uansport by PAM finorometr and the oxygen evolution of leaf discs, decrease more rapidly in 20% of oxygen in compare to 10% indicating an inlnqfitory effect of oxygen on photosynthesis. We have compared the activity of the Mehler reaction and photorespiration to determine the contn~oufion of both oxygen dependent processes to the protection of photosynthetic apparatus. We suggest that the Mehler reaction is not saturated even at 10 % of oxygen, supported by the measurements of the amount of peroxide lipids in stressed plants and by the treatment with scavengers of oxygen radicals. The involvement of singlet oxygen molecules was also established. We conclude that not only the photorespiration but the also the extend of the Mehler reaction and subsequent production of harmful species affect photosynthsis.

P-22-013

P-22-016

OVER-PRODUCTION OF THE Dl:2 MAKES SYNECHOCOCCUS CELLS MORE TOLERANT AGAINST PHOTOINlilBITION OF PHOTOSYSTEM II

PHOTOREDUCTION OF CYTOCHROME b-559 AND PHOTOINHIBITION OF PHOTOSYSTEM lI FROM HIGHER PLANTS: EFFECT O F C U O D INHIBITION

A. Soitamol, G. Zhou2, A. Clarke2, G. Oquist2, P. Gustafsson2 and E-M. Aro l IDept,ofBiology, University of Turku, BioCity 6A, FIN-20520 Turku, Finland 2Dept. of Plant Physiology, University of Ume~ S-90187 Ume~, Sweden

I. Ymela, J.J. Pueyo and 1L Picorel Estacibn Experimental de Aula Dei, CSIC, Apdo. 202, E-50080 Zaragoza, Spain

Over-expression of the DI protein (D1:2) in the Synechococcus sp. PCC 7942 was studied using tac-promoter and laclQ system. The over-expression of D 1:2 protein was induced with 40 Ixg ml"t IPTG for 12 hours at growth light (50 I.tmolphotons m'2s'l). Over-expression of tac-psbAlll was light induced and doubled the amount of psbA11/l11 mRNA and D 1:2 protein in cells at growth light. Concurrently, the amount ofpsbAl messages and D 1:1 protein was decreased. Total content of both D1 and D2 PSII proteins remained constant at low light indicating that the number of PSII centres in the tylakoid membranes was not affected. When the cells were photoinhibited in high light the amount of D1:2 protein remained at higher level in cells in which over- production of Dl:2 protein had been induced by IPTG. Those cells were also less prone to photoinhibition of PSII. It was proved that PSH centres with Dl:2 protein in the heterodimer are more tolerant against photoinhibition than cells with D1:1 protein.

The effect of Cu(H) inhibition on the photoreduction of cytochrome (cyt) b-559 in PSH-enriched membranes was measured by light-induced absorption changes at 560 nm. The positive band centered at this wavelength decreased in Cu(H)-inhibited samples. The photoreduction kinetics of eyt b-559 in the second range and the oxygen evolution activity were measured in aerobic and anaerobic photoinhibitory conditions. In the presence of oxygen the kinetics ofcyt b-559 photoreduction were comparable both in intact and Cu(II)-inhibited samples and the loss of oxygen evolution activity increased in the samples treated with CuC12but no effect on D1 degradation was observed. In the absence of oxygen no differences in the activity curves were observed. The role of cytochrome b-559 and the influence of Cu(II)inhibitory effect on the photoinhitionprocess will be discussed.

P-22-017

P-22-014 UV-B INDUCED DEGRADATION OF THE REACTION CENTRE II D1 PROTEIN IN PHOTOSYSTEM II MEMBRANE PREPARATIONS

INHIBITION OF PHOTOSYSTEM II BY ULTRAVIOLET-B RADIATION AND THE MECHANISM OF RECOVERY

, 1~. Hideg& I. Vass nstltute f Plant Biology, Biological Research Center, P.O. Box 521, Temesvari krt. 62, H-6701, Szeged, Hungary

Carolin Ihle & Hendk Laasch Institute of Ecological Plant Physiology, Heinrich-Heine-Universitat, Universit~tsstral~e 1, 40225 D0sseldorf, Germany

The role of the quinone electron acceptor (OB) in the UV-B induced loss and fragmentation of the Photosystem II (PS II) reaction centre D1 protein has been studied in isolated spinach thylakoids. A 20 kDa Cterminal fragment is detected both in intact samples, which contain functional QB, and in thylakoids from which QB has been depleted by heptane/isobutanol extraction. Furthermore, the UV-B induced loss of D1 protein is similar in the presence and absence of QB. Irradiation of the samples at 50 or 7 ~E/m2/s UV-B intensities induces the same fragmentation pattern and the same dose-dependence of D1 protein loss. This indicates that our results are relevant to in vivo studies conducted under lower intensity irradiation. It is concluded that, contrary to earlier assumptions, the cleavage of D1 protein does not absolutely require the presence of QB as UV-B sensitizer. Instead, the PS II donor side components are most likely involved in the degradation process.

The thalloid liverwort Conocephalum conicum is extremly sensitive to ultraviolet (UV)B radiation. 50% inhibition occumd within 10 minutes of irradiation. In comparison, photosynthesis of sunflowers was unaffected even after 5 h of similar irradiation. In the liverwort, photosystem (PS) II was the major target of UV-B radiation. The ratio of variable and maximal chlorophyll (Chl) fluorescence at room temperature, F~Fm, was linearly related to photosynthetic 02 evolution. Much lesser inhibition of F,,/Fm was found when photosynthetic electron flow was inhibited with DCMU after UV-B irradiation. This indicated that elctron flow from QA to Q, is not rate-limiting and suggested that Fm was not obtained even in saturating light. A comparable relationship between the emperical fluorescence index AF/Fm' and the quantum yield of CO2 fixation was obse[ved when electron flow was adjusted by light flux or by UVB inhibition. Recovery from UV-B damage was most effective when the thalli w e r e stored at low light. Recovery was temperature dependent and was prevented by addition of streptomycin, an inhibitor of protein synthesis. The recovery from UV-B induced damage and photoinhibition by visible light appeared to be comparable.

188

Poster

P-22-018 PHOTO-TOLERANT MUTANTS OF SynechocystJs PCC 6803 OBTAINED BY IN VITRO RANDOM MUTAGENESIS OFpsbAH ~,zy. Narusaka, ~M. Saeki, ~'3H. Kobayashi and ~'2K. Satoh (XNIBB, 2Okayama University a n d 3University of Shizuoka)

In vitro r a n d o m mutagenesis by PCR u n d e r a condition for reduced fidelity of amplification was applied to psbA in order to analyze the molecular mechanism underlying the photosensitivity of PSII reaction center. A fragment of psbAIl of S3~nechocystis PCC 6803 containing KpnI-HinclI (nucleotides of 538-1071 in the coding region) was subjected to the mutagenesis and then returned into the strain CmA-1 to replace the endogenous gene, in which psbAl a n d psbAll h a d been inactivated (Debus et al., 1988). The transformants subviving at 324tie p h o t o n flux density, where the control strain becames photo-bleached, were screened. In cnsequence 32 photo-tolerant mutants have been obtained from a population of transformants over 500,00. The result of mechanistic analysis of photo-tolerance caused by amino-acid displacements on D1 will be discussed in this poster.

P-22-019 DEGRADATION OF D1 AND D2 PROTEINS BY EXPOSURE OF PSII C O M P ~ S TO SINGLET OXYGEN K. Okada, N. Yamamote & M. Miyao-Tokutomi Lab. Photosynthesis, NIAR, Kannondai, Tsukuba 305, J a p a n Singlet oxygen (102) is active oxygen species generated inside PSII under photoinhibitoryillumination. When isolated PSII complexes were exposed to 102 generated by photesensitizing reaction of rose bengal, the D1 and D2 proteins were degraded to specific fragments. The pattern of fragmentation of the D1 protein was almost the same as that under photoinhibitery ilh]rnluation as reported previously (Mishra & Ghanotakis (1994) Biochim. Biophys. Acts 1187, 296300), and fragments of 22(N), 16(C) and 7.9(C) kDa were generated. The D2 protein was degraded to fragments of 24, 17, and 10 kI)a. The degradation of proteins was suppressed by histidine (a scavenger of 102) and n-propyl gallste (a scavenger of hydroxyl and alkoxyl radicals). When PSII complexes which had been completely solubilized with SDS were exposed to 102, fragments of the D1 protein of 22-24 and 16-18 kDa were generated, while no fragment of the D2 protein was detected. This observation suggests that the D1 protein has amino acid sequences specifically susceptible to cleavage by active oxygen.

P-22-020

CLEAVAGE OF P S I I D1 PROTEIN BY ACTIVE OXYGEN M. Miyao-Tokutomi, K. Ol~da & N. Y a m ~ o t o Lab. Photosynthesis, NIAR, Kannondai, Tsukuba 305, Japan

Under illumina~on, the D1 protein of PSII reaction center is cleaved on the loop that connects membrane spanning helixes IV and V, giving rise to the N-terminal and C-terminal fragments of 22-24 kDa and about 9 kDa, respectively. Exogenous active oxygen also degrades the protein to fragments of 22 and 9 kDa. The C-terminal fragments of the D1 protein were compared to investigate the cleavage sites. Strong illumination of PSII membranes gave rise to two different C-terminal fragments of 7.9 and 9.3 kDa. Treatment of PSII membranes with H202 in darkness also gave rise to two C-terminal fragments that were the same size as those produced by strong light. Exposure of PSII complexes to 102 generated by photesonsitizing reaction of rose bengal also gave rise to the same two C-terminal fragments. In this case, the 7.9-kDa fragment was much more abundant than the 9.3-kDa fragment. These results suggest that strong light and exogenous active oxygen both cleave the D1 protein at the same sites on the loop that connects helixes IV and V, and that the cleavage occurs at two different sites apart by about 10 amino acid residues.

P-22-021 ZEAXANTHIN FORMATION AND FLUORESCENCE QUENCHING IN INTACT LEAVES OF TRIAZlNE-RESISTANT AND SUSCEPTIBLE CHENOPODIUM ALBUM PLANTS RELATED TO PHOTOINHIBITION V.B. Curwiel & J.J.S. van Rensen Oept of Plant Physiology, Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands The triazine-resistant biotype (R) of Chenopodium album is in vivo more sensitive to photoinhibition (PI) than susceptible plants (S). Different mechanisms protective against PI in R and S which were grown at low (LI) and high irradiance (HI) have been studied. Grown at HI, both biotypes do not differ in qN, but R has a higher amount of zeaxanthin (AZToT). This result is partially explained by photorespiration, which is more important in R than in S for HIgrown plants. Grown at LI, the two biotypes have a higher qN at high actinic irradiance than the HI-grown plants, but they do not differ in AZToT. The HI-grown plants show a linear Stern-Volmer relationship between qN and AZToT which indicates a common regulatory phenomenon, the ApH. Recent experiments have shown more insight in the relationship between AZToT, ApH and qN.

P-22-022 pH-DEPENDENT PHOTOREACTIONS OF HP AND LP FORMS OF CYTOCHROME b559 DURING PHOTOINHIBITION Josd M. Orm~a. Manuel Herv~s,Miguel A. De laRosa and Manuel Losada

lnstituto de [~ioquimica Vegetaly Fotosfntesis, Universidad de Sevilla-CSIC, Apartado 1113, E-41080Sevilla, Spain The role of cytochrome b559 (Cyt b559), which is an intrinsic component of the PSIIreactioncentre,remainsunclear.Thiswork reportsthe photoresponses of the high- (HP) and low-potential(LP) forms of Cyt b559 ifiactivePSII preparationsunder photoinhibitoryconditions,both on the donor and acceptor sides,pH-depundentphotooxidationof H P Cyt b559 isinducedby red lightat room ~ m p e m ~ when the electron flow from water is impaired. The process takes place only at pH values higher than 7.5. At pH 8.5 and in the presence of 1 ixM of CCCP, such a photooxidation of the I-IPform is accompanied by its partial transformation into the LP form. On the other hand, red light-induced partial photoreduction of LP Cyt b559 is observed under aerobiosis when the electron transfer flow through quinones is impaired by strong irradiation in the presence of 40 }xM DCMU, a process which is enhanced at acidic pH values. To the best of our knowledge, this is the first time that both photoreductionand photooxidation of Cyt b559 is described under photoinhibitory conditions using the same experimental system. A model is proposed in which Cyt b559 could protect PSII against donor and acceptor side photoinhibition by a mechanism involving both redox and acid-base processes. Work supported by grants PB91-85 and PB93-0922 from DGICYT (Spain)

P-22-023 TKh R01~ OF C ~ A N D Cl" I(I~S IN DC~C~ SID~ INDUCED PHOTOINHIBITI(]~ C~ PHOTOSYSTEM II F. ~amedov, R. Gadjieva, N. Ferimazova & R. Gasanov BiophYsics Lab., Institute of Botsay, Azerbo Acad. of Sci., Patamdar sh. @0, Baku 370073, Azerbaijan The phctoinhlbition kinetics (1000 ~/ms. 20°C) of electron transport (H20 ~ ~pBQ, H20 ~ D~IP, D~C -, DPIP) and delayed fluorescence (DF) were investigated in PS II particles, lacking q8 kDa(i), 18,24 kDa (ii) and 18,2~,33 kDa(iii) peripheral proteins in presence of different concentration of Ca2~and CIions° The peripheral proteins removal (i) and (li) didn't effect of H20 -~ ~!oBQ reaction photoinhibition kinetics. Additicm of Ca~'and C1-ions (30 m~) increased sensitivity to photoinhibition H20 ~ PpBQ reaction in (i),(ii) particles, but not shown increasing of sensitivity of DI~ @ DPI~ reaction for any particleso The direct correlation between dicreasing of fast component of DF and H2C @ I pBQ reaction in pres enoe of C az+and Cl-"ions w&s showm. •I~ is suggested that electron transfer from ia-cluster to Tyrz may be i n h i b i t e d , C ~ : C l - i o n s have p r o m o t e d t h i s p r o c e s s °

189

Poster

P-22-024 DOES PHOTOINHIBITION OCCUR CONDITIONS IN A CYANOBACTERIAL

UNDER

NATURAL

FIELD

LICHEN?

J. Leisner, W. B i l g e r & O.L. Lange J u l i u s - v o n - S a c h s Institut mit B o t a n i s c h e m D a l l e n b e r g w e g 64, 97082 W~rzburg, Germany.

Garten,

Mittlerer

In a local x e r o t h e r m i c steppe formation, c h l o r o p h y l l fluoresc e n c e of the c y a n o b a c t e r i a l l i c h e n P e l t i a e r a r u f e s c e n s was m o n i t o r e d c o n t i n u o u s l y along with the m i c r o c l i m a t e d u r i n g the c o u r s e of an e n t i r e year. F r o m these data the time c o u r s e of p h o t o c h e m i c a l a c t i v i t y of this p o i k i l o h y d r o u s o r g a n i s m was derived, t o g e t h e r with incident p h o t o n flux d e n s i t y (PFD). The a c c u m u l a t e d time periods of p h o t o c h e m i c a l a c t i v i t y were ca. 30% l o n g e r in the w i n t e r as c o m p a r e d to the summer, w h i l e the l i c h e n r e c e i v e d o n l y about i0 to 15% of photons. The d a i l y m a x i m u m PFD r e c e p t e d in the active state d u r i n g s u m m e r c o u l d reach 30 times that of winter, but a p e r s i s t i n g d e p r e s s i o n of p h o t o s y s t e m II q u a n t u m y i e l d was not observed. O n l y e x p e r i m e n tal t r a n s p l a n t i n g of a lichen from d e e p shade into full sun r e s u l t e d in s l o w l y r e v e r s i b l e p h o t o i n h i b i t i o n a l r e s p o n s e s . A c c l i m a t i s a t i o n of the p h o t o s y n t h e t i c a p p a r a t u s to d i f f e r e n t light i n t e n s i t i e s c o r r e l a t e d with changes in the c o n t e n t s of chlorophyll a, h-carotene and canthaxanthin, but not zeaxanthin. It is c o n c l u d e d that p h o t o i n h i b i t i o n at the natural c o n d i t i o n s u n d e r c o n s i d e r a t i o n occurs o n l y u n d e r e x c e p t i o n a l circumstances.

P-22-025 ATP-DEPENDKNT PROTEOLYSIS OF A TRUNCATED D1 PROTEIN S. Schrader2, U. Alienfeld2 & U. Johanningmeier1 1 Biochemie der Pflanzen, Albert-Ludwigs-Universitit, Schinzles~r. 1, 79104 Freiburg, Germany; 2 Biochemie der Pflanzen, Ruhr-Universit~t, Universit~tsstr. 150, 44780 Bochum, Germany

The D1 subunit of photosystem II is damaged in the light, degraded and replaced by a newly synthesized protein. The mechanism of this turnover process is not understood. To learn more about degradation processes in the chloroplast in general and about DI degradation in particular, we have analysed the fate of a truncated DI protein (tD1) in a CTdmnydomonas mutant obtained by deleting an internal part of the psbA gene. The tDl protein consists of the 152 N-terminal amino acids, fused to 30 "foreign" amino acids at the C-terminus. It does not accumulate, but is detected by pulsu-labulling cellular proteins. Pulse-chase experiments show that tDl a half life below 3 rain and proteolysis is ATP-dependent. We will discuss the possible involvement of Clp- and Lon-like proteases recently identified in chloroplasts and mitochondria, respectively.

P-22-026 D E G R A D A T I O N OF A N T E N N A C H L O R O P H Y L L - B I N D I N G P R O TE IN CP43 DURING PNOTOINNIBITION OF P N O T O S Y S T E M II Yasusi Yamamoto and Tatsuya Akasaka Department of Biology, Faculty of Science, Okayama University, Okayama 700, Japan When PS II membranes from spinach were treated with Tris (0.8M, pH9.0) and illuminated with white light (5,000 pE m "2 s "1) under aerobic conditions at 25°C, not only were the reaction centerforming D1 and D2 proteins degraded but the antenna chlorophyllbinding protein CP43 was also degraded. Three products of the degradation of CP43, with molecular masses of 17.0, 15.5 and 14 kDa, respectively, were identified by sodium dodecyl sulfate/uraa polyacrylamide gel electrophoresis and Western blotting with a specific antibody. Degradation products of CP47 were not detected under the same conditions. Concomitant with the damage to the D1 and D2 proteins and CP43, cross-linked products of the D1 protein, CP43 and CP47 were formed. The relationship between the degradation of CP43 and cross-linking of the polypeptides of PS II in the damage-repair cycle of PS II will be discussed.

190

P-22-027 PHOTOSYNTHESIS AND PHOTOPROTECTION IN MANGROVES |. M. Cheeseman1, L. B. Herendeen I, A. T. Cheeseman1, Uwe StolzI & B. F. Clough2 IDept. of Plant Biology, University of Illinois, 505 S. Goodwin Ave. Urbana IL 61801 USA; 2Australian Institute of Marine Science, PMB 3, Townsville, Queensland, Australia. Gas exchange and fluorescence techniques were used to evaluate photosynthetic performance in Rhizophora stylosa in Western Australian. With temperatures >45"C and irradiances >1800 grnol m -2 s-1, CO2 exchange generally fell far short of the potential indicated by fluorescenceestimated electron transport. Yet, as Fv:Fm was ~0.8 within 30 rain of sunset, evidence of photointdbition was lacking. To partially characterize Rhizophora photoprotective systems, ascorbate, dehydroascorbate and glutathione levels were determined in R. stylosa leaves collected in the field; glutathione reductase (GSR) and superoxide dismutase (SOD) activities were determined in R. mangle leaves from greenhouse plants. Total ascorbate was >50 mmol m -2 (~9 mM), with 45% in reduced form. Glutathione levels were -500 fold lower. SOD activites were high, ca. 900 U m -2, with more than 90% being CN-insensitive (presumably cytosolic). GSR activities were only ca. 1 U m -2. These results indicate that while SOD can contribute significantly to protection against active oxygen in both the chloroplast and cytosol, enzymatic cycling of ascorbate is probably far less important than non-enzymatic scavenging in these species.

P-22-028 STRATEGIES OF CHLOROPLAST PROTECTION AGAINST DAMAGE BY SINGLET MOLECULAR OXYGEN. EVIDENCE BASED ON PHOSPHORESCENCE STUDIES. A.A. Krasnovsky, Jr. Biology Department, Moscow State University and A.N. Bakh Institute of Biochemistry, Russian Academy of Science, Moscow, 117071, Russia Mechanisms of singlet oxygen production and deactivation in chloroplasts have been reconsidered using measurements of pigment phosphorescence caused by the pigment triplet states and delayed luminescence caused by singlet oxygen (~02) formation. The data indicate that: (1) the short wavelength chlorophyll forms are the most probable ~O2generators in chloroplasts; (2) the 102 quantum yield in thylakoids is about 0.1%; (3) the *02 lifetime is about 70 ns and (4) the yield of ~O2-induced chlorophyll photodestruction is 106-105. The data suggest that the defense strategy of chloroplasts involves: (1) trapping the chlorophyll singlet states by the reaction centers and probably, long wavelength pigment forms not capable of tO2 generation; (2) quenching of triplet chlorophyll by carotenoids; (3) physical quenching of 102 by carotenoids and chlorophylls; (4) reparation biochemical processes. Relative efficiencies of these defense lines have been estimated.

P-22-029 ACCLIMATION TO ULTRAVIOLET-A RADIATION IN DUNALIELLA PAR VA. Leland S. Jahnke, Department of Plant Biology, University of New Hampshire, Durham,NH 03824, USA. The green hypersaline alga, Dunaliella parva was grown in visible light (400-700nm) with and without moderate ultraviolet-A (UVA) radiation. The inclusion of UVA (80mmol m"2 s'l), initially reduced growth, but growth rates recovered to control rates following <48h. Variable fluorescence and light limited photosynthesis was lowered by UVA exposure, but recovery from subsequent short term higher UVA exposure was more rapid in UVA grown cells (i.e. acclimated cells) compared to controls. The chloroplast protein synthesis inhibitor erythromycin eliminated the protective advantage found in the acclimated cells. No UV screening pigments (i.e. mycosporines) were found either before or aider acclimation. Acclimated cells showed a 24% increase in ascorbate and a 70-250% increase in Monodehydroascorbete reductas¢. Smaller changes were noted in soluble Ascorbate Peroxidase and no significant changes were measured in Glutathione reductase activity.

Poster

P-22-030

P-22-033

PROPERTIES OF THE ENERGY DEPENDENT QUENCHING OF FLUORESCENCE IN CHLORINA F2 AND WILD TYPE BARLEY. SIGNIFICANCE FOR CONTROL OF PSII IN VIVO.

CHLOROPLAST TRANSLATION ACTIVITY IS N O T REQUIRED FOR LIGHT DEPENDENT DEGRADATION OF THE D1 PROTEIN IN Chlamydomonas reinhardtii

F. RIZZA. D. RUELLE & B. GENTY Groupe " P h o t o s ~ et Environcement" Laboratoire d'Ecologie V(~36tale, I~t. 362, Universit6 Paris XI, CNRSURA 1492 ; 91 405 Orsey cadex (FRANCE)

S. Shochat, H. Zer and I. Ohad. Dept. Biological Chemistry, Hebrew University, 91904, Jerusalem, Israel

In leaves, PSII photochemical yield is reversibly down modulated by a mechanism of non radiative dissipation (NRD) which is reflected in the energy-dependent quenching of fluorescence, qE. The origin of this mechanism was studied using comparative measurements on chlorine f2 mutant (lacking the peripheral LHC2) and wild type barley. The involvement of the xanthophyil cycle, Mg and the peripheral LHC2 was investigated by measurements of yield of 02 per single turnover flash, absorbanoe and fluorescence in protoplests and thyiakoids. Data indicate that NRD processes do not involve primary photochemical events or charge recombinations at reaction centre but occur within the antennae pigment complexes. Neither the peripheral LHC2 nor Mg, nor violaxanthin conversion were required to observe qE even if qE was largely decreased in the mutant type.

P-22-031

Based on inhibition of D1 protein degradation by chloramphenicol (CAP) in Lemna it has been recently proposed that a rapidly turning over chloroplast encoded protease may be involved in this process (H. Gong.1995. Biochim. Biophys. Acta. 1188:422-426). Similar observations in cyanobacteria, were interpreted as a coordination between the degradation and synthesis/insertion of new D1 protein molecules (J. Komenda and J. Barber, personal communication). We have previously reported results indicating rapid degradation of the D 1 protein m the presence of CAP in Chlamydomonas reinhardtii. To test whether in these cells the observed rates of degradation were not lower than expected due to inhibition of chloroplast translation, the degradation of D 1 protein was tested in cells pulse-labeled with 35S methionine exposed to high light intensity (chase) in the presence of CAP or in its absence. CAP did not affect the rate of degradation in these experiments. No effect was observed on the rate of D1 protein degradation detected by immunoblot assays in cells preincubated in low light in the presence of CAP for up to 7 hours. We conclude that in C. reinhardtii the protease involved in the light dependent degradation of the D1 protein does not turnover rapidly. (supported by a INSF grant).

P-22-034

ARE SUPEROXIDE RADICALS INVOLVED IN ACCEPTOR SIDE INDUCED PHOTOINHIBITION?

CORRELATION BETWEEN PSII DAMAGE INDUCED BY LIGHT AND BACK-REACTIONS IN PSII

Hide~. 1~,, Spetea, C. and Vass, I. Instit~ate of Plant Biology, Biological Research Center, P.O.Box 521, H-6701 Szeged, Hungary

N. Keren 1,2, P. J. M. van Kan 3, A. Berg 2 H. Gong4 S. Shochatl, 2, H. Levanon 2, S. Styring 3, B. Andersson 3 & I. Ohad 1. IDept. of Biological Chemistry, and 2physical Chemistry, The Hebrew University of Jerusalem, Jerusalem, 91904 Israel and 3Dept. of Biochemistry, Stockholm University, Stockholm, Sweden; 4Dept. Biology, Oslo University, Oslo, Blindern, Norway

Superoxide anion radicals were detected in isolated spinach thylakoids and in oxygen evolving Photosystem (PS) II on the basis of the reaction between 0 2" and Tiron (1,2-dihydroxybenzene-3,5-disulphonate) which results in the formation of stable, EPR detectable Tiron radicals. We found that qQ~"was produced in illuminated thylakoids but not in PS II preparations. e amount of the radicals was about 70% higher under photoinhibitory conditions than under moderate light intensity. Superoxide production was inhibited by DCMU and enhanced4-5 times by methyl-viologen. These observations suggest that the 02" in illuminated thylakoids are from the Mehler reaction occurring in~PS I and are not related to the electron transport modifications brought about by photoinhibition. Although artificial generation of O 2" from riboflavin accelerated the photoinduced degradation of the PS II reaction centre protein D1, the protein fragmentation pattern demonstrated that this added O 2- did not enhance the fragmentation process brought about by photoinhibition but introduced an additional pathway of damage featuring different D1 fragments. On the basis of the above observations we propose that 0 2- radicals are not the main promoters of acceptor side induced photoinhibition.

P-22-032

PHYSIOLOGICAL SIGNIFICANCE OF THE ASCORBATE REGENERATING SYSTEM OF CHLOROPLASTS IN HIGHLIGHT TOLERANCE

H. Yamasakil, R. Heshiki 1, T. Yamasu2, Y. Sakihama 1 & N. Ikehara 1 1Dept. Biol., University of the Ryukyus, Nishihara, Okinawa 903-01, Japan; 2Dev. Gen. Educ., University of the Ryukyus, Nishihara, Okinawa 903-01, Japan

Ficus microcarpa L. f. cv. Golden Leaf (Golden Lea0 is a novel mutant of F. microcarpa L. f., a tropical fig (Yamasaki et al., 1995, J. Plant Res., 108 in press). Its shade-leaves are identical to those of wild-type but the sun-leaves show characteristic yellow color due to little amount of photosynthetic pigments. The lamella structures of thylakoid membranes were almost destroyed in the sun-leaves of Golden leaf, We found that Golden Leaf lacked dehydroascorbate reductase activity (DHAR), a component of ascorbate regenerating system of chloroplasts. Results suggest that chloroplasts of Golden Leaf are impaired by high-light irradialion because of the ascorbate deficiency of chloroplasts due to lacking DHAR activity. A physiological significance of DHAR in the tolerance of chloroplasts to high-light irradiation is proposed.

LOW

Series of light flashes given at intervals compatible with the half life time of back reaction from QB- to the S-states induce D1 protein degradation in C. reinhardtii cells in darkness. Oscillations of the QB- level induced by repetitive series of multiple (1-4) flashes correlate with oscillations in the degree of D1 protein degradation (Keren et al., J. Biol. Chem, (1995) 270:806-814). Spinach thylakoids exposed to series of single turnover flashes at time intervals permitting back-reaction from QB- (40 sec) exhibit loss of electron transport, thermoluminescence signals and EPR signals (Sig. Ilslow, Pheo-, QA--Fe2+). No loss (thermoluminescence) or only partial loss of the EPR signal intensity occurred if the same amount of flashes were given at 10Hz. Thus the damage to PSII is related to the flash regime and not to the total amount of energy absorbed. These result support the hypothesis that back reactions from QB" to the S-states, which occur in cells exposed to low light, are involved in the inactivation of PSII and degradation of the RCII-D1 protein. (Supported by a grant from the Israeli Science Foundation awarded to N. K. & and I. O.)

P-22-035 PHOTOSYSTEM II DAMAGE AND REPAIR IN D U N A L I E L L A SALINA. ACCLIMATION TO GROWTH IRRADIANCE and Anastasios Melis Dept. of Plant Biology, 411 Koshland Hall, University of California, Berkeley, CA 94720-3102, USA. The effect of growth irradiance on the photodamage to the D1 protein of photosystem rl was studied in the green alga Dunaliella salina. The time course of D1 photodamage was followed by immunoblot analysis upon addition of lincomycin to acclimated cell cultures. Both D1 and D2, as distinct 32 and 34 kD protein bands, respectively, decreased exponentially with time, with similar kinetics, and with rate constants that were a direct linear function of growth itradiance. The loss of the 32 and 34 kD protein bands correlated with the transient accumulation of a 160 kD reaction center (RC) complex. In the absence of lincomycin, cells grown at different irradiances contained different amounts of the 160 kD complex, the steady state level of which depended linearly on growth irradiance. When high-light grown cells were transferred to low light, the cellular content of the 160 kD complex gradually decreased to reach the normal level. We suggest that the 160 kD complex represents photodamaged photosystem H RCs that accumulate under irradiance stress, retaining a damaged D1 protein, which is tagged for proteolysis and replacement.

191

Poster

P-22-036 PSIl PHOTOINACTIVATION ABOLISHES THE REGULATORY ROLE OF THE QB SITE IN THE D1 PROTEIN DEGRADATION H. Zer, N. Keren & I. Ohad Dept. of Biological Chemistry, The Hebrew University of Jerusalem, 91904, Jerusalem, Israel

P-22-039 SELECTIVE AND SPECIFIC MODULATIONS OF THE DI PROTEIN INDUCED BY THE BINDING OF PSII INHIBI. TORS: MODEL OF D I PROTEIN DEGRADATION BY PHOTOINHIBITION y. Nakaiima 1, 2, S. Yoshida 2, Y. Inouel, K. Yoneyama 3 and T. trio l lSolar Energy Research Group, 2Regulation of Plant Functions Laboratory, The Institute of Physical and Chemical Research (RIKEN), Wake, Saitama 351-01, Japan, 3Weed Science Center, Utsunomiya University, Utsunomiya, Tochigi 321, Japan

In C. reinhardtii cells, DCMU retards the light induced degradation of the photosystem II (PSII) core proteins, D1, D2, CP43, but not of CP47. RCII-D1 photoinactivated in the presence of DCMU is irreversibly modified and can be subsequently degraded after a prolonged photoinhibitory treatment or removal of DCMU by washing. The modified protein can be replaced in cells transferred to low light or darkness (turnover). D1 protein degradation is retarded in photoinactivated mutant cells deficient in light dependent plastoquinol oxidation. Degradation ensues upon subsequent exposure of the cells to conditions allowing reoxidation of PQH2 by ambient oxygen in low-light or in darkness. DCMU added at that stage does not protect the modified D1 protein from degradation. In vitro, DCMU protects D1 protein from tryptic digestion of thylakoids obtained from low light exposed cells but not from cells photoinhibited in the presence of DCMU. We conclude that photoinactivation induces sequential changes in the acceptor side of PSII exposing the cleavage site of the D 1 protein to degradation and abolishing the regulatory role of the QB site occupancy by plastoquinone or analog ligands such as DCMU on the cleavage process. (Supported by SFB-184, GIF and INSF j~rants).

We studied the effects of photosystem II (PS 1/) inhibitors that interact with the QB-site on the DI protein degradation by photoinhibition. Among the PS II inhibitors, some phenol-type inhibitors showed enhancement of the degradation of the D1 protein and formation of aggregates between D 1 and other PS II proteins during photoinhibition. Phenol-type inhibitors having one hydroxyl group on a benzene ring induced the aggregates under complete darkness. Notably, PNO8(N-octyl-3-nitro-2,4,6-trihydroxybenzamide) that contains three hydroxyl groups on a benzene ring induced the selective cleavage of the D1 protein to form 23 and 9 kDa fragments under complete darkness. Interestingly, the resulting aggregates and fragments were quite similar to those induced by photoinhibition. From these observations, modulations of the D1 protein by the phenol-type inhibitors have been discussed in relation to the photo-degradation of the D1 protein.

P-22-037 REVERSIBLE DISSOCIATION OF t E E PROTEINS FROM THE LUMENAL SIDE OF THE THYLAKOID MEMBRANES DURING PHOTOINHIBITION AND RECOVERY OF PSII ACTIVITY

P-22-040 L I G H T - I N D U C E D D E G R A D A T I O N OF T H E N E W L Y DISCOVERED 6.1 kDa SUBUNIT OF PHOTOSYSTEM I I REACTION CENTRE IN HIGHER PLANTS.

Y. Domovich 1, T. S. Mot I , R. Oelmuller 2, R. G. Herrmann 2 and I. Ohad 1 IDepartment of Biological Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel; 2Institute of Botany, Ludwig-Maximilian University, Munchen, Germany.

~sa Ham-nan.Lan-Xin Shi, Bertil Andersson & Wolfgang P. SchrOder Dept. of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S- 106 91 Stockholm, Sweden.

Light dependent D1 protein degradation in C. reinhardtii cells induces dissociation of the MSP-33kDa and 23 kDa proteins of the t E E complex from the lumenal side of PSII. The t E E proteins are not degraded under these conditions and persist for up to 8 hours in the dissociated form. MSP does not dissociate from photoinactivated PSII if RCII-D1 protein degradation is prevented by addition of DCMU or in mutants deficient in plastoquinol oxidation. The free MSP reassociates with the PSII complex following de novo chloroplast protein synthesis and in absence of cytosolic protein synthesis. Reassociation correlates with synthesis of the D1 protein and recovery of PSII activity. These results demonstrate a dynamic state of the t E E proteins interaction with the thylakoid membranes. We propose that the persistence of the dissociated t E E proteins is essential in the process of recovery of PSII activity facilitating rapid reassembley of donor-side active PSII. (Supported by SFB-184 and GIF grants).

P-22-038 ENHANCED TOLERANCE TO HYDROGEN PEROXIDE IN TRANSGENIC TOBACCO OVEREXPRESSING ASCORBATE PEROXIDASE IN THE CHLOROPLASTS. L. Slooten 1, S. Kushnir2, I. Caplir 1, M. van Montagu 2 and D. Inz62'3. 1: Vrije Universiteit Brussel, Lab. Biofysica, Pleinlaan 2, B-1050 BRUSSEL, Belgium; 2: Lab. Genetika, and 3: Laboratoire Associ6e d'INRA(France), Universiteit Gent, Ledeganckstraat 35, B-9000 GENT, Belgium. We generated two independent transgenic lines (TP3 and TP5) of Nicotiana tabacum vat SR1, which overexpress a cytosolic ascorbate peroxidase (APx) from Arabidonsis thaliana in the chloroplasts. Both lines are homozygous and contain a single-locus T-DNA insertion. Tolerance to light-dependent oxidative stress was assessed in leaf disk assays. The tolerance of APx-overexpressing plants to chilling-induced photoinhibition, or to singlet oxygen (photoproduced by eosin) was not enhanced. The tolerance to superoxide (photoproduced by methyl viologen) was slightly enhanced. Hydrogen peroxide, produced during photorespiration in the presence of the catalase inhibitor, aminotriazole (AT), inactivated endogenous, chloroplastic APx and the PSII reaction centre in control plants, but not the overexpressed APx in transgenic plants. In the transgenic plants, the overexpressed APx lowered the level of hydrogen peroxide, produced during photorespiration in the presence of AT, sufficiently to prevent inactivation of the PSII reaction centre, but not sufficiently to prevent inactivation of endogenous, chloroplastic APx. The latter enzyme is more sensitive to hydrogen peroxide than the PSII reaction centre.

192

It has recently been discovered that isolated PSI] reaction centres in addition to the DI-, D2-protein, cyt b559 and the p s b I gene product contain an additional nuclear encoded subunit (SchriSderet al., see this abstract book) of 6.1 kDa (psb W gene product). It is well known that during photoinhibifion of PS I/reaction centres in vivo or in vitro there is a degradation of the D1 protein and to some extent the D2 protein. In this study we show that the 6.1 kDa subunit can undergo substantial degradation after subjecting isolated spinach thylakoids to strong illumination. The rate and extent of degradation is somewhat lower as compared to that of the D1 protein but more" pronounced than that of the D2 protein. The significance of this observation with respect to the problem of photoinhibition will be discussed.

P-22-041 HIGH-LIGHT TOLERANCE OF PHOTOSYSTEM II OF NOVEL S I T E S P E C I F I C D1 P O L Y P E P T I D E M U T A N T S O F S Y N E C H O C Y S T I S 6803 Pirkko M ~ e n p ~ t, Gaza Salih 2, Ronney Wiklund 2, Christer Jansson 2 and Eva-Mari

Aro i

IDept of Biology, Univ of Turku, FIN-20500 Turku, Finland; 2Dept of Biochemistry, Univ of Stockholm, S10691 Stockholm, S w e d e n We have constructed the novel D1 polypeptide mutants DI03I, E104Q, D103I+E104Q, F186Y, K238Q and K238R in Synechocystis 6803. Growth rate and tolerance of oxygen evolution of PS II to high light irradiance have b e e n a n a l y z e d . N e t decrease in the amount of the D1 polypeptide h a s b e e n followed during the high-light treatment by i~ununoblotting. The photosynthetic growth rate of F186Y is remarkably slower compared to the control. Oxygen evolution activity f r o m PS II of F186Y is only half of that of the control. Also DI03I+EI04Q h a s only 65% of the oxygen evolving activity of the control w h e r e a s PS II activities of the rest of the strains are almost comparable to t h o s e of the control. They also show similar high-light tolerance as the control. The photoinhibitory behaviour of FI86Y and DI03I+E104Q will be discussed.

Poster i|

P-22-045

P-22-042 DEGRADATION PATTERN OF DI PROTEIN IN v I V a

THE QU2~ITUM YIELD ZNTENSITY

R. Kettunen. Tyystjiirvi, E. & Aro, E.-M. Dept Plant Physiol & Mol Biol, University of Turku, 20500 Turku, Finland

Photoinhibition was observed to cause the cleavage of the Photosystem II reaction center protein DI at two distinct sites in intact pumpkin leaves. The main cleavage occurs N-terminally from the DE-loop thus generating a 18 kDa N-terminal and a 20 kDa C-terminal degradation fragment of the DI protein. The other, less frequent cleavage occurs at the DE-loop and generates the well-documented 23 kDa N-terminal DI fragment. These results give indirect evidence that the main photoinhibitiun mechanism in viva is "donorside" photoinhibition, and that the "acceptor-side" photoinhibitiun is only a side path, although the possibility that one photoinhibition mechanism leads to two different degradation pattem can not be excluded. The molecular sizes of DI fragments are difficult to estimate by SDS-PAGE as the mobility of the fragments seems to largely depend on their conformation despite of SDS solubilization and urea treatment.

P-22-043

OF pHOTOZNHIBXTION

IS

XNDEPIDIDENT

OF L~OHT

E. Tyystj~rvi and E-M. Aro, Dept. of Biology, Univ of Turku, Plant Physiology, Bioeity A 6th floor, FIN-20520 Turku, Finland Pumpkin leaves were illuminated under photon flux densities ranging from 6.5 to 1500 ~mol m'2s"I in the presence of lincomycin, an inhibitor of chloroplast protein syntheaia. Under all light intensities, photoinhibition of PSII was a first-order reaction, and its rate constant was directly proportional to photon flux density. This means that each photon absorbed ceuees photoinhibition of PSlI with the same probability irrespective of light intensity. The constancy of the quantum yield of photoinhibition under different photon flux densities strongly suggests that photoinhibition in vivo occurs by one dominant mechanism. Furthermore, it is unlikely that this mechanism is the acceptor side mechanism, because double reduction of Q^ most probably does not occur at low light. The loss of PSII activity and the loss of the D1 protein from thylakoid membranes were strictly correlated at low light. This correlation reflects the fact that when photoinhibition is slow, there is no accumulation of p h o t o i n h i b i t e d PSII centree with the D1 protein waiting to be degraded. The excellent kinetic agreement between D1 protein degradation and the inactivation of Photosyatem II indicates that the degradation of the D1 protein is preceded by a light-induced inactivation both under low and high light.

P-22-046

REVERSIBLE PHOSPHORYLATION OF D1 REACTION CENTRE PROTEIN IS MODULATED BY PHOTOINHIBITION OF PSII

ADAPTIVE RESPONSE OF DUNALIELLA SALINA TO OXIDATIVE STRESS

E. Rintam[iki & E.-M. Aro Dept of Biology, Univ. of Turku, FIN-20500 Turku, FINLAND

Barbara Sigala and Kleoniki Gounaris Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, U.K.

Reversible phosphorylation of the D1 protein of PSII was studied in pumpkin, wheat and pea leaves. Non-phosphorylated and phosphorylated D1 protein (DI*) were separated in SDS-PAGE and quantified by immunoblotting. In all species 70 to 80 % of D1 protein was phosphorylated during 30 min illumination of leaves at growth or stronger irradiances. After short exposure to strong light (no photoinhibition of PSII) the half-time of D 1" dephosphorylation (1.5 h) was similar both in darkness and at 40 , E m 2 s4. Notably, however, photoinhibitory illumination of leaves markedly slowed down DI* dephosphorylation in darkness; the more severe photoinhibition, the slower was DI* dephosphorylation. The degradation of damaged DI* was also blocked in darkness but started immediately upon transfer of the leaves to low light. The results suggest that i) D 1 protein phosphatase per se is functional both in darkness and at low irradiance but ii) dephosphorylation and/or degradation of the photodamaged DI* protein are light-dependent reactions in viva.

The response of the halotolerant green alga DunalieUa salina to sress induced by oxidants was investigated. We observed that D. salina is capable of adapting to hydrogen peroxide and to agents generating the superoxide anion. Thus, pretreatment of algae with low concentrations of hydrogen peroxide causes adaptation and renders the organism resistant to otherwise lethal concentrations of peroxide. The adaptive process is dependent on the age of the cells and requires de nova protein synthesis from the chloroplast genome. Cultures adapted to hydrogen peroxide additionally become resistant to treatment with agents generating the supemxide anion. Cultures adapted to superoxide stress, however, are only partially resistant to hydrogen peroxide. Furthermore, hydrogen peroxide adapted cultures exhibit partial resistance to heat stress and to other agents which cause oxidative damage. We have carded out two-dimensional eleca'ophoresis of 3aS-labelled cultures and will report on the results obtained on protein synthesis during the adaptive process and subsequent challenge with oxidants. We have also examined the pattem of protein phosphorylation in adapted cultures.

P-22-044

SPINACH CHLOROPLASTIC AND CYTOSOLIC CuZn-SODs ARE LOCALIZED AT THE SITES OF SUPEROXIDE GENERATION ~ 1 , S. Kanematsu2 & K. Asada3 ~u ,ep. ~ . , Fac. Sci., Kyoto Univ., Kyoto 606-01, Japan; 2Dep. Food Sci. tecnunl., Minami-Kyushu Univ., Takanabe, Miyazaki 884, Japan;" Res. Inst. Food Sci., Kyoto Univ., Uji, KyotO 611, Japan.

Spinach contains chloroplastic and cytosolic CuZn-SODs. In order to reveal localizations of the enzymes, immuno-electron microscopic analyses using the antibodies against the chloroplastic and the cytosolic CuZn-SODs were performed. Spinach leaves were fixed and substituted by a rapid freezing and substitution method that allows visualization of intact chloroplasts. The embedded sections were immuno labeled with the antibodies against CuZnSODs by the immuno-gold method. Chlomplastic CuZn-SOD was not uniformly distributed in the slroma but was localized on the stromal-faces of thylakoid membranes where the PS l-complex, the site of generation of 02", is localized (Ogawa et al., 1995,Plant Cell PhysioL, in press). Similarly, cytosolic CuZnSOD was not uniformly distributed in cytosol but was localized in the cytosol near vacuoles, the nuclei and the apoplast. Considering the nature of O2-, cytosolic CuZn-SOD is likely to be localized at the site of superoxide generation just as chloroplastic one is. Actually, the site of localization of cytosolic CuZnSOD was associated with the lignifying m o o n s where generation of 02- is required for supplying H20 2.

P-22-047 Effect of the substitution of glycine 2 1 5 for tryptophan in the D2 protein of Synechocystis PCC 6 8 0 3 on the characteristics of Photosystem II G Schansker 1, WFJ Vermaas 2 and JJS van Rensen 1 ~Dept of Plant Physiology, Wageningen Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands; 2 Dept of Botany and Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe Az, U.S.A. A Photosystem II (PSII) acceptor side mutant of Synechocystis PCC 6803 was characterized to investigate its higher sensitivity to light. The effect of the substitution of glycine 215 for tryptophan in the D2 protein of PSII (G215W strain) is characterized by: a faster charge recombination, a shift of the equilibrium between QA and QB/O.B= towards QA (1:40 in the reference strain compared with 1:2 in G215W), modified fluorescence induction kinetics and a higher sensitivity to formate compared to the reference strain. The relationship between these changes and the 4-times higher sensitivity to photoinhibition of the G215W strain is dicussed.

193

Poster

P-22-048

P-22-051

D E P E N D E N C E OF PHOTOINHIBITION OF PHOTOSYNTHESIS IN B E A N L E A V E S ON DAILY PHOTON RECEIPT

RUBISCO ACTIVITY AND GENE EXPRESSION IN TREE SPECIES SUBMITTED TO ABIOTIC STRESSES.

Dennis H. Gree5 Horticulture and Food Research Institute, Private Bag 11030, Palmerston North, New Zealand.

D. Afif, M. Cabanr, V. George, J-M. Favre & P. Dizengremel Laboratoire de Biologie Foresfi/'~re (associ6 INRA), Universit6 H. Poincar6 Nancy-I, B.P. 239, F-54506 Vandceuvre Cedex, France.

P h a s e o l u s vu/garis plants were grown in controlled environments at a photon flux density (PFD) of 800 rtm01 m 2 s "+for 6 and 16 h, daily photon receipt being 17 and 46 m o l m 2. Diumal photosynthesis was measured to determine net carbon gain and the extent of feedback inhibition. Leaves of plants from both treatments were also exposed to PFDs of 800 and 1400 ~mol m 2 s + at 14 and 20°C to determine susceptibility to photoinhibition (PI), in the presence and absence of the inhibitors chloramphenicol (CAP) and Dithiotreitol (DT'r). The PFD responses of photochemical and non-photochemical quenching were also determined for each treatment. Results show that although photosynthetic rates were similar, plants grown in short days were more susceptible to PI than long-day plants. Both CAP and DI-I" exacerbated PI though more so in plants grown in short days. Differences in susceptibility to PI will be discussed in terms of light interception, leaf morphology, energy dissipation and carbohydrate accumulation.

The elevation of phytotoxic air pollutant ozone (O3), and the rising atmospheric carbone dioxide (CO2) concentrations are considered to be major environmental problems. Generally, net photosynthesis decreased under 03, and increased under high CO2 concentrations. Little is known about the effect of a combination of both factors on CO2 fixation in evergreen tree species. We investigated whether high CO2 concentrations could prevent negative effects of 03 on photosynthesis and what are the response mechanisms of trees to both factors. Spruce (Picea abies) were long-term exposed, in climatic chambers, to 03 (200ppb) and CO2 (700ppm) alone or in combination. Net photosynthesis was measured and the rubisco activity, quantity and expression of genes coding for the large (rboL) and the small (rbeS) subunits were studied. The response of rubisco and its regulation by other proteins in these stress conditions will be discussed.

P-22-049

P-22-052

UV-B LIGHT EFFECTS ON SCENEDESMUS LF-1 AND SOME SYNECHOCYSTIS PCC 6803 MUTANTS

LIGHT4NDUCED DAMAGES OF PHEOPHYTIN A AND CHLOROPHYLL A IN THE ISOLATED PHOTOSYSTEM n REACTION CENTER D1/D2/CYT B-559 COMPLEX

F. Rigoni, R. Barbato, S. Chiaramonte and G.M. Giacometti Deparment of Biology - University of Padova, via Trieste, 75, 35121 Padova, Italy

Tinq-Yun Kuang, Jian-Min Hou+ Oe-ChuanPeng. Chong-Oin Tang, Pei-Song Tang Lab. of Photosynthesis+ Inst of Botany. Academia Sinica Be@rig 100044, P R China

The effect of 25 p.moles of UV-B photons on photosystem II of Scenedesmus LF1 and two insertion mutants of Synechocystis PCC 6803 with lesions on psbO and psbH genes has been studied. Loss of PSII activity was measured by electron transfer following DPIP reduction with H20 or DPC as donor, by 02 evolution with different acceptors, by variable fluorescence and single turnover flash. The results indicate damage to both sides of the PSII. Immunological analysis with anti-D1 protein polyclonal antibodies suggests that loss of the D1 protein is linked with damage to the donor side. The effect of the D1 protein degradation on the PS II structure has been investigated by sucrose gradient centrifugation and Deriphat-PAGE

The isolated Photosystem II (PSII) reaction center (RC) DIID2/Cyt ;o-559 complex from higher plants is very sensitive to illumination and damaged easily. Photoinduced damages of pigment molecules including chlorophyll a (Chla) and pheophytin a (Pheo) in the isolated PS II RC under the exposure to strong light had been investigated by high performance liquid chromatography. Our previous note reported that photodarnage of Pheo in the PSII RC complex (T.-Y. Kuang. J.-M Hou. D.-C Peng, C-Q Tang, P.-S. Tang, 1995, Acta Botanica Sinice, 37(5), I-4). In this paper we found that the Pheo and primary electron donor P680 occurred one by one. in which Pheo and followed P680 is damaged by illumination The damaged Pheo. which is not associated w,th the primary photochemical activ,ty, may be attributed to bJnd to D2 polypeptide based on the homology between the RC of PSII and that of purple bacteria (J.-M Hou. T-Y Kuang, D-C Peng, C-Q Tang, P -S Tang. 1995. Acta Botenica Sinica, 37(5). 5-8). Here we proposed the photoprotective hypothesis of the Pheo in the PSII RC against the photodamage by dissipating excess excitation energy in vivo.

P-22-050 UV-B

SENSITIVITY

SCREENING

AMONG

pHYTOPLANKTON

SPECIES

1'2 3' , F . L e d e r e r 1'2 , J. L u k a v s k y 2 & L.Nedbal 1 Institute of M i c r o b i o l o g y , 379 81 Trebon, Czech Republic; 2University of South Bohemia, C.Budejovice, Czech Republic; 3 j i a n g s u A g r i c u l t u r a l University, People's Republic of China M o r e than 20 species of algae and c y a n o b a c t e r i a were u s e d in the the s y s t e m a t i c s c r e e n i n g of the UV-B s e n s i t i v i t y of their photosynthetic apparatus. The species were selected to represent d i f f e r e n t natural environments: A n t a r c t i c s n o w and high e l e v a t i o n lakes on one extreme and shaded thermal springs on the other extreme. A n o t h e r set of algal and c y a n o b a c t e r i a l species was s e l e c t e d to represent the organisms used in the l a r g e - s c a l e open solar bioreactors. The algae were e x p o s e d at their optimal c u l t i v a t i o n t e m p e r a t u r e to 0.6 W / m 2 of UV-B radiation (weighted for g e n e r a l i s e d plant damage) from Philips TLI2 UV-B fluorescent tube screened by cellulose acetate filter. The response of the algae was m o n i t o r e d by changes in Chl f l u o r e s c e n c e parameters(Fo, Fv/Fm, qP, qNp and ~IIs) and in p h o t o s y n t h e t i c o x y g e n e v o l u t i o n rate as well. The UV-B i n d u c e d changes in the p h o t o s y n t h e t i c pigment c o m p o s i t i o n and in the content of the U V - B screening compounds were m e a s u r e d using a b s o r p t i o n s p e c t r o s c o p y and HPLC analysis. Role of the known p r o t e c t i v e m e c h a n i s m s in the elevated U V - B resistance of some of the species was investigated.

194

P-22-053 PHOTOINHIBITION AND psbA TRANSCRIPTION S. Constant, I. Perewoska and D. Kirilovsky URA 1810, CNRS, E.N.S., 46, rue d'Ulm, 75230 Paris Cedex 05, France We observed that, during photoinhibition of Synechocystis 6714 cells, not only the rate of transcription of psbA -mRNA was increased by the high light but also that of degradation. The increase in the rate of transcription was larger than that of degradation. As a result, when the cells were exposed to high light, there was a rapid increase of the level ofpsbA -mRNA (1.2 kb) Then, a progressive accumulation of degradation products Of psbA -mRNA was observed. When the photoinhibition was reversible, the degradation products dissapeared during the recovery process under low light. The degradation products were also eliminated in the dark although the PSII activity was not recovered. When the photoinhibited ceils were unable to recover PS II activity due to a prolongate light stress, they did not eliminate the degration products of psbA -mRNA when transferred to low light. Despite the high level of the psbA -mRNA in these cells, they lost the ability to replace the damaged Dh This suggests a post-transcriptional regulation of D1 synthesis.

Poster

EXCZTATION P R E 8 8 U I ~ DET'J~P~(ZI~8 PHC~EOS~3BE S 18

TO~CE

TO

P-22-054 PHOTOZN][XBZTZONOF

G.R. Gray & N.P.A. Huner Department of Plant Sciences, The University of Western Ontario, London, ON N 6 A 5B7 C A N A D A Cold-hardened winter rye (Secale cereale L.) grown at low temperature (5"C) and moderate irradiance (250 ~unol m -2 s -I) displays a significant increase in maximal rates of photosynthesis and an increased tolerance to p h o t o i n h i b i t i o n in comparison to plants grown at the same irradiance at 20"C. We demonstrate that these changes are not a response to low temperature growth, but instead, reflect responses to increased excitation pressure on p h o t o s y s t e m II (PSII). Excitation pressure, which reflects the redox poise of intersystem electron transport and carbon metabolism, is estimated l-qp (QA reduced:Q^ total). Plants grown at 5"C/250 ~unol m -2 s -I are subjected to an excitation pressure 1.6-fold greater than typical non-hardened control plants grown at 20"C/250 pmol m -2 s -z, even though absolute irradiance is identical. A comparable excitation pressure can be achieved at 20"C but requires an irradiance of 800 ~mol m -2 s -I. These plants grown under high excitation pressure at 20*C also exhibit increases in maximal photosynthetic rate and increased tolerance to photoinhibition. Thus, the thermodynamic constraints of low temperature growth provide a means for increasing excitation pressure on PSII, even at moderate irradiance, and this in turn is responsible for the tolerance to photoinhibition observed in plants grown at low temperature.

P-22-055 COMPARISON OF ABSORPTION CHANGES OCCURRING DURING PHOTOINHIBITION OR PHOTOACTIVATION OF WATER OXIDATION IN MANGANESE-DEPLETED PHOTOSYSTEM H Paul Megerdiehian, Anna Bergonia, Sun-HeePark, and Dept. of ~ i s t r y and Biochemistry,Utah State University,Logan, UT 84322 Mn-depleted PSII were subjected to either donor-side photoinhibtionor photoassembly oftbe water-oxidizingcomplex and were analyzed in terms of the spectroscopic changes that occur during the ilhimination (40 lie m-2 s-l). During photoinhibidonin the absence of any additiens, both chlorophylla (Chl a) and carotenoids(Car's) exhibited bleaching, with -20 Chl a molecules per PSII being lost with a half-time of 15-20 rain, while a total loss of Cat's ocotrred with a half-time of-200 rain; Chl b was unaffected. Loss of Chl a is tentatively attributed to a gross modification of a Chl abinding protein, possibly the 43 or 47 kD protein. The loss of Car's parallels the loss of a site of eloctron donation by exogenouseleelren donors and suggests that Car's may be a site of electron d~ation to PSII. Additionof DCIP alone partially protected against the pigment bleaching with preferential protection to the Car's. This protection is attributed to a cycling of eleca-onsback to the PSII donor side by reduced DCIP. By contrast, during photoactivation (Mn2+, Ca2+ and DCIP present) both Chl a and Od b were bleached, with Cbl b bleached to a greater extent than Chl a (the Cbl a/b ratio increased). This bleaching of Chl b during photoaetivation is observed as a specific bleaching at 649 ran that is not observed during photoinhibitionin the absence of Mn. The loss of Chl a is attributed to photninhibitionoccurring during the photoactivation process; the non-bleachingof Car's is atUibutedto protection by DCIE,the apparent loss ofChl b appears to be correlated with assemblyof the Mn complex.

P-22-057 FUNCTIONALITY OF PHOTOSYSTEM II IN PEA LEAVES AFTER PHOTOINHIBITION: INTERPRETATION OF CHLOROPHYLL FLUORESCENCE PARAMETERS

W. S. Chow, Y.-I. Park I and Jan M. Anderson CSIRO, Div. of Plant Industry, GPO Box 1600, Canberra, Australia 2601. 1present address: Research School of Biological Sciences, Australian National Univ., GPO Box 475, Canberra, Australia 2601 Leaves of pea plants grown in 250 ~mol photons m-2 s-1 (12 h/day) were allowed to take up either water or inhibitors (lincomycin, nigericin or dithiothreitol) and then illuminated at various irradiances and durations, in air with 1.1% CO2 at 25°C. The number of functional photosystem (PS) lls, assayed in vivo with repetitive single-turnover flashes, declined with increase in photon exposure. There were concomitant changes in chlorophyll fluorescence parameters: Fo, for open PS II traps; Fm and Fv, maximum and variable fluorescence. Combined data from all treatments shows that, as the number of functional PS IIs declined with increasing photon exposure, (1) despite the initial loss of ca. 25 % functional PS II at low photon exposure, Fv/Fm remained constant: it is, therefore, a poor indicator of PS II functionality at low photon exposure; (2) Fo was increased markedly with nigeriein (+ lincomycin), indicating reaction-centre malfunction; and (3) the decrease in 1/Fo - 1/Fro (trapping rate constan0 was linear.

P-22-058 Ascorbate peroxidase, electron transport, and H202 - dependent fluorescence quenching in chloroplasts and thylakoids: effects of KCN. S.C. Grace and C.B. Osmond. Research School of Biological Sciences, Insitute of Advanced Studies, Australian National University, GPO Box 475, Canberra 2601, Australia Addition of H202 to pea chloroplasts and thylakoids prepared in the presence of ascorbate, and assayed with 5ram ascorbate, led to photochemical (qp) and nonphotochemical (qN) quenching of fluorescence (cf. Neubauer and Sehreiber. 1989. Z. Naturforsch. 44c, 262-270) which relaxed with time constants well correlated with ascorbate peroxidase (APX) activity. Thylakoids prepared in the absence of ascorbate lost this activity and H202 - dependent fluorescence quenching could not be restored by adding excess soluble APX (cf. Miyake and Asada. 1992. Plant Cell Physiol. 33, 541-553). We conclude that thylakoids and chloroplasts contain a vast excess of membrane bound APX because KCN concentrations needed cause 50% inhibition of linear electron transport were about 103 x greater, ofH202 - dependent qN were about 102 x greater, and H202 -dependent qp were about 10 x greater than those needed to inhibit APX itself. The significance of these responses for photoprotection and H202 scavenging will be discussed.

P-22-056

P-22-059

EXCESSIVE HIGH LIGHT CAUSES CHANGES IN THE PROTEIN COMPOSITION, THE SPECTROSCOPIC FEATURESAND THE PIGMENT COMPOSITION OF PSI

PHOTOSYSTEM II IS A PHOTON COUNTER

B. Hettmann & C. Schafer, Lehrstuhl Pflanzenphysiologie, UnlversitOt Bayreuth, 95440 Bayreuth, Germany

Jan M. Anderson 1, Y.-I. Park 1,2 and W. S. Chow1 tCooperative Research Centre for Plant Science, GPO Box 1600, Canberra, Australia 2601; "~Researeh School of Biological Sciences, Australian National Univ., GPO Box 475, Canberra, Australia 2601

Marchanfla polyrnorpha suspension culture ceils were exposed to a 30 h high light treatment. This treatment results in moderate reductions in PSII photochemical efficiency and in the contents of PS II proteins. The present study demonstrates that also PSi is affected. Separation of PSI pigmentprotein complexes from mildly solubilized thylakoid membranes by sucrosegradient ultracentrifugation yielded one PSI band in control cells but two bands in high light treated cells. The same proteins were present in both bands, but the ratio of antenna to reaction center proteins was increased in the additional PSI form. The 77 K chlorophyll fluorescence emission spectrum was different as weir. Emission at 680 nm was strongly increased in the additional band, pointing to a modified energy transfer within the complexes, High light related changes in the PS { protein associations were also detectable when stroma thylakoid proteins were separated by isoelectric focussing. Plgment analysis of the eluted complexes indicates that the hlgh light PSI forms had increased xanthophyll cycle pigment contents. Changes in PS I properties were accompanied by pronounced reductions in the PSI electron transport capacity. Correspondingly, an increased In vlvo reduction state of QA was measured after high light treatment

Loss of PS II function with photon exposure in pea leaves was assayed by Fv/Fm ratios and content of functional PS II. There was a reciprocity of irradiance and illumination duration, i.e. PS II inactivation depends on total photons absorbed and not the rate of photon absorption. At low photon exposure, although Fv/Fm ratios were unaltered, 25% of functional PS Hs were inactivated in lincomycin-treated leaves, demonstrating a PS II heterogeneity in terms of functional stability. Photoinhibition occgrs at low through to high light: 1 PS II was inactivated for 3x10" photons absorbed under high tight exposure, while the less stable (25%) PS Hs require only 2x10° photons for inactivation. Under low photon exposure the effictency of PS II inactivation was modified by photoprotective strategies: D1 protein synthesis > transthylakoid pH gradient > violaxathin cycle. In high photoB exposure, the more stable PS II complexes were inactivated by ~ 10' photons in both inhibitor-treated and control leaves, demonstrating that the photoprotective strategies were overwhelmed. We conclude that PS II is a photon counter under low and high light.

195

Poster

P-22-060

P-22-063

PROTECTS

Photochemical response to excess light as influene~l by the developmental history of the plant

Y.-I. Park 1, W.S. Chow and Jan M. Anderson CSIRO, Division of Plant Industry, GPO Box 1600, Canberra, Australia 2601. tpresent address: Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, Australia 2601

M. Berger, P. Maur~, S. Guy, F. Mojayad and C. Planchon Laboratoire de Biotechnologie et Amdioratiou des Plantes, 1.N.P.- E.NS.A.T., 145 Avenuede Muter, F-31076 Toulouse,France

ELECTRON TRANSPORT TO PHOTOSYSTEM 1I IN PEA LEAVES

OXYGEN

Leaves of pea plants grown in 250 ~mol photons m-2 s-I (12 h/day) were illuminated at various intensifies and durations, in various gas mixtures at 25°C. The relative photosystem (PS) II electron transport rate, assayed in vivo by chlorophyll fluorescence, was oxygendependent. Furthermore, non-assimilatory electron transport to oxygen induced non-photechemical quenching. The inactivation of PS II (measured by repetitive flash-induced Oz yield and Fv/Fm) was dependent on the total number of absorbed photons and also the reduction state of the primary quinone electron acceptor (QA)- With increasing photon exposure, non-photoehemieal quenching increased; the increase was greater at higher 02 concentration, corresponding to less inactivation of PS II. Our data demonstrates that oxygen-mediated electron transport is one of the photoprotective mechanisms in leaves.

P-22-061

The objective was to showthe decisive role of the dovelopmentalhistory of the plants (differing nitrogen source or water stress acclimation) ou the photochemical response to stress-inducedexcess light. Sunflowerand soybeanwere subjected to drought and chilling stresses, respectively. The subsequentdecrease in the use of photochemical energy generated photoinhibitory eenditions. The extent of photoinhibition was measured through the decrease in the photochemical efl:icieacy of PSII (Fv/Fm). Under chilling stress, the photoch~nical energytransfer (q~PSI1)stronglydecreased in soybean, but Fv/Frn decreased more in Nz-fixing than in nitrate-fed soybean. The latter showed higher phoCoprotective dissipation by nonpliotochemical quenching (NPQ). In contrast, under water stress, OPSll was much less affected in drought acclimated than in non acclimated sunflower.Acclimmedsunflowerdissipated part of excess light through photochemicaluse end ultimately (severe water stress) increased NPQ As a result of the dissipative processes, drought acchmated sunflowerdid not show any decrease in Fv/Fm. A total lack of photoprotectiou in N2-fixing soybean (probably linked to high photeaymhate roquiremmts of Nz fixation) and in non drought acclimated sunflowerenhanced photedamagoto the photochemicalapparatus as l-qP increased.

P-22-064 IMPACT OF UV-B ENHANCED IRRADIATION ON PHOTOSYNTHESIS

I S O E L E C T R I C FOCUSING OF PHOTOSYSTEM II CORE PREPARATIONS ISOLATED FROM PHOTOINHIBITED AND CONTROL PLANTS Paulina Geiier & Cecilia Sundby Dept. of Biochemistry, University of Lund, PO Box 124, S-221 00 Lund, Sweden

Raymon A. Donahue 1, Mary E Poulson ~, John Whitmarsh ~2. Dept Plant Biology~, Photosyn Res Unit ARS\USDAz, Univ lUioois, Uthana, IL 61801 USA We measured the effect of enhanced ultraviolet-B (UV-B) (0, 1, 2, and 3 xs ambient levels) on photosynthesis and growth of plants. Photochemistry, whole leaf photosynthesis, and growth patterns were investigated for Cucumis sativus cvs Poinsatt 76 and Ashley, Pisum sativu..m cv Little Marvel, and flavonoid-deficient mutants of Arabidopsis thaiiana var Landsberg erecta. UV-B absorbing compounds increased with UV-B dose for both Pisum and Cucumis; chlorophyll concentration and a/b ratios were not affected. For C. sativus cv. Poinsett 76, interveinal chloresis developed after leaf emergence and accounted for 10-15% of mature leaf surface area for plants grown in 3xs ambient UV-B. Chlorosis and decreased stomatal conductance decreased maximum rates of whole leaf photosynthesis 20% at high UV-B. Leaf expansion also decreased markedly for Cucumis. Electron transport as measured by Oz evolution and chlorophyll fluorescence was unaffected for Pisum and nonchlorotic leaf tissue of Cucumis. Biomass accumulation was not affected for Pisum or C. sativus cv. Ashley. For Arabidopsis mutants, increased UV-B decreased photosynthesis, reduced biomass accumulation, and induced severe morphological changes.

Photosystem II-enriched membranes (BBY) can be further fractionated by the use of the detergent dodecylmaltoside. It has previously been shown that following treatment in 1 % dodecylmaltoside, a variety of different populations of Photosystem II-core can be recovered by isoelectric focusing (Giardi et al 1993, Planta, 190:107-113). We have applied this approach to investigate in vivo photoinhibition and the effects at a molecular level on the innermost part of the photosystem II chlorophyll-protein complex, isolated from leaves that are pretreated in various ways which affect the degree of photoinhibition. Data will be presented which correlate the degree of leaf photoinhibition and Dl-protein turnover in vivo with the existence of different Photosystem II core populations in terms of polypeptide composition and de novo-synthesis, degree of phosphorylation, rate of Dl-degradation in vitro and secondary structure as determined by CD-spectroscopy.

P-22-062

P-22-065

POSSIBLE ROLE OF THYLAKOID MEMBRANE UNCOUPLING IN THE PROTECTION FROM PHOTOINHIBITION

EFFECTS OF OZONE ON THE BIOLOGICAL SYSTEMS OF TWO TOBACCO CULTIVARS

M. Richter, D. T. Nguyen and A. Wild Institute of General Botany, Iohannes Gutenberg-University, 55099 Malnz, Germany

P. Batini. S. Pasqualini, L. Ederli and M. Antonielli Dept of Vegetal Biology, University of Perugia, Borgo XX Giugno, 74Perugia-Italy

Illumination. of spinach leaves led to partial uncoupling of the thylakoid membrane. Uncoupling was large following strong illumination and smaller but still significant in thylakoids from leaves kept at growth light conditions when compared to thylakeids from dark treated leaves. A decrease in ATPsynthesis was observed which corresponded to light induced uncoupling. On the other hand the whole chain electron transport was stimulated by uncoupling. Together with the observed parallel increase of the activation state of NADP-malate dehydrogenas¢ uncoupling could thus open a non photosynthetic pathway for electrons. NADPH exported from chloroplasts through malate transport into the eytosol could finally serve as a snbstrate for light enhanced mitochondrial respiration. One important result of this NADPH to ATP-transformation would be a shift towards higher ATP/NADPH-stoichiometries with increasing light intensities. Furthermore, the desdbed mechanism includes partial heat dissipation of excessively absorbed light energy via shortcurcuiting of the t, pH and it could also help to prevent over-reduction of the electron transport chain artd consequently protect from aeceptor side mediated PSII photoinhibition.

196

Atmospheric ozone causes formation of various highly reactive intermediates (e.g. peroxyl and superoxide radicals, H202, etc.) in plant tissues. The effects of ozone on photosynthesis were investigated in two tobacco cultivars. The decrease in net carbon fixation may result from inhibition of the electron transport system or desturbing the biochemical processes by from inactivation of the enzymes of the Calvin cycle. To investigate the first way the ratio of NADPH/NADP and the quantum yield in leaves fumigated with ozone was determined. For the second way, the following two parameters were investigated in isolated, intact chloroplasts: 1- the content of the sulphydrilic group, because the activation state of the enzymes of the Calvin cycle depends on the ferredoxin-thioredoxin system: 2- activity of NADP-dependent malate-dehydrogenase because its activation state only depends on the availability of reducers in chloroplasts.

Poster P-22-066 C H A N G E S I N D U C E D BY O Z O N E IN P R O T E I N P A T T E R N O F T W O T O B A C C O CULTIVARS S. Pasqualini, P. Batini, L. Ederli and M. AntonieUi Dept of Vegetal Biology, University of Perugia, Borgo XX Giugno, 74 -Perugia- Italy Ozone (O3), a widespread atmospheric pollutant in the industrialized regions of the world, is a powerful oxidant. Plants that are exposed to this oxidizing agent undergo several physiological and biochemical changes that often result in foliar injury, premature senescence and reduced productivity. 03 enters living ceils through the stomata and, producing oxygen radicals, alters primary, secondary, and tertiary structures and causes the proteins to be more sensitive to proteolytic hydrolysis (Davies etal., J. Biol. Chem., 1987, 262, 9908-9913). Two tobacco cvs. with different O3-sensitivity (Havana 425, O3-tolerant and Bel-W3, O3-sensitive) were exposed to ozone fumigation (150 ppb for 2 h) and the soluble and thylacoid proteins were examined. Measurements taken immediately after O3-treatment (until 1 h) in the stressed plants showed a reduced soluble protein content. The soluble proteins returned in the following hours to the control levels. The ozone effect was also tested on thylacoid proteins, one of the main targets of O3. The denaturing electrophoresis (SDS-PAGE) protein pattern is presented.

P-22-067 Regulation of Nonphotochemical Quenching in ABA-defieient Mutants of Arabidopsis thaliana That Do Not Contain Epoxy-Xanthophylls. Vauahan M. Humt. Wah Soon Chow, Jan M. Anderson & C. Barry Osmond. Cooperative Research Centre for Plant Science 0/MH), CSIRO Division of Plant Industry 0NSC & JMA), Research School of Biological Sciences (CBO). Canberra, ACT 2601, Australia. Abeeisic acid defcient mutants of Arabidopsisdo not synthesise the epoxy-xanthophylls antheraxanthin or violaxanthin and therefore do not contain the complete xanthophyll cycle. However, thylakoid preparations from these mutants contain levels of zeaxanthin three to four times those found in wild-type (WT). This large increase in zeaxanthin sensitises the leaves to the development of nonphotochemical quenching (NPQ) during the first 2 to 4 min following a dark-light transilJon. However, steady state levels of NPQ are similar in the mutants and WT over a wide range of irradiance. Steady state trapping efficiency, photochemical quenching and the yield of electron transport are also similar in the mutants and VVT.When leaves of the mutants and WT are exposed to twice growth irradianco for up to 3 h, the mutants developed lower maximal levels of NPQ and suffered more photoinhibition, measured both as a decline in F~Fm and as a loss of active PSII centres, determined from oxygen flash yield measurements, than did WT leaves. Further, the in~al kinetics of NPQ development during high light exposure is similar in both the mutant and WT leaves, despite the high inifal level of zeaxanthin in the mutants. Thus, these results show that high levels of zeaxanthin in the mutants in the dark sensifses these leavesto the development of NPQ upon illuminal~on. However, they also show that increasing the zeaxanthin content of thyiakoids does not necessarily lead to an increase in thermal dissipation or to an increase in photoprotecflon and down-regulation of PSII.

P-22-068

P-22-069 INCREASED XANTHOPHYLL CYCLE ACTIVITY AS AN IMPORTANT FACTOR IN ACCLIMATION OF THE PHOTOSYNTHETIC APPARATUS TO HIGH-LIGHT STRESS AT LOW TEMPERATURE O.Y. Koroleval, A. Thiele 2 and G.H. Krause2 1Dept. of Photosynthesis, Komarov Botanical Institute, Prof. Popov Str. 2, 197376 St. Petersburg, Russia; 2Institute of Plant Biochemistry, Heinrieh Heine University DQsseldorf, Universt~tsstr. 1, D-40225 DQsseldorf, Germany Changes in xanthophyll cycle activity related to cold acclimation under moderately excessive light were studied with the chilling-tolerant species Oxyria digyna L., an arctic/alpine plant, and Spinacia oleraeea L. (spinach). In O. digyna, 5 weeks of acclimation to 9°C (light flux, 250-300 ttmol m-2 sa) compared to growth at 1825°C caused a strong increase (factor 2-3) in the pool size of xanthophyll cycle pigments (sum of viola-, anthera- and zeaxanthin per reel chlorophyll). Moreover, zeaxanthin formation in high light at low temperatures (40C) was considerably faster, and a higher proportion of violaxanthin was available to deepoxidation in cold-acclimated leaves. Similar results were obtained with field-grown spinach acclimated to low temperatures during winter, as compared with non-acclimated plants. We conclude that the presence of very high levels of zeaxaathin in excess light essentially contributes to the decrease in susceptibility to photoinhibition observed upon cold acclimation of ehilling-tulerant herbacious plants.

P-22-070 THE R O L E OF D1 PROTEIN T U R N O V E R IN P H O T O S Y N T H E S I S AND PHOTOINHIBITION A W e n d y Russell 1, Ursula L Schiefthaler 2, Harald R Bolh~rNordenkampf 2 & Christa Critchley 1 1Dept of Botany, The University of Queensland, QId 4072, Australia; 2Institute for Plant Physiology, University of Vienna, Althanstr 14, Wien A1091, Austria Results from experiments integrating biophysical, biochemical and physiological measurements on mature leaves of Scheffiera arboricola clearly demonstrate that turnover of the PS II reaction centre protein D1 is controlled by more variables than simply light. Factors influencing D1 turnover include long-term irradiance acclimation state and carbon assimilation rates. Our results indicate that D1 turnover is an essential regulatory process in leaves which saturates at low irrediance and is modulated by feedback mechanisms which sense the balance between electron transfer and carbon fixation reactions. D1 turnover may play a central role in the interconversion of active and dissipative photosystem II centres.

Burkhard Jak.Qb and Ulrich Heber Julius-von-Sachs-institut fiir Botanik und Pharm. Biol.,Wtlrzburg (Germany)

P-22-071 TRANSIENT ASSOCIATION OF A NEW PROTEIN WITH SPIRULINA MAXIMA PHYCOBILISOMES IN RELATION TO LIGHT INTENSITY. Florence GARNIER, Anne-Lise ETIENNE, Jean-Claude THOMAS Photortgulation et Dynamique des Membranes Vtgttales, URA CNRS 1810 Ecole Normale Suptrieure, 46 rue d'Ulm, 75230 Paris Cedex 05, FRANCE

The light-dependent production of hydroxyl radicals by thylakoids and chloroplasts of Spinacia oleracea was investigated using dimethylsulfoxide (DMSO) as trapping agent. Maximum rates of OH* formation by thylakoids were observed under aerobic conditions in the absence of added electron accepters. They approached 3 pmol (mg chl * h)-z. Saturation of OH* formation occured at the low photon flux density of 100 lamol m-2s-L Dichlorophenyldimethylurea (DCMU), which binds to photosystem II, inhibited OH" formation. Methylviologen (MY), which stimulates linear electron flow to O 2, increased OH* formation only in the presence of Fe 3÷. In intact chloroplasts, OH* formation became appreciable only when KCN was added to inhibit effective H202 scavenging by ascorbate peroxidase. It was stimulated by ferrisulfate, but not by ferricyanide which does not penetrate the chloroplast envelope. The data are interpreted on the basis of a Fenton-type reaction which produces OH* from I-t202 and Fe z*, when MV is electron acceptor, and from H202 and reduced ferredoxin or Fe-S-centres, when 02 is electron aeceptor in the Mehler reaction. Trapping of OH* by DMSO decreased, but did not eliminate loss of the activity of photosystems I and II in photoinactivation experiments.

In Spirulina maxima, an increase in light intensity caused a decrease in the phycocyanin (PC) content with a loss of the linker polypeptide of 33 kD. The rods were shortened. We evidenced a transient association of a new protein (p47) with the PBS in relation to light intensity, p47 is found specifically associated with the PC fraction. Using an antibody anti-P 47 of S. max/ma, we determined its specificity with PBS containing organisms (procaryote and eucaryote). The p47 was found in two cellular fractions, associated in various amounts to the PBS fraction and in the soluble proteins. For the "low light "PBS, all the linkers and phycobiliproteins were present, with a weak band at 47 kD. For the "high light" PBS, the linker 33 kD was missing, three polypeptides clearly unrelated to linkers were present, among them the p47, in very obvious quantity. An increase of p47 associated to the PBS also occttred during nitrogen starvation or heat shock. We propose that p47 has a stabilizing role for the PBS structure devoidod of the terminal disk of PC. We consider p47 as a particular chaperon protein because of its high affinity for ATP and its possible association in a permanent manner with PBS when the cells are submitted to extreme conditions. F. GARNIER, J.P. DUBACQ and J.C. THOMAS (1994) Plant Physiology106:747-754

F O R M A T I O N AND SCAVENGING O F HYDROXYL RADICALS B Y T H Y L A K O I D S AND INTACT CHLOROPLASTS

197

Poster P-22-072 PHOTOINHIBITION OF PHOTOSYNTHESIS RELATED TO XANTHOPHYLL CYCLE AND D1 PROTEIN TURNOVER IN HIGHER PLANTS A. Thiel¢l, K. Winter2, and G.H. Krause1 Institute of Plant Biochemistry, Heinrich Heine University D0sseldorf, Universit~tsstr. 1, D-40225 D0sseldoff, Germany; 2 Smithsonian Tropical Research Institute, Panama City, Apartado 2072, Balboa, Rep. of Panama Two plant systems with leaves of different susceptibilities towards high-light stress were used to study the role of xanthophyll cycle activity and D1 protein turnover during photoinhibition. Previous investigations with greenhouse-grown spinach revealed two distinct stages of inhibition which were reflected in different kinetics of recovery in low light. A first fast recovery phase (1 h) of unknown origin was followed by a slow phase (several h) which was suggested to be based on resynthesis of the D1 protein in the photosystem II reaction center. With young and mature canopy leaves of tropical forest trees and cold-acclimated and non-acclimated spinach, we found a close relationship between epoxidation of zeaxanthin via the xanthophyll cycle and fast recovery. When inhibited to the same degree, affected D1 protein contributed less to photoinlfibition in young than in mature canopy leaves and in cold-acclimated than in non-acclimated spinach. The results suggest that acclimation to excessive sun exposure, which includes increased xanthophyll cycle activity, may serve to avoid D 1 protein inactivation.

P-22-075 FUNCTION OF LEAF HAIRS AND LEAF WAXES IN THE PROTECTION OF THE PHOTOSYNTHETIC APPARATUS AGAINST PHOTOINHIBITION M Lung, C. Schindler, H. K. Lichtenthaler Botanical Institute II, University of Karlsruhe, D-76128 Karlsruhe, Germany. In the succulent plant Senecio medley-woodii Hutchins, which contains a dense layer of leaf hairs, and in leaves ofAeonium hawothii Webb et Berth, which possess a surface wax layer, we studied the effect of a 30 min high-light treatment (1300 pmol m-2 s" 1) on the variable chlorophyll fluorescence and the light-induced zeaxanthin accumulation. Senecio leaves, where the hairs were wiped off, accumulated more zeaxanthin, but were sensitive to photoinhibition (decrease in Fv, Fv/Fm and Fv/Fo as well as increase in qFo and qN ) and photooxidation of pigments, whereas leaves with hairs were not. In a similar way Aeonium leaves, where the surface wax layer was removed, exhibited a higher photoconversion rate of violaxanthin into zeaxanthin and showed a higher photoinhibitory damage and a higher rate of pigment photooxidation. The results indicate, that leaf hairs and leaf waxes protect leaves against photoinhibitory damage and photooxidation of pigments. Under the given conditions zeaxanthin did not prevent photoinhihition.

P-22-073

P-22-076

INFLUENCE OF THE STRUCTURE OF THE D 1 PROTEIN ON THE PSII ACTIVITY AND PHOTOINHIBITION

A PROTEOLYTIC ACTIVITY INVOLVED IN ACCLIMATIVE DEGRADATION OF LHCII

1D. Kirilovsky, IS. Constant, lI. Perewoska, 2L. Nedbai and IA-L. Etienne 1URA 1810, CNRS, E.N.S., 46, rue dTllm, 75230 Paris Cedex 05, France; 2Inst. of Microbiology, Academy of Sciences, 37981 Trebon, Czech Rep..

Dan-Hui Yank. Marika Lindahl and Bertil Andersson Department oi' Biochemistry, Arrhenius Laboratories for Natural Sciences, Stock_holm University, S-106 91 Stockholm, Sweden

We have studied PS II activity in Synechocystis PCC 6714 cells which carried the mutation A251V in the QB pocket of the DI protein. This mutation eonfered to the PSII an increased sensitivity to high light. The A251V mutation also resulted in a modification of electron transfer between QA" and QB and a destabilization of the S 1 state due to a better accessibility of the oxygen evolving complex to eytusolic reductants. Double mutants carrying the A251V mutadon plus another one (F211 S, $264A or N266T) in the QB pocket of the DI protein are more sensitive to high light than the simple mutant. The double mutants presented a wild type PSII donor side behaviour. So, we can conclude that there is no direct relationship between the S 1 destabilization and the increased sensitivity to high light. In all the studied mutants, the reoxydation of QA" was slower than in the wild type. Results about the relationship between the sensitivity of the PSII to high light and QA- reoxidation rates will be presented.

The relative amount of the major light-harvesting chlorophyll a/b protein (LHCII) is reduced when plants acclimate from low to high light intensities. We have identified a proteolytic activity degrading LHCII in vitro once spinach leaves have been subjected to increased irradiances for approximately 2 days. This proteolysis can be followed in both isolated chloroplasts and thylakoid membranes. The proteolysis is strictly ATP dependent. The protease is of serine or cysteine type. The proteolytic activity can be abolished by washing of the thylakoid membranes with high salt and reconstituted b.y readdition of the desalted wash supematant, which suggests that the protease *s extrinsically bound to the outer thylakoid surface. Identification, isolation and purification of the thylakoid bound protease is currently under way.

P-22-074 PHOTOINHIBITION IN

IN

PHOTOAUTOTROPHIC

TOBACCO

PLANTLETS

VITRO

I. T i c h ~ D e p a r t m e n t of Plant V i n i ~ n ~ 5, C Z - 1 2 8 4 4

OBLIQUUS Physiology, Charles University, Praha 2, C z e c h R e p u b l i c

W i t h the a i m to o p t i m i z e h i g h e r p l a n t s in vitro growth, t h e p h y s i o l o g i c a l e f f e c t s of t w o light t r e a t m e n t s h i g h light (HL) and low light (LL) w e r e s t u d i e d in p h o t o a u t o t r o p h i c a l l y in vitro grown tobacco p l a n t l e t s at s a t u r a t i n g CO 9 ½eve~s in a c o n t r o l l e d e n v i r o n m e n t . In HL (200 p m o l ~s- ) c h l o r o p h y l l a + b c o n t ~ t s l w e r e d o w n r e g u l a t e d as c o m p a r e d to LL (60 p m o l m s ). The c h l o r o p h y l l f l u o r e s c e n c e p a r a m e t e r F /F of d a r k • . . m a d a p t e d leaves w h l c h xs an e s t x m a t e for The p h o t o c h e m i c a l e f f i c i e n c y of P h o t o s y s t e m II and an i n d i c a t o r of p h o t o i n h i b i t i o n , was r e d u c e d in HL i n d i c a t i n g that p h o t o i n h i b i t i o n occurred. P h o t o i n h i b i t i o n in HL could be p r e v e n t e d by a d d i t i o n of s a c c h a r o s e (3 %) to the MS m e d i u m at the b e g i n n i n g of the e x p e r i m e n t . A d d i t ion of s a c c h a r o s e s t i m u l a t e d growth, c h l o r o p h y l l content as w e l l as p h o t o s y n t h e t i c c a p a c i t y in HL and LL.

198

P-22-077 N O N - P H O T O C H E M I C A L F L U O R E S C E N C E Q U E N C H I N G IN W I L D T Y P E AND P I G M E N T MUTANTS OF SCENEDESMUS llona Heinze, Markus Htihn, Holger Dan and Horst Senger FB Biologie/Botanik, Lahnberge, D-35032 Marburg, Germany Wildtype and several pigment mutants of the unicellular alga Scenedesmus obliquus were preilluminated with different light intensities (154600 pE m -2 s-1) for 15 h. (1) The capability to develop ApH-dependent non-photochemical quenching (qE) increases with increasing light intensity of the preillumination. (2) Cells preilluminated with high intensifies contain an increased amount of the xanthophyll cycle pigments (zeaxanthin, antheraxanthin, violaxanthin). (3) In wildtype and some of the mutants the extent of qE was correlated to the zeaxanthin content of the ceils. (4) The fluorescence parameter (F'm-F)/Fm is linearily related to the polarographically determined oxygen yield. (5) In uncoupled thylakoid membranes qE is initiated by a medium pH between 6.25 and 5. (6) A chlorophyll b-free mutant and a mutant without ct-carotinoids, which both lack LHCIl-trimers, show energy quenching with the same characteristics and cssentially the same extent as the wildtype. This finding indicates that LHCIl-trimers are not essential for qE formation.

Poster P-22-081

P-22-078 ULTRAVIOLET.B RADIATION INDUCED DAMAGE TO THE FUNCTION AND STRUCTURE OF PHOTOSYSTEM II

L I G H T S T R E S S A N D REACTIVE O X Y G E N SPECIES: C O N S E Q U E N C E S FOR P H O T O S Y N T H E S I S IN EUGLENA GRACILJS

1, C. Spetea 1, L. Sass1, I~. Hideg 1 & V. Petrouleas 2 f Plant Biolog),, Biological Research Centre, Szeged, Hungary. 2Inst. of Materials Science, NCSR "Demokritos", Athens, Greece

Peter Landgraf, Maria Doege, Erich Ohmann and Henning Tschiersch, Dept. of Biology, Inst. for plant end cell physiology, Martin-Luther-Univ., 06108 Halle/S, Germany

The damaging effects of UV-B light on the electron transfer and protein structure of Photosystem II (PSII) have been studied. UV-B irradiation of spinach PSII membrane particles results in the rapid inhibition of oxygen evolution in parallel with the loss of the multiline EPR signal from the S2 state of the water-oxidizing complex. These effects are followed by slower decline of EPR detectable QA-Fe2+, Tyrosine-D ÷ and Fe 3+, fluorescence detectable QA', the amount of the D1 reaction centre protein and membrane-bound manganese. A gradual conversion of cytochrome b-559 from the high potential form to the low potential form is also observed as a consequence of UV-B irradiation. It is concluded that UV-B light affects the photosystem II redox components to different extent. The primary damage occurs at the water-oxidizing complex which interrupts electron transfer from the catalytic manganese cluster to Tyrosine-Z ÷ and P680+. This is thef°ll°WedFby2+inactivation or modification" ' of the redox-active tyrosines, QA e acceptor complex, cytochrome b-559 and degradation of the D1 protein component of the PSII reaction centre.

Light stress induces both in whole cells of Eugtena gracilis and in isolated chloroplasts and thylakoids inactivating and damaging processes of membrane structures and stromal enzymes. The kinetics of inactivation of PS II is nearly identical with those of stromal enzymes. In both cases it could be shown that highly toxic oxygen species were involved. This light mediated oxidative stress leads to a specific degradation of Dl-protein and Rubisco as could be shown by western blotting. These damaging processes could be prevented in the presence of vaflous radical scavengers, antioxidants, and enzymes of radical defence. Degradation processes of proteins were enhanced in the presence of different radical-generating substances. The date indicate a special role of *OH-radicals and suggest that PS II is the site of origin for radical formation involved in photoinhibition.

P-22-079 RESPONSE TO LIGHT OF BEECH SEEDLINGS GROWN UNDER NATURAL FOREST SHADE G. Agati l, J.D. Johnson2, P. Mazzinghi l , M. Michelozzi 3 , S. Pinzauti 3 & R. Tognetti 3 list. Elettronica Quantistica - CNR, Via Panciatichi 56/30, 50127 Firenze, Italy; 2School Forest Resources & Cons., Univ. Florida, Gainesville, FL 32605, USA; 3Ist. Miglioramento Genetico Piante Agrarie - CNR, Via Vannucci 13, 50134 Firenze, Italy Beech seedlings grown under the forest shade were irradiated by a 1000 p.mol m-2s-1 light from halogen lamps. Net photosynthesis, chlorophyll (Chl) fluorescence induction kinetics and the Chl fluorescence ratio, F685/F730, were monitored on two leaves of each of four different plants, during the light treatment up to nine hours. The Fv/Fm ratio decreased rapidly with irradiation time indicating that the plants experienced photoinhibition. Also F685/F730 rapidly decreased with irradiation time. The photosynthesis rate increased to a maximum after 6 hours of irradiation and then declined. Our data indicate that, beside the Fv/Fm parameter, photoinhibitory effects can also be detected by the F685/F730 Chl fluorescence ratio.

P-22-080 ANTIOXIDANT EFFECT OF REDUCED PLASTOQUINONE IN THYLAKOID MEMBRANES. Torill Htmdal. Patrik Forsmark Andr6, Lars Emster and Bertil Andersson Department of Biochemistry, Arrhenius laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden. The effect of strong illumination on intact thylakoid membranes was studied. Three different parameters were measured; 1. electron transport, 2. chlorophyll a/b ratio and 3. malondialdehyde, MDA, formation ( as indicator of lipid peroxidation ). The electron transport activity was totally inhibited after 90 minutes illumination whereas bleaching of chlorophyll and MDA formation were not induced until the electron transport was reduced by appr. 90%. Chlorophyll a/b ratio was rapidly decreased and MDA formation were stimulated after addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to the incubation medium. In contrast, the addition of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) inhibited these two parameters but did not inhibit electron transport. Two different phases separated in time occur during strong illumination.First, inactivation of electron transport activity and secondly, destruction of thylakoid membrane lipids with concomitant loss of pigments and formation of lipid peroxidation products. Lipid peroxidation is prevented by a component located between PSII and PSI, most probably the reduced form of plastoquinone. For thylakoid membranes the stimulatory effect of DCMU and the inhibitory effect of DBMIB can be explained by the redox state of the plastoqulnone pool, namely, oxidized in the presence of DCMU and reduced in the presence of DBMIB.

199

Poster

P-23-003 LONG-TERM STRESSSYNDROME: DROUGHT INDUCES STRUCTURAL AND FUNCTIONAL REORGANIZATION OF PHOTOSYSTEM II

Poster session 23

A. Cona, T. Kucera, J. Masojidek, A.K. Mattoo* and M.T. Giardi IBEV-CNR. Via Salaria kin. 29.3. 00016 Montetotondo Scalo. Italy (EC). *Department of Agriculture, Beltsville, 20705 Maryland. USA

Water deficiency, salt and chemical stresses

To study the effect of drought stress and its combination with high irradiance on Photosystem II, in vivo and in vitro techniques were used in pea (Pisum sativum L.) seedlings. As shown by the separation of the thylakoids into grana and stroma-exposed regions and further fractionation of the PSII particles, drought led to a considerable depletion of the PS II core. The remaining PS II complex appeared to be functional and reorganized, with the PSII unit size twice greater than well-irrigated plants.The rate of 3SS-methionine incorporation into the D~ protein in droughted plants increased threefold as compared with control plants. On the contrary, droughted plants further exposed to high irradiance showed a decreased rate of methionine incorporation. We suggest that long-term water stress is a combination of two events: a reduction of the number.of active PSll centers caused by a physical disassembly of the PSII core and a PSII reorganization with enhanced protein turnover to counteract the core depletion.

P-23-001

- P-23-053

P-23-001 A Q U A P O R I N S AND S A L I N I T Y T O L E R A N C E Manabu lshitani, Shigehiro Yamada, Maid Katsuhara and Hans J. Bohnert. Department Biochemistry, University of Arizona, Tucson, A Z 85721, USA Homologs of :animal a q ~ exist in plants in two families of related sequences, one subfamily targeted to the tonoplast, the second to the plasma membrane. We have isolated several transcripts for each sub-family f~om the halophyte Mesembryamherawn crysrallinum L which we termed N i p for major intrinsic protein.

Their~ o n

characteristics in stressed(500 m M NaCI) and

unstressed plants and during plant development will be presented. Functional assays of two plasma memlmme NIP showed that they function in f~ilitsting water movement. Their cell- mad tissue-specific x ~ ion in Meaembryamhemum willbe described. In addition, of one of the M. crysta///num MIP-prommers in transgenie tobacco has been analyzed. This immmter is active in a number of cells which are involved in the movement of water, The complexity of this gane family, f ~ a function that has only recently been identified as imtmttant in plants, seems to indicam tissue and develolmamtal specificity and functional redundancy. Differences in the exlaeSsion of Mip-traascdpts under salinity stress may be a character that distinguish glyeophytes and halophytes.

P-23-002 T H E A P P L I C A B I L I T Y O F C H L O R O P H Y L L F L U O R E S C E N C E AS A S E N S I T I V E I N D I C A T O R O F D E S I C C A T I O N IN A L I V E R W O R T , PORELLA PLA TYPHYLLA M. Marsehall, S. Dulai, I. Molnar Botanical Department, Eszterhazy T.T. College, 3301 Eger, PL 43. Hungary It is well-documented that the function of thylakoid membranes and the processes of PS II are sensitive to several environmental stresses. With the help of chlorophyll fluorescence measurements we have quantified the changes in the photosynthetic activity under desiccation. Desiccation is one of the most extreme physical changes that cells can survive. The photosynthetic tissue of the leafy liverwort, Porella platyphylla, can endure high levels and periods of desiccation and can recover after remoisturising. We have measured chlorophyll fluorescence induction in Porella platyphylla under different levels of desiccation, compared with fresh and rehydrated tissue, using a modulated fluorimeter. The analysis of the fast and the slow fluorescence kinetics phases in Porella with different water status (fresh, lightly desiccated, highly desiccated and rehydrated) provided information about the degree of desiccation tolerance and indicated the optimal hydration range for the function of photosynthesis.

200

P-23-004 R . E S P | g A T O R Y AND PHOTOSYNTHF:I'|C. RESPONSE IN SALT STRESSED S Y N E C H O C Y S T I S P C C 6803 k)m~ Ardelemt and Malta Lui~a Flonta Institute of Biology, Splaiul hJdependentei 296, POB 56-53 Bucharest 79651. ROMANIA ttere we preso~t tim results concelrfing die suategies developed by S~hh~3~s PCC 6803 to grow trader 0.5 M NaCI- supplemented B G ~ The results obtained wath oxygen probes and tnechated ainperometry Uanlg hydrophilic (e.g ferrycianidei a~ well as lila)phihc q~benzxgtuinone) redo'~ mediatoes show the tbllowmgs: i)the increase in respiratory, a c t i v i ~ both under photoauti~ and photoheterotrophtc conditions; ii)the co-operation between rcspirathm and photosynthesis fit order 1o increase the flux ofunergy reqmred thr actree exclusion of Na*. I'he results are intlx~rlant to letter under,~mnd the intelpiny ielween re,~l)lr~holl a i ~ pltotosymlmsis under normal aim salt stress coJ'tditions.

P-23-005 LEAF PHOTOSYNTHESIS AS AN INDEX OF DROUGHT SENSITIVITYIN WHEAT " K.C. Bensal S. Nagarajen & P.N. Gambhir Division of Plant Physiology, end Nuclear Research Laboratory, Indian Agricultural Research Institute, New Dethi-ll0012, India Response of leaf photosynthesis to water stress was compared between two wheat species, i.e. Triticum aest~vum (a hexaploid cultivated species) end T. kotschyi (a tetraploid, drought resistant species). Plants of both species were grown in pots of size 35 cm diameter, 45 cm height at two levels of soil moisture potential, ~3.03 MPa and -0.2 MPa. Leaf photosynthesis on intact youngest fully expended leaves using infra red gas enalyser (LI 6200, Licor, USA) end water relation parameters were measured at different stages of plant growth. Plants of T. ae~tivurn (cvs HD 2329 end C 306) had lower rate of photosynthesis at all stages compared to T kotschyi. The difference between the two species was maximum at enthesis stage. At this stage, T. kotschyi experienced a minimum reduction of 35% in photosynthesis due to water stress as compared to 69% end 85% in 1". aest~vum cultivars HD 2329 and C 306, respectively. Higher leaf photosynthesis in stressed plants of T. kotschyi was found related to higher values of stomatal conductance, internal CO2 concentration, relative water content, leaf water relaxation time (T1) and leaf water activity. The possibility of using intact leaf photosynthesis as an index of drought sensitivity and as a selection trait in a inter-specific hybridisation programme will be discussed.

Poster P-23-006 I14FLU~qCZ OF HF.AVY ~ T A L S

P-23-009

0~q P H O T O S ~ T Z C PARAbRTEB8 UI~DER OF 8 ~ M $ ~ 8 M.

DZFI~I~l~L~ LIGHT O~qDZTZO~'S Zlq C I ~ S

L. Fodorpataki & M. Trifu Babes-Bolyai University, Dept. of Plant Kogalniceanu i, Cluj-Napoca, RO-3400, Romania

Biology,

Str.

Micromolar and m i l l i m o l a r a m o u n t s of lead (pb2+), mercury (Hg2+), copper (Cu 2+) and cadmium (Cd;+) induce changes in the photosynthetic oxygen evolution, the kinetics of fast chlorophyll fluorescence, the chlorophyll a/b ratio, the carotenoid pigment content, the rate of cell divisions, the protein content of cells and in the biomass accumulation of the green microalga Scenedesmus acutus M. in axenic cultures illuminated 13 hours/day with low and high photon flux density. Cd influences mainly the p h o t o s y n t h e t i c pigment content of the cells, especially in dim light, while Cu has a more pronounced effect on the p h o t o s y n t h e t i c oxygen evolution and on the dynamics of chlorophyll fluorescence related to the electron transport through p h o t o s y s t e m II, both in dim and high light. Hg inhibits protein accumulation m a i n l y in low light, while Pb reduces the rate of cell divisions and biomass production irrespective of light conditions. The negative influence of Pb and Hg occurs in considerably lower amounts than those registered in the case of Cd and Cu, but in higher concentrations than usually reported for higher plants. Photosynthesis of this alga continues even in the presence of relatively high amounts of heavy metals in the medium, probably because of an efficient self-protection with phytochelatins.

P-23-007 EFFECTS OF HEAVY METALS ON PHOTOSYNTHETIC CUCUMBER (Cuoumle estlvux L) SEEDLINGS

APPARATUS

IN

F. I_~o, ~. S~'v~lri, Z. Szigsti, F. Fodor and E. Cseh Department of Plant Physiology, E0tv0s University, H-1445, Budapest, P.O.B. 330., Hungary Effects of Pb and Cd were investigated on the photosynthetic apparatus in the presence of FeEDTA and Fe-citrate. The concentration of iron was 4 I~M, that of heavy metals was 10 p.M in the modified Hoagland solution of 1/4 strength. Cd strongly ioldbited, but Pb hardly influenced the growth of 6-week-old seedlings. Chlorophyll concentration was very low (15-25 % of the control) in the Cd-treated plants, while Pb inhibited the chlorophyll accumulation only a small extent. The amount of chlorophyll in PSI complex, in PS II core complex as well as in the LHC II were highly affected by Cd, but only a small effect was found in the case of Pb. Photosynthetic activity was characterized by in vivo CO2 fixation and fluorescence induction parameters. Cd inhibited the photosynthetic activity dramatically in the presence of both Fe-complexes. Photosynthetic parameters in Pb treated plants were similar to the control values. Fe transiocation experiments showed that Cd caused a strong iron deficiency in the shoot. It is concluded that Cd may inhibit the Fe tranalocation from the root to the shoot because the symptoms observed are very similar to tho~e of a strong iron deficiency.

M

EFFECTS OF PACLOBUTRAZOL ON STABILrI4TION PHOTOSYNTHETIC COMPONENTS IN SENESCING BEAN LEAVE8 E w a Madef, 8ibdas Qhosh =, John E. Thompson*, Erwin B. Dumbrofl~''

~Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 8G1; =Department of Bielogicel Sciences, Univers/tyof W'~ooasin-Whitewater, Whitewater, W'meoasin,USA 53190; 'Keanedy-LelghCentre for Horticultural Research, Faculty of Agriculture, Hebrew Univendty of Jerusalem, P.O. Box 12, Rehovot, Israel 781000. Paclobutrazol, a triasole derivative and synthetic plant growth regulator, was applied as a root drench at a concentrations ran~n~ from 0.01 mM to 1.0 mM per pot, each containing three, 7-d~-oid ~,edllno,~ of bean (Phasealus vulgaris L.). Levels of total protein, chloropl~ylland carotenoids in primary leaves of 14-, 18- and 22-day-eld plants increased markedly following peelobutrasol treatment. Variable f l u o r e s e s ~ measurements indicated higher quantum elTtcienciesof photosystem H in the primary leaves of treated plants compared to those of controls, and the activities of phesphoenolpyruvate carbo~lase and ribuloas-l, 5-bisphosphate carboxylase per unit leaf area were si£,ninmntly higher in the prknary leavea ofthe treated seed,he, at all stages of development. In toto, these data suggest that de/eyed senescence in response to paelobatrazol treatment may, in part, be a function of enhanced photosynthetic eft]derides.

P-23-010 EFFECTS OF WATER STRESS ON RUBISCO ACTIVITY OF Setaria sphacelata (C4) J. Maroues da Silva & M.C. Arraba~a Departarnento d e Biologia Vegetal, Faculdade de Ci~ncias d a Universidade de Ltsboa, 1700 LISBOA, Portugal Setaria sphacelata was grown for two m o n t h s in a c h a m b e r with full water supply. Afterwards, w a t e r stress was induced by withholding watering, and relative water content (RWC) of leaves was determined. Leaf s a m p l e s were collected a n d i m m e d i a t e l y frozen in liquid nitrogen. Ribulose 1.5 B i s p h o s p h a t e Carboxylase / Oxygenase (RUBISCO. E.C. 4.1.1.39) was extracted and its activity determined In vitro t h r o u g h incorporation of labeled bicarbonate. The degree of activation was determined comparing the activities with and without pre-incubaUon with CO2 (bicarbonate) and Mg2+. The enzyme activity w a s expressed in several basis: per dry weight, leaf area, soluble protein a n d chlorophyll. Wathever the m e t h o d used, we found a statistieally significant linear decrease of the RUBISCO activity with decreasing RWC. However, the best correlation between activity and RWC was found when activities were expressed in a dry weight basis, the correlation being poorest in a chlorophyll basis. Apparently an increase in the activation state of the enzyme occurs at moderate water stress and a decrease at very severe water stresses. The quantification of RUBISCO-protein is currently being done.

P-23-008 • FFIOT OF DROUGHT OF WHZTE CLOVER

ON

PHOTOBYNTHZSZ8

OF

TWO CULTZVARB

OF

P-23-011 EFFECT OF WATER-STRESS ON PHOSPHOENOLPYRUVATE CARBOXYLASE.

MAIZE-LEAF

Phillnn~ Grien, Christophe Robin and h r m a n d Guckert Agronomie et Environnement, INRA-ENSAIA, BP 172, F-54505, Vandoeuvre les Nancy Cedex, France.

M. Rodriouez-Penaoos. & R.A. Mufioz-Clares. Dept. of Biochemistry, Faculty of Chemistry, UNAM 04510 Mexico City, Mexico.

White clover plants (Trifolium repens L., cv. Crau and cv Huial were grown from seedlings in controlled conditions and then subjected to an edaphic drought by withholding watering. A strong reduction of leaf gas exchange due to stomatal closure in the early stage of water deficit was observed. Between -0.5 and -1.5 MPa of water deficit, the net CO 2 assimilation rate and the transpiration rate were higher for cv Huia than for cv Crau. The consequence is that cv Huia is more sensitive to drought. For the 2 cultivars, chlorophyll a measurements demonstrated that the photochemistry of PSII, the light driven elect~on transport and the enzymatic reactions requiring ATP and NADPH in chloroplasts were not affected by plant water status. Moreover, oxygen evolution showed that the capacity of the photosynthetic apparatus was not altered by the stress. The photosynthetic activity is probably limited by a reduction of CO 2 allocation to chloroplasts, and this allocation is different for the 2 cultivars.

In plants, phosphoenolpyruvate carboxylase (PEPC) activity is involved in several metabolic pathways. The activity and/or synthesis of this enzyme have been reported to be modified by several environmental factors such as light, temperature and nitrogen and water availability. Leaves of facultative CAM plants respond to osmotic stress with increases in PEPC-mRNA and protein, and with changes in the kinetic properties of the enzyme. We have studied the effect of water stress on the activity and content of PEPC in leaves of a C4 plant: maize. We found that subjecting either whole plants or leaf-discs to water-stress results in decreases in S0.5 (PEP-Mg) and in increases in Vmax, which closely correlate with the increases in the content of leaf PEPC. Neither the molecular weight of the PEPC subunit, nor its aggregation state are changed by means of the water-stress treatment. A new PEPC-isoform of pl. 6.5 was observed in extracts from water stressed leaf-discs. At present we are purifying this more alcaline isoform in order to study its kinetic and molecular properties.

201

Poster

P-23-015

P-23-012 THE INFLUENCE OF LEAF AGE AND DROUGHT ON PHOTOSYNTHESIS TROPICAL EVERGREEN TREE Curatella americana L.

OF THE

M. A. Sobrado Depto. Biologla de Organismos. Universidad Sim6n Bol~var. Apartado Postal 89.000. Caracas, VENEZUELA. C. americana is an evergreen tree growing scattered within Venezuelan savannas where the climate has marked rainfall seasonality. Gas exchange and fluorescence parameters were studied in five leaf age classes,from expanding to senescent leaves,in plants growing naturally in the field. Haximtun rates of carbon assimilation (A), transpiration rate (E) and stomatal conductance (gs) were lowered during drought in all age classes, except in growing leaves. A/E ratios increased during drought Thus, season indicating improved efficiency for use of water. A in function of gs relationship was linear except in oldest leaves during wet season,with very high gs and low A. Ratio of variable to m a x i m u m fluorescence (Fv/Fm) increased rapidly during leaf maturation from 0.64 to 0.76 and remained unchanged during drought for all leaf age classes. In conclusion, C. americana adjusted to drought with a different control of gs in growing {sink) and full grown (aource) leaves snd with this maintained A, improved water use efficiency and continued leaf growth.

PHOTOSYNTHESIS IN TRANSGENIC PSSU-IPT TOBACCO PLANTS AS A F F E C T E D BY W A T E R STRESS. H. Synkov& J. Pospigilovh, R. Valcke*

Inst. of Experimental Botany, Na Karlovce la, 160 O0 Praha 6, Czech Republic LUC, Dept. of_Plant Physiology, Universitaire Campus, B-3590 Diepenbeek, Belgium* Introduction o f ipt-gene coupled with Pssu-promotor into N. tabacum L. cv. Petit Havana SRI resulted in 10 times increased endogenous cytokinin content and severely affected electron transport around photosystem 1. Transgenic plants grown as gratis and rooted plants o f first autogamic generation sufferred from wilting especially during light period. Low stomatal conductance and lower water potential found mainly in older leaves indicated considerable water stress in spite o f closed stomata. Ineffective gas exchange (i. e. CO 2 deficiency) is probably the reason for slow growth of transgenic plants. Correlation with concentration o f abscisic acid in leaves was found.

P-23-013

P-23-016

RECOVERY OF PHOTOSYSTEM I1 ACTIVITY DURING REHYDRATION OF THE DESICCATION TOLERANT LICHEN CLADONIA CONVOLUTA

A CHLOROPLASTIC DROUGHT-INDUCED PROTEIN OF $OLANUM TUBEROSUM SHOWS HOMOLOGY WITtt A SALMONID PROTEIN.

2 Z. Tuba, 2 and I. Vass 1 ~Institute1 Zs.ofCsintalan, Plant Physiology, Biological Research

CEA, DPVE, Laboratoire d'Ecophysiologie de la Photosynth~se, Centre

G, Provot, S. Cuint, N. Gault, G. Peltier & P. Rey

Center, Szeged, Hungary. 2Agricultural University of GtdSllt, G t d t l l t , Hungary.

The recovery during rehydration of photosystem II (PS II) activity in the lichen Cladonia convoluta with green algal phycobionts was monitored by thermoluminescence and fluorescence measurements. In the dry state, achieved with slow 24 hours desiccation, no PS II activity was observed. During rehydration, a rapid increase of PS II activity occurred as indicated by the appearance of variable chlorophyll fluorescence and thermoluminescence from the recombination o f the S2Q B- and S2QAcharge separation states. After 20 minutes rehydration in the light, the flash-induced thermoluminescence showed normal oscillation pattern with maxima after 2 and 6 flashes. In contrast, after rehydration in the dark, the oscillation showed maxima after 3 and 7 flashes. This indicates that in dehydrated Cladonia convoluta the water-oxidizin~ complex is in an unusually reduced state. PS II activity in this lichen is extremely resistant to UV-B radiation as compared to other plant species. This indicates the existence of an effective defence and/or repair mechanism in Cladonia

convoluta.

P-23-014

d Etudes de Cadarache, F-13108 Saint-Paul-lez-Durance, France. Using two-dimensional electrophoresis, the accumulation of a 32-kDa.stromal polypaptide (named cdp 32 for chloroplast drought-mauceu llrotem) was observed in chloroplasts of Solanum tuberosum plants subjected to a sev.ere drought stress (leaf relative water content lower than 70%). Western blot analysis using a serum raised against the ~ p 32 N-te.~'n~,, region ~.owed that, within the chlor~op.last, the cdp 32 protein was spc~ncauy .recap. m me stroma. The accumulation of cdp 32 was oemonstratea .to originate trom an active synthesis upon severe drought conditions. Syntbe=s of cdp 32 was also induced by high salinity, but not by low temperature and exogenous a.b.scisic acid. The cdp 32 protein was shown to be nuclear encoded. Rewatering ot severely wilted plants caused decreases in the synthesis and in the oa~ount ~f edp 32 down to the control levels. While the N-terminal seg.uence" o cap .~ showed no evident homology with known sequences, the amino aod sequence (30 residues) of a tryptie-digest peptide displayed 83% identity with a region of a salmonid growth hDrmone protein involved in osmoregulation during seawater adaptation. Based on this homology, the cdp 32 protein is pro.~s~i to play a role in the response to the increased osmolarity occurring m me stroma during water deficit or high salinity.

P-23-017

GAS EXCHANGE OF WATER-STRESSED WHEAT LEAVES AND LEAVES WITH SIMULATED PATCHY PHOTOSYNTHESIS

CHARACTERIZATION OF A NOVEL THYLAKOID DROUGHTINDUCED PROTEIN IN SOLANUM TUBEROSUM.

Biota Martin and Chdetel Rilllng, Department of Agronomy, Oklahoma State University, Stlllwatar, OK 74078, U.S.A.

G. Pruvot, S. Cuint, G. Peltier & P. Rev CEA, DPVE, Laboratoire d'Ecophysiologie de la Photosynt~se, Centre d Etudes de Cadaracbe, F-13108 SaJnt-Paul-lez-Durance, France.

It has bean reported that stomata close in patches when plants are exposed to drought stress. This causes a problem in calculating stomatal and nonstomatal Ilmitetlons to photosynthesis, because current methods depend on computation of the Intercellular CO= concentration (C~)that will be In error If the etomatal conductance (g,) Is distributed In a patchy manner. We simulated Increasing photosynthetic patchiness by applying stdpa of increasing widths of tape to the upper or both surfaces of wheat (Triticum eestivum L) leaves. Transparent tape impedes gas diffusion and was used to simulate stomatal patchlnsss with g, in the patches reduced to about 50% or 0% of untreated control leaves. A combination of patchy g, and mesophyil photosynthesis was simulated with opaque tape that also blocks out light under taped patches. Photosynthesis rate at ambient CO2 (Ax~o)and saturetlng COz (A==), C~, intdnelc leaf water-use efficiency (A~/g,), stomatal limitation, and other gas exchange properties of leaves of wheat exposed to different levels of water deficit stress wore compared with the same properties of artificially patchy leaves. From the dependencies of these pmpectlss on g= we conclude that water-stressed wheat leaves differed from leaves with the types of patchy phototynthesls we tested. The data show that A~0/g, first Increased when g, decreased at mild to moderate water stress, and confirm an eadler prediction that, upon further reduction of g, at severe water stress, A~/g, again deteriorated.

202

From analysis of two-dimensional patterns of ~ylakoid prote.in.s, a 34-kDa protein (named cdp 34 for chi.oroplastic drough.t-mdu.c~d ]zrot=n) was sno.wn to accumulate in $olamon tuoerosum ptants subjected to a progresstve water deficit. Sequences of the N-terminal extremity and of four tryptic-digest peptides did not reveal significant homology b e t w ~ the edp. 34 pro.tern an~ known proteins. In v/co labeling e x p , . ments ~ v ~ e n an acuve, synme~s. cap 34 from earl]/stages of the drought stress ano mrougn.out ~ e conswam. The cdp 34 protein was demonstrated to be nuclear enc~en. AHer rewatermg of severely water-stressed plants, the ~ s t e n c e ofonoticeab~ .edp 34 ..amoun~ was observed at least for two wengs, tmmanomot analysis : t chloroplastic subfractions, using a serum raised agmnst the cdp 3 N. , region, showed that the edp 34 protein was an extrinsic protein premrenuauy located in unstacked stroma thylakoids. By another way,.~e cdp 34 synthesis was induced in response to low temperature, high salinity .ariaexogenous abseisic acid. Based on its characteristics,the cdp 34 protein is suggested .to play a role in the stabilizationof photosynthetic membranes dunng osmotic stress.

Poster P-23-018 ~-CAROTENE SENESCENCE

PROTECTS DI PROTEIN AND WATER STRESS

OF THYLAKOID

DURING

B.Biswal & P.M.Deo S c h o o l of Life S c i e n c e s , S a m b a l p u r U n i v e r s i t y , Jyotivihar-768019,0rissa,India

BETAI~ EHI4~qlICgS S A L T TOLI~RAIM2E IN TI~qNSGEHIC CYANOBACTRRIUP~

M. Nc~Aura & Tetsuko Takabe BioScience Center, Nagoya University, Japan

C h a n g e s in the c o m p o s i t i o n of c a r o t e n o i d s and s t a b i lity of DI p r o t e i n of t h y l a k o i d s are e x a m i n e d in m a t u r e and s e n e s c i n g c o t y l e d o n s of c l u s t e r b e a n u n d e r w a t e r stress. S e n e s c e n c e b r i n g s a b o u t loss of c h l o r o phyll and a d e c l i n e in o x y g e n e v o l u t i o n w i t h o u t any loss e i t h e r in ~ - c a r o t e n e or DI p r o t e i n as p r o b e d by immunoblotting.~-carotene associated with reaction c e n t r e m a y t h e r e f o r e , c o n t r i b u t e to DI s t a b i l i t y d u r i n g s e n e s c e n c e . W h e t h e r m a t u r e or s e n e s c e n t c o t y l e dons,water stress treatment causes a quantitative loss of DI p r o t e i n w h i c h is c o r r e l a t e d to a d e c l i n e in ~ - c a r o t e n e l e v e l . T h e s t r e s s i n d u c e d loss in DI p r o tein is a r r e s t e d if c o t y l e d o n s are e x p o s e d to w e a k l i g h t that r e s u l t s in an e n h a n c e m e n t in the level of B - c a r o t e n e e v e n d u r i n g s e n e s c e n c e . T h e d a t a on the c h a n g e s in the c o m p o n e n t s of x a n t h o p h y l l c y c l e d o n o r s u g g e s t o p e r a t i o n of the c y c l e e i t h e r d u r i n g senes c e n c e or stress.

P-23-019 COz ASSIMILATION, STOMATAL CONDUCTANCE, TRANSPIRATION AND WATER USE EFFICIENCY IN TWO MULBERRY CDLTIYARS AS AFFECTED BY WATER STRESS

OF

RUBI~O

Cnikusa Nagoya 464-01

Glycine betaine is known to be a compatible solute to keep osmotic balance, and also act as an osmoprotectant in a wide range of organisms including bacteria, higher plants and animals. We introduced the E. cell bet genes encoding choline-glycine betaine pathway into the fresh water cyanobacteri~ Synechococcua sp. PCC 7942 and showed that bet containing cells produce glycine betaine from choline and acquire enhanced resistance to salt stress as reported previously (Nomura et al., Plant Physiol. 107:703-708, 1995) It was also found that glycine betaine stabilizes RuBisCO protein, whose level decreases relatively faster than other photosynthetic proteins under salt stress. We analysed the effect of glycine betaine accumulation on photosynthesis components under salt stress in details.

P-23-022 THE EFFECT OF WATER STRESS ON PHOTOSYNTHETIC CHARACTERISTICS AND SUGAR ACCUMULATION OF FIELD GROWN SWEET SORGHUM A. Masaacd I M.A. lalmelli', F. PieJadni', A. BatliateJli 2, S. MDscatello 2 & F. Lorato1 IC.NR.- Istituto di Bioehimiea ed Ecofisiologla Vegetali, via Salaria Ion 29, 00016 Monterotondo Scale. Italy; 2C.N.P,_ - ]stintto per l'Agroselvicoltura. Porano, I~aly

S. Ramanjulu & C. Sudhakar Department of Botany, Sri Krishnadevaraya University, Anantapur - 515 003, India. Two mulberry (Morus alba L.) cultivars, S-54 (drought sensitive) and S-13d--~ug-ht-tolerant) were subjected to different regimes of water stress by withholding the water. Exposure of plants to water stress for 5, ]0 and ]5-day intervals has caused a decrease in net photosynthesis (PN ), stomatal conductance (Gs ~ and transpiration (E) in both the c u l t i vats. ~ slight increase in intercellular CO z concentration (Ct ~ was noticed under severe stress conditions though was unaltered under mild and moderate water stress conditions in both cultivars. Involvementof non-stomatal components may be of limitation to P~ , was evident from the Ct levels under water stress conditions. However, the r e l a t i v e magnitude of decrease in these parameters was differing between two cultivars. Drought tolerant nature of S-13, ascertained from r e l a t i v e l y better PN , Gs and water use efficiency with low E during water stress.

P-23-020 ADAPTIVE RESPONSES TO MILD METAL S T U D Y I N SYNECHOCYSTIS PCC 6803

P-23-021 GLYCIRB PROTEIN

S T R E S S : A N IN VIVO

Sweet sorghum, a (2, plant, accumulate photosyndmtes as atcmse in the sam, dining panicle maturation. Photosynthesis of sweet sorghum plants cv. Korall was more tolerant to water slress than that of other ¢vs of sweet and fiber sorghum. Even under very high water stress (ff = -2.0 MPa) Korall leaves maintained a photosynthesis of about 20 lamol m': sq. However, stomatal opening daring ~he day was evident in inigated plants but almost absent in water stressed plants. Photosynthesis of water sU~essed leaves saturated at a lower CO: partial pressure and at a lower rate than that of inigated leaves. Water stress reduced the quantum yield of CO2 asaimllation and, to a bigger extent, the quantttm yield of linear electron transport. These results suggest that a reduced capacity for CO2 fixation may lower photosynthesis in plants grown under limiting water supply. The results also indicate that the electron transport is not perfectly down-regulated when carbon metabolism is inhibited by water stress. After anthesis, water stressed pisnts grew less than centrols. Conte~rarily, sucrose ac~,m,,lafion in the stem took place. The accumulation was anticipated in water stressed leaves but the total content of sucrose at panicle maturatiun was similar in water slTessed and control plants. Sucrose accumulation in the ~aex was lower and stopped earlier than in the other parts of the g e m probably because seed filling in the panicle is a suen8 competing sine In general, ot~ results indicate that in sweet sorghum plants sucrose acenraulation is a sink of photosynthates stronger than 8~owth and m m f f e ¢ ~ by water sirens.

P-23-023 WATER STRESS EFFECTS ON PHOTOS~TflESIS NEDITERRANEAN SHRUBS: A FIELD STUDY

ON

Swati Tiwari and Prasanna Mohanty School o f Life Sciences, Jawaharlal N e h m University, N e w Delhi, India.

A. Pastor, S. N o g u ~ s & L, A l e g r e Dept. B i o l o g i a Vegetal, U n i v e r s i t a t de B a r c e l o n a , Av. D i a g o n a l 645, B a r c e l o n a 08028, Spain.

Adaptive responses to a long term mild stress due to the presence o f sublethal levels o f cobalt chloride (10 u M ) in the growth m e d i u m have been studied in Synechocystis PCC 6803. Cobalt-grown cells s h o w e d upto 50 % stimulation in photosystem II (PS II) catalyzed Hill reaction w h i c h w a s not due to an increase in the PS II antenna size ['Fiwari and Mohanty (1993) Photosynth. Res. 38 : 463469]. T h e rate o f P S I supported D C P I P H 2 to M V electron transport showed about 20 % increase in cobalt-grown cells at saturating light intensifies. The functional antenna size o f PS I, as measured by the rate o f photooxidation of P700 in limiting light (25 ~Em--s" " I), showed about 25 % decrease in cobaltg r o w n cells. T h e r e was, however, no c h a n g e in the steady state levels o f psa A/B polypeptides as determined by immunoblotting with antibodies against psa A/B polypeptides. These findings suggest that there was no change in P S I content and PS II m a y be stimulated in cobalt-grown cells to o v e r c o m e the functional limitation at PS I. due to a smaller antenna, ,,..,hen cells are g r o w n under low light intensities, as in the ',resent case.

D i u r n a l c y c l e s of gas e x c h a n g e w e r e s t u d i e d in four autochthonous Mediterranean evergreen shrubs well a d a p t e d to s u r v i v e u n d e r c o n d i t i o n s of w a t e r stress. T h e s p e c i e s s t u d i e d d i f f e r e d in s c h l e r o p h y l l o u s leaf consistency: Rosmarinus officinalis has semis c l e r o p h y l l o u s l e a v e s a n d C i s t u s clusii, L a v a n d u l a stoechas and Phagnalon saxatile have less s c h l e r o p h y l l o u s leaves. T h e p o s s i b l e p l a n t b e h a v i o r of d r o u g h t a v o i d a n c e or t o l e r a n c e a n d w a t e r r e l a t i o n s w e r e o b s e r v e d at m o n t h l y intervals, f r o m J u n e to September, e i t h e r in w e l l w a t e r e d a n d or p l a n t s g r o w i n g u n d e r d e c l i n i n g soil w a t e r content. P r e v i o u s r e s u l t s s h o w e d a m i d d a y d e c l i n e d e p r e s s i o n in net C02 u p t a k e in w e l l w a t e r e d p l a n t s - v a l u e s of PN at 12 h 35% l o w e r t h a n at 8 h - in L. stoechas, lesser depresion was observed in the m o r e xeromorphic,

203

Poster

P-23-024 E F F E C T S O F Z I N C D E F I C I E N C Y ON L E A F P H O T O S Y N T H E S I S M. EvletterS1, V. Lomhaert 2, H. Bermis Taleb ] & R. Lannoye t ~ULB, Labo. de physiologic v6g6tale, Av. P.Heger 28CP169, 1050 Bruxelles, Belgium; 2SCPA, Centre de rechercbe, Aspach-Le-Bas 68700 Cemay, France. Zinc deficiency was induced in maize plants by hydroponic method. In this way, a good control of the zinc content o f both the leaf and the nutrient solution was obtained.This technique can be applied to the study of others micronutrient deficiency or toxicity. Our purpose was to detect zinc deficiency in leaves before the manifestation of visible symptoms (shortening ofinternodes, drastic decrease in leaf size often combined with chlorotic bands along the midrib on leaves). The early diagnostic is very important for the application of correetive treatment before the irreversible alterations of metabolism inducing yield decrease. At different days of culture, the carbon dioxide uptake was measured and a decrease oftbe net photosynthesis was observed. Proportion of oxidized P700 during the steadystate was determined and increased in deficient plants. The fluorescence parameters have not shown signifiant difference for this intensity of deficiency. Some of these changes can be explained by the zinc function in enzyme reactions ( carbonic anhydrase and dehydrogenase) or in steps in a metabolic pathwav (carbon reduction cycle).

P-23-025 WATER STRESS EFFECTS ON RIBULOSE-1,5BISPHOSPHATE CARBOXYLASE AND THE RELATIONSHIP TO PHOTOSYNTHESIS IN SUNFLOWER LEAVES [VI. Kanechi. E. Kunitomo, N. inagaki and S. Maekawa Fac. of Agric., Kobe University, 1, Rokko-dai, Nada-ku, Kobe, 657, JAPAN Water stressed Helianthus annuus L. plants were monitored to detect the relationships between photosynthesis, chlorophyll, total soluble protein, fibulose-l,5-bisphosphate eafl~xylase (mbisco) and leaf relative water content (RWC). The fight saturated photosynthesis and the quantum yield as a initial slope of photosynthetic light response curve decreased with decreasing leaf RWC. Rubisco activities (initial and total) and chlorophyll content were affected little by water stress. Total soluble protein and mbisco declined as soon as the leaf RWC was reduced below 75%. The dehydrated leaves showed a clear correlation between the light saturated photosynthesis and mbisco amount, indicating that decreased rubisco might have played an important role of non-stomatal contribution to the reduction in photosynthesis due to water stress.

P-23-027 PHYSIOLOGICAL ADAPTATION IN CELL DEHYDRATING AND REWETTING PROCESS OF A NEWLYISOLATEDCHLORELLASP. STRAINDT FROM TAIWAN Pei-Chung Chen and Ching-Long Lal, Department of Botany, National Chung Hsing University, Taichung, Talwan, ROC A new terrestrial species of green alga (Chlorella sp. strain DT) which could survive under extremely drought condition was isolated from an arid area of Talwan mountains. The water content of the alga natural cells dropped to less than 3 % after 3 hours' dehydration at 42°C. The dried ceils could regrow if they are rewetting again. The photosynthetic activity of the ceils ceased following 2 hours of dehydration, but respiratory activity was still detectable after 3 h desiccation. During the rewetting process, respiration increased immediately and then decreased to a steady level after five days of rewetting, whereas matching the net photosynthetic oxygen evolution. The cells had 37.6 % neoxunthin (1520 nmol/g dry vet.), 39.2 % hitein (1580 nmol/g dry wt.), and 13.8 % violaxanthin (575 nmol/g dry wt) of total carotenoids, but only 5 1 % 13-carotene(205 nmol/g dry wt.), 3.8 % s-carotene (152 nmol/g dry wt.) and trace amount of antheraxanthin and zeaxanthin. Upon dehydration, zeaxanthin and carotene contents in the cells increased and they would recover to the normal contents after 8 days of rewetting. The photoprotective xanthophyll cycle was found existing in these ceils. It appears that the energy dissipation process for preventing photo-damage is perhaps one of the dry-tolerant mechanism in this Chlorellastrain.

P-23-028 P h o t o s y n t h e t i c A d a p t a t i o n for Salt in the H a l o t o l e r a n t Cyanobacterium Aphanothece halophytica H.Ishikawa, T. Hibino & T. T a k a b e Dept. of C h e m . Fac. of Sci. & Technol., Meijo U n i v . , T e n p a k u - k u , Nagoya 468, J a p a n Aphanothece halophytica is a highly halotolerant cyanobacterium that can grow at high external NaCI concentrations up to 3 M concomitant with the a c c u m u l a t i o n of glycinebetaine. C h a n g e s of p h o t o s y s t e m stoiehiometry and photosynthetic activities of Aphanothece halophytica cells grown u n d e r various salt conditions were examined. It was found that the levels of Chl a and PSI increased in the cells grown with increasing of external salinity, whereas the contents of Cyt b6/f and PSII w e r e a l m o s t c o n s t a n t s and the p h y c o b i l i p r o t e i n s d e c r e a s e d . T h e f l u o r e s c e n c e e m i s s i o n spectra and photoacoustic m e a s u r e m e n t s also s h o w e d the a c t i v a t i o n of P S I - m e d i a t e d electron flow u n d e r high salt conditions.

P-23-029

P-23-026 CHARACTERIZATION OF OEC23 POLYPEPTIDE OF NACL ADAPTED PHOTOAUTOTROPHIC CULTURED CELLS OF TOBACCO

TORGOR-RELhTED SUNFLOWER.

LF,~F N g ~ S

AND

RADIATIO~I

USE

EFFICIENCY

IN

M a r c e l l c Guiducci and Paolo Benincasa.

F.Sato, Y.Kanda, M.Shiga, Y.Oshita, K.Murota 1 and Y.Yarnada, Dep. Agric.Chem., Kyoto Univ., Kyoto 606-01, 1Dep. Agric. Chem., Tokyo Univ. Agric., Tokyo 156, Japan A line of cultured tobacco cells (Nicotiana tabacum cv.Samsun NN) was established that was able to grow photoautotrophically in a medium that contained 0.2M NaC1 (K.Murota et al., 1994, Plant Cell Physiol., 35:107 - 113). Thylakoid membranes of the NaCl-adapted ceils had higher oxygenevolving activities, on the basis of chlorophyll, than those of unadapted cells. Furthermore, the oxygen-evolving activities of thylakoid membranes from NaC1adapted cells were more tolerant to high concentrations of NaC1 than those from unadapted ceils. Examination of the dissociation of extrinsic 23-kDa polypeptide (OEC23) of photosystem II at high concen~ations of NaCI indicated that the affinity with which the OEC23 was bound to thylakoid membranes of saltadapted ceils had been altered. OEC23 cDNAs were cloned from NaCl-adapted cells and expressed in E. coil Re,constitution experiment to examine the function of OEC23 polypeptide is going.

204

Istituto di Agronomia, Universit& di Perugia, Borgo XX giugno, 74, 06100 Perugia, ITALY. Plant water status g r e a t l y affects leaf posture in sunflower. This d e t e r m i n e s variations of light e n v i r o n m e n t inside the canopy, w i t h obvious influences on crop photosynthesis and radiation use efficiency. Intensity of transmitted, reflected and absorbed PAR, leaf angles distribution, and diurnal variations of leaf CO 2 a s s i m i l a t i o n rate at high p h o t o n flux densities were m e a s u r e d in a field experiment. A model was i n t r o d u c e d which attributes a relevant role to the t u r g o r - r e l a t e d movements of leaves on crop a d a p t a t i o n to water deficit. Because of t u r g o r - r e l a t e d leaf movements, light intensity upon leaf surfaces changed according to the actual leaf p h o t o s y n t h e t i c capacity. As a result, radiation use efficiency was quite insensitive to water availability. Further, under severe water stress conditions, leaf wilting c o n t r i b u t e d to g r e a t l y reduce the e n e r g y load on leaf surfaces, avoiding the chance of irreversible damages of p h o t o s y n t h e t i c apparatus.

Poster P-23-033

P-23-030 THE LOSS OF RuBPCase BY 24 h TREATMENT OF "RAIN" IN LIGHT M.Ishibash 1, I. Terashima2, H. Usuda3, K. SonoikeI & A. Watanabe 1 l-D--¢partment of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Hongo, Tokyo, 113 Japan; 2Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305 Japan; 3Laboratory of Chemistry, Faculty of Medicine, Teikyo University, Hachioji, 192-03 Japan. Chronic decrease of the capacity of photosynthesis in the primary leaves of Phaseolus vulgaris is induced by the treatment of simulated rainfall. By such a short treatment period as 24 hrs, the photosynthetic rate decreased to 55% of the original rate ( Ishibashi & Terashima, 1995, Plant Cell & Environment, 18). Through analyses of this newly found phenomena, we found that this decline is caused mainly by the decrease in the/n situ activity of RuBPCase. To our surprise, it was due to the decrease in the AMOUNT of the enzyme, and NOT due to the decline in the activation state of the enzyme as expected. The amount of RuBPCas¢ decreased even down to 54% and the photosynthetic rates at the ambient CO 2 concentration of the leaves before and after the "rain" showed a linear relationship with the amount of the enzyme, confirming the above results. Another inhibition site concerned with the decline of photosynthetic rate at saturation is expected to be present independent from the prescribed loss of RuBPCase. The mechanisms responsible for the inhibition of photosynthesis induced by "rain" and the condition in which the inhibition was induced will be discussed in detail.

PHOTOSYNTHESIS BY LITHOPHYTIC AND EPIPHYTIC PLATYCERIUM FERNS UNDER HIGH LIGHT AND WATER STRESS J.A.M. Holtum !, L.E. Keto 1, S. Robinson 2 & J.T. Christopher 1. ~James Cook Univ. Nth. QId., Townsville, Queensland 4811; 2R.S.B.S., Aust. National Univ., Canberra, A.C.T. 2600, Australia. The performance of t w o tropical Australian Platycerium ferns, a monsoon rainforest epiphyte (P. bifurcatum ssp. bifurcatum) and a dryland Iithophyte (P. b. ssp. veitchit), was compared in the field and in a glasshouse. In the field, the ferns grew in shade interspersed with periods of high light (~2,300 ~tmol phot. m -2 s l ) . In the glasshouse, well-watered ferns exhibited no reduced photosynthetic efficiency (Fv/Fm) after exposure to full sunlight for 35 d. Similarly, under 80% shade neither exhibited reduced efficiency after 35 d of drought. However, both subspecies exposed to full sunlight and drought for 35 d exhibited reduced photosynthetic efficiency. The reduction was more pronounced in ssp. bifurcatum than for ssp. veitchfi. This differential reduction in efficiency may be explained by differences in leaf reflectance, stomatal density and morphology, leaf angle and levels of protective pigments.

P-23-034

P-23-031 Cd/Fe I N T E R A C T I O N A N D ITS E F F E C T S O N P H O T O S Y N T H E T I C CAPACITY OF PRIMARY BEAN LEAVES

S A L I N I T Y T O L E R A N C E O F ARABIDOPSIS THALIANA TRANSFORMED WITH CHOLINE OXIDASE GENE

Z. Kruoa & A. Siedlecka Deparlmentof Plant Physiology, Instituteof Biology, Maria Curie--Sk;odowskaUniversity, Akademicka 19, 20-033 Lublin, Poland

H. Hayashi 1, P. Deshunium 2, M. Ida2and N. Murata 2 1Department of Chemistry, Ehime University, Mustuyama, Japan 2National Institute for Basic Biology, Oakazaki Japan

Cd/Fe interaction, induced by changing the content of both metals in the nutrient medium, was investigated in Phaseolus vulgaris L. seedlings.. Under Fe deficiency the toxic effects of Cd on plastid pigment levels, PSII and PSI activities, CO2-dependent O2 evolution and fluorescence induction parameters were generally much more pronounced than under n o r mal Fe supply. At 2 and 4 normal doses Fe decreased Cd accumulation in primary leaves and chloroplasts, preventing photosynthetic apparatus from Cd toxicity. However, at the highest dose Fe itself limited photosynthetic capacity. Our results also show that in relatively young plants the inhibition of photosynthetic electron transport by heavy metals is of an indirect nature and results from inhibited Calvin cycle. Accumulated excess of ATP and NADPH and high ApH sustained across thylakoid membranes may reversely influence the efficiency of light reactions.

Under high salt conditions some plants produce osmoprotectants and enhance their salt resistance. Glycinebetaine, a quaternary ammonium compound, is the osmoprotectant found in halotolerant plants and bacteria. We discovered that this compound protects the photosystem II complex from inactivation under high-salt conditions. To understand the in vivo effect of glycinebetaine on salinity tolerance, we transformed Arabidopsis thaliana with the codA gene for choline oxidase of Arthrobacter globiformis, which can produce glycinebetaine from choline. We examined the salt tolerance of the transformants at different stages of growth and found that they could germinate and grow in the medium containing 80 mM NaCI at higher rate than the wild type.

P-23-035

P-23-032 Effects of the water stress on the biochemisby and physiology of photssynthesis In sunflower. Tezara W. Habash D. Paul M.J. & Lavdor D.W. IACR-Rothamsted,Harpenden, Hertfordshire AL5 2JQ UK. Photosynthetic rate (Pn) of intact leaves of one month old sunflower (Helianthus annuus L.) was measured by gas exchange with slowly increasing water deficit caused by withholding irrigation, The aim was to establish the relative importance of stomatal and metabolic regulation in the reduction of Pn and in particular ribulose bisphosphate (RuBP) regeneration. The ~bPSii and coefficients of photochemical (qp) and nonphotochemical (qN) quenching were determined from chlorophyll fluorescence. Leaves were sampled by freeze clamping; RuBP, and the amounts of Rubisco and tota~soluble protein (tap) measured. Pnmax in air decreased from 27 to 0.5 tJmol m-2 s-" as ~P declined from -0.3 to -2.7 MPa, carboxylationefficiency decreasedby 95% and stomatal conductance (g) by 98%. C decreasedwith decreasing 'Y to -1.7 MPa but increased with further stress. The amount of tsp ncreasedwth decreasng H~, but the pmporton of Rubisco was unaffected, initial and total activity of Rubisco remained constant during moderate stress, but below -1.7 MPa decreased. RuBP concentration decreased progressively with increased stress. There was a strong positive correlation between a~eSiI and the apparent quantum yield of photosynthesis (4)CO2). 4)CO2 was-more cted by moderate stress than ~PSII' which decreased only at very severe stress, qp followed ~PS ' but qN increased progressively. Reduction of g with moderatestress may limit Pn because Ci and RuBP decrease. However,with ~P < -1.7 MPa, Ci increases but RuBP remains low. 14CO2 feeding showed a homogeneousreduction in uptakes,but no indication of stomatal patchiness, nor did application of CO2 at 2700 IJmolmol'" cause recovery of Pn, indicating non-stomatallimitation at severestress.

AND PHOTOSYNTHESIS IN AN ABA-UNSENSITIVE POPLAR CLONE : LACK OF STOMATAL CLOSURE AND INDUCTION OF SEVERE XYLEM EMBOLISATION WATER

STRESS

M. Ridolfi. H. Cochard and E. Dreyer Unit6 d'EcophysiologieForesti~re,INRANancy,F 54280 Champenoux,France Poplar curttings from the CV Peace (ABA unsensitive) and Robusta have been submitted to drought, and time courses of leaf water potential, stomatal closure, net CO2 assimilation and photochemical efficiency of PS II have been recorded, together with the onset of embolism in the xylem. In Robusta, stomatal closure occurred at moderate stress levels, resulting in rapid decreases in photosynthesis. In Peace, only a limited stomatal closure was recorded, and as a consequence, an important embolisation developed in the petioles at still high leaf water potentials, resulting in rapid leaf shedding. The youngest, ABA sensitive leaves survived alone, with closed stomata, till very severe stress intensifies. Vertical gradients of vulnerabilityto cavitation, sensitivity of stomata to diverse stimuli have been described, and related to the ability of the leaves to withstand drought. The relative role of stomatal vs mesophyll resistances to CO2 influx into chloroplasts has been analysed through indirect estimates of chloroplastic concentrations in CO2, and with I3C isotope discrimination in leaves. The consequences of this behaviour for stress tolerance will be discussed.

205

Poster P-23-036

TRANSFORMATION OF Synechococcua ap. PCC 7942 WITH T H E G E N E FOR C H O L I N E O X I D A S E E N H A N C E S T O L E R A N C E TO SALT STRESS

P. Deshnium. D.A. Los, H. Hayashi and N. Murata Department of Regulation Biology, National Institute for Basic Biology and Department of Molecular Biomechanics, The graduate University for Advances Studies, Myodaiji, Okazaki, 444, Japan Choline oxidase, which is isolated from the soil bacterium, Arthrobacter globiformis, converts choline to glycinebetaine (Ntrimethyl glycine) via a one-step reaction. We cloned the coda gene for this enzyme and introduced it into the cyanobacterium, Synechococcus sp. PCC 7942. The codA gene was expressed under the control of a strong constitutive promoter, and the transformed cells accumulated glycinebetaine at 80 mM. Consequently the cells acquired tolerance to salt stress, as evaluated by growth, chlorophyll accumulation and photosynthetic oxygen evolution.

P-23-039 SHORT AND LONG TERM ADAPTATION OF TOBACCO AND POTATO PLANTS TO WATER STRESS D. B0ssis, F. Kauder & D. Heineke Institut for Biochemie der Pflanze, Untere KarspQle 2, D-37073 G6ttingen, Germany For the examination of short and long term adaptation of intact plants to mild and severe water stress, tobacco and potato plants were grown hydroponically. After four weeks of growth different concentrations of PEG 6000 were added to one set of plants to induce water stress. In all stressed plants the photosynthesis rate was reduced accompanied by a reduction of stomatal conductance. The adaptation was biochemically characterised by measuring the contents of carbohydrates, malate, amino acids and abscisic acid (ABA). After induction of severe water stress the apoplastic ABA and malate concentrations increased rapidly. During the adaptation to mild water stress changes in the apoplastic concentrations of ABA were only transient, but in whole leaves the contents of ABA and amino acids (mainly proline) increased steadily. Mechanisms of the inhibition of photosynthesis are discussed.

P-23-040

P-23-037 STOMATAL CHARACTERISTICS AND CARBOHYDRATE CONTENTS IN FOUR XEROPHYTIC SPECIES FROM VENEZUELA FOLLOWING EXPOSURE TO ELEVATEDCO2 C. Donoso,A. Pleters, M. D. Fembndez M. Azkue & A. Herrera. Centre de Bot~nica Tropical, Universidad Central de Venezuela. Aptdo. 47577, Caracas 1041-A Venezuela Plants of four tropical xerophytes(Jatmoha aosvioifolia. C3; Ioomoeacamea. C3; Alternantheracrucis, C4, and Talinum trianaulaml inducible-CAM)were grown for 46 weeks duringthe rainyseasonin two open-topchambers,one at 560_+40ppm, and the other one at ambient CO2, and in a control plot. Sampleswere taken on week 40. Adaxialand abaxialstomatalindexwere unaffectedby CO2 concentrationin all species but Ipomoea (abaxialface). The response of stomatal density and pore length to elevated CO2 was not uniform among species; in some cases a significant chamber effect was found. The responseof epidermalcell densityto elevatedCO2 was speciesspecific and occurredin the same direction (increaseor decrease)in both leaf faces. No definedtendencywas detectedin eithersoluble carbohydrateor starch contents among treatments. No evidence was found for lack of starch mobilizationdue to elevatedCO2. Partlyfinancedby projectsEC CTIl*-CT91-0896(in cooperationwith F.I. Woodward,U. Sheffield) and CONICITRPVII-270078(Venezuela).

P-23-038

E F F E C T OF D I F F E R E N T C O N C I ~ f R A T I O N S OF A T R A Z ~ N E S Y N T H E S I S A N D C E L L D I V I S I O N IN A N A C Y S T I S G R O W N LIGHT.

ON D1 PROTEIN IN STRONG WHITE

F. K o e n i q B o t a n i s c h e s Institut, J.W. Goethe-Universit~t, Siesmayerstr. 70 D-60323 Frankfurt am Main, G e r m a n y As has b e e n p u b l i s h e d (Koenig, 1990, Photosynth. Res. 26, 2937) g r o w t h of A n a c v s t i s in the presence of certain sublethal c o n c e n t r a t i o n s (conc.) of atrazine (atr.) leads to r e d u c e d D1 p r o t e i n synthesis (synth.) per cell. A careful r e i n v e s t i g a t i o n revealed a d e p e n d e n c e on atr. cone. in form of an o p t i m u m curve. Unexpectedly, D1 p r o t e i n synthesis first increases with increasing atr. conc. to an o p t i m u m at 8-10 -7 M and o n l y thereas exafter d e c r e a s e s with further increasing inhibitor c o n c . pected. Cell d e n s i t y p l o t t e d against atr. conc. d u r i n g growth of A n ~ c y s ~ i s in strong light yields a v e r y similar curve: Cell d e n s i t y increases from 10 -8 M to an o p t i m u m around 8-10 -7 M and then d e c r e a s e s w i t h further increasing atr. in the medium. Relative to total 35S i n c o r p o r a t i o n per cell D1 p r o t e i n synth. c o n t i n u o u s l y increases with increasing inhibitor conc.: While cells kept in the presence of 10 -8 M air. invested only 11% in the synth, of the D1 protein, in the presence of 10 -5 M atr. 27% of the incorporated 35S was detected in the D1 protein.

P-23-041 PHOTO-

S P I N A C H PLANTS A D A P T TO SALT S T R E S S BY S T E P P I N G UP THE PHOTOSYNTHETIC SUPPLY OF R E D U C T A N T S .

SITES OF ACTION OF COPPER IN THE SYNTHETIC APPARATUS OF MAIZE LEAVES

George C. P a p a g ~ j and Norio Murata 2 'lnstititute of Biology, National Research Center Demokritos, Athens, Greece 153 10, and 2National Institute of Basic Biology, Okazaki, Japan 444

M. Moustakas °, G. Ouzounidou°, R. J. Slrasser 2). 1) Dept. of Botany2, Aristotle University of Thessaloniki, 54006 Thessaloniki, Greece. ) Lab. of Bioenergetics University of Geneva, CH-1254 Jussy-Geneva, Switzerland

Spinacea oleracea plants were grown hydroponically at controlled levels of salinization and were compared to similarly grown nonsalinized plants. Plants adapted to 120 mM NaCI were morphologially similar and yielded the same amount of thylakoids per unit fresh weight as unsalinized conntrol plants. The leaf tissue glycine betaine escaped during thylakoid isolation. Isolated thylakoids consisted of properly sealed membrane vesicles. Thylakoids from salinized spinach were 30-50 % more active in photosynthetic 02 evolution and more tolerant of alkaline pH (pH 8.4) than thylakoids from nonsalinized spinach. These results suggest an adaptive increase of photosynthetic efficiency which allows the plant to meet increased demands for photosynthetic reductants for glycine betaine biosynthesis during salinity stress.

206

Maize plants were grown on media containing 0 or 8.0 or 80 copper. The first visual effect is that copper reduces the chlorophyll content of leaves. In terms of photosynthelic capacities such as maximal or actual quantum yield for the primary photochemistry, the rate constant for photochemical trapping or the rate constants for nonphotochemical de-excitations in dark-adapted leaves was not influenced by the copper. If the leaves were exposed to continuous light, which allows the state Wansifion fi'om state 1 to state 2, a high copper dependency appears. Copper prevents the adaptation process from a dark-adaptate~cl to a light adaptated state with the consequences that all photosynthetic activity criteria become more inefficient in the presence of copper.

Poster

P-23-045

P-23-042 S C R E E N I N G C R I T E R I A F O R D R O U G H T T O L E R A N C E IN Nicotiana tabacum L D E R I V E D F R O M T H E P O L Y P H A S I C RISE O F T H E C H L O R O P H Y L L • F L U O R E S C E N C E T R A N S I E N T (O-J-I-P). Eaaenbero. P.*,Van Rensburg, L.**,KrOger, G.H.J. ~ and Strasser, R.J.* * University of Geneva, Bioenergetics Laboratory, CH-1254 JUSSY ** Potchefstroom University, Dept. of Plant and Soil Sciences, 2520-POTCHEFSTROOM, South Africa The use of chlorophyll a fluorescence from intact plant leaves has become a popular and unique nonintrusive method for monitoring photosynthetic events and judging the physiological state of the plant. In the present investigation the drought-stress induced changes in the fast phase fluorescence induction kinetics are measured, and indices derived thereof are used to compare four tobacco cultivars of different, but known drought tolerance. Results as presented here may serve as a calibration method for the in vivo screening of the drought tolerance of different cultivars by fluorescence techniques.

SOME CHARACTERIZATION OF GRADUALLY INDUCED BY Fe-DEFIClENCY

S.Q. Lin, X.G. Wen, S.Y. Li, C.D. Chang, C.Q. Tang & T.Y. Kuang Photosynthesis Lab. Institute of Botany, Academia Sinica, 100044 Beijing, P.R. China Gradually inactive-PS II was found in increasing Fe-deficientthylakiod membranes in pea leaves from bottom to top attached to the plants cultivated in the Fe-free Hoagland medium. Although the charge seperation of PS II had already existed in the most upper leaves, the photosynthetic electron transport was almost blocked and the proton gradient could not be established evidenced from almost lack of fluorescence quenching. An inhibition site was assumed to be at Qa-Qb. Only a small amount of LHC n had been synthesized forming a small size of PS II antenna in the upper leaves but it could not migrate induced by Mg 2+. The Mg2+-induced heterogeneity of PS II was examined in different Fedeficient samples (Lin: in Res. in Photosyn. II, 769-772, 1992). The characterization of the inactive PS II induced by Fe-defioiency was discussed.

P-23-046

P-23-043 LIMITATION TO CARBON ASSIMILATION OF SUN AND SHADE LEAVES IN HOT AND DRY ENVIRONMENTS. T. Faria 1, J. I. Garcia-Plazaola 1, R. Besford2, D. Willkins2, M.M. Chaves and J.S. Pereira 1 (I) Instituto Superior de Agronomia, Lisbon, Portugal, (2) Horticulture Research Institute, Litleharnpton, England. We studied the limitation to photosynthesis of sun and shade leaves of adult Quercus suber trees. Measurements were done in a hot, dry and sunny day and in a mild cloudy day. The trees were severely water stressed (predawn leaf water potential -17.2±2.41MPa). Shade leaves had lower photosynthetic capacity and lower Rubisco activity than sun leaves. In sun leaves photosynthetic capacity, net photosynthesis and stomatal conductance, showed a midday depression in both days. Maximal photochemical efficiency showed only some decrease in sun leaves at midday in the sunny day. Shade leaves, showed a midday depression on net photosynthesis and almost no diurnal variation in photosynthetic capacity, O2 quantum yield and stomatal conductance. In conclusion, stomatal and non-stomatal limitations occur at midday in sun leaves, whereas the negative carbon balance of shade leaves at midday may result from higher respiration rates due to high temperatures.

CHLOROPHYLL FLUORESCENCE UNDER Cu STRESS IN TWO CVS. OF Tritlcum durum Desf. WITH OIFFERENT DROUGHT SENSITIVITY. CiSCato1, R. Valcke2, K. Van Loven2, H. Clijsters2, F. Navad-lzzo I . himica Agrada, Universit& di Pisa, via S. Michele degli Scalzi 2, 56124 Pisa, Italy; 2Limburgs Universitair Centrum, Dept. SBG, Universitaire Campus, B3590 Diepenbeek, Belgium. In order to investigate the effect of Cu on the photosynthetic apparatus, vanous chlorophyll a fluorescence techniques have been applied on ten days old Cu stressed seedlings of two cvs. of 7". durum, grown in nutritive solution or in nutritive solution added with CuSO4 to achieve final concentrations of 3.57 pM and 20.3 pM Cu. Fluorescence induction analysis has been performed, in presence or not of DCMU, with a P.E.A. fluorometer (Hansatech, UK); fluorescence quenching analysis has been performed with a P.AM. fiuorometer 0Nalz, D). Besides, LM and TEM morphological investigations have been also carried out on beth roots and shoots. Although the amount of Cu used caused toxic effects on growth, the PSII activity didn't seem to be affected. Nevertheless a slight reduction of the variable fluorescence and a reduced area above the fluorescence induction curve were observed due to the Cu (beth with or without DCMU), indicating a possible effect of Cu at the Q^ level. The effects observed on the fluorescence quenching kinetics suggest a complex regulation of the electron tranport, probably related to an inhibition of the Calvin cycle enzymes; the observed starch accumulation in the chloroplasts, possibly related to the reduced growth, could account for that inhibition.

P-23-047

P-23-044

RESPONSES OF PHOTOSYNTHESIS TO WATER STRESS UNDER FIELD CONDITIONS IN GRAPEVINES ARE DEPENDENT ON IRRADIANCE AND TEMPERATURE Os6rio, ML., Os6rio, J., Pereira, IS. and Chaves, M.M Instituto Superiorde Agronomia, Tapada da Ajuda,1399. Lisboa codex, Portugal.

Gas exchange and PSII activity of leaves with different orientations in the canopy from well-watered (qJpd= -0. lMPa) and water-stressed (q'pd= -057 MPa) grapevines were studied under natural conditions. The measurements were done in a hot, sunny day and a cooler, cloudy day. On the sunny day, an aitemoon depression of net photosynthesis (A) and stomatal conductance (gs) was observed in both weil-watered and water-stressed plants, the inhibition being more pronounced in east facing leaves.- In these leaves mesophyll limitations to A (increased photorespiration?) seem to play an important role in A depression under water deficit. On the cloudy day, the afternoon decline in A and gs was only observed in water-stressed plants. Maximal photochemical efficiency of PSII declined during the sunny day, more drastically in the horizontal leaves of water-stressed plants. Recovery took place overnight in all cases. No diurnal changes on Fv/Fm were observed on the cloudy day.

INACTIVE PS II

STUDY OF THE EFFECTS OF ALUMINUM AND CALCIUM DEFICIENCY ON PHOTOSYNTHESIS. H. Bennis Taleb 1, R. LarmoyeI and J. F. Ledent 2 Univ. Libre de Bruxelles, Labo. de Physiologie V~g~tale, av.Paul H~ger 28 CP 169, 1050 Bruxeiles; 2Univ. Catholique de Louvain, Laho. d'Ecolegie des Grandes Cultures, Pl. Croix du sud, 2 btel 1,1348 Louvaln-La-Neuve, Belgium. The effects of aluminum and calcium deficiency on some chlorophyll fluorescence parameters of maize leaves (Zea moys) were studied. They showed a decrease of quantum yields for photochemistry (~bp).maximal quantum yield for photochemistry in PS2 (4>pop,)and photochemical fluorescence quenching (Qp) on both traltements whereas relative variable fluorescence (V) was found to increase. These physiological effects were correlated with structural alterations. Indeed, the ultrastructure of mesophyll chloroplasts in all traitements was examined by electron microscopy. Aluminum toxicity was characterized by disruption of the chloroplast membranes and by appearance of globules chloroplasts. Calcium deficient plants showed an accumulation of globules and the envelope was sometimes ruptured. The assessment of injury as evaluated by evapotrenspiration, shoot and root fresh and dry weights showed a reduction of all these parameters in both traltements.

207

Poster

P-23-048 T H E R O L E OF S U C R O S E - P H O S P H A T E SYNTHASE AND SUCROSE SYNTHASE THE DESICCATION T O L E R A N C E OF T H E R E S U R R E C T I O N PLANT,

IN

Craterostigma Plantagineum Hochst. J. Inqram I, L. Gallagher I, R. Elster I G. Bianchi 2 & D. Barrels 1 IMax-Planck-Institut f6r Z~chtungsforschung, K61n, FRG; 2University of Pescara, Italy

Craterostigma plantagineum (Scrophulariaceae)

is rare amongst higher plants in being able to withstand extreme protoplastic desiccation. It is probable that synthesis of the compatiblesolute sucrose partly accounts for the maintenance of cellular function. Sucrose-phosphate synthase (SPS) and sucrose synthase (SS) activities increase during drought in C. plantagineum. For SS, one mRNA type is present constitutively, while another is abscisic acid- and drought-inducible. Thermodynamic considerations suggest that SS would be less likely to participate in the sucrose synthesis accompanying desiccation, and it is therefore suggested that the enzyme is in preparation for rehydration. For SPS, two mRNA-transcript types show subtle yet distinct responses to abscisic acid and drought. The regulation of the sucrose synthesis in C. plantagineum probably rests with SPS, and thus the build-up of osmoprotectant sucrose is at least controlled via selective alteration in the expression pattern of that enzyme.

P-23 -049 OXIGEN EVOLUTION BY CHLAMYDOMONASREINHAP.DTIILIVING CELLS IN PRESENCE OF Cu =÷ ~M.J. Vflchez,'I. Garbayo/ C. Vflchez,U.M. Vega 'Departamento de Qufmica y CC.MM. Univ. de Huelva. Spain ='Inst. de Bioqufmica Vegetal y Fotosfntesis. Univ. Sevilla. Spain

Chlamydomonas reinhardtii is a green microalga capable to assimilate and/or accumulate different molecules normaly present in wastewaters, like nitrate, ammonia, S and P-compounds or divalent metal ions (Cu 2÷, Cd2+, Zn=+, etc). One of the main goals in our research is to design a living microalga cell system that should be effective for metal removal from wastewaters. In order to get it, preliminary studies characterizing the cell behaviour in presence of increasing metal concentrations (Cu2+, down to 1 raM) were done. The cell growth was followed and photosynthetic and respiratory activities were determined, as parameters that allowed us to know the state of the cells at the described conditions. By the other hand and to get clear if the cupper accumulation cccured, Cu2+-uptake was measured by atomic absorption. Supported by Grantsfrom University of Huelva, AIQB, and DGICYT PB930735.

P-23-050

P-23-051 SHORT TIME EFFECTS OF A SALT SHOCK ON ENERGY DISTRIBUTION TO THE PHOTOSYSTERS OF SYNECHOCYSTI$SP. PCC 6803

Arne Schoor, Hendrik Schubert & Martin Hagemann FB Biologie, UniversitAt Rostock, Zellphysiologie, 18051Rostock Complete adaptation to high salt results in an enhancement of PSl and respiratory activity in Synechocystis sp. PCC 6803. We have investigated the short time effects of a salt shock of 580 mM NaC] on energy distribution between PSll and PSl as well as on PSII and PSI yield with respect to the interaction between photosynthetic and respiratory electron transport chains (ETC). A dramatic decrease in PSII yield and a parallel increase in PSI efficiency occured immediately after a salt shock. Changes in PSI efficiency reflect decreased donor limitation. Additionally, state transition and inhibitor experiments led us to the conclusion, that changes in the interaction between respiratory andJphotosynthetic ETC as well as redistribution of excitation energy are the most important alterations of photosynthesis of Synechocystis sp. PCC 6803 immediately after a salt shock.

' P-23-052 EARL~ ETIMULATION OF LEAF ACID IEVEETAEE PRECEDEE REDUCTION OF GROWTH AND PHOTOETNTHEEIS IN MAIZE PLANTS SUBMITTED TO MILD WATER ETREBE. GENGTTPE VARIABILITT.

S. Pelleschi, D. Ruelle, J.P. Rocher and J.L. Prioul Inatitut de B i o t e c h n o l o g i e des p l a n t e s . ( U R A CNRS 1128) Universit~ Paris-Sud, 91405 Orsay, France

B&t

630

Water shortage two weeks after seedling, induced w i t h i n 3-4 days a 2-3 times increase of acid invertase correlated w i t h that of hexoses, in the m a t u r e 4th leaf of maize. The m o d i f i c a t i o n of soluble sugars precedes any changes in leaf water status. At the s a m e time, b o t h A D P g l u c o s e - p y r o p h o s p h o r y l a s e and e u c r o s e - P synthase activities were depressed. Then, plant growth, m e a s u r e d by plant height and dry matter, was slowed down. Rates of leaf photosynthesis and transpiration declined progressively in d r o u g h t - p l a n t s (5-6 days). In parallel, atc~atal resistance and leaf or x y l e m A B A content increased. The genetical v a r i a b i l i t y of these responses was further examined, as a preliminary for a genetical analysis of water stress in maize, using the so-called Q T L a p p r o a c h (Quantitative Trait Loci}. Two parental lines (F2 and IO) and 100 of their 6th-generation recombinant inbred lines w e r e c o m p a r e d after a 9-day water-stress. The effects on acid invertase, h e x o s e c o n t e n t and other traits were confirmed. The l a r g e g e n e t i c a l v a r i a b i l i t y a l l o w e d to e s t a b l i s h g e n e t i c a l relationships between the different responses and to map QTLn.

P-23-053

IRRIGATION AND GRAPEVINE PHOTOSYNTHESIS

SALT STRESS INDUCED PROLINE ACCUMULATION PROTECTS THYLAKOID MEMBRANES AGAINST PHOTODAMAGE

E. Delgado, J. Vadell, F. Aguilo, and H. Medrano Institut d'Estudis Avancats de les IUes Balears (CSIC/Universitat Illes Balears). Ctra. Valldemossa Km 7.5. 07071 Palma de Mallorca. Spain.

Ali_....aa', P. Pardha Saradhi ~ & Prasanna Mohanty= ~Centre for Biosciences, Jamia Miliia Islamia, New Delhi-1 l0 025, INDIA, 2Schooi of Life Sciences, Jawaharial Nehm University, New Delhi-1 l0 067, INDIA

Vineyard's for wine making are traditionally cultivated in Spain under non-irrigated conditions, since there are strong regulations for irrigation of this crop. Although grapevine plants are well adapted to the hot and dry mediterranean conditions, irrigation clearly increases yield. However the effects on grape quality and thus wine making are not as clear. The present work belongs to a project where the effects of irrigation on the photosynthetic characteristics, assimilate partitioning, and grape quality are studied. In this communication we show the photosynthetic characteristics of two cultivars of Vitis vinifera L. under moderate irrigation and compared to non-irrigated conditions in two consecutive years. The results sho~ed how under severe drought conditions, where CO2 assimilation is strongly reduced, moderate irrigation improves the mesophyll characteristics for CO2 assimilation and thus carbohydrate production. This also had important effects on grape quality.

208

Thylakoids isolated from seedlings of Brassicojuncea raised under salt stress are more prone to photodynamic damage than those from controls. Peroxidation of thylakoid membranes (monitored in terms of diene conjugate and malondinldehyde levels) during exposure to high light intensity was "3 to 4 fold higher in thylakoids from stressed seedlings as compared to thylakoids from control. As proline level in the cotyledonary leaves of stressed seedlings was "23 fold higher than controls, the role ofproline on lipid peroxidation ofthylakoids was investigated. The presence ofproline (1 M) reduced the production of both diene conjugates as well as malondialdehyde by the thylakoids. The extent of proline induced reduction in these cytotoxic compounds was significantly higher in thylakoids from stressed plants than those from unstressed plants. These results reflect that proline has an adaptive role in protecting thylakoids against photodamage. Further experimets showed that thylakoids from stressed seedlings upon exposure to high light intensity generate significantly higher levels of superoxide and singlet oxygen radicals than those from controls. However, the presence of proline reduced the levels of singlet oxygen radicals, but failed to alter the generation of superoxide radicals. These results support our hypothesis that proline is involved in reducing the light-induced peroxidative damage to thylakoids by lowering the levels of singlet oxygen.

Poster P-24-003 EFFECT OF HIGH TEMPERATURE ON PHOTOSYNTHESIS

Poster

session

Temperature

P-24-001

C. Pastenes 1 & P. Horton2 tFac. Cs. Agrarias. Universidad de Chile, Casilia 1004, Santiago, Chile; 2Robert Hill Inst. University of Sheffield, P.O. Box 594 Sheffield, UK.

24

stress - Virus infection

- P24-039

P-24-001 UV-B EXPOSURE OF LEAVES AT 2 ° AND 22°C: EFFECTS ON Fv/Fm, R E L A X A T I O N OF THE E L E C T R O C H R O M I C SHIFT, AND THE LEVELS OF cab mRNA TRANSCRIPTS.

The effect of high temperature on photosynthesis, from 20 to 35 °C, has been examined in two bean varieties: Barbucho (Chilean) and Blue Lake (commercially available in the UK), characterising the response of electron transport and carbon assimilation in non-photorespiratory conditions. The Q10 for 02 evolution and CO2 assimilation decreased maintaining constant the proportion of the quantum efficiencies of PSII and O2 evolution. The minimal Q10, observed from 30 to 35 °C, coincided with an increase in the proportion of PSII-beta centres, state transitions and the ratio phospboglycerate/triose phosphates, the latter suggesting a limitation in the assimilatory power. No indication of photoinhibition was detected at any temperature. From analysis of adenylate concentration, transthylakoid energisation and, indirectly, NADPH/NADP+ ratio, it was concluded that the assimilatory power was limited due to an oxidation of the NADPH/NADP + pool. Also, in the range 30-35°C, the quantum yield of PSI increased whereas that of PSII maintained its value. It is concluded that the reorganisation of thylakoids observed at 30 to 35°C, increased the excitation of PSI, inducing an increase in cyclic electron transport, and a decrease in the supply of NADPH, seriously limiting carbon assimilation.

P-24-004 EFFECTS OF ELEVATED TEMPERATUR~ ON THE RESPONSES OF SYMBIOTIC DINOFLAGELLATEE

PHOTOSYNTHETIC

~, Iolesias-Prieto

/tke Strid 1, W.S. Chow2 and Jan M. Anderson 2 IDept of Biochemistry and Biophysics, G6teborg University, Medicinaregatan 9c, S-41390 G6teborg, SWEDEN; 2CSIRO Div Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia

Departamento de Ecologfa, Centro de Investigaci6n Cientifica y Educaci6n Superior de Ensenada, Kin 107 Carretera TijuanaEnsenada, Ensenada B.C. 22860 M4xico.

Detached pea leaves were exposed to supplementary UV-B radiation at either 2° or 22°C for up to 4 h. After exposure, the leaves were either analyzed directly or left in darkness for up to 4 h at either the exposure temperature or were transferred to the other temperature. At 2°C the UV-B-induced increase in relaxation rate of the electrochrornic shift (518 nm) was partially prevented as long as the leaves were kept at 2°C. However, were they transferred to 22°C after exposure, the relaxation rate increased to a similar level as in leaves that had been kept at 22°C. This indicates a temperature-independent triggering as well as a temperature-dependent development of the effect. In contrast, the Fv/Fm parameter was decreased to a greater extent in leaves that had been UVB-exposed at 2°C than in those exposed at 22°C. The levels of cab mRNA transcripts was about 2.5-fold higher in leaves kept at 2°C compared to those kept at 22°C. This possibly indicates that mRNA breakdown was more temperature-dependent than transcription. Also, UV-B-induced decreases of cab mRNA were smaller at 2°C than at 22°C.

Elevated temperature (28-34*C) has been implicated as the primary cause of the loss of color in symbiotic coral reefdwelling invertebrates. This phenomenon, termed "coral bleaching", has been observed on a word-wide scale over the past 15 years. Brief exposures (<60 min) in the dark of cultured symbiotic dinoflagellates to temperatures above 30"C resulted in reductions in the quantum yield (Fv/Fm) of photosystem II in relation to controls maintained at 26"C. This reduction resulted from an increase in Fo values without any detectable change in Fm. In contrast, the same treatment in the presence of subsaturating light resulted in smaller, but significant, reductions in Fv/Fm in relation to controls. Changes in Fv/Fm were due to losses in Fm without variation in Fo relative to controls illuminated at 26°C . Longer exposures (12 h) to elevated, but physiological, temperatures (31-33°C) under subsaturating illumination resulted in reductions in Fv/Fm comparable to those obtained in the dark after short exposures to thermal stress.

P-24-002 DIFFERENT MECHANISMS FOR EXCITATION DISSIPATION AT HIGH AND LOW TEMPERATURE IN BARLEY Gunnar Oouist l, Vladimir Spunda2 and Christina Ottander 1 1Department of Plant Physiology, University of Umdt, S-901 87 Ume~t, Sweden, 2Department of Physics, University of Ostrava, Czech Republic To test the hypothesis that qN is mediated through different sites at high and low temperatures, chlorophyll fluorescence excitation and emission spectra at 77 K were compared for barley leaves under growth conditions and after photoinhibition at 5 and 20 °C. There is almost no antennae quenching of fluorescence after photoinhibition at 5 °C, since Fv/Fm was inhibited 45 % as compared to only 5 % inhibition of antennae to reaction cenlre energy transfer. In contrast, after photoinhibition at 20 °C, Fv/Fm and energy transfer were similarly inhibited. Upon photoinhibition at 5 °C, stable inactive reaction centres accumulate. The model of inactive reaction centres as dissipative units does not, however, apply at 20 °C where D1 turnover is rapid. Instead, at 20 °C, formation of quenching centres within the light harvesting complex has an important regulatory role. Zeaxanthin is formed at both temperatures and does not explain why antennae quenching is more important at 20 °C. Photoinhibition is a protective down-regulation of PS II under high light stress, but barley leaves respond with different mechanisms for dissipating excess excitation at high and low temperature.

P-24-005 EFFECT OF WHEAT VIA

HIGH TEMPERATURE ON SOME CHLOROPHYLL FLUORESCENCE.

VARIETIES

OF

F.Babani Dept.of Bioch. and Biophys.,Institute of B i o l o g i c a l R e s e a r c h , A c a d e m y of S c i e n c e s , Tirana, Albania. H i g h t e m p e r a t u r e e f f e c t s the f u n c t i o n a l i n t e g r i t y of the photosynthetic apparatus p a r t i c u l a r l y thylakoids membranes and reactions associated with its. The chlorophyll f l u o r e s c e n c e p r o v i d e s e s s e n t i a l i n f o r m a t i o n on the state a n d f u n c t i o n of p h o t o s y n t h e t i c p r o c e s s in plants. The a i m of this r e s e a r c h was to c h a r a c t e r i z e the e f f e c t of h e a t stress on photosynthesis of s o m e v a r i e t i e s of w h e a t via some chlorophyll fluorescence parameters. The values of F 6 9 0 / F 7 3 5 ratios at m a x i m u m and steadystate fluorescence determined by fluorescence induction kinetics using LITWaF, the parameters of chlorophyll fluorescence emission spectra measured by CCD-OMA spectrofluorometer, the chlorophyll fluorescence ratios Fo/Fm, Fv/Fo and AF/Fm' as well as photochemical and non-photochemical quenching determined by PAM fluorometer were used to estimate the thermotolerance of a n a l y z e d v a r i e t i e s of w h e a t .

209

Poster P-24-009

P-24-006 AN ANALYSIS OF THE RELAXATION OF STRESS-INDUCED NON-PHOTOCHEMICAL QUENCHING IN C4 SPECIES. David Blowers and Neil Baker, Department of Biology, University of Essex, Colchester CO4 3SQ, U.K. Upon illumination the quantum efficiency of leaf photosynthesis is reduced by quenching processes associated with the antenna of PSII. When leaves are placed in the derk this quenching relaxes to restore the high photosynthetic efficiency. In C3 plants initial kinetics of this relaxation of quenching appear to be monophasic, however in several C4 species studied during dark recovery the quantum efficiency of PSII rose rapidly initially then fell to a minimum before a sustained increase occurred. Such changes appear to be associated with the activities of the xanthophyll cycle since the ratio of (zeaxanthin + antheraxanthin):violaxanthin fluctuated during the recovery. These complex kinetics of the relaxation of quenching are lost when leaves are placed in low light (30-50 lamol m"2 s'l), however on subsequent return to complete darkness the complex kinetics can be seen. Decreasing leaf temperature prolongs the timecourse over which the effect is manifested in complete darkness. Analyses of this phenomenon using dithiothreitol to block the activity of the xanthophyll cycle before the high light period or before the beginning of the relaxation period and chloramphenicol to inhibit chloroplast protein synthesis will be presented.

FATTY-ACID DESATURATION AFFECTS THE RECOVERY FROM LOW-TEMPERATURE PHOTOINHIBITION Z. G o m b o s t, H. W a d a 2, N. Murata 2 1Biol. Res. Center o f Hung. Acad. o f Sciences, Szeged, Hungary 2National Institute for Basic Biology, Okazaki, Japan Cyanobacterial cells can be used as a simple in vivo model system to study the correlation o f membrane structure to thermal behaviors of photosynthesis. To examine how the fatty-acid unsaturation is related to environmental stress tolerance o f photosynthesis we developed a unique experimental system in which the desaturation of fatty acids can be eliminated. The complete elimination o f polyunsaturated fatty acids from Synechocystis P C C 6 8 0 3 cells by genetic manipulation did not alter the low-temperature tolerance o f transformant cells. However, the susceptibility to low-temperature photoinhibition enhanced remarkably. W e separated the process o f photoinduced inactivation from the recovery. Our experiments with isolated thylakoids emphasized that the damage site o f photoinhibitory process is not affected by the elimination o f the polyunsaturated fatty acids. Nevertheless, the recovery process, which involves the synthesis of photosynthetic reaction-center proteins and their insertion into the photosynthetic membranes was severely affected by the modification of fatty acid composition in the photosynthetic membranes.

P-24-010

P-24-007 THE O C C U R E N C E A N D S T R U C T U R A L - B I O C H E M I C A L F E A T U R E S OF T H E C4-ALPINE P L A N T S OF THE PAMIKS MOUNTAINS.

V.Pyankov 1,E.Voznesenskaja 2 1 Dept o f Plant Physiology, Ural State University, 620083, Ekaterinburg; 2Komarov Botanical Institute, 1970022, Sankt-Petersburg, Russia. The occurence, altitudinal distribution, fight and electron microscopy, biochemical features o f fo tosyntesis o f Ca-plants o f the Pamirs mountains (34-400 NL, 68-75 o EL) were studied. There are 38 plants with C4photosynthesis (21 species o f Chenovodiaceae. and 17 species o f Poaceae.) which habitate on elevation more 1700m. Eight species with C4 -photosyntesis on the East Pamirs on the altitudes 3800--4200m.-were detected (all belong to Chenepodiaceae. family only and annual life forms). All C4-alpine species demonstrated Kranz-anatomy, formation o f malate and aspartate as primary products o f ~4CO2 fixation, high relation o f PEPC (phosphoenolpuruvate carboxylase) activity to Rubisco (ribulose-l,5-bisphosphate carboxylase). At the same time C4-alpine species demonstrated more high level o f activity and amount o f R u bisco in comparision with typical desert Kranz-species.

P-24-008 Effects o f Long-Term Combinations

Chilling

ott Oenothera

with

Differetlt

Plastome/Genome-

DIFFERENCES AMONG THREE RICE CULT1VARS DURING CHILLING S T R E S S AND SUBSEQUENT RECOVERY PERIOD H.-S. Par~kt, I. H. Park ~, B.-Y. Moon :~, C.-B. Lee~ & C.-tt. Lee ~ 1Dept of Molecular Biology, Pusan National Univ, Pusan 609-735; ~Dept of Biology, Dong-A Univ, Pusan, 604-714; '~Dept of Biology, lnje Univ, Kimhae, 621-749; adept of Biology, Donguei Univ, Pusan, 614-714, Korea Fluorescence induction kinetics were measured from leaf slices taken from 3 week-old rice seedlings chilled at 4°C for 2 days and leaf slices during 72 hr-recovery period at 25°C under light (25 !amol • m ~ • s 1). For the measurement, leaf slices were dark-adapted for 20 min at 25~C. Among 5 rice cultivars, 3 cultivars, which showed large differences in Fr (maximum slope in D-P rise curve) and (Fv)m/Fm under chilling stress, were selected. Dongjin-byeo was the most resistant, Chilsung-byeo was in the middle, and Hyangdo-byeo was the most sensitive to chilling stress. The degree of photoinhibition during the subsequent recovery periods was in the reverse order. Fo did not change significantly. Similar experiments were done using leaf slices on wet filter papers with similar results, showing these results were not mainly due to water stress. The differences among cultivars were much less when the parameters were measured at 4°C, suggesting that they differs mainly in their sensitivities to photoinhibition during recovery period after chilling. The 3 cultivars did not show significant differences in their extent of the unsaturation of glycerolipids in thylakoid membranes.

P=24-011 FEEDBACK LIMITED PHOTOSYNTHESIS IN LOW TEMPERATURE STRESSED AND COLD ACCLIMATED SPRING AND WINTER WHEAT.

B.DAUBORNAND W.BRClGGEMANN

L.V.Savitch and N.P.A.Huner. Dept. of Plant Sciences, Western Ontario, London, Canada N6A 5B7.

Since the beginning of the 20th century Oenothera is an important object in plant genetics. Today it is regarded as a plant systom that stated for the study of ~ interactions of genome and plastom¢. Becauseofsora¢ unusual features -blparental transrmsslonof plasnds, the occurence of complex-betcrozygosityetc.- a large number of combinationsbetween.plastomeand gcnome have been created. There ate three basic genotypes (A, B, C) and five wdd type plastomes (I - V). Presumably plastome IV is the common ancestorand the most .primitive one..In their ~.~nal region of distribution (North and South America) Oenothera species are found m many di eJ~at habitants. For characterizingphotosyntheticreactions f~m plants grown in the gfcen/~ouse(control plants) and after cold acclimation (14 days at 2 C, 50t~mol quanta m-'~ s " , I0/14h) we used homozygouslines with diffea'entl~lastomes: Oe. clara (AA/I - IV) from the western half of the North American continent wUh RS dry envtronment and Oe..grandiflora (BB/III,IV) which origins from the wetter climate of the South-East. FurthcT siudi¢~ are planned on a plastomemutante from Oe. data and on SlX~es with the BC-genome. We have looked for several physiological parameters as a function of temperature, e.g. DI v/vo chlorophyll fluofescen_ce quc'achingIlm'amctct's,kinetic ~.~ ' ¢ s of p~fied rubi.set and.C.O-2-saml .'a.tcd..photosy..n.. thesis."In co~ol plants the temperature eepem~ces o~ t,,Tevol~ a m imotocnemc= qu~cmn$ vane~ between different genomes, wbereas It seems that the diffeaeat plastomes rum troy ,me or no infineace. Cold aeelimatioa did not lead to siginfi.c~t,changes in most of the parameters, but ".m some lines an increase in e n e r ~ , ~ quenching could be observed. We suspect that this enuld be a consequenceof the diffexentplastomes.

Carbon metabolism in s p r i n g and w i n t e r wheat was studied with respect to low temperature stress, cold acclimation, and growth irradiance. Under low temperature stress, CO2 assimilation rates declined in both cult~.vars as a result of feedback ]imitation of photosynthesis. This was reflected in a decreased stroma| and cytoso]ic FBPase and SPS a c t i v i t y , and was associated with trioseP accumulation and decreased ATP/ADP ratio. Cold acclimation resulted in a similar restrictSon of photosynthesis. However, w i n t e r wheat adjusted C09 assimilation rates to the control values, while s p r i n g c u l t i v a r d i d not. We show t h a t this adjustment was due to 3-fold increased SPS a c t i v i t y . When plants were grown o r shifted to low temperature t h e y exhibited a similar r e s t r i c t i o n of photosynthesis i n d e p e n d e n t of growth irradiance (50 o r 250 PPFD). However, when plants were grown at 20°C and e i t h e r 50, 250 or 700 PPFD photosynthesis became feedback limited o n l y at high i r r a d i ance. Feedback ]imitation was always reflected in a high excitation pressure (1-qn). In addition, wheat grown u n d e r similar 1-qp were morpho]ogicalT'y comparable even though plants were grown u n d e r the d i f f e r e n t l i g h t and temperature conditions.

Institute of Ecological Plant Physiology, Heinrich-Heine University, Universitatsstr. 1 40255 Dttsseldorf, F.R. G.

210

Univ. of

Poster

P-24-012 EFFIV/TB O F L O W T I ~ P B R A T ~ R E ~ G R O W T Z O N T R J PEPAPORIP*~CE OF TRX PXOTOBTRTI[ETIC APpJUUI&TurBO F I~%IZE LIULVIS.

Y- Fracheboud I, P. Haldimann = and P. Stamp I Swiss Federal Institute of Technology, Zurich, Switzerland; 2 Lab. bioenergetique, university of Geneva, Switzerland The effect of chilling temperatures on the performa/~ce of the photosynthetic apparatus was examined by the mean of chlorophyll fluorescence in maize leaves developed at 25oC and 15oc. In both leaf types the photosynthetic oxygen production strongly decreased with progressive cooling from 25 to 4oc under moderate light. In leaves developed at 25oC this decrease correlated with a decrease (qp), whereas in in photochemical Chl fluorescence quenching leaves developed at 15°c q, was largely insensitive to temperature. Leaves developed at 15oc were also able to maintain QA more oxidised than leaves developed at 25oc when exposed to strong light at 26°C. In both conditions (low temperature or strong light), leaves developed at sub-optimal temperature exhibited a substancial decrease of the ground level of fluorescence Fo, indicating the presence of an energy dissipation mechanism absent in leaves developed at near-optimal temperature. Furthermore, leaves developed at 15°c were able to recover faster than leaves developed at 25°c after photoinhibitory treatment, as measured by the photochemical efficiency of PSII (Fv/Fm). The possible protective role of the xanthophyll zea>~nthin, only present in 15°C grown leaves, is discussed.

P-24-013

P-24-015 HIGH

SICON

LIGHT STRESS AT LOW SPECIES DIFFERING IN

IN LYCOPERSENSITIVITY

TEMPERATURE

CHILLING

J.H. V E N E M A & P.R. V A N H A S S E L T Department of P l a n t Biology, Univ. of G r o n i n g e n , P.O. B o x 14, 9 7 5 0 A A H a r e n , T h e N e t h e r l a n d s The susceptibility to photoinhibition and photobleaching was studied in L y c o p e r s i c o n e s c u l e n t u m cv. A b u n d a and the chilling tolerant L. p e r u v i a n u m LA 385 before (non-chilled) and after a chilling (chilled) treatment o f 7 d a y s at 10°C. A t a p h o t o inhibitory irradiance of 5 0 0 ~ E m -2 s -L a t 5°C, n o n chilled L. p e r u v i a n u m e x h i b i t e d a greater resistance to photoinhibition than non-chilled L. e s c u by the photochemical efficienty lentum as measured of photosystem II. C h i l l e d L. p e r u v i a n u m s h o w e d a n increased resistance to photoinhibition and photobleaching whereas chilled L. e s c u l e n t u m d i d not. The higher resistance t o h i g h l i g h t s t r e s s in c h i l led L. p e r u v i a n u m c o u l d be e x p l a i n e d by a reduced formation of t o x i c o x y g e n s p e c i e s , because a lower reduction s t a t e of t h e e l e c t r o n t r a n s p o r t chain was observed in t h i s h i g h a l t i t u d e w i l d s p e c i e s .

P-24-016

M E C H A N I S M OF X A N T H O P H Y L L D E P E N D E N T NONP H O T O C H E M I C A L Q U E N C H I N G OF C H L O R O P H Y L L A FLUORESCENCE AT LOW PHYSIOLOGICAL TEMPERATURES

TRIENOIC FATTY ACIDS OF CHLOROPLAST MEMBRANE LIPIDS PLAY AN IMPORTANT ROLE IN CHILLING ACCLIMATION OF HIGHER PLANTS

~ 1 , Govindjee 1 & Olle BjOrkman2 1plant Biol., UIUC, Urbana, IL 61801;2Carnegie Inst., Stanford, CA 94305

H. Kodama, T. Hamada, G. Horiguchi, T. Nishiuchi, M. Nishimura & K. Iba Dept of Biology, Fac of Science, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-81, Japan

When photosynthesis is limited, excess light triggers protective heat dissipation in PSH through the formation of a trans-thylakoid pH gradient that, in turn, stimulates formation of zea- and antheraxanthin. These xanthophylls when combined with protonation of antenna pigment-proteins increase heat loss an0 quench Chl a fluorescence. Here we show (1) that low temperatures increase the ratio of Chl a fluorescence quenching relative to the coupled electron transport or ATP hydrolysis rates [Gilmore and Bj0rkman, unpublished data]; (2) xanthophyll-dependent quenching of Chi a fluorescence is mediated by an antenna complex with a fixed fluorescence lifetime of 400 ps independent of [xanthophyll] [Gilmore et al. (1995) PNAS 92; 2273-2277]; and (3) the extent of quenching is temperature independent between 0 and 30 ° C. These data suggest (a) that low-temperatures decrease the ratio of thylakoid membrane proton leakage to coupled proton pumping and (b) that xanthophyll-dependent Chl a fluorescence quenching is not controlled by temperature-sensitive diffusive collision rates. The mechanism of xanthophyll-dependent photoprotection ever the physiological temperature range will be discussed.

P-24-014 CHILLING SENSITIVITY OF PHOTOSYNTHESIS: EVIDENCE FOR DIFFERENTIAL OXIDATIVE STRESS IN TWO L¥COPERSICON SPECIES OF DIFFERENT CHILLING TOLERANCE 3~_]~igg.¢.lIla~, P. Linger, K. Maas-Kantel; Institute of Ecological Plant Physiology, Heinrich-Heine-University, Universit~tsstr. 1, D-40225 Diisseldorf The cultivated tomato, Lycopersicon esculentum, and high-altitude lines of its wild relative, L. peruvt~num, differ significantly in their sensitivity towards long-term chilling stress under non-photoinhibiting conditions. While the latter species is hardly affected by. such stress treatments, L. esculenmm reacts with a permanent partial inhibition of photosynthesis in the chillingexposed leaves. This inhibition is Caused by an oxidation of cystein residues ofribulose-1,5-bisphosphate caxt~xylasu/oxygenase (rubisco), leading to ruoisco-limitation of photosynthesis in chilled L. esculentum plants. Possible reasons for the differential in v/vo-sensitivity of rubisco in the two species during chilling have been found in different temperature dependences of QA reoxidation and of energy dissipation by the ql~. mechanism. With respect to both processes, the probability of formation ofreactive oxygen species by Mehler reaction and by increasing lifetime of triplet chlorophyll appears much higher in L. esculemum than in L. peruvianum.

One of the remarkable characteristics of the chloroplast membranes is that they have unusually high contents of lipids containing trienoic fatty acids (TAs) such as a-linoleic and hexedecatrienoic acids. This high level of fatty-acid unsaturation has been assumed to be important for the low temperature fitness of higher plants~ Recently, a cDNA (FADT) encoding chloroplast (0-3 desaturase, which is the key enzyme for the formation of TAs, was isolated from Arabidopsis thaliana (Iba et al., 1993, J. Biol. Chem., 268: 24099-24105). The overexpression of the FAD7 cDNA in tobacco plants resulted in the significant elevation of TAs levels, and in such transgenic plants chilling injury was reduced. The physiological significance of TAs in the chilling tolerance of higher plants will be discussed.

P-24-017 FAST RECOVERY OF PHOTOSYNTHESIS FROM LOW-TEMPERATURE PHOTOINHIB1TIONIN CHILLING-RESISTANTPLANTS Byoun= Yong Moont & Norio Murata2 1Department of Biology, lnjc University, Kimhae 621-749, Korea, 2Departmant of Regulation Biology, National Institute for Basic Biology, Okazaki 444, Japan Recovery of photosynthesisfrom low-temperaturephotoinhibition was studied in leaves of spinach, a chilling-resistant species,and of squash,a chilling-sensitive one. Inhibition and recovery of Photosystem II (PS II) was monitored by fluorescence measurement. During strong light illumination at low temperature, the ratio of variable to maximum fluorescence (Fv/Fm) decreased faster in squash than in spinach. However, the two types of plant showed a comparable decrease in Fv/Fm in the presence of lincomycin, an inhibitor of the chloroplast-encoded protein synthesis. Recovery of Fv/Fm under various light intensities was much faster in spinach than in squash.These results suggest that chilling sensitivity depends on the rate of recovery of photosynthesis from low-temperature photoinhibition. This possibility was examined by directly comparing the recovery of Fv/Fm from lowtemperature photoinhibition between leaves of wild-type and transgenic tobacco plants, whose degree of chilling sensitivity was genetically modified by means of fatty acid unsaturation of the chloroplastic membrane lipids. We propose that recovery process, including the repair of DI protein, has an important role in the tolerance of chilling-resistant plants against low-temperaturephotoinhibition.

211

Poster

P-24-021

P-24-018 EFFECT

OF

CHILLING

STRESS

ON

PHOTOSYNTHESIS

IN

RBCOVBRY OF pHOTOSYNTHISSIS

FROM WINTER

STRB8$

IN

INTAC'T P L A N T S

CUCUMBER

AND DETACHEDTWIGS OF CONIFERS

~.-S. Jun l, J. M. Kimt, B. Y. Moon2 and C. B. Lee 1 Department of Biology, Dongeui University, Pusan 614-714; 2Deparmaent of Biology, Inje University, Kimhae 621-749, Korea

M.

The effect of chilling stress in the light on the subsequent photosynthesis of cucumber was compared with that in the dark. After treating whole plants at 4"C for.variable time, the photosynthetic activity was monitored, using leaf discs taken from chilled plants, by the shnultsneous measurement of 02 evolution and fluorescence..Photosynthasis was gradually decreased after chilling in light (50 limol/m~/sec) or in the dark. However, the inhibitory effect of chilling stress occurred more rapidly in the light. 02 evolution was decreased 80-90% after chilling treatment for 4 hrs in the light or for 24 hrs in the dark. Fv/Fm was also decreased accordingly. Chloroplasts isolated from chilled plants which were either light-chilled (4 hrs) or dark-chilled (?_A hrs), had 40-50% lower photosynthetic activity than those from control plants, indicating that photosynthetic apparatus was harmed. Results with reconstituted chloroplasts showed that thylakoids isolated from light-chilled plants were damaged while thylakoids from dark-chilled plants were not. In contrast, stroma from dark-chined plants was less functional than that from light-chilled plants. Moreover, NADP reduction was decreased by more o than 70% in thylakoids from light-chilled plants, indicating electron transport chain has been harmed by chilling treatment in the light. However, NADP reduction was not affected by dark-chilling. These results suggest that chilling stress in the light and in the dark is a different pl"OCeSS.

Recovery from winter s t r e s s w a s compared in intact plants and detached twigs of Picea abies and Pinus cembra experiencing winter stress in the field. After distinct weather conditions twigs of young and adult plants were detached and transferred together with potted young plants to a climate chamber (17/12°C, 40 pmol photons m -1 s d, 12 h photoperiod I . Detached twigs were either supplied with water or a solution of benzyladenlne (44 ~M). The rate of CO2uptake and the potential efficiency of PS II {Fv/FM) were recorded. Photosynthetic CO, uptake increased during the first two days of recovery in all samples, but afterwards only in intact plants. In detached twigs the rate remained low or even decreased, irrespectively the twigs were supplied with water or cytokinin. Recovery in detached adult twigs was always poor. In contrast to CO 2 uptake the fluorescence parameter Fv/F M improved in all samples in a similar way up to summer values. Thus, recovery of photosynthetic productivity from winter stress cannot be ~tudied using detached twigs, although the efficiency of PS II recuperates like in intact plants.

Loidolt-Nauele,

F. G a p p

& n.

Institute of Botany, University A-6020 Innabruck, Austria

Bauer Innsbruck,

SternwarteetraRe

15,

P-24-022

P-24-019 EFFECTS OF H I G H T E M P E R A T U R E ON P H O T O S Y N T H E T I C S Y S T E M S IN H I G H E R P L A N T S

PHOTOSYNTHESIS

Y. Yamane, Y. Kashino, H. Koike and K. Satoh Dept. Life Science, Fac. Science, Him@ Inst. Tech., Harima Science Garden City, Hyogo 678-12, Japan

Jean-Marc Routaboul. Michele McConn and John Browse Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340.

It is known that photosynthetic systems in higher plants are most sensitive to high temperature treatments. However, it has not been well established that where and how the chloroplasts are damaged by the tTeatment and how the damages can be detected easily. In the present study, we used spinach leaves and intact chloroplasts and mainly measured changes in fluorescence parameters during and after high temperature treatments. When spinach leaves were exposed to increasing temperatures, the F o level started to increase at around 42°C and reached maximum at around 5 0 ~ , and then decreased gradually at higher temperatures. This F o increase was partly reversible. In the case of intact chloroplasts, F o changed in a similar way as in detached leaves. The relationship between the changes of these fluorescence parameters and the changes in thylakoids will be discussed.

P-24-020

REQUIRES

TRIENOIC

FATTY ACIDS

FOR LOW

AND HIGH TEMPERATURE SURVIVAL

Depending upon the plant species, trienoic fatty acids (C18:3 and C16:3) constitute up to 80% of the fatty acids in chloroplast membrane lipids. A triple mutant of Arabidopsis ~aliana lacks both linolenic (C18:3) and hexadecatdanoic acids (C16:3) in its membrane lipids. Chlorophyll fluorescence measurements were used to monitor photosynthesis. Under standard conditions the altered membrane lipid composition had no effect on Fv/Fm and on the quantum yield of Pail electron transport (d)PSll). By contrast, when the mutant was grown at 3oC or 33oC up to 30 days, Fv/Fm and d)PSII were strongly inhibited in mutant compared to wild-type. Mutant tissues developed at both 3oC and 33°C became chlorotlc and died after 15 days at 33°C. Chilling inhibition of photosynthesis were related to the amount of trlenoic fatly adds present in the membrane lipids. Dudng the chilling stress of the mutant, d)PSii was preferentially inhibited and reduced quinones accumulated suggesting an inhibition of the reducing side of PSII. During heat treatment both Fv/Fm and OPSii were similarly inhibited suggesting an inhibition of the mutant PSII complex. These observations show that if trienoic fatty acid membrane Iipids are not necessary In maintaining normal photosynttletic functions, they directly affect the stability to heat or cold of photosynthetic membranes.

P-24-023

T W O C O M P O N E N T S ARE INVOLVED IN T H E DETERMINATION OF C H I L L I N G SENSITIVITY IN C O M M O N BEAN

EFFECTS O F L I G H T AND CARBOHYDRATES ON ACCLIMATION O F PHOTOSYICrHESIS IN SPINACH TO L O W TEMPERATURE

IC Sonoike, M . Ishibashi & A. Watanabe Department of Biological Sciences, Graduate School of Scicnco, University of Tokyo, Bunkyo-ku, Hongo, Tokyo 113, Japan

A. Battistelli l, W. Martindale 2 and R. C. Leegood 2 llstituto per l'Agroseivicoltura, CNR, Porano, Italy. 2Robert Hill Institute, University of Sheffield, Sheffield, U K .

It was recently shown that photoinhibition occurs in PSI but not in PSII w h e n

leaves of Cucum/a sat/Pua L , a chilling-sensitive plant, is exposed to chilling temperatures (Terashima et al., 1994, Planta, 193, 300-306). In isolated thylakoid membranes, the PSI photoinhibition can be induced even in a chilling-tolerant plant, spinach, without exposing to chilling temperatures (Sonoike, 1995, Plant CellPhysioL, in press). TherefOre, we have concluded that chilling-sensitive step is not PSI itself but some components protecting PSI in vivo. In the present study, another chilling sensitive plant, common bean (Phaaeolua vulgaris L.), was grown under different light environment. The results obtained were: 1) Susceptibility of PSI photoinhibition in yiyo increased when plants were grown under weak light. 2) The change ot the susceptibility did not correlate with the ratio of PSI/PSII or of Rubisco/chlorophyll. 3) The photoinhibition by weak light and that by moderate light were different regarding the response to the growth irradi.'ancos or to temperature du~n_g the photo.inhibitory .treatment. From these results, we concluded that there are two chilling sensiUve components m common cean. It was shown that one component loses its activity at 3.5 C, and exposure to 1.5°C or isolation of thylakoid membranes inactivates both components.

212

The effect of light intensity and of leaf carbohydrates on acclimation of photosynthesis to low te~nperature was studied in normal and sink-limited spinach plants. Control plants and plants reduced to only two mature leaves were transferred from 25°C to 10°C under either 300 ~mol quanta m -2 s-I (hi~ light) or 40 pmol quanta m "2 s-1 (low light) and aspects of the acdimatory process (assimilation rate, carbohydrates, Rubisco, ADPglucose pyrophosphorylase) were examined during the following I0 days. Light was shown to be necessary for photosynthetic acclimation to low temperature. Acclimation of photosynthesis was associated with an increase of leaf

carbohydrate content, particularly of glucose. These results indicate that the increase of leaf carbohydrate aRer t r a n s i t of plants to low temperature represents a positive signal for the acclimation of photosynthesis, rather than acting to down-re,relate photosynthetic capacity and gene expression, as occurs at 1~gher temperatures.

Poster

P-24-024 PROPERTIES O F F o - Q U E N C H I N G C H I L L E D M A I Z E PLANTS

(qo)

OCCURRING

1N

P-24-027 D E T E C T I O N OF PLANT C H I L L I N G SENSITIVITY BY T H E F685/F730 C H L O R O P H Y L L FLUORESCENCE RATIO.

T. Janda l, G. Szalai l, Z. Szigeti 2 & E. Paldi l ~Agricultural Research Institute of the Hungarian Academy of Sciences, H2462 Martonvhs~ir, P. O. B. 19, Hungary; 2Department of Plant Physiology, EOtvOs Lor~ind University, H-1445 Budapest, P. O. B. 330, Hungary

M. Lipucci di Paola l, G. Agati 2, F. Fusi2, P. Mazzinghi 2 lDipartimento di Biologia delle Piante Agrarie, Universitb. di Pisa, Viale delle Piagge 23, 56124 Pisa, Italy; 2Istituto di Elettronica Quantistica - CNR, Via Panciatichi, 56/30, 50127 Firenze, Italy

During the measurement of the slow chlorophyll fluorescence induction curve in cold treated maize plants not only the variable fluorescence (Fv), but also the initial fluorescence (Fo) was quenched. The level of qo depends on the irradiance and temperature of the cold treatment: the higher the irradiance and the lower the temperature the more pronounced the F o quenching. However, alter a certain chilling period (below a certain F v / F m level) qo also starts to decrease. The level of qo also depends on the actinic light intensity and the temperature at which the measurement took place. The quenching of F o is reversible: F o decreases in the light and increases in the dark DCMU and NaF may inhibit F o quenching.

The chlorophyll (Chl) fluorescence ratio, F685/F730, of Pisum sativum and Phaseolus vulgaris, differing in chilling sensitivity, was monitored as the environment temperature was decreased to 4 °C. The plants were grown in a controlled chamber at 20 °C and 100/.tmol m-2s-1. After 20 days, the chamber temperature was set at 4 °C and F685/F730 was measured continuously and contemporary on an attached leaf of both species by two portable fiber-optics fluorometers. The temperature of both leaves was also measured by two infrared thermometers. During the first 5 hours, when the leaf temperature passed from 24 to about 4 °C, F685/F730 in the bean decreased by a 30%, while in the pea F685/F730 slightly increased. Our results suggest the potential use of the F685/F730 Chl fluorescence ratio as discriminatory parameter for the evaluation of chilling-tolerance in plants.

P-24-025 C O L D - T O L E R A N C E , O X I D A T I V E STRESS T O L E R A N C E AND A N T I O X I D A N T E N Z Y M E L E V E L S IN N O N - T R A N S G E N I C AND Mn-SUPEROXIDE DISMUTASE OVEREXPRESSING MAIZE. ,I-M. Stassart 1, L. Slooten 1, F. van Breuseghem2, J. Botterman 4 and D. Inz~2, 3 lVrije Universitcit Brussel, Lab. Biofysica, Pleinlaan 2, B-1050 BRUSSEL, Belgium; 2Lab. Genctika, and 3Laboratoire Associ6e d'INRA (France), Universiteit Gent, Ledeganckstraat 35, B-9000 GENT, Belgium; 4plant Genetic Systems, J. Plateaustraat 22, B-9000 GENT, Belgium. Oxidative stress is involved in many types of physiological stress, including drought, cold, heat and waterlogging. In order to elucidate the role of supcroxide dismutase (SOD) in oxidative stress tolerance, two transgenic lines of Zea mays line H99 were generated which overexpress a mitochondrial manganese SOD from tobacco in the chloroplasts. Both lines contain a single-locus DNA insertion. The line with the highest overexprcssion level exhibited (a) enhanced tolerance to methyl viologen dependent, light-induced oxidative stress in leaf disc assays after growth at 25 °C, and (b) enhanced tolerance to moderate temperatures, as indicated by growth rates at 17 °C. Four other, non-transgenic maize lines (CP 101 to 104) were investigated with respect to cold sensitivity. CP 103 and untransformed H99 exhibited symptoms of chilling-induced stress after growth at 17 °C. There was no simple correlation between cold sensitivity and antioxidant enzyme levels in leaf extracts, except that high endogenous SOD levels were indicative of experienced stress, rather than of stress tolerance.

P-24-026

P-24-028 AN INVESTIGATION OF HIGH-TEMPERATURE REGION OF CHLOROPHYLL FLUORESCENCE TEMPERATURE CURVE P. Ilik 1, J. Nau~ 1, D. Cik,~ek 1, V. ~;punda2, M. Caj~laek2, IL Novotn~,3, D. ;~ik 1 IDepartment of Experimental Physics, Palack~ University, ti'. Svobedy 26, 771 46 Olomouc; 2Department of Physics, Ostrava University, Br~fova 7, 701 03 Ostrava; 3Center of Microscopic Methods, Palaclo) University, I. P. Pavlova 35, 775 20 Olomouc, Czech Republic Chlorophyll fluorescence temperature curve (FTC) is defined as a teanperatare dependence of chlorophyll fluorescence intensity during linear heating of a leaf segment in region 25 - 75 °C under weak light excitation. We have studied still controversially interpreted increase of FTC between 60 and 70 °C. Leaves of barley grown under intermittent light were used as the model samples having high value of this fluorescence increase under preferential chlorophyll a excitation. Under preferential chlorophyll b excitation the increase was expressively reduced. As resulted from fluorescence excitation spectra at 77K the energy transfer from carotenoids and chlorophyll _bto chlorophyll _adecreased within this part of FTC. At these temperatures an appearance of new condensed structures of thylakoid membranes was observed by transmission electron microscopy.

P-24-029

S E A S O N A L C H A N G E S IN P H O T O S Y S T E M II O R G A N I S A T I O N AND P I G M E N T C O M P O S I T I O N IN P I N U S S Y L V E S T R I S .

INFLUENCE OF TEMPERATURE ON PHOTOSYNTHESIS AND RESPIRATION OF PRIMARY BARLEY LEAVES

Christina Ottander, Douglas Campbell, Gunnar Oquist Department of Plant Physiology, University of Ume~t, S-901 87 Ume/t, Sweden

Tilt P&rnik1, Peeter Talts I , Per Gardestr6m 2 and Olav Keerberg 1 ~lnstitute of Experimental Biology, Estonian Acdemy of Sciences, EE3051 Harku, Estonia;2Department of Plant Physiology, University of Ume&, S-90187 Ume&, Sweden

The evergreen Pinus sylvestris encounters considerable combined stress of low temperature and high light during winter. These stresses coincide with major seasonal changes in PS II organisation and pigment composition. The earliest changes occur in September, before any freezing stress, with initial losses of chlorophyll, the Dl-protein of the PS II reaction centre and of LHC II proteins. Dl-protein content and PS II photochemical efficiency (Fv/Fm) eventually drop 90% while chlorophyll drops 40-50 % during winter. The carotenoid composition varies sea~nally with a two-fold increase in lutein and the carotenoids of the xanthophyll cycle during winter, while the epoxidation state of the xanthophylls decreases from 0.9 to 0.1 from October to January. The loss of chlorophyll is complete by October and during winter much of the remaining chlorophyll is reorganised in aggregates of specific polypeptide composition which apparently efficiently quench excitation energy through non-radiative dissipation. In April and May recovery of the photochemistry of PS II, protein synthesis, pigment rearrangements and zeaxanthin epoxidation occur concomitantly and without net increase in chlorophyll. The fall and winter reorganisation allows Pinus sylvestris to maintain a large stock of chlorophyll in a quenched, photoprotected state, allowing rapid spring recovery of photosynthesis.

Components of steady state photosynthetic and respiratory CO2 exchange in the barley primary leaves (Hordeum vulgare, vat. Golden Gunilla) were studied at three temperatures (25, 14 and 7°C) and at two oxygen concentrations (15 and 210 roLL"1 02). Photorespiration decreased more than photosynthesis at low temperatures. Specifity factor of Rubisco was 79, 127 and 135 at 25, 14 and 7°C, respectively. C O 2 evolution at the expense of end products of photosynthesis increased progressively at low temperatures. ATP/ADP ratios increased only slightly in the chloroplast (1.1 at 25°C and 1.4 at 7°C), and much more in extra-chloroplast fraction (3.3 at 25°C and 6.0 at 7°(3). Oligomycin, an inhibitor of mitochondrial ATPase, reduced the rates of photosynthesis in leaves by 28+2% at all three temperatures. Therefore, it is likely that mitochondrial respiration in the light is activated at low temperatures but it does not influence photosynthesis more than at higher temperatures.

213

Poster P-24-030

P-24-033

THE EFFECTS OF COLD ACCLIMATION ON THE PHOTOSYNTHETICCARBON METABOLISM IN LEAVES OF WINTER RYE (SECALE CEREALE L.)

CARBOHYDRATE METABOLISM IN LEAVES OF P A S P A L U M DILATATUM ACCLIMATED TO LOW TEMPERATURE

O. Keerberg 1, P. Gardestr6m 2, H.Ivanova1, H. Keerberg 1, T. P/irnik 1 & El. (3quist2 1Institute of Experimental Biology, EE3051 Harku, Estonia; 2Dept of Plant Physiology, University of Umea, S-901 87 Umea, Sweden

A.M.Cavaco and M.C. Arrabaqa Departamento de Biologia Vegetal, Faculdade de Ci~ncias da Universidade de Lisboa, Campo Grande, 1700 Lisboa, Portugal.

Leaves of winter rye grown at 5°C (coldhardened plants) and 25°C (nonhardened plants) were exposed to ~aco2 and the kinetics of 14C incorporation into the products of steady-state photosynthesis was determined. From kinetic data the rates of carbon fluxes and the pool sizes of metabolites were calculated. Efficiency of the regeneration of ribulose 1,5-bisphosphate in the reductive pentose phosphate cycle and the rate constant of a reaction of the glycolate cycle, glycine --> serine, increased in response to cold acclimation. Syntheses of C3- and Ca-acids and of protein were enchanced. Starch synthesis slowed down. The rate of sucrose synthesis was equal in hardened and nonhardened plants. The described metabolic shifts allow to elucidate changes in the activity of enzymes catalyzing partial reactions of the biochemical system of CO 2 assimilation.

Carbohydrate metabolism in leaves is influenced by low temperature in several ways. Plants of Paspalum dilatatum cv Raki, a C4 gramineae semi-tolerant to low temperature, were grown for 1 month at 25/18 oc. After this time, half plants were kept at these conditions (control), and the other half was acclimated for 20 days to a temperature of 10/8 o c (acclimated plants). Contents of glucose and sucrose increased in leaves of acclimated plants by 6 and 3-fold, respectively. Fructose was not detected in both cases and starch was only detected in leaves of acclimated plants. Fruetans and hexoses-P are being determined. In what concerns the enzymatic assays, results suggest that the activity of sucrose synthase is the same in leaves of control and acclimated plants, while the activation state of sucrose-phosphate synthase (Vmax and limiting assays) showed a tendency to be higher at the lower temperature. Neutral invertase was not detected in both cases. In respect to acid invertase assay results point to a decrease of activity at the lower temperature.

P-24-031 PHOTOSYBTEM I Z RESPONSB TO B I O T I C AND A B I O T I C S~R]©SS

P-24-034 DEGRADATION OF PHOTOSYNTHETIC ACTIVITY INDUCED BY TEMPERATURE SHIFT-DOWN IN PALISADE CELLS OF SAINTPA ULIA

M - B a r 6 n 1, J.Rahoutei z, J.L~zaro ] & I. Garc~a Luque 2 IDept of Plant Biochemistry and Cell and Molecular Biology, Est. Exp. Zald~n CSIC, Granada, Spain; O p t of Plant Biology, CIB, CSIC, Madrid Biotic and abiotic stress conditions may exert their effect on photosynthesis by inhibiting Photosystem II (PSII). Two examples of these stresses are pathogens that invade plants spreading systemically and heavy metal toxicity. We have compared the effect of virus infection and copper toxicity on PSII composition and functionality. N i c o g i a n e b e n g a m i a n a plants, infected with two strains of pepper mild mottle virus (PMMV-S) that induce symptoms of different severity, and Zea msys plants, grown under high Cu concentrations, were used as model system. Light-induced electron transfer and v a r i a b l e fluorescence were m e a s u r e d in leaves, thylakoid membranes and PSII preparations from both plants. Changes on PSII polypeptide pattern induced either by infection or toxicity were also analysed. In addition, the association of the viral coat protein (cP) with t h y l s k o l d and PSII fraction was studied.

J. G. Yun I , T. Hayashi ] , S. Yazawa ] , Y. Yasuda 2 & T. Katoh 3z Faculty of Agriculture, Kyoto Univ., K~oto 606-01; z Institute Pasteur de Kyoto, Sakyo-ku, Kyoto 606; Dept. Botany, Faculty of Science, Kyoto Univ., Kyoto 606-01, Japan With the irrigation of cold water on leaf, the palisade cells of Saintpaulia stop to photosynthesize instantly and irreversibly. This was accompanied by a gradual decrease (not increase) of chlorophyll fluorescence to nearly 1 / 3 as high as the original level (close to F0 level) without any changesin the emission spectrum nor in the

composition of photosynthetic pigments. Electron microscopically an extensive disarrangement of grana stacking was seen with broadening of lumen spaces. This phenomenon occurs only in the palisade cells of some Gesneriaceae and Acanthaceae plants, and never in the spongy cells adjacent below the palisade tissues.

P-24-035

P-24-032

OF LAND PLANTS

THE HEAT TOLERANCE OF PHOTOSYNTHESIS IN THE CYANOBACTERIUM, Synechococcus ap. PCC 7002

PHOTOSYNTHETIC ACCLIMATION TO TEMPERATURE AND LIGHT

Y. Nishivama. H. Hayashi and N. Murata Department, el Regulation Biology, National Institute for Basic Biology, Okazaki 44~, Japan

Alaka Srivastava and Reto J. Strasser, Laboratory o f Bioenergefles, University o f Geneva, CH-1254, Jussy, Geneva, Switzerland

When plants and cyanobacteria acclimate to high-temperature environments, their photosynthesis becomes more tolerant to high temperatures. We have lately shown that the acquired heat tolerance of oxygen evolution is preserved in the thylakoid membranes in the cyanobacterium, Synechococcussp. PCC 7002. When the vesicular structure of the heat-tolerant thylakoid membranes was disrupted with a low concentration of Triton X-100, the heat stability of oxygen evolution was remarkably decreased. The heat stability could be restored by reconstituting the membranes with the components dissociated by Triton X-100. Moreover, the components were able to increase the heat stability of the heat-sensitive thylakoid membranes from non-adapted cells. These results indicate that the factors responsible for the heat tolerance of oxygen evolution are dissociated from the thylakoid membranes by Triton X-100. Using chromatography, we determined that one of the factors for heat tolerance was a lowpotential cytochrome c550.

The in rive photochemical activity o f PSI] was inferred from fast and steady state Chl a fluorescence in intact leaves o f pea plants to environmental stresses (heat, light) applied separately or in combination. The results show that the low light (about 40 W m -2) is a primary requirement to protect the plants against heat slress. Once the plant has been stressed by heat, it can be recovered slowly by further low light treatment. Heat protection was also lriggered by exposing the leaves to moderately elevated temperature (35°C) for 15 min before exposing them to high temperature. Moderate heat prelreatment also protected the photoinhibition o f PSII showing the existence o f an adaptative mechanism that cancels the synergism between heat and strong light and markedly enhances the resistance o f PSI/ to photodamage.

214

Poster l lm

P-24-036 EFFECTS OF HIGH TEMPERATURE AND WATER STRESS ON THE POLYPHASIC CHLOROPHYLL a FLUORESCENCE TRANSIENT OF POTATO LEAVES Berouba. Guiss6 A. Srivastava and R.J. Strasser, Bioenergetics Laboratory, University of Geneva, CH-1254 Jussy, Switzerland. High temperature around 44°C, transforms the polyphasic (O-J-IP) rise o f the chlorophyll a fluorescence transient of potato leaf discs (Solarium tuberosum L.) to an O-K-J-I-P transient with a new intermediate step around 2001Is, (K-step). The appearance of this new step is accompanied by a big dip after J or K, depending on the severity of the stress. These phenomena were correlated- to severe injury in the water splitting system and a partial inhibition of the electron flow before (Qa). Simultaneous treatment o f high temperature and drought did not provoke a K-step. We suggest that water stress protects the PSII activity against high temperature injury. The experimental signal for the K-step might be a practical measure for the stress intensity in the plants.

P-24-039 FUNCTIONAL

AND

STRUCTURAL

DESTRUCTIONS

OF

CHLOROPLASTS

IN

VIRUSINFECFEDTOBACCOPLANTS

A. Alm~si I, Z. Szigeti2, B. B6ddi 2, M. Ek6s 3, R. GAborjhnyi1 1plant Protectio,,n Inst. Hung. Acad. Sci. Herman O. u. 15. Budapest H1022 Hungary aDept, Plallt Physiol. E6tv0s Univ. Mfizeunl krt. 4/a H1088 Budapest, Hungary; 6Dept, Plant Anat. E6tv6s Univ. Mfizeum krt. 4]a H-1088 Budapest, Hungary; Tobacco (Nicotiana bentharniana) plants infected with three different viruses were investigated. Tobacco necrosis virus (TNV) caused local hypersensitive necroses in inoculated leaves. Plum pox virus [PPV) caused systemic mosaic symptoms and the plant survived the infection. Tomato spotted wilt virus (TSWV) i/lfection resulted in slight chlorotic spots on the leaves and after 2 weeks the plants suddenly wilted and died. Electron microscopic results revealed that TSWV caused the strongest destruction of chloroplast structures but only at the latest stage of symptom development. All three viruses changed the relative 77 K fluorescence intensities of the chlorophyllprotein complexes, Fluorescence induction measurements proved that TNV o_ud PPV partially inhibited the electron transfer between QA and QR in the PSII complex in early stages, before visible symptoms o~ the inTection. TSWV, however, started to inh~bit the above processes only in wilting leaves.

P-24-037 ~

I

C

PAR~r~

OF VIRUS INF~'~u GRAPEVI~ IEAVES

(1)G~.V~radi, (2)B.B~Io, (3)E.Papp, (4)B.B~ddi, (5)D.Poly~k (1)Research Institute for viticulture and Enology, 60CO Kecskem~t, Kisf~i 182., (2)Res. Inst. Vitic. ar~Enol., 3301 Eger, P.O.B.83., (3)Plant Health and Soil Conservation Station, 6001 Kecskem~t, P.O.B. 171., (4)E~tv~s Lor~md University, 1445 Budapest, P.O.B. 324., (5)university of Horticulture and Food Industries, 1502 Budapest, P.O.B. 53., Hi,GARY Photosynthetic parameters were followed during the growing season of 1994 in a Vitis vinifera L. cv. Chardonna!1 vineyard artificially inoculated by Grapevine Fanleaf Virus (GFLV) in 1982. Net photosynthesis (measured by ICA3 Infrared Gas Analyzer) of green and partly yellow infected leaves was similar to that of control, however, the photosynthetic activity of wholly yellow infected leaves dropped to almost zero. Photosynthetic electron transport rate (tested by Hansatech PEA Flourometer) did not present similar dramatic drop in the case of yellow infected leaves. Low temperature fluorescence spectra of isolated thylakoids and the xanthophyll cycle also were studied. Preliminary results suggest that the complex effect of GFLV on the photosynthetic apparatus of Chardcnnall vine leaves appears not exclusively in the light reaction of the photosynthesis.

P-24-038 HEAT-INDUCED MODULATED CHLOROPHYLL A FLUORESCENCE CHANGES IN ATRAZINE-RESISTANT AND SUSCEPTIBLE BIOTYPES OF Conyza CaaadenMs (L.) Cronq. Sfmdor Dulai I , Istviin Mohutr I , Endre Lehoczki2 1 Botanical Dept., Eszterh~izy Teachers College, H-3301 Eger, POB, 43.; 2 Research Group of the Hungarian Academy of Sciences, Dept. of Botany, JATE University, 11-6701 Szeged, Hungary The effects of temperature on the yield of in vivo chlorophyll a fluorescence and CO2 fixation were measured in intact leaves of atrazine-resistam and susceptible biotypes of Conyza canadensis. Analysis of the temperature-dependence of the initial fluorescence emission (Fo) and of both the photochemical and nonphotochemical quenching clearly indicated an enhanced heat-sensitivity of the photosynthetic apparatus in the alrazine resistant Conyza canadensis leaves. The three critical temperature values of the Fo-T curves were 33_+0.6; 38.6+0.4; 44_+1.1 and 37.5_+0.06; 41.1_+0.6; 47.8_+1.1 °C for atrazineresistant and susceptible biotypes, respectively. The heat-induced changes in effective quantum yield and CO2 fixation correlated closely. The role of the xanthophyll cycle activity in the thermal stability of photosystem II is suggested. 215

Poster

P-25-003

Poster

session

THE CHLOROPLAST-NUCLEUS COOPERATIVE INTERACTION AND CHANGE OF PLANT MORPHOGENESIS CAUSED BY ETHEPHON T. Selga & M. Selga Institute of Biology, 3 Micra st. Salaspils LV- 2169, LATVIA

25

Cucumber (Cucumis saavus L.) plants were sprinkled with camlx:rsan M (ETHEPHON) in a concentration of a 4x10-3M (after ETHEPHON). This caused inhibition of plant growth, overaecumulation of starch and plastoglobules in chloroplasts, and reduction of thylakoids in the mesophyll of mature leaves of the main stalk 2-7 days after the treatment. Afterwards in these cells, disappearance of starch from chloroplasts, renewal of their fission and increase of heteromorphysm/heterogeneity of the fund of chloroplasts were typical, determing further promotion of branching and blossoming of plants. During the change of growth phases, the sticking of chloroplasts around nuclei which contained large nucleoli in the form of a "mushroom" in the periphery of nuclei were observed. The "legs" of nucleoli were directed towards the chloroplasts comaeting the nuclei. The narrow space between organelles was fulfilled with ribosomes. This structural chain serves for a common reversion of nuclear and plastidal cycles, quicker two way exchange of macromolecules between the nucleus and chloroplast, and repair of damaged thylakoid membranes.

Intra- and inter-cellular exchanges P.25-001

- P-25.018

P-25-004

P-25-001 Binmaas pm-Utioning and carbon al]ocaUon i n tnmsgenle tobacco plants expressing the movement protein of tobacco mosaic virus as influenced by environmental stress factors. Suchandra Balachandran I, Richard J Hull2 , Shmuel Wolf3 , Yoash Vaadia 4, william J Lucas 1 Iplant Biology, Universityof Callfornla, Davis, CA 95616; 2 Plant Sciences, University of Rhode Island, Kingston, RI 02881; 3 Vegetable Crops, & 4 Botany, The Hebrew University of Jerosalem, Rehovot 76-100, Israel. IYansgenic tobacco plants expressing the movement protein of tobacco mosaic virus (TMV-MP) show a reduction in blomass partitioning to roots and decreased rates of export of photoassimflates from the mature leaves during the day. We imposed various environmental stresses that are known to alter biomass partitioning and carbon allocation within plants, in order to understand the ways in which the TMV-MP alters biornass partitioning in transgenic plants. Drought stress overrides the effects of the MP and restores partitioning of biomass to shoots and roots to that of vector control plants [increase in root -to-shoot ratio). However, a nitrogen independent effect of the movement protein was observed in nitrogen stress studies. Also, light and photoperiod affect the ways in which TMV-MP alters biomass partitioning. We propose that the TMV-MP interferes with a signal trafficking pathway via plasmodesrnata that may be involved in the integration of carbon allocation between sink tissues. The mechanism(s) through which TMVMP could mediate its effects on carbon partitioning wil] be discussed.

P-25-002 ULTRASTRUCTURAL EVIDENCE OF EFFLUX OF LIGHT R E A C T I O N PRODUCTS F R O M T t i Y L A K O I D S A V O I D I N G STROMA M. Sel$~.& T. Selga Institute of Biology, 3 Micra st. Salaspils LV- 2169, LATVIA The prevalence of the light reactions, chloroplast supersaturation with starch g a i n s and plastoglobules, growth inhibition and promotion of blossoming was induced by cultivation of cucumber (Cucumis saavus L.) plants at high irradiance (250 W/m 2 for 16h) and lowered root nutrition (Helriegel's solution with 0,1 NPK). The decreased membrane system of chloroplasts was studied in leaf mesophyll of 30 d-old plants. The tips of stroma and grana thylakoids enlarged resembling dictyosome cisterns and were observed close to: 1) electronmicroscopically light compartment of saccharides encircling the starch grains, 2) plastoglobules, 3) chloroplast envelope. That suggests several pathways of effiux o f light reaction products and the chloroplast membrane decay products from the surface and interior of thylakoids. They can enter into saccharide, protein and lipid depots of chloroplast, as well as into vacuoles and cytosol, avoiding the stroma directly or by various forms ofpinocytosis.

216

MOLECULAR PROPERTIES OF iON TRANSPORT PROCESSES IN THE CHLOROPLAST iNNER ENVELOPE MEMBRANE. ~. FUK$ and F. HOMBLE. Plant Physiology C.P. 206/2, Universit~ Libre de Bruxelles, B-1050 Brussels, Belgium. The inner envelope membrane of chloroplasts (IEM) is a selective barrier for solutes. We have investigated the diffusion of ions through the glycolipid matdx and ion channels of the IEM. The contribution of glycolipids to the IEM permeability has been obtained from electrical measurements in planar lipid bilayers (PLB) (Fuks and Hombl~, Biophys J 1994,66:1404). Their PH* value is extremely high as compared to PK* and Per and indicates that the lipid matdx could be the pathway for H+ diffusion across the IEM. Our data suggest that free oleic acid produced in the chloroplast cannot be an efficient H+ carrier but that the voltage.dependent transport could occur by H+ jumps along water wires in the chloroplast lipid matrix. Lightscattering experiments with swollen intact chloroplats in the presence of valinomycin have been performed to characterize a highly selective CI" transporter in the IEM. We describe its selectivity, temperature-dependenco and inhibition by ZnSO4. Two ion channels were also reliably observed with purified IEM vesicles reconstituted into PLB. One channel has a conductance of 130 pS in 150 mM KCI and is K+ selective. The molecular properties of a weakly anion selective channel were also characterized (Fuks and Hombl6, J Biol Chem, in press). Its conductance of 525 lOS in 150 mM KCI increases linearly with salt conductivity. The channel shifts to a longlived substate at positive potentials and is mainly closed at negative potentials. The sucoinylation of the protein modifies its gating and reverses its selectivity. Our results indicate that this protein is a porin-like channel.

P-25-005 EFFECTS OF TEMPERATURE ON EXPORT AND INTRACELLULAR PARTITIONING OF 14C-PHOTOASSIMILATES. B. Grodzinski,& Y. Lee, Univ. of Guelph, Guelph,

N1G2W1, Canada.

Protoplasts of m e s o p h y l l cells form exporting fully e x p a n d e d leaflets of Pisum s a t i v u m cv. I m p r o v e d Laxton's Progress have photosynthetic rates of 55, 112 and 140 # m o l e s ~4C-fixed" m g chl ~- h 1 at 15, 25 and 35°C, respectively. Release of 14C-photoassimilates, primarily ~4C sucrose, at 15, 25 and 35°C was 8%, 18% and 26% respectively of the ~4C assimilated during 20 min. Protoplast intactness was over 90%. The subceUular distribution of ]*C-assimilates a m o n g chloroplast, mitochondria and cytosol show that from 15 to 35°C more ]4C-sucrose was "exported" from the protoplasts w h i l e more was recovered in the cytosolic and vacuolar fractions. Less ~4C was sequestered in chloroplast starch and more ]4C was in organic and amino acids (eg., ]4C-serine) in mitochondria and cytosol indicating that increased flux of C & N through the photorespiratory pathway occurs at increased leaf temperatures supporting higher export rates.

Poster

P-25-006

P-25-009

THE 2-OXOGLUTARATE/MALATE TRANSLOCATOR OF C4 BUNDLE-SHEATH MITOCHONDRIA

MOLECULAR STUDIES ON THE REGULATION OF THE NITRATE/ NITRITE TRANSPORTER OF SYNECHOCOCCUSSP. PCC7942.

M. Tallieuchi and T. Sugiyama Dept. o f Appl. Biol. Sci., Sch. of Agric. Sci., Nagoya Univ., Nagoya 464-01, Japan

T. Omatal, M. Kobayashi 1, R. Rodriguez2, M.G. Guerrero 2, C. Lara 2 1Dept. Applied Biol. Sci., Sch. Agric. Sci., Nagoya Univ., Chikusa, Nagoya, 464-01 Japan; 2Inst. Bioquim. Vegetal y Fotosintesis, Fac. Biol., Univ. Sevilla-CSlC, Apdo. 1113, 4 1 0 8 0 Sevilla, Spain

The bundle-sheath mitochondria in NAD-malic enzyme-type CAplants are functionally differentiated into photosynthetic organelles, containing some enzymes that operate in the (24 dicarboxylate cycle. In these mitochondria, high capacities of translccators are required to accommodate the high rates of exchange of metabolites needed in photosynthesis. We have isolated cDNA clones for 2-oxoglutaratodmalate translccator (OMT) from leaves of Panicum miliaceum (proso millet), an NAD-malic enzyme-type CAplant. The deduced amino acid sequence was highly homologous with that of bovine mitochondrial OMT. Western analysis showed that the OMT proteins were localized in mitochondrial membrane. The OMT protein expressed in E. coli showed preferential transport of malate and 2-oxoglutarate. The steady-state level of OMT mRNA was much higher in green leaves compared to non-photosynthetic tissues. The mRNA that corresponded to the obtained eDNA was accumulated only in bundle sheath cells and its expression was light-inducible in a similar manner with other genes for photosynthetic carbon assimilation enzymes. These findings suggested a possibility that mitocbondrial OMT in P. miliaceum has a role as a translccator in CAphotosynthetic carbon assimilation.

P-25-007 IDENTIFICATION AND LOCALIZATION OF THE ENVELOPE PROTEINS FROM SPINACH CHLOROPLASTS M. Yoshio, Y. Kashino, H. Koike, K. Satoh Dept. Life Science, Fac. Sci. Himeji Inst. of Teeh., Harima Science Garden City, Ako, Hyogo 678-12, Japan The chloroplast envelope functions not only as a simple barrier to separate between stroma of chloroplast and cytoplasm, but is also the site of the transport of inorganic ions and metabolites between chloroplast and cytoplasm. The envelope proteins of which functions have been identified so far are very few, while the amino acid sequence data of the envelope proteins whose functions are not elucidated are accumulating rapidly due to quickly developed techniques of molecular biology. However, assignment of some proteins is not clear yet. We thus separated and isolated the outer and inner envelope membranes by linear density gradient centrifugation and examined the distribution of some particular proteins by SDS-PAGE. We also identified some proteins by N-terminal amino acid sequencing and allocated them to the particular membranes. The results will be discussed focusing on the location of the particular proteins.

P-25-008 sLrBCELLULAR COMPARTMENTATION OF METABOLITES IN PLANT CELLS

T h e n i t r a t e / n i t r i t e t r a n s p o r t e r of Synechococcus sp. PCC7942 is a n ABC t r a n s p o r t e r e n c o d e d by t h e f o u r g e n e s nrtABCD, w h i c h is u n i q u e a m o n g the prokaryotic ABC t r a n s p o r t e r s in h a v i n g a n extra d o m a i n linked to one of t h e ATP-binding s u b u n i t s (C-term i n a l d o m a i n of NrtC). A m u t a n t (NC2) lacking the C-terminal d o m a i n of NrtC utilized low concentrations of nitrate a n d nitrite, s h o w i n g t h a t t h e extra d o m a i n is n o t r e q u i r e d for t h e t r a n s p o r t of t h e s u b s t r a t e s . While n i t r a t e / n i t r i t e t r a n s p o r t b y w i l d - t y p e Synechococcus was i n h i b i t e d u p o n a d d i t i o n of a m m o n i u m to t h e c u l t u r e m e d i u m , t h e t r a n s p o r t by NC2 was only partially inhibited by a m m o n i u m . These findings s h o w e d t h a t t h e C-terminal d o m a i n of NrtC is involved in the a m m o n i u m - p r o m o t e d inhibition o f t h e n i t r a t e / n i t r i t e transporter.

P-25-010 ADAPTATION OF POTATO PLANTS TO AN ANTiSENSE INHIBITION OF BOTH THE ADP-GLUCOSE PYROPHOSPHORYLASE AND TRIOSEPHOSPHATE TRANSLOCATOR ~). Heineke 1 A. Hattenbach 1, B. MC=ller-Rober2 & H. W. Heldt 1 Institut fflr 13iochemie_der Pflanze, Untere Karsp01e 2, D-37073 G6ttingen, Germany;Zlnstitut for genbiologische Forschung, Ihnestr. 63, D-14195 Berlin, Germany The inhibition of the triosephosphate-transtocator (TPT) in potato plants lead to redistribution of phosphorylated compounds in leaf cells, which usually inhibits photosynthesis and favour starch formation (D. Heineke et al., Planta 193, 1994, 174-180). However, as the photosynthesis rate under growth conditions and the tuber yield were unaltered, one possible explanation was that assimilates were transiently stored as starch, degraded hydrolytically and exported from the chloroplast as glucose. To proof this hypothesis, an antisense inhibition of the ADP-glucose pyrophosphorylase (AGPase) was added to TPT antisense plants (AT plants). In these AT plants the starch content was correlated to the degree of inhibition of the AGPase. In the strongly inhibited plants the chlorophyll content was reduced and the photosynthesis rate in saturating light and CO 2 was identical with the photosynthesis rate under growth conditions indicating a limitation of photosynthesis caused by reduced triose phosphate utilisation.

P-25-011 THE MEMBRANE OF LEAF PEROXISOMES CONTAINS A PORIN-LIKE CHANNEL MEDIATING THE PASSAGE OF THE PHOTORESPIRATORY METABOLITES

K. Leidreiter t, A. Kruse 1, B. Rienst, H. Winter, D. Robinson2, D Heineke x & H.W.Heldt 1 llnstitut fur Biochemie der Pflanze der Universitat, Untere Karsptile 2, 37073 GOttingen, Germany, 2Pflanzenphysiologisches Institut der Universititt, Untere Karsp01e 2, 37073 GOttingen, Germany

Si.qrun Reumann 1, E. Maier2, R. Benz2, H.W. Heldt 1 1 Institut•r Biochemieder Pflanze, UniversitBtGOttingen,Untere Karsp01e2, D37075 G0ttingen, F.R.G.2 Lehrstuhlfor Biotechnologie,Theodor-Boveri-lnstitutder Universit~ttWBrzburg,Am Hubland,D-97074WBrzburg, F.R.G.

In order to reveal general principles concerning the subcellular compartmentation of metabolites in leaves, we analyzed in leaves of barley, spinach and potato the distribution of major metabolites by means of nonaqueous fractlonation in the cytosolic, chloroplastic and vacuolar fraction of the frozen and lyophilized leaf material. By morpbometric analysis we determined the volumes of the various subcellular compartments in the above mentioned leaves. The corresponding data will be presented. From these results, the concentrations of various metabolites in the different subcellular compartments have been determined. A comparison of the data shows for the different lants a general distribution pattern, As to be expected, nitrate was found to e almost exclusively located in the vacuole. Of amino acids the concentrations in the vacuole are far less than in the cytosol and in the plastids. Whereas sucrose was found to be accumulated in the cytosol, the hexoses were found to be almost totally sequestered in the vacuole. The implications of these findings are discussed.

The results are conflicting as to whether peroxisomes of yeast and rat liver contain a pore-forming protein or specific translocators for the transport of metabolites. We decribe for the first time a channelforming protein in the membrane of leaf peroxisomes (Spinacia oleracea L.). This channel is distinctly different from the porin channels of leaf mitochondria and chloroplasts. In lipid bilayer experiments the porin-like channel reveals a single channel conductance of 0.35 nS (in 1 M KCI), is slightly anion-selective and not voltage-regulated. It allows the passage of the metabolites of the photorespiratory metabolism. The molecular identity of the poreforming protein is not known yet. We show our recent results of protein purification by hydroxyapatite chromatographie and gel filtration

~

217

Poster

P-25-012 TRANSPORT OF PHOSPHATE AND SUGARPHOSPHATES ACROSS THE ENVELOPE MEMBRANES OF TOMATO (L YCOPERSICON ESCULENTUM) FRUIT PLASTIDS

P-25-015 PHOTOSYNTHESIS, PHOTOSYNTATE TRANSPORT AND S T O R A G E IN SPECIES OF SUBARCTIC ZONE, A.V.Siutkina, Y.V.Gamalei Botanical Institute RAS, 198376, Prof. Popov str. 2, St.-Petersburg, Russia.

Danja Schtinemann, Sieglinde Borchert, Hans Walter Heldt Institut ~ r Biochemie der Pflanze, Untere Karspiile 2, 37073 GOttingen The phosphate translocators of envelope membranes from various plastids are different with respect to their substrate specificities. Tomato fruits are a suitable object for experiments with plastids undergoing a developmental process. Envelope membranes have been isolated by sucrose density centrifugation starting from a total membrane homogenate. For transport experiments membrane proteins were reconstituted in liposomes preloaded with Pi, G6P, G1P, DHAP and 3-PGA. A transport ofPi, DHAP and 3-PGA was observed with liposomes containing envelope proteins from tomato leaf, fruit chloroplasts and chromoplasts. Additionally, fruit plastids transport hexosephosphatas in counterexchange with Pi, indicating that they contain a hexosephosphate translocator. First experiments to elucidate the structure of the different phosphate translocators in leaf and fruit plastids indicate, that fruit plastids possess beside a hexosephosphate translocator also the ,,classical" phosphate translocator present in leafmesophyll chloroplasts. "

The specifity of photosyntesis/photosyntate transport/storage interaedons of different groups of plants of Cola peninsular are studied. Two types of balance in cold climate conditions can be distinquished. Low temperature does not stop photoassimilate translocation via apoplast. Apoplestic species (the majority of species in flora of subarctic region) are charaeterised by comparably low levels of starch accumulation in mesophyll (starch grains occupy usially not more then 30-40% of chloropast volume), the photosyntesis rate is enough high. In symplastic species low temperature affects strongly the transport part of the balance, closing the plasmodesmatal connections between cells, so the photosyntates have to be stored mainly as starch, which reaches 60-70% volume of mesophyll chloroplast causing the depression of photosynthesis. While low temperature suppresses photosyntesis in apoplastic species only directly (slowing all chemical reactions), the disfunction of intercellular connections make the additional limitative factor (feedback effect) in symplastic species.

P-25-013

P-25-016

THE E24 PROTEIN OF SPINACH CHLOROPLAST ENVELOPE OUTER MEMBRANE

A COMPARISON OF THE RELATIVE RATES OF TRANSPORT OF ASCORBATE AND GLUCOSE ACROSS THE THYLAKOID CHLOROPLAST AND PLASMALEMMA MEMBRANES OF THE PEA

Emeline Teyssier, Maryse A. Block, Roland Douce & Jacques Joyard Laboratoire de Physiologie Cellulaire Vtgttale, URA CNRS 576, Dtpartement de Biologie Molteulaire et Structurale, Centre d'Etudes Nueltaires de Grenoble et Universit6 Joseph Fourier, 38054 GRENOBLE cedex 9, France.

LEAF MESOPHYLL

E24 is one of the most abundant protein of the outer membrane of chloroplast envelope, however its function is still unknown. This protein has been previously shown to be accessible from the cytosolic side of the chloroplast by immunoagglutination and immunofluorescence analyses of intact chloroplasts. In addition, this protein is susceptible to thermolysin treatment of isolated intact chloroplasts. A eDNA encoding E24 has been obtained by screening an expression library of spinach eDNA with polyclonal antibodies. Analysis of the protein sequence and comparison with the thermnlysin susceptibility of E24 in the chloroplast in situ suggest that E24 is a transmembrane protein and show that its NH2 terminal end is located on the cytosolic side of the chloroplast. Comparative analysis of the protein sequence with databases and studies of the protein expression in the plants are in progress in order to enlighten the function of this protein.

P-25-014 CO2-UPTAKE BY INTACT LEAVES INDICATES REGULATION OF THE CHLOROPLAST STROMA

FAST

PH-

M. Hauser 1, H. Eichelmann2, V. Oja2, A. Lalsk 2 & U. Heber 1 lJ.-v.-Sachs-Institut, Mittl. Dallenbergweg 64, 97082 W0rzburg, Germany 2Chair of Plant Phys., University ofTartu, Riia 181, EE2400 Tartu, Estonia Intact leaves of C3 plants (Brassica oleracea, Helianthus annuus and Nicotiana rustica) were exposed for 20 s to CO2 (0.5 to 16%) to simulate cellular acidification for the investigation of pH regulation, pH values were calculated from the bicarbonate accumulation in the stroma where carbonic anhydrase is located. Buffer capacities in the chloroplast stroma were much higher than expected. Proton consumption by rapid bicarbonate formation of darkened leaves ranged between ca. 35 and 60 mM when the CO2 concentration in the gas phase was 16%. It was increased up to 120 mM by illuminating the leaves and by prolonging the exposure times. Repeated titrations of leaves with CO2 decreased apparent buffering in the dark, but not in the light. The observations indicate that under acid stress protons are not only consumed by chemical buffering but are also rapidly (within seconds) exported from the chloroplasts, apparently in exchange for cations which serve as counterions for bicarbonate. This transport occurs first across the chloroplast envelope and then, obviously, also across the tonoplast of leaf cells.

218

Christine H Foyer1 and Maud Lelandais2. 1Department of Environmental Biology, IGER, Plas Gogerddan, Aberystwyth, Dyfed SY23 3EB, UK; 2Laboratoire du M6tabolisme, INRA, Route de Saint-Cyr, 78026, Versailles Cedex, France. Mesophyll protoplasts, chloroplasts and thylakoids were prepared from young pea leaves. Transport of ~4C-ascorbate and ~4C-glueose across the plasmalemma, chloroplast envelope and thylakoid membranes was measured in the presence or absence of dehydroascorbate. The plasmalemma and chloroplast envelope membranes transport both ascorbate and glucose but the thylakoid membranes appear to have no transport system for either compound, permeability resulting from diffusion alone. Dehydroascorbate inhibited ascorbate transport but not glucose transport across the chloroplast envelope membrane. Similarly no effects of ascorbate on glucose transport or glucose on ascorbate transport were observed suggesting completely independent carriers for these compounds into chloroplasts. Unlike animal membranes where the glucose transporter has also been found to transport dehydroascorbate the plant counterpart does not, neither in the chloroplast envelope or plasmalemma membranes.

P-25-017 GENETIC MODIFICATION PERMEABILITY

OF

PLASTID

ENVELOPE

Kerry J. Blissett and John C. Gray Department of Plant Sciences, Downing St, Cambridge CB2 3EA. U.K. Starch is synthesized by higher plant plastids to sequester carbon fixed during photosynthesis. In chloroplasts synthesis occurs during the day when photosynthesis is maximal. At night starch is broken down to triose phosphate and exported to the cytosol via a triose phosphate:phosphate translocator in the plastid inner membrane. After translocation to the non-photosynthetic parts of the plant such as tubers, endosperm and roots, hexose phosphates, principally glucose 6-phosphate and glucose 1-phosphate, are selectively taken up by amyloplasts for starch production. The export of photoassirailate to the cytosol in photosynthetic tissues and the import of hexose phosphates into amyloplasts may be pivotal with respect to carbon partitioning between organs and biosynthetic pathways. To investigate how changing plastid envelope permeability would perturb this relationship we have created a chimeric gene construct for targeting a bacterial glucose 6-phosphate:phosphate translocator protein to the plastid inner membrane. Constructs have been made which encode the triose phosphate translocator import signal, envelope targeting signal and the bacterial translocator. The constructs are being used for expression in vitro in chloroplast uptake studies and in vivo in the production of transgenic tobacco and potato for metabolic studies.

Poster P-25-018 NADPH FLUORESCENCE IN I N T A C T C H L O R O P L A S T S : DETECTION, KINETICS AND ENHANCEMENT EFFECT. M. Broglia Ente per le Nuove Tecnologie, l'Energia e l'Ambiente (ENEA), Centro Ricerche Energia Casaccia, Settore B i o t e c n o l o g i e e Agricoltura SP026, P.O. Box 2400, 00100 Roma A.D., ITALY Laser induced fluorescence of NADPH has been detected in thylakoids, chloroplasts and intact leaves, simultaneously with chlorophyll fluorescence (Cerovic et al., 1993, Photosynth. Res., 36 193-204). In this work a frequency modulated Argon ion laser is used to probe NADPH in active chloroplast suspensions. Possible artifacts due to the concentration and quality of chloroplast suspensions and to the laser probe characteristics are considered and operation in the linear dynamic range is strictly observed. Kinetics of the NADPH fluorescence signals in different experimental conditions are detected on-iine, together with chlorophyll fluorescence, and compared to the published biochemical results on the redox state of the NADP/NADPH chloroplastic pool (Takahama et al., 1981, Biochim. Biophys. Acta, 637 530-539). With this high sensitive technique and very diluted, high quality chloroplast suspensions the E m e r s o n enhancement on NADPH production in intact chloroplasts is detected for the first time.

219

Poster

P-26-003 IN VIVO K . FOR CO2 0Kp) OF PHOSPHOENOLPYRUVATE CARBOXYLASE (PEPC) AND MESOPHYLL CO2 TRANSPORT RESISTANCE ( r , ) IN LEAVES OF ZEA MAYS L. P o s t e r s e s s i o n 26

M. Pfeffer & M. Peisker Institut for Pflanzengenetik und Kulturpflanzenforsehung, D-06466 Gatersleben, Germany

C O 2 diffusion and concentration mechanisms

Enzyme parameters are important for models of C4 photosynthesis. Since extraction distorts factors relevant for enzyme activity, data measured in vitro do not necessarily reflect the in v/vo situation. However, Kp and, in addition, rm can be derived from combined in vitro PEPC assays and gas exchange measurements (Peisker et al., 1988, Biochem Physiol Pflanzen, 183 351-354). The method assumes that intraceUular resistance (rl) equals the sum of mesophyll resistance, and carboxylation resistance (r=), where ro corresponds to the fraction Kp/Vp (Vp, maximum PEPC activity). Hence, rl = rm+(Kp/Vp). Zea mays L. plants were adapted to a range of light intensities. The correlation between ri and 1/Vp was examined by measuring maximum PEPC activity in crude enzyme extracts and the initial slopes of the relationship between net CO2 uptake rate and intercellular CO2 concentration of leaf segments. The slope and intercept of first order regression corresponded to lump values of Kp and r=, respectively. To consider the substrate specificity of PEPC, CO2 gas phase concentrations were related to cytosolic bicarbonate. The validity of a uniform Kp value for plants under different growth conditions and altered measuring environment is discussed. Further, the obtained ratios of r , to r~ are critically evaluated.

P-26-001

- P-26-024

P-26-001 Effect of dissolved inorganic carbon on oxygen evolution and f l u o r e s c e n c e quenching by Selenastrum minutum adapted to ambient air and CO2-enriched air E. D e m i d o v & D. C a n v i n , * tnst.of Soil Sci.& Phot., Pushchino,142292, Russia; *Dep.of Biol. Queen's Univ.,Kingston K7L 3N6, C a n a d a . Addition o f DIC a t t h e CO2 c o m p e n s a t i o n point to highCO2 c e i l s resulted in a increase in non-photochemical quenching (qn} which peaked at 0.5-1 mM D I C , w h e r e a s the addition of DIC to low-CO2 cells caused a suppression of qn. Examination of kinetics of relaxation o f qn e i t h e r upon turning off illumination or after pretreatment with nigericin showed that the m a i n c o m p o n e n t o f qn i n low-CO2 c e l l s was the energy dependent quenching (qe). The h i g h a f f i n i t y f o r DIC and the induction of changes i n qn w e r e c o m p l e t e l y induced within 4-7 h after transferring high-CO2 ceils to ambient air. Mass-spectrometric measurements of 02 exchange and effect of [02] a n d GA ( i n h i b i t o r of Calvin cycle) indicated that oxygen photoreduction was not responsible for the changes in fluorescence parameters. The v a r i a t i o n in fluorescence quenching in high and low-CO2 cells may b e d u e t o t h e o p e r a t i o n of cyclic electron flow, which could provide energy to t h e CO2 c o n c e n t r a t i n g m e c h a n i s m i n low-CO2 c e l l s .

P-26-002

P-26-004 T H E P Y R E N O I D STARCH SHEATH F O R M A T I O N IN H I G H COz-REQUIRING MUTANT O F CHLAMYDOMONAS REINHARDTll E. del Camoo, M.P. Plumed, M. Jimenez del Rio, G. Garcia-Reina & Z. Ramazanov Instituto de Algologia Aplicada, ULPGC/ITC, Muelle de Taliarte s/n, 35214 Telde, Las Palmas, Spain The effect of external CO~ concentrations on protein synthesis and the starch sheath formation around the pyrenoid in a wild type strain of Chlamydomonas reinhardtii was compared with that of a new high CO2-preferring mutant. 35SO4 labeled wild type and pyr-45 cells exhibited up-regulation of two polypeptides (42-45 kDa) when tranferred from high CO2 (5% CO2 in air) to low CO~ (0.03%) conditions. The wild type induced three new polypeptides (21, 36, 37 kDa), while no induction of these proteins occured in mutant cells. The K05(CO2) decreased when pyr-45 cells were switched from high to low CO2, but not to the extent of wild type cells. Immune-gold labeling showed that almost all of Ribulose-l,5-bisphosphate carboxylase/oxygenase was located in the pyrenoid in both wild type and mutant. A pyrenoid starch sheath appears in response to low CO2in wild type and inpyr-45. Results suggests that the low COs inducible polypeptides are not involved in the pyrenoid starch sheath formation in C. reinhardtii.

P-26-005

CARBONIC ANHYDRASE OF PROCHLOROPHYTES AND ITS ROLE IN PHOTOSYNTHESIS

I N D U C T I O N O F A CO z C O N C E N T R A T I N G M E C H A N I S M IN STARCHLESS MUTANTS O F M I C R O A L G A E .

M. L. Dionisio-Sese I , A. Shimada 2, T. Maruyama 2 & S. Miyachi 2 1 Institute of Biological Sciences, College of Arts and Sciences, University of the Philippines at Los Bafios, College, Laguna 4031, Philippines; 2Marine Biotechnology Institute Co. Ltd., Shimizu Laboratory, 1900 Sodeshi-c_,ho, Shimizu-shi, Shizuoka 424, Japan

M. P. Plumed E. del Campo, G. Garc~a-Reina & Z. Ramazanov Institute of Applied Algology, ITC, ULPGC, Muelle de Taliarte s/n, 35214 Telde, Las Palmas, Spain.

The presence of carbonic anhydrase (CA) in Prochloron and Prochlorothrix was studied. Both prochlorophytes exhibited CA activity largely associated with the cell surface. CA activity in Prochlorothrix was about haft of that observed in Prochloron. Both species exhibited the same sensitivity to the CA inhibitor ace~azolamide with 150 values in the range of 10-4 M, similar to the values observed for intracellular CA of green plants. Examination of the effect of acetazolamide on the rate of photosynthetic oxygen evolution of Prochloron at varying NaHCO 3 concentration revealed that the inhibitor decreased the apparent K1/2 for photosynthesis. The results indicate that CA on the cell surface increases the affinity for inorganic carbon during photosynthesis at low inorganic carbon concentration.

220

In a unicellular green algae, the starch sheath formation specifically arounfi the pyrenoid was observed in response to a decrease in the CO2 concentration in growth medium (from 5 to 0.03%). The pyrenoid starc.h sheath synthesis was shown to be correlated with the induction of a CO 2 concentrating mechanism (CCM). In this work, effect of the external CO2 concentrations on photosynthesis and the ultrastructural changes in wild type are compared with that of a starch-less mutant strain of the same microalgae. 35SO4 labeling and Western blot protein analysis showed that mutants and wild type ceils induced the same polypeptides under low CO2. A pyrenoid starch sheath appeared in response to low CO2 in wild type, but not in mutants. The functional role of the starch sheath around the pyrenoid in the CCM will be discussed.

Poster

P-26-006 OS TBB IM)SSIBI~ITT OF ~ I J ~ I C ~ IP]~2IFIC COa C O N C ~ T I O N ~ ZN ~ O P I ~ B T E OF C3 PIJLBTS

P-26-009 THE EFFECT OF DARK RESPIRATION ON CO2 COMPENSATION CONCENTRATION IN C3, C4, AND C~-C4 INTERMEDIATE PLANTS

N

L.E. Fridlvand Institute of Experimental Botany, Academy of Sciences of Belarus, Minsk, 220072, Belarus

M. Peisker l, P. Apel =& I. Tich/t2 tlnst, for Pfinnzengenetik und Kulturpflanzenforschung, D-06466 Gatersleben, Germany; 2Department of Plant Physiology, Charles University, Vinicn~ 5, CZ-12844 Praha 2, Czech Republic

Mathematical models are developed for functioning of CO2 concentration mechanisms (CCM) in mioroalgae. Cases for both the active bicarbonate transport through plasaualemHaa and passive c0a diffusion with subsequent concentration of CO2 inside the pyrenoid are analyzed. The possibility of existence of a specific CCM with the participation of membrane-bound carbonic anhydrases is computer simulated for chloroplast of terrestrial C3 plants. When this CCM operates, there is a passive C02 diffusion through chloroplast envelope with subsequent C02 concentration to the center of chloroplast (Fridljand and Kaler 1986, Gen. Physiol and Biophys, 6, 617-636). Decrease in the changes rate of photosynthesis and specific fluorescence concentration in were observed at supraoptimal COz several wheat species. Characteristic distribution of carbonic anhydrase in wheat chloroplast is shown. These results can be explained by the possible existence of the discussed CCM in wheat.

In photosynthesizing leaves, the CO2 compensation concentration (F) depends on processes related to CO2 fixation (to be expressed by a component Fp) but is also influenced by their interaction with dark respiration. Thus, F is assumed to be given by the equation F = Fp + ~ 'r~ "RD (rl = intracellular resistance for CO2 uptake, P,D = respiration rate measured in darkness, I~ = factor describing the extent to which dark respiration continues during illumination). In C3 plants, Fp is determined by the specificity of Rubisco for its substrates COs and O5. Significant correlations between F and the product rl "RD have been found in various species with different mechanisms of CO2 fixation. Values of I~ < 1 are interpreted as indicating an inhibition of dark respiration by light. However, with leaves of Polygonurn lapathifolium differing in insertion level p. > 1 was obtained. These results may be interpreted as due either to an enhancement of dark respiration by light or to changes in Fp, i. e. to shiRs in the relations between processes connected directly with CO2 fixation.

P-26-010

P-26-007 NET CO2 A S S I M I L A T I O N A N D INTERNAL TO CO2 IN TREE LEAVES.

CONDUCTANCE

D. Epron 1,2, B. Genty I & R. Liozon 1 1Lab. Ecologie V6g6tale, Univ. Paris Sud, Bat 362, 91405 Orsay ce0ex, France; 2Inst. Sci. Tech. Environ., P61e Universitaire, BP427, 25211 Montb61iard cedex

PHOTOSYNTHETIC ADAPTATIONS IN LEAVES POTAMOGETONACEAE (MONOCOTYLEDONS).

All members of the Potamogetonaceae are ubiquitous aquatic plants. In aqueous environments the photosynthetic yield may be limited by the availability of CO2, and functional adaptations are required to ~ v i v e this imposed stress. The plant habit (with respect to the partial or full submersion of the photosynthetic organs) may have some bearing in the modulation of the adaptative responses. The physiological mechanisms in the photosynthesizing cells may be supported by structural devices. A comparative study of photosynthetic behaviour and fine structure of the leaves has been made in P. nodosus Poiret which has floating leaves bearing stomata on the adaxial epidermis and P. crispus L., P. pectmatus L., Groenlandia densa L. with submerged leaves. These latter species share both physiological behaviour (they are typical "HCO 3- users") and structural specializations, aimed at improving the exchange between the external and intracellular environments. P.nodosus seems to behave as a species able to use both HCO 3" and CO 2 to carry out photosynthesis.

P-26-01l

P-26-008 THE FLUORESECENCE E X C U R S I O N AND DURING GAS TRANSIENTS

M. P i n t o & A. R i q u e l m e Fac. de Cs. A g r a r i a s y Forestales, C h i l e , C a s i l l a 1004, S a n t i a g o , C h i l e

Universidad

SOME

Baldan B., Zuppini A., Dalla Vecchia F., Rascio N. and Mariani P. Dipa~nento di Biologia, via Trieste 75, 35121 Padova (Italy)

Simultaneous measurements of chlorophyll fluorescence and leaf gas exchange were made on attached leaves in an open system and used to estimate the partitioning of photosynthetic electron flow between carboxylative and oxygenative processes. Using this approach, the apparent CO2/O2 specificity factors were estimated for the leaves of two deciduous tree species. These low values (50 M M -1) contrasted with those estimated for herbaceous species and were ascribed to a drop in CO2 molar fraction between the intercellular airspace and the catalytic site of Rubisco, due to a low internal conductance for CO2 (gi--0,1 tool m -2 s-l). The relative limitation of net CO2 assimilation which can be ascribed to internal resistance to CO2 transfer was of a same ordre of magnitude than those imposed by stomata (25-30 %).

RELATIONSHIP BETWE~ THE C02 ASSIMILATION

OF

de

T h e f l u o r e s c e n c e e x c u r s i o n i n d u c e d b y t h e r e m o t i o n of C02 (0F) a n d t h e C02 a s s i m i l a t i o n (A) w e r e a n a l y z e d in b e a n l e a v e s . M e a s u r e m e n t s of b o t h p a r a m e t e r s w e r e d o n e o n t h e s a m e l e a f at d i f f e r e n t l i g h t i n t e n s i t i e s , C02 concentrations and temperatures. At low light i n t e n s i t i e s 0 F w a s p r o p o r t i o n a l to A b u t w h e n A b e c a m e l i g h t s a t u r e d a n a b r u p t d e c l i n e in 0F w a s o b s e r v e d suggesting a shift in the mechanisms of energy dissipation. When inductions of ~F were done d e c r e a s i n g t h e CO2, b y s u c c e s s i v e f r a c t i o n s , f r o m i 0 0 0 to 0 ppm, a c l o s e c o r r e l a t i o n b e t w e e n 0F a n d A w a s also observed. Proportionality between both parameters was also mantained under different leaf temperatures. As a consequence variations in photosynthetic p a r a m e t e r s l i k e A m a x a n d t h e i n i t i a l s l o p e of t h e A / C i c u r v e w e r e w e l l r e f l e c t e d in t h e O F v a l u e s . T h i s l e a d to t h e c o n c l u s i o n that goods estimations of t h i s parameters can be obtained from OF excursions.

PHOTOSYNTHETIC PLANTS

BEHAVIOUR OF LEAVES AND STEMS IN SOME AQUATIC

F. Dalla Vecchia, A. Zuppini, P. Marianl & N. Rasclo Dipartlmento di Biologia, Unlversit~ di Padova, via Trieste 75, 1-35121, Padova, ITALY The behaviour of leaves and stems, in order to supply themselves with the different forms of inorganic carbon available in the aquatic environment to carry out photosynthesis, has been studied in an entirely submerged (Ranunc~oA / ~ e ~ o p ~ L.) and in three partially emerging ( C o ~ c h g 6~gn~ Scop., N~tw%_ tium officinale R. Br., Vcro~ga anagallis aquatica L.)plants. In the submerged species the leaves, devoid of stomata, show to be able to utilize both CO 2 and H C 0 3 present in the water. This capability is coupled with some ultrastruetural features of the epidermis cells, which exhibit a transfer-cell organization and a cell wall texture that improve the apoplastic flux and the water-cytoplasm exchanges. The submerged leaves of the three patti ally emerging species have stomata and do not show any ultrastructural specialization. They use the environmental CO 2 as the only source of inorganic carbon. The stems of all the studied plants can photosynthesize, but they essentially depend on their own gaseous C02, accumulated in the intercellular spaces of aerenchymatie tissues, to carry out this process.

221

Poster

P-26-015

P-26-012 CARBONIC

$ O L A ~ ] ~ ~ 0 ~ E R O S ~ M L. : PURIFICATION O F TWO C Q M P A R ~ N T - e P E c I F I C ISOFO~.

AN~ZDRASE

CIARACTERIEAT/ON

ZN

AND

D. Rumeau, S. Cuing, L. Fina & G. Peltier CEA-CNRS; Laboratoire d'Ecophysiologie de la Photosynth&se; DPVE; Centre de Cadarache; F-13108 Saint-Paul-lez-Durance; France

The enzyme Carbonic Anhydrase (CA, EC 4.2.1.I) catalyses the reversible hydration of CO2 to bicarbonate. CA has been involved in the early steps of photosynthesis in many photosynthetic organismes such as cyanobacteria, microalgae, C, and CAM plants. In C3 plants, many aspects of its localization and physiological role still remain to be elucidated. The intracellular compartmentation of CA has been investigated in potato leaves. A l t h o u g h enzyme activity was m a i n l y located in chloroplasts, significant activity was also identified in cytosol. The compartment-specific CA isoforms were purified to homogeneity and some biochemical properties of both isoenzymes were characterized. Antibodies raised against a synthetic polypeptide corresponding to the N-terminal aminoacid sequence of the chloroplastic isoform were obtained. The antibodies which cross-reacted with the cytosolic CA isoform were used for in~nunocytolocalization experiments. The distribution of CA in the stroma and cytosol of the potato mesophyll cells was confirmed.

P-26-013 INTRACELLULAR CARBONIC ANHYDRASE OF CHLAMYDOMONAS REINHARDTII Jan Karlsson, Thomas Hiltonen, H. David Husic, Zakir Ramazanov, G6ran Samuelsson Department of Plant Physiology, Ume/~University, S-901 87 Ume~, Sweden An intracellular Carbonic Anhydrase (CA; EC 4.2.1.1), was purified to homogeneity from cells of Chlamydomonas reinhardfii, (CW 92). Internal CA activity . sedimented with the membrane fraction after disruption of cells, and was dissociated after treatment with a Tris/HC1buffer pH8.2 containing 200rnM KC1. The CA was precipitated with (NH4)2SO4, and further purified by anionic exchange and hydrophobic interaction chromatography. The resulting fraction had a specific activity of 1260WAU/mg protein and was inhibited by acetazolamide with an 150of 12nM. Final purification was accomplished as a result of desalting and concentrating the sample with a Centricon-10 rnicroconcentrator. The retentate and eluate contained little CA activity. The filter with absorbed CA was rinsed either with SDS-PAGE sample buffer containing 0.1% SDS and then revealed a single polypeptide of 29.5kDa, or 50mM Tris/HC1 pH8.0 with 1.5M (NH4)2SOawhich gave an eluent displaying CA activity. N-temainal and internal amino acid sequences from the 29.5kDa polypeptide showed 67% identity in an intema/ conserved region, and 40% identity in the N-terminal region in comparison with human CAII. We postulate that the 29.5 kDa polypeptide is an internal CA in C.reinhardtii and that the enzyme is closely related to the s-type CAs.

P-26-014 CLONING AND CHARACTERIZATION OF ¢DNAs ENCODING CARBONIC ANHYDRASE FROM Populus tremula x tremuloMes Susanne Larsson 1, Olof Olsson2 and G6ran Samuelsson 1 1Department of Plant Physiology, Ume~ University, S-901 87 Ume~t, Sweden 2Department of Forest Genetics and Plant Physiology, Swedish Agricultural University, S-901 83 UmeL Sweden Carbonic anhydrase (CA; carbonate dehydratase, EC 4.2.1.1.) is a zinc metalloenzyme which catalyses the reversible hydration of CO2 to bicarbonate. Several cDNA clones encoding carbonic anhydrase were identified from a Populus tremula x tremuloides (hybrid aspen) leaf cDNA library, using a CA cDNA from Pisum sativum (pea) as a probe. Two different poplar CA cDNAs were isolated and sequenced. The DNA sequences studied differed from each other at 12 nucleotide positions. In addition, one of the clones was 74 nucleotides longer than the other in the non-coding 3"-region. A structural comparison of the deduced amino acid sequences from the poplar CAs with those of pea, spinach, tobacco and Arabidopsis thaliana showed a considerable degree of identity. These data, describing for the first time CA sequences from a tree, will be further discussed. Finally, northern blot analysis of mRNA isolated from poplar leaves displayed a transcript of 1300 bases hybridising to the poplar CA probe.

222

LEAVES USING ~O L a m ~

C ~ AR) kmSS S

~

L. Cournac, V. Despax, B. Dimon, L. Firm, D. R~reau & G. Peltler. CEA, Laboratoire d'Ecophysiologie de la Photosynth&se, DPVE, Centre de Cadarache, 13108 Saint Paul Lez Durance, France. Carbonic anhydrase (CA) activity of leaves was measured in vivo using mass spectrometry, by following the exchange of leO atc~s between 180-enriched CO2 and leaf water. A model of gas diffusion and exchange was used to interpret the evolution of CO2 isotopic species and allowed simultaneous estimation of CA activity and CO~ diffusion kinetics. The original model {Gerster 1970, Planta 97:155-172) was modified in order to account for situations where photosynthesis is active. CA activity values frcra in vivo measurements were consistent with in vitro assays of leaf extracts, and were affected in the same way by ethoxyzolamide, a specific inhibitor of CA. Experiments achieved either on intact or stripped (epidermis removed) leaves of Commelina and Pisum spp, showed that CA activity was unchanged during light to dark transients and was little affected by environmental conditions such as dehydration or ABA treatment. Data on CO2 diffusion resistance were co,oared to those obtained by usual methods involving measurement ef H20 and C O 2 fluxes. Analysis of i~O exchange kinetics appears as an original method to follow the in vivo activity of an enzyme and as an alternative method to study 002 transfer within leaves.

P-26-016 PHOTOAFFINITY LABELLING OF CHLAMYDOMONAS REINHARDTII W I T H A C A R B O N I C A N H Y D R A S E - D I R E C T E D P H O T O A F F I N I T Y LABEL. H. David Husic I, Heather H. Chalfant =, Stephanie Y. Hugghins2 and James V. Moroney2 Department of Chemistry, Lafayette 'College, Easton, PA 18041 USA~ and Dept. of Plant Biology, Louisiana State Univ., Baton Rouge, LA 70803, USA~. A carbonic anhydrase (CA)-directed photoaffinity label, 12Sl-(N-(paminomethylbenzenesulfonamide)-4-azidosalicylamide, specifically labels an insoluble intracellular 30 kDa polypeptide in C reinhardtii. The analysis of mutants deficient in efficient C, utilization has verified that the labeled 30 kDa polypeptide is not observed in a variety of lines believed to be deficient in intracellular CA based on physiological analyses. Photoaffinity-labeling of the 30 kDa polypeptide in (7. reinhardtJi extracts is inhibited by ethoxyzolamide, with an Is0of about 0.2 IJM. Furthermore, the 30 kDa photoaffinity-labeled polypeptide is localized within isolated chloroplasts from C reinhardtii cells. These results provide support for the identification of the labeled 30 kDa polypeptlde as a chloroplastic CA that is an important component of the C~ concentrating system in C reinhardtii. Supported by NSF grants DMI~9117563 (HDH) and IBN-9304662 (JVM).

P-26-017 IDENTIFICATION OF TWO PROTEINS INVOLVED IN REGULATION OF INORGANIC CARBON UPTAKE IN THE CYANOBACTERIUM Synechocystis PCC6803. S.B~du, M. Hisbergues, P. Pozuelos and F. Joset. U. M~tabolisme Energ~.tique LCB-CNRS, 13402 Marseille cedex 20, France. Growth of Synechocystis PCC6803 in limiting inorganic carbon (Ci) leads to the establishment of a high affinity Ci uptake system. A mutant impaired in this system, but still capable of growth under low Ci conditions, was isolated. Two ORFs, possibly organized in an operon, were identified in the wild-type genomic region corresponding to the mutation site. Deletion of the downstream gene (hatR) gives rise to a phenotype close to that of the original mutant. The deduced HatR protein, which possesses characteristics of the E. coli CbeY sub-family of response regulators, might be the regulator of a twocomponent regulatory system controlling activity of the high affinity transport process. Its phosphorylation capability will be presented. Deletion of the second ORF (hatA) prevents the strain to grow under Ci limiting conditions. The deduced HatA protein showed no significant homology with any known protein; it possesses a putative signal peptide. The mature form of the protein, its cellular localisation and its role will be detailed.

Poster P-26-018 PYRENOID MORPHOLOGY IN RUBISCO AND CO2 CONCENTRATING MECHANISM MUTANTS OF CI-II..AMFDOMONAS REINHARDTII

Margaret C. Heak1, Mamta Rawat2, Stephanie Y. HugghinsL3, Lara L. Lavigne"3, Ziyadin Ramazanov2, Catherine B. Mason= and 1Department of Microbiology, 2Departmeat of Plant Biology, 3Department of Biochemistry, Louisiana State University, Baton Rouge, LA 70803, USA The pyrenoid is a roughly spherical structure residing within the chloroplast of Ch/amydomonas re/nhardz/i. It is the site of CO2 fixation, and previous immunoloc~liT~fion studies have shown that most of the cell's rubisco is localized within it. Transmission electron microscopy reveals that the wildtype pyrenoid consists of an anastomosing, tubular, membranous network with internal elements, all of which is embedded in a matrix of granular material. We have compared wild-type pyrenoid ultrastructure with that of pyrenoids from a number of mutant strains, including some with mutations in the chloroplast rbcL gene as well as some without a functional CO2 concentrating mechanism. Ultrastructural differences range from changes in the starch sheath surrounding the pyrenoid, to alteration of the pyrenoid proper, to the complete absence of the pyrenoid from the cell.

P-26-021 ESTIMATION OF INTERCELLULAR CO2 CONCENTRATION DURING NON UNIFORM LEAF PHOTOSYNTHESIS BY MAPPING OF PHOTOCHEMICAL YIELD OF PSlI.

S. MEYER & B. GENTY Groupe "Photosynth6se et Environnement" Laboratoire d'Ecologie V~j6tale, bat. 362, Universit0 Paris XI, CNRSURA 1492 ; 91 405 Orsay cedex (FRANCE) Umitation of photosynthesis is usually investigated by the relationship between net CO2 assimilation (A) and intercellular concentration (C|).Validity of Ci calculated from gas exchange measurements has bean questioned when photosynthesis is spatially heterogeneous. A valid Ct has been estimated using map of local C= computed from images of photochemical yield of photosystem I1. When non uniform photosynthesis was induced by feeding a Rose leaf with abscisic acid (ABA), the A vs valid C= relationship remained unchanged. This result shows that ABA primarily affects conductance to CO2. The origin of the overestimation of Ci calculated from gas exchange during patchy photosynthesis is discussed.

Supported by the National Science Foundation

P-26-019

RIBISCO, PEPCarboxylaseAND THE plt-POIJ~REACTION: THEIR ROLE IN PHOTOSYNTHETICBICARBONATEUTILISATION IN Elodea canadensis

Lucina C van Ginkel, Ingeborg Sch~tz and Hidde BA Prins, Univ. of 6roningen, Dept. of Plant Biology (Ecotrans), Kerklaan 30, 9750 AA Haren, The Netherlands. Availability of inorganic carbon and low light intensities are often a problem for submerged aquatic macrophytes. To overcome the problem of inorganic carbon shortage for photosynthesis C02 concentrating mechanisms are found in several species. Examples are a C, like metabolism, a pHpolar reaction, apoplastic and cytoplasmetic carbonic anhydrase a c t i v i t y or direct bicarbonate uptake. Here we show that for Elodea ¢anadensfs three of these mechanisms are active under low CO~conditions: The pHpolar reaction, apoplastic carbonic anhydrase a c t i v i t y and a low RUBISCO/PEPCasea c t i v i t y ratio. The RUBISCO/PEPCaseratio increases i f plants are grown under low nutrient levels, whereas the pH-polar reaction is not affected.

P-26-022 ANALYSIS OF SPATIAL HETEROGENEITY OF LEAF CO2 ASSIMILATION DURING INDUCTION OF PHOTOSYNTHESIS BY LIGHT.

E. BRO, S. MEYER and B. GENTY Groupe "Photosyntldme et Environnement" Laboratoire d'Ecologie V(~j6tale, bat 362, Universit6 Paris XI, CNRSURA 1492 ; 91 405 Orsey cedex (FRANCE) Spatial distribution of photosynthesis was analysed during light-dark transition using chlorophyll fluorescence imaging. Images of leaf photochemistry displayed a large heterogeneity of photosynthesis during the early stages of induction which progressively disappeared to become evenly distributed after a duration varying upon conditions and species. Similar behaviour was seen in photorespiratory and nonphotorespiratory conditions at low irradiances. The attribution of this asynchronous induction to a heterogeneous stomatal response or to a heterogeneous induction of metabolism was investigated using leaves with the epidermis stripped off.

P-26-020 Q U A N T I T A T I V E MAPPING OF LEAF P H O T O S Y N T H E S I S USING CHLOROPHYLL FLUORESCENCE IMAGING.

P-26-023 INFLUENCE OF DISSOLVED][NORGANICCARBON(DIC) CONCENTRATIONON PHOTOSYNTHESISIN GREEN ( UL VA SP. ), BROWN( F u c u $ SgRRA r{:S ) AND RED PALMARIA PALMATA ) MARINE MACROALGAE.

B. GENTY & S. MEYER Groupe "Photosyntl~se et Environnement" Laboratoire d'Ecologie V~3g6tale, bat 362, Universit6 Paris XI, CNRSURA 1492 ; 91 405 Orsay cedex (FRANCE)

G Doucet & G Levavasseur Station Biologique, CNRS UPR 9042, Univ Paris VI, 29680 Roscoff (France)

A method has been developed for routine, non-invasive monitoring of the topography of leaf photochemistry. The method used video images of leaf chlorophyll fluorescence, taken during steady state photosynthesis, (I), and during a transitory light saturation of photochemistry, (In~, to construct, pixel by pixel, in almost real time, an image , (12), of the photochemical yield of PSII, ~p,s2. ~PS2 was estimated by (1..¢,/¢,m), where ~ and '~m are relative fluorescence yields obtained for each related pixel in I and Im, respectively. The effectiveness of the method was shown by mapping the heterogeneous distribution of photosynthetic activity after treatment with either an herbicide (DCMU), abscisio acid, or during the course of the induction of photosynthesis. This approach provides a powerful tool to probe the spatial distribution of leaf photosynthesis over a wide range of scale from single cell to whole leaf photosynthesis.

There are conflicting reports as to whether the inorganic carbon concentration in seawater is saturating for photosynthesis In this work, we examine light response curves of photosynthetic 02 exchange in seawater, in CO2-freeseawater and in DIC-supplemented-seawater Light responses curves of photosynthetic 02 exchange as a fonction of DIC concentration in seawater are established as well In natural seawater, the concentration of HCOf is much higher than that of CO2. Thus marine algae are able to utilize HCO3" for their photosynthetic needs The mechanisms of HCOf uptake are still not clear So we considere the eventual mechanisms involved in DIC uptake through the effects of selective inhibitors (AZ : acetazolamide, DIDS : 4,4'-diisothiocyanostilbene-2,2'-disulfonate) on photosynthesis of studied species There is DIC limitation of photosynthesis under bright light according to algae and season Both AZ and DIDS inhibit photosynthesis, suggesting that at least two mechanisms are involved in DIC uptake

223

Poster

P-26-024

MEASUREMENT OF THE COz/O2 SPECIFICITY OF RUBISCO IN LEAVES

Astrid Sumbera and A. Laisk, institute of Molecular and Cell Biology, University of Tartu, Riia str. 181, Tartu, EE2400, Estonia In literature, the specificity factor Ks, has been calculated from the CO 2 photocompensation point, which h ~ been found from the intercept of the (CO 2 exchange rate P) vs. (intercellular COo concentration Ci) curves measured at different light intensities. W-e suggest to calculate the specificity coefficient from the slope of the plot o f the (CO, compensation point) vs. (intracellular O~ concentration), which also elTminates the effect of the nonphoto-reswation but is simpler to measure. The values of K in leaves of different C_ species at 23"C varied from 85 to 98i-0.7 (theS~rror was found from th(~ repetitive measurements on one and the same leaf). Kso was variable also in different leaves of one species. The K valuers found from the 02 dependence of the usual CO, compensa~on point better agree with the in vitro results than ValLTescalculated from the CO 2 photocompensation point. It is concluded that in intact leaves Ks is variable between species and dependent on conditions. P

224

Poster

P-27-003 CARBON PARTITIONING IN THE LEAVES OF CAM PLANTS J.T. Christopher, T. Wooley & J.A.M. Holtum James Cook University, Townsville, Queensland 4811, Australia

P o s t e r s e s s i o n 27

C a r b o n partitioning a n d p r o d u c t i v i t y P-27-001

- P-27-023

In plants with Crassulacean acid metabolism (CAM) 75% of the carbon in malic acid is supplied by storage carbohydrate (CHO). Some species store starch/glucans in chloroplasts, others store sugars or polysaccharides extrachloroplastically. In 12 species from a range of taxa we measured the proportions of the fluctuating CHO pools attributable to starch and the activities of the regulatory enzymes of carbohydrate metabolism, phosphofructekinase (PFK), PPi fructose-6-P 1-phosphotransferase (PFP), cytoplasmic- and chloroplastic- fructose 1,6-bisphosphatase (FBPcyt and FBPchl). Starch was the major fluctuating CHO in Bryophyllum tubiflorum, t9. pinnatum, Kalanchoe daigremontiana and Peperomia rankii. In these species PFP activity was generally < PFK and FBPcyt and FBPcyt < FBPchlor. Starch was not the major fluctuating CHO in Aloe vera, Ananas comosus, Portee petrapolitana, Hoya cafnosa, Agave guadalajarana and Vanilla fragrans. In these species, with the exception of Aloe, PFP > > PFK and FBPcyt and FBPcyt > FBPchI.

P-27-004

P-27-001 IN VITRO PHOTOAUTOTROPHIC GROWTH OF SOME MEDICINAL PLANTS : LIGHT, SUGARS,AND CO2 EFFECTS ON PHOTOSYNTHESIS.

J. Creche1, J.C. ~ l e r c 2 • MI Ridean1 lLaboratoire de Bioteehnologie v~g~tale, Facult~ de Pharmacic, Av. Mooge 37000 Tours ; 2 Laboratoire de Biologic V~g6tale, Eeophysiologie, Facult4 des Sciences 42023 Saint Etienne C&lex2. Two models were tested : multiple-shootcultures of Ruta graveolens, and callus cultures of Choysia ternata. During growth of the cultures, oxygenand carbondioxide were continuously and simdteaneously measured. The growth is photomixotrophicat the beginningof cultares either with 166 mM or 41 mM glucose. If cultures are started at 41raM, a phytomixotrophie/phntoautntrophictransition is possible, when the glucose is rather completely used, and a photosynthesishigher than respirationrate is observed. The CO2 compensationpoint regularly decreases with culture ageing. The photoantotrophiestate is more rapidly reached with high light than low light in Ruta. In Choysia cultures we observed an incidence of CO2 concentration even under limiting lights. Also, Choysia is sensitive to a CO2 sehoek, mad is able to growth in cell suspensionssubculturedduring severalmonthswithoutsugarsbut with

2 ~ c~.

The relations between light intensity and CO2 concentrations, and the problems about photomixoto photoantotrophytransition,will be discussed.

P-27-002 PHOTOSYNTHETXC POTENTIAL OF FXVg GgNOTYPES C o £ £ e a PIERRE

canlphora

E. C a m p o s t r i n i I & M. Maestri 2 IState U n i v e r s i t y of the North Fluminense, CCTA, Campos dos Goytacazes, RJ, 28015-620, Brasil, E-mail: [email protected]; 2Department of Plant Biology, Federal U n i v e r s i t y of Vigosa, MG, 36570-000, Brasil T w e l v e - m o n t h - o l d plants of Coffea canephora Pierre grown under field c o n d i t i o n s were used. Five genotypes were obtained from selected m a t r i c e s for low (2 clones), average (2 clones) and high p r o d u c t i v i t y (i clone). The five genotypes were found to have s i m i l a r b e h a v i o r in regard of their quantum yield. The average p r o d u c t i v i t y clones tended to show h i g h e r m a x i m u m net p h o t o s y n t h e t i c potencial w h i c h was u n r e l a t e d to productivity. No r e l a t i o n s h i p b e t w e e n leaf m o r p h o l o g i c a l c h a r a c t e r i s t i c s and p r o d u c t i v i t y was observed. All genotypes p r e s e n t e d the same e f f i c i e n c y c o n c e r n i n g the p h o t o s y n t h e t i c apparatus e v a l u a t e d a c c o r d i n g to the fluorescence emission.

PECULIARITIES OF HIGHLY PRODUCTIVE WHEAT PHOTOSYNTHESIS AND USAGE OF PHOTOSYNTHETIC SIGNS IN SELECTION J_A_Aliev and E.G.Kazibekova Institute of Botany, Azerbaijan Academy of Sciences, Patamdar shosse, 40, Baku, 370073, Azerbaljan Republic Highly productive varieties are characterized by higher stability, i.e. by longer period of active photosynthesis in crops with multiple peaks during vegetation. Dynamics of rate of biosynthesis of sucrose, total glyein+serin fund, correlation of carboxylase-oxygenase activity of Rubiseo and CO2 assimilation intensity contribute to parallel change of photosynthesis and photorespiration intensity in leaf ontogenesis. Under light, CO2 evolution rate is higher in highly productive varieties at the expense of photorespiration. Values of ratio of photorespiration to true photosynthesis in genotypes with different intensity of CO2 assimilation are close and equal in average to 1:3 in ontogenesis. Assimilated carbon losses during photorespiration are equal to 30%. High intensity of true photosynthesis and photorespiration, more active carbonic anhydrase, Rubisco, phosphoenolpyruvate carboxylase and high activity of primary photochemical processes together with the established optimal architecture of crops determine high productivity of wheat genotypes.

P-27-005 Changes in photosynthetic carbon metabofism in leaves after interaction with a fungal pathogen, as visualized by chlorophyll-a-fluorescence imaging.

Petra Esfeld, Katharina Siebke & Engelbert Weis Institute of Botany, Univ. Miinster D-48149 Mtlnster (Germany) Small spots on leaves of a resistant cultivar of Cicer arietinum L. were inoculated with the fungal pathogen Ascochyta rabiei. The subsequent elicitor-induced defense reactions where accompanied by differential changes in the photosynthetic carbon metabolism over the leaf as visualized by quantitative Chl.-fluorescence imaging (Siebke & Weis 1995, Planta 196). In mesophyll cells below elieitation spots the assimilation capacity slightly decreases while the induction of photosynthesis after a dark period under reduced CO2-concentration was stimulated, compared to that in a control plant. Dark respiration increased. In contrast, the induction of photosynthesis was hampered in the surrounding mesophyll cells. No such changes were seen in a sensitive (non-resistant) plant. Photosynthesis imaging in conjunction with biochemical studies leads to the assumption, that in a resistant plant elicitation of mesophyll cells induces mobilization of carbon metabolites and that a 'source-sink relationship' develops between elicitated and surrounding, non-elicitated mesophyll cells.

225

Poster

P-27-006

P-27-009

PHOTOSYNTHESIS IN RELATION TO SACCHARIDES IN WHEAT AND MUNGBEAN LEAVES

THE RESPONSE OF PHOTOSYNTHESIS AND PARTITIONING IN POTATO TO ELEVATED CO 2.

M.C. Ghildiyal & Poonam Sharma-Natu Division of Plant Physiology, Indian Agricultural Institute, New Delhi 1|0 012, India

Adcock, M.D., Brooks, A. Leegood, R.C. and Quick, W.P. University of Sheffield, Sheffield, U.K.

Research

The reducing, non-reducing sugars and starch content in the leaves of wheat (T. aestivam) CVo Sonalika and mungbean ( ~ radiata ( " L ~ . ) ~ k ) cv. Pusa Baisakhi were examined a - ~ " 6 " - a . m , and 3 p.m. There was a marked accumulation of saccharides in the leaves during the day in both the genotypes. Mungbean accumulated more starch whereas, concentration of sugars was more in wheat. In s p i t e of a marked accumulation of saccharides in the leaves, the photosynthetic rate remained more or less unaffected in mungbean but showed a decline in wheat. The study indicated a d i f f e r e n t i a l s e n s i t i v i t y of these species to feedback effect and could be one of the a t t r i b u t e s f o r t h e i r observed d i f f e r e n t i a l response to elevated CO2.

An increase in atmospheric CO2 may have profound effects on plant photosynthesis, growth and harvest index. Elevated CO2 may increase the plants' capacity to produce carbohydrate. However, any increase in source capacity can only be sustained if the carbohydrates produced are utilised by the plant to generate additional sink capacity. In this study we have investigated the growth response of Solatmm tuberosum (potato) in elevated CO2. Our main observations were; i) Rubisco amount and activation state were reduced, suggesting a down-regulation of photosynthesis in elevated CO2. ii) Carbohydrates accumulated in the leaves of plants grown in elevated CO2 during the day, but predawn amounts were similar in ambient and elevated CO2. iii) The activation state of the main enzymes of the carbohydrate pathway complemented the observed changes in carbohydrate content of the leaves, iv) The biomass production of plants grown in elevated CO2 increased, particularly with respect to tubers. Even though acclimation to CO2 was observed, we conclude that elevated CO2 will increase tuber yield and harvest index in potato.

P-27-007 PHOTOSYNTHESIS AND NITROGEN RELATIONSHIPS IN SUN AND SHADE LEAVES OF FOUR TREES SPECIES OF A CLOUD FOREST IN VENEZUELA. Hernandez, R. and Medina, E. Centro de Ecolvgia- IVIC. Aptdo 21827. Caracas 1020 A. Venezuela. The relationship between Amax and leaf nitrogen content (N) was determined for four trees species, Asvidosverma fendlerii, Graffenrieda latifolia, Richeria grandis and Tetrorchidium rubrivenium. The relationship between photosynthesis and leaf N content was more significant on a leaf weight basis than on leaf area basis, due to the large range of specific leaf areas. The curve relating maximum photosynthetic rates per unit weight and N content approach saturation at leaf N concentrations around 1 mmol/g T. rubrivenium stands out because its higher SLA, Amax and N content. _A. fendlerii y R. flrandis showed large differences in SLA between sun and shade leaves, therefore their chlorophyll and N contents, and their photosynthetic rates were larger in sun leaves on an area basis, while the contrary was true on a weight basis.

CARBON

P-27-010 CHANGES IN THE RELATIONSHIPBEltNEEN THE QUANTUM EFFICIENCIES OF PSll PHOTOCHEMISTRYAND CO= ASSIMILATIONIN MAIZE DURING THE EARLY PART OF THE GROWING SEASON Kevin Oxborouuhl James R. Andrews and Nell R. Baker, Dept. Biology, University of Essex, Colchester, CO4 3SQ, U.K. A linear relationship between the quantum efficiencies of PSII photochemistry (~PSH) and CO2 assimilation ((~CO2) has previously been demonstrated within maize leaves. Exposing chamber-grown maize plants to relatively short (4 h - 24 h) periods of high PPFD (800 iJ.mOl m-2 s"t ) plus low temperature (5 - 8°C) lowered the quantum yield and maximum rate of COrfixation but had no measurable effect on the relationship between 4,PSII and ~bCO2. Conversely, field grown maize plants which had developed at sub-optimal temperatures displayed ~bPSII to #CO2 ratios close to those for chamber grown plants at high or low PPFDs, but much higher ratios at moderate PPFDs (50 - 300 ttmol m2 st), inducing a curvilinear relationship. These data suggest that a much smaller proportion of the reducing equivalents dedved from.charge separation at PSII are being utilised for CO2 assimilation and, consequently, that one or more alternative electron sinks compete effectively with CO2-assimilatlon within these field-grown plants. The curvilinear aspect to this relationship between ~bPSIIand ~bCO2decreases over a few clays at higher growing temperatures,

P-27-011

P-27-008 EFFECT O F PHOSPHATE DEFICIENCY ON MOBILIZATION OF N A N D P A N D ON TWO-DIMENSIONAL PROTEIN PATTERNS

BALANCING OF STEADY STATE P H O T O B ~ B I S PLANT MATERIAL.

H. Usuda Lab. Chem. Teikyo Univ., 359 Ohtsuka, Hachioji, Japan

Karen Van Loven, Roland Valcke and Luc Vleeschouwers. Limburgs Universitair Centrum, dept. SaG, Universitaire Campus, B-3590 Diepenbeek, Belgium.

Previously we found that P deprivation has a detrimental effect on the rate of photosynthesis in maize accompanied by reduced contents of enzymes involved in photosynthetic carbon metabolism and postulated that the P status of leaves might be involved in the regulation of leaf senescence through regulation of the remohilization of N in maize. To investigate this postulate, the effects of P deprivation on the changes in the contents of N and P and the changes in the two-dimensional protein patterns within the fully expanded second leaf blades were investigated from 12 to 30 days after planting (DAP). P deprivation greatly decreased the contents of acid-soluble Pi (5% of control (C)), esterified P (4% of C), CId (9% of C), total N (42% of C), soluble protein N (9% of C), and insoluble protein N (21% of C) at 30 DAP. Soluble leaf proteins were resolved into more than 450 spots, many of which were common to all samples. However, 11 polypeptides decreased in relative abundance and 18 polypeptides increased in relative abundance during P-deprivation-aecelerated senescence. In low-P plants, remobilization of P from the older leaf blades to young tissues occurred continuously, and the total P in the older leaf blades decreased to an ext~mely low concentration. These results suggest that the P status of leaves is involved in the regulation of leaf senescence in maize.

In transgenic tobacco plants containing the chimeric Pssu-ipt gene construction, the endogenous cytokinin content can be increased by light induced ipt-gene expression. Chlorophyll fluorescence kinetics of the transgenic plants have been analyzed upon a dark-light transition, following an inductive wake up procedure. In contrast to non-transformed plants, this procedure did not provoke a typical dampening oscillatory behaviour of the fluorescence signal in the Pssu-ipt transgenic plant material. The non-photochemical quenching, ~, as determined by the pAM-instrument, was very low in these plants. In transgenic grafts that produce a high cytokinin level, the coupling status and the ATP-production were also reduced. A comparison of re-oxidation kinetics of P700 upon a multiple turnover flash and after a period of actinic illumination suggested that the accumulation of stromal reducing equivalents (NADPH; cf. Asada et al., Plant Cell Physiol. 1993, 34: 39-50) was not affected. The highly disturbed build up of a proton motive force in the transgenic plant material coincides with an enormous starch accumulation in the leaf tissue and a disproportional shoot/root growth. This might give an explanation for the effect of cytokinins on the sink strength.

226

IN PSBU-IPT

TRANBGENIC

Poster ii

P-27-015

P-27-012 EFFECTS OF NITROGEN NUTRITION AND LIGHT INTENSITIES ON RUBISCO ACITIVASE CONTENT AND PHOTOSYNTHESIS IN RICE LEAF • 12 H 2 2 2 1,2 ", . Fukayama • H. Koshimura T. Azuma and T. Yasuda • . . • 2 Faculty ofAgncu ture, Kohe University, Rokko, Kobe 657, Japan, Graduate School of Science & Technology, Kohe University, Rokko, Kobe 657, Japan

The amounts of Rubisco acitivase and Rubisco, and photosynthetic 02 evolution rate (OER) in rice plants grown under different nitrogen (N) concentrations and light intensities (full sunlight, 42% of full sunlight) were investigated. The amount of Rubisco acitivase increased linearly with increasing N content regardless of the growth light conditions, whereas that of Rubisco increased slightly under the shade condition• A close, curvilinear correlation was observed between the OER and the amount of Rubisco with tendency of saturation at the higher Rubiseo content (>3g/m2). On the other hand, the regression curve between the OER and the amount of Rubisco activase for shaded leaves differed from that for sun leaves: the OER in shaded leaves saturated at lower level of Rubisco acitivase.

COLD HARDENING OF SPRING AND WINTER WHEAT (Triticum aestivum L.) AND RAPE (Brassica napus L.) RESULTS IN DIFFERENTIAL EFFECTS ON CARBON METABOLISM AND CARBOHYDRATE CONTENT Asa Strand I , Vanghan Hurry 2, Per GardestrOmI and Gunnar ~quist 1 lDepartment of Plant Physiology, Umeh University, S-901 87 Ume~t, Sweden 2Cooperative Research Centre for Plant Science, GPO Box 475, Canberra ACT 2601• Australia The effects of long-term growth at low temperature (5° C) on photosynthesis and carbon metabolism were studied in spring and winter cultivars of wheat (Triticum aestivum) and rape (Brassica napus). The high photosynthetic rates observed in cold grown winter cultivars were associated an increase per unit protein in both stromal and cytosolic fructose-1,6-bisphosphatase and in sucrose phosphate synthase activity. Neither of the two spring cultivars increased enzyme activity per unit protein. These changes in enzymatic activity following cold hardening were correlated with a relative increase in the metabolite pools and with a relative accumulation of soluble sugars. The data are discussed with respect to the regulation of enzymes involved in carbon metabolism at low temperature and their possible role in accumulation of cryoprotective sugars and the development of freezing tolerance.

P-27-016

P-27-013

REGULATION OF EXCITATION PRESSURE BY PHOTOSYNTHETIC CARBON METABOLISM IN CHLORELLA VULGARIS.

PllO'gOlllfllTin~I¢ DmPitlIBBI~! IN /.J~VIIB OF PitOST-IUUIDEInmIVY IB CAUMID BY A P ] B D BACK I N ] [ I B I T I O N

R. pamer & H. Bauer Institute of Botany, University A - 6 0 2 0 Innsbruck, A u s t r i a

V I A JUJBZMIZ,ATB ACCDNUZ3~ION

15,

N.P.A Hurter, L.V. Savitch, and D.P. Maxwell. The University of Western Ontario, Dept of Plant Sciences, London, Canada N6A 5B7

The aim of this study was to prove whether an impairment of photosynthetic activity following frost hardening is associated with an acc~aaulation of soluble carbohydrates usually occurring during the develoI~nent of frose tolerance. Rates of photosynand~ evolution and contents of glucose, fructhetic C~uptake t o s e and sucrose in ivy leaves were d e t e r m i n e d in plants of Hedera helix with varying source/sink relations prior and after hardening for four w e e k s at 5/0°c day/night temperatures. The effect of Pi feeding via the petioles on photosynthesis and the r e c o v e r y at 20/15°c w e r e also recorded. P h o t o s y n t h e s i s was red u c e d by about 30% after hardening accompanied by a strong increase in the content of the sugars (sucrose 2x, glucose and fructose 10x I . P h o t o s y n t h e s i s and sugars t e n d e d to normalize afte~ three days at 20/15ec. P h o t o s y n t h e t i c d e p r e s s i o n in frosth a r d e n e d leaves was Bim£1ar at ambient and saturating cc~ and irrespective of the s o u r c e / s i n k relations. F e e d i n g the leaves with Pi did hardly r e s t o r e their photosynthetic activity. Therefore, w e conclude that the photosynthetic impairment was not the result of a feed back inhibition vla assimilate accumulation.

('hlorella was grown under conditions designed to cause PSII excitation pressure of 0.10 (27°C/150 PPFD or 5°C/20 PPFD) and 0.75 (27°C/2200 PPFD or 5°C/150 PPFD). Regardless of absolute temperature or irradiance, Chlorella grown under either high or low excitation pressure exhibited similar cell ultrastracture, pigment composition, LHCII and cab mRNA abundance. ( "hlorella grown at high excitation pressure had a decreased ATP/ADP ratio as a result of restricted ATP synthesis which result from limited triose-P utilisation However, cells grown at 27°C/2200 PPFD had a 2.7-fuld higher excitation pressure than cells grown at 5°C/150 PPFD. We suggest that this is due to a differential restriction in carbon metabolism. Cells grown at 27°C/2200 PPFD exhibited a preference for the eytosolie sucrose biosynthetic pathway In contrast, growth at 5°C/150 PPFD favoured the stromal, starch biosynthetic pathwayWe suggest that photosynthetic acclimation to growth at either high or low PSII excitation pressure reflects adjustment in response to the redox poise of intersystem electron transport and photosynthetic carbon metabolism.

Innsbruck,

SternwartestraBe

P-27-014 PHOTOSYNTHESIS OF WINTER AND SPRING CULTIVARS OF Brassica napus RESPONDS DIFFERENTLY TO SUCROSE OR PHOSPHATE FEEDING AFTER COLD HARDENING R. H~k, P. GardestrOm and G. Oquist Dept. Plant Physiology, University of Umdt, S-901 87 Ume~t• Sweden Photosynthetic capacity (Pmax) measured as the rate of carbon dioxide- and light-saturated oxygen evolution increases in a cold hardened winter eultivar (CHW) while it decreases in a cold hardened spring cultivar (CHS) of Brassica napus in comparison with non-hardened plants (NH). CHW plants accumulate high levels of sugars and starch during low temperature acclimation (see also Strand et al. in this volume). NH and CHW plants exhibit strong inhibition of Pmax after sucrose feeding to leaf discs, which is the expected reaction of "starch storers" to excess of sucrose. Pmax of the more cold sensitive spring cultivar is after cold hardening less inhibited by sucrose (or even sucrose-stimulated) which more resembles the "sucrose storer" response. In addition, Pmax of CHS plants is increased after phosphate (P) feeding. Based on our data we speculate that feeding sucrose to CHS could help to restore the exhausted metabolite pools and stimulate Calvin cycle regeneration capacity. Stimulation of Pmax by P feeding might imply that a relatively high proportion of P is bound in sugar phosphates. We are now on the way to elucidate the mechanism of different responses of winter and spring cultivars to cold acclimation and different tolerance to externaly fed sucrose.

P-27-017 Spatial

distribution of assimilation chlorophyll-a-fluorescence imaging.

control

in leaves

by

quantitative

K. Siebke & E. Weis Institute of Botany, Univ. M~nster, D-48149 MUnster Images of the assimilation rate have been derived pixel-by-pixel from chlorophyll-a-fluorescence by a computer-supported video system (Siebke & Weis 1995, Planta 196, in press). One image point represents fluorescence emitted from about four palisade cells. Under optimal conditions the assimilation rate is homogeneously distributed over the leaf area. Specific patches or microscopic domains of assimilation appears during secondary kinetics and oscillations as initiated by perturbations of external conditions: (1) Gas exchange compartments ('alveoli') appeared in images of stomatarelated oscillations (period around 15 min). Each individual compartment oscillated independently over hours• (2) Minor-vein distribution appear in images of photosynthetic oscillations (period around 1 min). Oscillations were low and most damped in cells adjacent to minor-veins while the highest amplitudes and periods occurred in most distant cells. We discuss the control of photosynthesis in relation to (1) spatial distribution of CO2, (2) sugar transport and 'microscopic' source-sink interactions in the mesophyll.

227

Poster

P-27-021

P-27-018 G R O W T H CONDITIONS OF CO2 AND Nll4 ALTER THE CELL SIZE AND PHOTOSYNTHETIC CHARACTERISTICS OF DUNALIELLA SALINA M. Giordan• 1 • G. Bowes2 ZFaculty of Sciences, University of Ancona, Via Brecce Bianche, 60131 Ancona, Italy; 2Dept. of Botany, University of Florida, Gainesville, FL 32611, U.S.A. Dunaliella salina cells grown at low [CO2] were 40% smaller than those acclimated to high (5%) [CO2]. On this basis, the cell Chl and glycerol concentrations were 78% and 120°6 greater, respectively, but rubisco protein concentration was not changed. The CO2 concontrating mechanism of the low-CO2 cells may obviate the need for more mbisco. Cells grown on NH4÷ had greater volumes than those on NOa, and the Praax and affinity for CO2 were higher. These changes were associated with 1.5- and 3.5-fold increase in pigment and rubiseo protein concentrations. However, the glycerol concentration was decreased, and the carbon allocation pattern was altered. Supported in part by the USDA/NRICG Photosynthesis Program.

P-27-019 MANIPULATING RAPESEED CARBOHYDRATE PARTITIONING S.P. Kin~ L2'3,J.E. Lunn2, M.R. Badger I'3 & R.T. Furbank 1'2 1 Cooperative Research Centre for Plant Science; 2 CSIRO Plant Industry, PO 3 Box 1600, Canberra, 2601, Australia; Research School of Biological Sciences, The Australian National University, PO Box 475, Canberra, 2601, Australia.

Characterisation of rapeseed (Brassica napus L.) carbon assimilation in leaf and silique source tissues is being carried out prior to genetically engineering perturbations. Plants were grown in a naturally-lit glasshouse (25°C day/18°C night). Photosynthetic rates during the photoperiod have been measured. Diurnal changes in carbohydrate contents and 14CO2 primary partitioning have been determined for source tissues after 12h of illumination. Starch primarily accumulates during the photoperiod. Silique walls also amass large amounts of free glucose and fructose. Within the first hour of light, both source tissues immediately partition higher amounts of sucrose than starch. In contrast to leaf, silique walls continue to partition more ~4CO2into sucrose near the end of the photoperiod. Plmtis/overexpressing sucrose phosphate synthase (SPS) are being produced to perturb wild-type carbohydrate metabolism.

P-27-020 PRIMARY PARTITIONING OF PHOTOSYNTHATE IN C 4 PLANTS J.E. Lunn & M.D. Hatch CSIRO Division of Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. A radiotracer technique has been developed to measure the primary (initial) partitioning of photosynthate between sucrose and starch in leaves under physiological conditions. In a survey of partitioning behaviour in leaves from plants grown under similar conditions, C 4 species showed ratios of primary partitioning of photosynthate between sucrose and starch ranging from about 0.5:1 in those species that accumulate mostly starch in the leaves during the day to 8:1 in those species that accumulate mostly sucrose. In most of the species examined partitioning into starch was greater at the end of the photoperiod than at the beginning. Photosynthate partitioning in maize (Zea mays L.) leaves was shifted, favouring sucrose synthesis, when the leaves were illuminated at lower irradiance. However, varying the sucrose and starch content of the leaves had little or no effect on the partitioning ratio. The regulatory mechanisms involved in control of photosynthate partitioning in C 4 plants are being investigated.

228

PHOTOSYNTHESIS AND C A R B O N PARTITIONING PLANTS GROWN UNDER BLUE OR RED LIGHT

IN RADISH

N.G. Bukhov. V.V. Bondar, I.S. Drozdova, A.N. Kara, A.A. Kotov, S.N. Maevskaya, A.A. Vasil'ev, S.Yu. Voevudskaya, P.Yu. Voronin & A.T. Mokronosov K.A. Timiriazev Institute of Plant Physiology Russian Academy of Sciences, Botanicheskaya 35, Moscow 127276, Russia The tuber fresh weight to foliage fresh weight ratio was found to be similar in radish plants grown under blue (BL) or red (RL) light only till the day 13 after germination. Contrary to RL, BL stimulated the normal tuberization. The rates of photosynthesis were found to be similar under BL and RL irradiances corresponded to ones maintained during the plant cultivation. The diurnal changes in the rate of photosynthesis corresponded to the starch accumulation were observed only in plants grown under RL. The rate of translocation of labelled carbon from leaves to roots or tubers was 2.5-times higher in BL plants. The low rate of starch hydrolisis rather than ineffective photosynthesis seems to prevent the normal development of tubers in radish plants under RL.

P-27-022 DRY MATI'ER AND ENERGY PARTITIONING IN PLANTS H.R.Bolh~r-Nordenkampf, D.Donis, M.H.Meister, K.Nemeth and W.F.Pestl Institute of Plant Physiology, University of Vienna, Austria During ontogenesis plants distribute assimilates quite differently among their organs. In case of high sink capacity energetically cheap storing compounds such as carbohydrates and / or organic acids are formed, whereas during periods with low demand proteins and lipids may be accumulated. These substances can be remobilised during periods of promoted growth or when pod and grain filling starts. Remobilisation of lipids consequently followed by formation of carbohydrates can give rise to an increase in dry weight. This phenomenon of growth without photosynthesis is a process which can occur several times during ontogenesis when sink capacity is changing. During growth and development such phases hidden by normal photosynthesis can be traced up using the relation of increments in energy and dry matter called storing index. Besides of ontogenesis, drought and increased CO z are able to modify sink capacity and by this transients in the partitioning pattern are induced. During the last 5 years wheat, sugar beet, chicoree, corn, lupine, lettuce, Fatshedera, Schefflera as well as tropical grasses Brachlaria and Eulalia were investigated to verify the statements above.

P-27-023 EFFECTS OF COMPARTMENTATION OF ACID INVERTASE ON CARBOHYDRATE METABOLISM IN MATURE LEAVES and C.J. Pollock, IGER Aberystwyth, UK Photoassimilate produced by mature leaves is usually loaded into the phloem as sucrose. Transport may involve an apoplastic step or be entirely symplastic depending on species. Acid invertase is both soluble and cell wall bound, and so could regulate photeassimilate utilisation by a) hydrolysis of apoplastic sucrose in the transport path or b) determining sucrose availability for transport/storage resulting from intraeellular futile cycling between cytoplasm (synthesis) and vacuole (degradation). Tissue level loealisation of acid invert,am was investigated by tissue printing, which establishes protein distribution within a leaf. Antiserum, raised against a synthetic peptide corresponding to the active site region, crossreacted with both forms of acid invertase. Species-specific differences between Fuchsia, l~sum and Hordeum were identified which correlate with differences in carbon assimilation rate and presence of oligosaccharides in leaf extracts. We propose that acid invertase loealisation is implicated in species-specific differences in photoassimilate partitioning.

Poster P-28-003 HISTORICAL TRENDS OF PHOTOSYSTE| II COMPONENTS OF CEREAL CULTIVA£S N. |atanabe & A. Ogawa Faculty of Agriculture. Gifu University, 1-1Yanagido, 6 i f u 501-11 JAPAN

Poster session 28

Considerable i n t e r e s t has been paid to the comparison of photosynt h e t i c capacity of old and new c u l t i v a r s of c e r e a l s . The h i s t o r i c a l wheat and barley c u l t i v a r s , r e p r e s e n t a t i v e of those widely c u l t i v a t ed over the l a s t century, fro= s e v e r a l regions of the world were assessed for chlorophyll a/b r a t i o and photosyste= I I coeponents. In Australian wheat c u l t i v a r s , the i n c r e a s e in l e a f nitrogen and photos y n t h e t i c r a t e i s most probably due to i n t r o d u c t i o n of the ~ b t dwarfing genes. Counter t h i s , d e c l i n e s in r a t e of e l e c t r o n t r a n s p o r t per unit chlorophyll, chlorophyll a/b r a t i o and photosystem I I count per unit chlorophyll have been observed. | e observed the s i t i l a r trends for chlorophyll a/b r a t i o in the h i s t o r i c a l c u l t i v a r s of wheat and barley frog the s e v e r a l regions of the world.

Photosynthesis and ecosystems productivity P-28-001

- P-28-035

P-28-004

P-28-001 PHOTOSYNTHETIC AND RESPIRATORY ACTIVITY OF KARWINSKIA WITH HUMBOLDTIANA, SPECIES PRODUCING SUBSTANCES ANTITUMOURAL EFFECTS Elena Masamvi#.ov& Alexander Lux, Department of Plant Physiology, Comenius University, Mlynsk~ dolina B-2,. Sk-842 15 Bratislava, Slovsk Republic

Katwinskia humboldliana [Zucc. ) is a Mexican shrub, producing the anthraconone substances with antitumoural effect. One of them was later identified and named as "toxin T-514". Pmeented results follow up on the structural analysis and in vitro cultivation of this species. Two years old plants were grown in greenhouse conditions and daily CO2 exchange (photosynthesis, respiration) was measured outdoors from May untill August 1994. Gasometrical method and simple assimilation chamber for short time exposition were used. Simultaneously with physiological measurements basic microclimatic factors (irradiance, air temperature and air humidity) were registered. Time of day and irredisnce at which the values of maximal net photosynthetic rate was attained were determined. The response curves for CO2 uptake to irmdianco were evaluated by boundary line method. The values of dark respiration rate were also found. Above mentioned photosynthetic parameters may be also used for determination of leaf productional activity with regard to the "toxin T-514" production and accumulation in Karwinskia humbolo~'ana plant organs and tissues.

P-28-002

PHOTOSYNTHETIC PERFORMANCE OF EMILIANIA HUXLEYII IN A FLUCTUATING LIGHT CLIMATE SIMULATING VERTICAL MIXING Jacco Kromkamp, Jan Peene and Inez Flameling NIOO-CEMO Vierstraat 28, 4401 EA Yerseke, The Netherlands The marine coccolithophoric alga Emi/iania hux/eyii was grown in light-limited continuous cultures with a constant and fluctuating light climates simulating vertical mixing. The total daily light dose experienced by the unicellular algae during the total photoperiod was the same for all light climates. PSm,= decreased in the afternoon in the cultures with a fluctuating light climate. PAM-analyses showed that this decrease in p a = coincided with an increase in nonphotochemical quenching. The cultures with a fluctuating light climate showed hysteresis of photosynthesis during the day, partly due to qN-quenching. Fv/Fm-ratio's were similar for all light climates. From relative rates of linear electron transport (¢,p.I) and oxygen evolution rates at different light intensities (I), a measure of the functional cross section of PSII (ops,,) was calculated. Ops, did not seem to depend on the light climate.

P-28-005

EFFECTS OF ELEVATED CO z CONCENTRATION O N THE THYLAKOID MEMBRANES OF DIFFERENT MAIZE GENOTYPES

PHOTOSYNTHETIC CHARACTERISTICS OF SOME VARIETIES A N D M U T A N T S OF W H E A T AS T H E Y I E L D C O M P O N E N T S .

Zhoo Qi, Tang Chong-qin, Kuong ung-wn, Boi Ke-zhi, Fu Kai ( InstItute of Botony, Ac~dernia Sinica, Beijng, 100044, Chino)

E. Konui ika Dept. of Physiology, I n s t i t u t e of B i o l o g i c a l Research, A c a d e m y of Sciences, Tirana, Albania.

Maize (Zea r,x~s L. ) hybrid ~ - d a n 14 (Mo17 x Zhooglhuong 64) were grown in normd CO2 (350 _+ 1096 ppm) or elevated CO2 (700 + 10% ppm) concentration. Companson of some photosynthetic charocteristics of chloroplasts among three genotypes were analysed by means of cl~omphyl content, obsoqotion. fluorescence induction kb'let~ ~ excitationenergy ~ between PSI and PSII with isolated thyId¢oicl membranes. It was proved thot chlorophyll content was increased by elevated CO2 concentTction among three genotypes, the chlorophyU of hybrid was the highest. The ffattenr~, of absorpticn spectrum was shown in hybrid. end female, alx,orption of rnde was increased. In addition, potential activity (Fv/Fo) Oral convermon efficency (Fv/Fm) of prrnar,/ li@%t energy of PSII were increased except male, the excitation energy dist(txltion between PSI1 and PSi was eq~liboum by elevated CO2. All these results were benefit to absoqation, transferring and conversion of solar energy in plants.However, effects of ciffrent species or dfferent genotypes of the same speaes were not equalby elevated CO2 concentration.

The main way to increase the plant productivity is the creation of optimal conditions of the photosynthesis. The estimation of a new genetic materials of wheat to the physiological characteristics is based on the genetic variability. The aim of this study is the comparison of the physiological parameters of eight varieties and four mutants of wheat. The assimilation rate, stomatal conductance, internal CO 2 concentration and transpiration rate are measured by infra-red gas analyser; the quantity of photosynthetic pigments by spectrophotometry. The surface and specific density of the leaves are measured also. The coefficients of correlation between the photosynthetic parameters themselves and between these and productivity parameters are calculated. The possibility of using these parameters as a selection criterion is discussed.

229

Poster P-28-006

C H A N G E S O N CHLOROPHYLL FLUORESCENCE EMISSION IN Vicia faba CULTIVARS U N D E R HERBICIDE TREATMENT

|. Martinez Guijarro, D. Vidal & E. Sim6n Dept. of Plant Biology, Faculty of Biology, University of Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain. Chlorophyll fluorescence was evaluated in spring (Histal) and a u t u m n (Luz de otoflo) cultivars of Vicia faba to determine the effect of two r e c o m m e n d e d doses of methabenzthiazuron, 0.25 and 0.4 g . m a. A decrease on relative q u a n t u m yield (Fv/Fm), effective q u a n t u m yield (F/F,'), and photochemical (qp) and nonphotochemical quenching (on), as well as, an increase in minimal (F0 a n d F0') a n d variable fluorescence (F, a n d Fro') were observed during the first weeks after plant emergence in both cultivars. These fluorescence parameters were fully recovered in L.otofio plants only thirteen days after plant emergence, while Histal plants showed a similar recovery three weeks after plant emergence. The quick plant development of L.otofio plants during the firts m o n t h s respect to Histal plants gave a better avoidance of herbicide damage.

P-28-009 DIFFUSE-LIGHT DUAL MODULATION FLUORIMETER: MONITORING OF ELECTRON TRANSFER REACTIONS IN CYANOBACTERIA CULTIVATED IN INTERMITTENT LIGHT Ladislav Nedbal laz and Filip Lederer I~ ~Institute of Microbiology AV CR; "~Photosynthesis and Global Climate Research Center, 37981 T?ebofi, Czech Republic; 3Laboratoire des Biomembranes ENS, 46 rue d'Ulm, 75230 Paris Cedex, France Diffuse light dual-modulation fluorimeter was designed to measure fluorescence emission from suspensions of cyanobacteria (modification of the instrument for conifer shoots will be presented at the satellite meeting in Bordeaux). Both the measuring and the actinic light are provided by arrays of light emitting diodes in square pulses of adjustable duration (-~ 10 p,s). Timing sequence is user defined and consists of generally non-periodic series of measuring and actinic flashes separated by intervals ranging 12 las - 1O0 s. The single-turnover actinic flashes are saturating the QA reduction. The instrument was applied to monitoring of electron transfer reactions in cyanobacteria cultivated in intermittent light (see parallel contribution by Tithe, et.al.). The experiments were designed to identify mechanisms resulting in the dependence of oxygen evolution and of growth rate on the intermittent light frequency.

P-28-007

P-28-010

IS THE MEASURE OF PSH QUANTUM YIELD BY MEANS OF INV1VO CHL A-FLUORESCENCE REALLY A DIRECT MEASURE OF PHYTOPLANKTON PRIMARY PRODUCTION?

P H O T O S Y N T H E T I C C H A R A C T E R I S T I C S OF LEAVES OF THE FIRST AND SECOND GROWTH FLUSHES OF BEECH SAPLINGS (FAGUS SYLVATICA L.)

C. Wilhelm, M. Lohr, J. Bida and A. Domin. Institut ffir Botanik, Universitat Leipzig, Johannisallee 21, D-04031 Leipzig, FRG

D. Epron 1,2, R. Liozon I , M. Mousseau 1 & B. Thiebaut 3 1Lab. Ecologie V6g~tale, Univ. Paris Sud, Bat 362, 91405 Orsey cedex, France; 2Inst. Sci. Tech. Environ., P61e Universitaire, BP427, 25211 Montl~liard cedex, France; 3Inst. Botanique, 163 rue A Broussonet, 34000 Montpellier, France

The effect of intermittent photoinhibiton on productivity was measured by the use of homocontinuous cultures the diatom Phaeodactylum trtcornutura. The controls were illuminated continuously with 19 ~tE/(mZsec), whereas the probe was photoinhibited for a period of 2 or 5 hours per day, respectively. The production was measured on the basis of chlorophyll, cell number and dry matter. Photoinhibition was characterised by the changes in variable fluorescence, PSII quantum yield and light saturation parameters at different stages of the experiment. Photoinhibition reduced the variable fluorescence to about 20%, the apparent quantum yield for the oxygen evolution to about 30%, but the productivity was exactly the same as that of the controls. Intermittent photoinhibition decreases the susceptibility of the cells against subsequent light stress. The results also indicate that photostress modulates the carbon acquisition efficiency under light saturation.

P-28-008 PHOTOSYNTHETIC RESPONSES TO LIGHT AND EXTENT OF PHOTOINHIB1T1ON IN ULVA SP.FROM THE "GREEN TIDES". A COMPARATIVE STUDY WITH ITS BENTHIC HOMOLOGS.

Guy Levavasseur & Dominique Le Guen Station Biologique, CNRS UPR 9042, 29680 Roscuff (France) In Spring and Summer, eutrophication could induce massive proliferations of Ulva along the Brittany coast (France) At the mercy of tides, a great biomass of free-living thalli invades very shallow waters and could be deposit on sandy beaches In contrast with benthic Uh,a, few studies deal with the photosynthetic behaviour of this planktonic form P/I curves were realized to quantify photosynthetic activity and quantum-yield Chlorophyll fluorescence measurements (Fv / F.) were used as a probe during onset and recovery of photoinhibifion The "green tide" thalli exhibit an higher maximum photosynthesis than the benthic ones, independently of the season They also appear to be less sensitive to bright light (stress of 1700 p mol m"2 s-I for 3 hours) and afterwards to have better capabilities for recovery than their benthic homologs All these results suggest that this planktonic Ulvo is well adapted to survive and to proliferate under such drastic conditions.

230

The leaves of the second growth flush of beech exhibited a lower chlorophyll content per unit nitrogen than the leaves of the first growth flush. This decrease in the proportion of nitrogen dedicated to chlorophyll and hence, to light trapping, together with an increase in the photosynthetic capacity par unit chlorophyll may indicate that the leaves of the second growth flush expressed more clearly the characteristic of sun leaves that did leaves of the first growth flush, despite the fact that both were sunlit leaves. It is also shown that the variations in leaf nitrogen contents almost fully accounted for the variations in leaf photosynthetic capacity among leaf types and along the growing season.

P-28-011 PHOTOSYNTHETIC PROPERTIES OF CRYPTOMONAS OVATA IN CULTURE DURING PERIODS OF INCREASING POPULATION DENSITY F. Schanz, M. Zbinden & R. Bachofen institute of Plant Biology, Limnological Kilchberg, Switzerland

Station, Seestr. 187, CH-8802

CrvDtomonas ovate is abundant in Lake Z0rich during the spring bloom. Its ability to grow exponentially allows it to dominate the biomass within one to two days. Investigations were carried out into the physiological causes of this species' growth capabilities. A batch experiment was conducted to follow the species' adaptation to dark during the period of cell density increase. Within ihe growth period of 39 days, the cell density increased to 4.5 • 105 ml"1 while the light intensity inside the cuRure decreased from 47 to 7 i.tmot quanta m -2 s"1. An increase in the antenna size of both PSli (from 2000 to 2900 tool Chl a tool 1 RCII) and PSI (1700 to 2000 tool Chl a tool 1 RCI) was noted. As a consequence, an increase in light uti(ization, ct (from 4.5 to 6.5 , 10 "14 moles 02 m2 cell "1 rain"1) and a decrease in the minimum quantum requirement (from 23 to 10 moles quanta tool-1 02) was observed. During the growth period, the number of RCII per cell stayed constant, whereas the number of RCI per cell diminished, resulting in an increase in turnover time, "¢ (from 7.3 to 9 ms). C.ovata thus exhibits a highly flexible response to fluctuations in light intensity.

Poster

P-28-012

COMPUTER-CONTROLLED PHYTOPLANKTON ANALYSER BASED ON CHLOROPHYLL FLUORESCENCE QUENCHING ANALYSIS USING 4 DIFFERENT EXCITATION WAVELENGTHS J. Kolbowski & U. Schreiber Lehrstuhl Botanik I, Universittit Wiirzburg Mittlerer Dallenbergweg 64, D-97082 Wtirzburg, Germany A new instrument is described which allows detailed analysis of phytoplankton composition and properties in natural surface waters on the basis of 4-wavelengths chlorophyll fluorescence excitation in combination with saturation pulse quenching analysis. Fluorescence is excited by ps-pulses of differently colored light-emitting-diodes in order to differentiate between the 3 major classes of phytoplankton (green algae, diatoms and cyanobacteria). Saturating light pulses of 60ms duration are applied in order to asses the effective quantum yield of photosystem II. Computer-assisted data analysis provides the following information on the 3 different phytoplankton classes: (i) chlorophyll concentration, (ii) apparent photosynthetic activity and (iii) light saturation characteristics. The new instrument should be useful for assessment of primary production in surface waters and early detection of algae blooms.

P-28-013

A MECHANISTIC MODEL FOR PREDICTION OF THE INTERACTIVE EFFECTS OF ELEVATED CONCENTRATIONS OF CO 2 AND 03 ON WHEAT PHOTOSYNTHESIS

M. J. Martin, S.W. Humphries, P.K. Farage, I.F. McKee & S.P. Long. Biology Dept., University o f Essex, Colchester, CO4 3SQ, Essex, UK. A mechanistic model is presented which uses data from acute ozone exposure studies on winter wheat to predict the effect o f elevated ozone concentrations on photosynthesis in terms o f the "effective ozone dose", that is, the dose above a threshold flux. This model is also used to predict stomatal conductance relative to ozone dose, and the results are consistent with the hypothesis that ozone affects stomatal conductance via its effect on photoassimilation rate.

P-28-014 UTILIZATION OF NITROGEN AND CARBON SOURCES OF SUNFLOWER LEAVES UNDER DI_~IqSRENTLIGHT CONDITIONS AND NITROGEN AVAILABILITY Kiyomi ONO, Akira WATANABE, Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan Sunflower plants were grown hydropgnically in an environmentally controlled chamber under nigh (450 /zmol m-2 s-1) and low (150 /zmol m-2 s- 1) light, and high (8.0 raM) and low (0.8 raM) nitrogen conditions. With the growth of the plants, nitrogen content of the first leaves per unit area decreased early under nigh light or low nitrogen. Changes in nitrogen content per dry weight basis did not differ between plants grown under nigh or low light conditions, if nitrogen supply was the same. Light conditions thus appear to limit primarily the total growth of plant that affects the utilization of nitrogen. Total carbon content of the first leaves per unit area was higher in high light than in low light independent of nitrogen availability. The content of metabolizable carbohydrate (glucose + sucrose + starch) decreased extensively after full expansion of the first leaves in nigh nitrogen supply, and the extent of decrease seemed to depend on the amount of nitrogen available to the plant. More scarce resource (nitrogen in low nitrogen supply or carbon in low light) limits thegrowth of plants as expected. These observations suggested that utilization of the two resources in older leaves tightly couples, and utilization of more abundant one (carbon in high light or nitrogen in high nitrogen supply) is determined by the availability of the other.

P-28-015 EFFECT OF LIGHT WAVELENGTH FOR H Y D R O G E N PRODUCTION BY PHOTOSYNTHETIC BACTERIA S. Nishikata, Y. Asada& j. Miyake ResearchInstituteof InnovativeTechnologyfor the Eallh (RrI~) / National Instituteof BiosciencemidHuman-Technology(NIBH), 1-1-3Higashi, Tsukuba,Ibaraki305, JAPAN Effect of light wavelength for the hydrogen production by photosynthetic bacterium Rhodobactor sphaeroides RV was examined. The light penetration in a photobioreactor and its relation to hydrogen production was analyzed. The penetration depends on wavelength which affects the hydrogen production. Light around the absorption maximum of the bacterium was lost in the reactor, perfectly. Only light which does not contain the absorption maximum reached in the deep region and has much contribution on hydrogen evolution. Hydrogen evolution rates were also measured by several light sources to clarify the effect of wavelength. At the illumination with tungsten lamp which contain the light mainly 850 nm, hydrogen evolution rate and conversion efficiency of light to hydrogen were 2.0 l/h/m2 and 5.7 % (106.6 W/m2), 1.8 l/h/m2 and 5.2% (103 Whn2) under illumination with halogen lamp which contain mainly 700 nm, respectively. These results show that photosynthetic bacteria can use the light except the absorption maximum for hydrogen production.

P-28-016 C O M P A R I S O N OF E M P I R I C A L L E A F P H O T O S Y N T H E S I S AND STOMATAL CONDUCTANCE MODELS A l b e r t Olioso, Bioclimatologie INRA, F-78850 Thiverval-Grignon, France Olivier Bethenod, Bioclimatologie INRA, Grignon, France Serge Rambal, CEFE-CNRS, Montpellier, France Marc Tchamitchian, Bioclimatologie INRA, Avignon, France Leaf photosynthesis and stomatal conductance data were acquired on a water stressed soybean crop at the same time as incident PAR, leaf water potential, leaf temperature and leaf vapour pressure deficit (VPD). These data are used to compare different models of stomatal and photosynthesis response through a bootstrap analysis. Different response curves to PAR and to VPD are tested. A 3 parameters response function to PAR gives a good description of stomatal conductance, while a 4 parameters response function is prefered for photosynthesis. Linear responses of photosynthesis and stomatM conductance to VPD, but no temperature effect, are found. Leaf photosynthesis and stomatal conductance models may be used to study the effect of water stress at leaf level. They may also be used at canopy level, if they are introduced in a model of canopy energy and mass fluxes.

P-28-017

REMOTE SENSING OF CHLOROPHYLL FLUORESCENCE LIFETIME OF MAIZE DURING DIURNAL CHANGES I. Moya!, Y. Goulasl, 2.M. Briantais 2, L. Camenen 1, M. Gorbunov4 and Z.G. Cerovic 1. lLURE - CNRS, Universit6 de Paris-Sad, 91405 Orsay Codex, France ; 2Labomtoire d'Ecophysiologie, URA 1492, 91405 Orsay Codex, France; 4 Moscow state Universsity, 119899 Moscow, Russia

A LIDAR system developed by LURE for lifetime (x) measurements, was shooting on maize plants from a distance of 15 meters. Measurements were performed every 5 minutes, day and night, for several days. In addition the leaf temperature, PAR and the relative fluorescence yield (~) were measured. The results can be summarized as follows: i) The proportionality of x and ~ was confirmed for the plants under well watered conditions, ii) Water stress induced a quenching of the lifetime (xs) at midday, which decreased from 0.7 - 0.8 ns to 0.4 - 0.5 ns. The same quenching effect was present in the fluorescence signal (Fs) from a PAM fluorimeter, iii) The maximal fluorescence (Fmax) was strongly quenched at midday. The effect is stronger under water-stress conditions. Interestingly the quenching of the lifetime (xmax) was much lower, indicating the presence of static quenching.

231

Poster

P-28-018

LIGHT ACCLIMATION OF FAST- AND SLOW-GROWING GRASSES.

P-28-021 SOME

PHOTOSYNTHETIC INDICATORS OF L E A V E S AT DIFFERENT A G E S OF T W O B E A N V A R I E T I E S . I.CHLOROPHYLLS, SOLUBLE SUGARS AND PROTEIN CONTENTS.

U. Sundin and E. Ogren Dept. of Plant Physiology, Univ. of Ume~, S-901 87 Ume~, Sweden

M. C a s t r i l l o , Ao F a r i ~ a s , C. D o n o s o , N. S u a r e z ; A. K a z a n d j i a n o Depto. Biolog~a Organismos. Universidad Sim6n Bol~var Apdo. Postal 89000. Caracas 1080-A Venezuela.

Photosynthetic light acclimation was investigated in four species of grasses, the fast-growing Hordeum vulgare and Phalaris arundinacea originating from productive habitats and the slowgrowing Festuca ovina and Nardus stricta, from unproductive habitats. Plants, grown under the low irradiance of 200 pmol m-2s -1 , were transferred to 1000 pmol m -2 s-1 and were analysed over the next 10 days by measurements of net CO2 uptake, stomatal conductance and activation and amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Differences in photosynthetic acclimation will be discussed in relation to the ecological habitats of the plants.

The c h l o r o p h y l l a, b a n d a + b c o n t e n t s a n d R a / b , t o t a l soluble and reducing sugars and protein contents were a n a l y z e d in l e a v e s of d i f f e r e n t a g e s in p l a n t s of f o r t y six to f i f t y t h r e e d a y s o l d of two v a r i e t i e s : Tacarigua and Montalban. T h e l e a f age w e r e established as f o l l o w s : a d u l t l e a f t o t a l l y e x p a n d e d 3 4 - 3 8 d a y s o l d (i); e x p a n d i n g y o u n g l e a f 1 8 - 2 2 d a y s o l d (2) a n d v e r y y o u n g l e a f 2-6 d a y s o l d (3). The chlorophyl a, b a n d a + b i n c r e a s e w i t h l e a f aoe. T h e R a / b is h i g h e r in t h e e x p a n d i n g y o u n g l e a f (2). The total soluble sugars and reducing sugars decrease in t h e y o u n g e x p a n d i n g l e a f in the M o n t a l b a n variety w h i l e in the T a c a r i g u a variety these contens decrease w i t h l e a f age° T h e p r o t e i n c o n t e n s i n c r e a s e w i t h leaf age in b o t h v a r i e t i e s .

P-28-019 PHOTOSYNTHETIC LIGHT-RESPONSE CURVES IN MARINE BENTHIC PLANTS FROM THE THAU LAGOON Adam Lata~a Institute of Oceanography, Gdafisk University, AI. Pi~'sudskiego 46, 81-378 G-dynia, Poland The irradiance-photosynthesis curves in plants collected from the ropes with oyster cultures and also from the bottom of the lagoon (1 and 5 m depth) were determined by the volumetric method. Based on photosynthetic light-response eta-cos, values of compensation point, Ik, saturation point, maximum photosynthesis and dark respiration were calculated. The studies comprised the measurements of gas exchange intensities in 33 species belonging to green (I0 species), brown (3) and red (18) algae and vascular plants (2). The highest maximum photosynthetic and respiration rates were found in green (Enteromorpha lima, Cladophorapygraaea) and red (Antithamnionplumula, Dasya hutchiniae) algae. The lowest values were noted in red algae Gracilaria verrucosa, Laurentiapinnatifida and Chylocladia verticillata. The present data were compared with the results obtained by the same method for algae from two other geographic zones: Arctic region (S-W Spitsbergen) and cold-temperate region (S Baltic).

P-28-020 E F F I C I E N C Y O F P R I M A R Y R E A C T I O N S OF Y O U N G B E E C H E S (FAGUS S Y L V A T I C A L. ) IN T H E U N D Z R S T O R E Y O F M A T U R R B E E C H T R E E S U N D E R LIGHTFLECK- AND CONSTANT IRRADIATION

P-28-022 A COMPARISON OF THE DIURNAL FLUCTUATIONS IN PHOTOSYNTHESIS AND ASSOCIATED METABOLISM IN VITIS V1N1FERA LEAVES GROWN IN SEMI-ARID AND TEMPERATE CLIMATES. M. Chaumont 1, M.L. Osrrio 2, M.M. Chaves2, H. Vanacker, J-F. Morot-Gaudry 1 and C.H. Foyer 3. 1Laboratoire du Mrtabolisme et de la Nutrition des Plantes, INRA, Route de St Cyr, 78026, Versailles, France; 2Instituto Superior de Agronomia, Universidade Trcnica de Lisboa, Tapada da Ajoda, P-1399 Lisboa Codex, Portugal; 3Department of Environmental Biology, IGER, Plas Gogerddan Aberystwyth, Dyfed, SY 23 3EB, U.K. Fernao Pires and Pinot noir grapevines grown in field-sites in Lisbon and Versailles respectively, both showed substantial inhibition of photosynthesis from mid-morning onwards. Prior to the period of measurement, the Lisbon site had received no rain for 45 days while the Versailles vines were watered daily. In neither case was photoinhibition observed, the Fv/Fm ratio remaining relatively constant from dawn until dusk. The foliar zeaxanthin content showed a marked diurnal variation being maximum at mid-day and at minimum values at dawn and dusk. Interestingly, the total xanthophyll pool was much greater in the leaves of Versailles vines than the Lisbon site. The maximum zeaxanthin levels of the former at mid-day were three times those of the latter. In contrast, the foliar pools of ascorbate and glutathione either remained constant or increased slightly during this period. The pools of glycine and serine in the leaves declined from mid-morning onwards at both sites suggesting that photorespiration was declining simultaneously with CO2 assimilation. Nevertheless, assimilatory power (measured here as the ratio of 3-PGA/FP) remained constant throughout the photoperiod indicating that energy supply and utllisation in the leaves were well-balanced throughout the photoperiod despite the decrease in CO2 assimilation.

P-28-023 DIFFERENT MODES OF LIGHT LIMITATION OF TURBIDOSTAT CULTURES O F RHODOBACTER CAPSULATUS

U. Hansen ~ & V. H0flich Inst. of Biophysics, University of Hannover, Herrenh&user Str.2, D-30419 Hannover, Germany; ~present address: EC-JRC, Environment Institute, T.P.051, 1-21020 Ispra (Va.), Italy

A. Tsygankov & T. Laurinavichene Inst. of Soil Sciences and Photosynthesis RAS, Pushchino, Moscow Region, 142292 Russia

The aim of the study was to compare the extent of down regulation of photosynthetic primary reactions under constant non photoinhibitory irradiation and under conditions where the light phases (10 s) with the same intensity were interrupted by dark phases of the same duration. Chlorophyll fluorescence measurements were carried out on attached uninduced leaves of young beeches growing at their natural site in the understore~ of a beech forest in the Solling area, Germany, where sunflecks contribute considerably to the total incident irradiation. A lower non-photochemical fluorescence quenching was found under conditions of alternating light intensities. Also the apparent electron transport rate, calculated from fluorescence and irradiance data, showed a slight but insignificant increase. Whether the higher carbon gain found during brief lightflecks is due to primary processes rather than t o post-illuminatio n carbon fixation is discussed.

Quantum consumption for biomass production (QCBP) and physiological peculiarities of Rh. capsulatus grown in turbidostat regime with different degrees and modes of culture light limitation were analyzed comparatively. Light limitation was achieved by decreasing of incident light intensity at low biomass concentration (up to 0.5 g of dry biomass/l) or by increasing the biomass concentration (up to 3 g/l) at constant incident light intensity (80 W/m2). In both the cultures QCBP was minimal at highest light limitation. Cells grown at the same degree but different modes of light limitation were characterized by equal phosphorous content and by equal ratio of antenna to reaction center bacteriochlorophyll. However, cultures grown at low biomass concentration exhibited the lower QCBP (as low as 0.2 einstein/g). Moreover, these cultures were differed by the biomass reductance degree, by C/N ratio and by bacteriochlorophyll content in cells. Reasons for differences of cells grown at these modes of light limitation are discussed

232

Poster

P-28-024 SHADE TOLERANCE PHOTOSYNTHETIC PHOTOINHIBITION.

IN OAK CAPACITY

AND BEECH SEEDLINGS • AND SENSITIVITY T()

P-28-027 PHOTOSYNTHETIC PROPERTIES OF THE ARCTIC KELP, LAMINAR1A SOLIDUNGULA, GROWN IN A LIGHT-DARK CYCLE OR CONTINUOUS DARKNESS

E. Dmyerl, E. Manrique2, A Sanchez2, J M Chico2 & B Hubert l Unit6d'EeophysiologieForesti~re,INRANancy,F 54280 Champenoux,France, 2 Biologia Vegetal II, Facultad de Farmacia,UniversidadComplutense,E 28040 Madrid, Spain. Young seedlings of oak (Quercus robur) and beech (Fagus sylvatica) were grown under 3 irradiance levels (100%, 45% and 15%) in a nursery near Nancy. Effects on growth, leaf anatomy, chlorophyll content, photosynthetic capacity, in situ net assimilation rates and tolerance to methyl viologen were recorded. Growth was substantially reduced at the lowest irradiance. The photosynthetic capacity of shade leaves was lower than in sun leaves. Half the plants of the shade treatments were then exposed to full irradiance, and the consequences were recorded during several weeks. A significant decrease in the predawn values of PS II efficiency was recorded during the first week on shade plants, with a gradual recovery thereafter. Shade leaves presented a high sensitivity to methyl viologen, that decreased gradually after reacclimation to high irradiance. Treatment-induced differences in the xanthophyll cycle were recorded and related to the ability to dissipate excess irradiance. Significance of these results for the acclimation of the two species to contrasted light regimes will be discussed.

P-28-025 SOME ECOLOGICAL AND PHYSIOLOGICAL IMPLICATIONS OF PHOTORESPIRATION IN MARINE PHYTOPLANKTON C. Leboulangerl,2, H. Jupinl and C. Descolas-Gros 2 1Lab Biologie V6g6tale, Univ Perpignan, 66860 Perpignan cedex, 2Lab Hydrobiologie Marine et Continentale, URA 1355, cc 093, Univ Montpellier, 34095 Montpellier cedex 5 Marine phytoplankton as C3 terrestrial plants is submitted to photorespiration, and a rapid method to show it consists in measuring the amount of glycolate released in seawater. We developed two methods (HPLC and CPV) allowing a precise determination of glycolate in the field as in laboratory, with an error less than 10 %. We found in ocean, as in lagoon and culture samples, concentrations of dissolved glycolate that are consistent with a general repartition of photorespiration among phytoplanktonic populations. In oligotrophic waters, total dissolved glycolate represented an equivalent carbon stock with estimated daily primary production. Such a release of orgb.nic carbon is an energy-cost process, but seems to be counterbalanced by ecological avantages and/or nocturnal reassimilation. Ecological avantages could be at first a protection against high irradiance, sustain of a given C/N/P ratio inside the cell, etc... Nocturnal reassimilation is involved in nitrogen fixation, extracellnlar glycolate providing energy that misses from photosynthesis. Photorespiration appears as an important process regarding to the global carbon cycling.

P-28-026 VARIATIONS OF PRIMARy PRODUCTION MEASUREMENTS LAGOON (TRAU LAGOON, FRARCE) DURING ONE YEAR

IN

A

MEDITERRANEAN

E_ W~uil]and, A. V a q u e r and C. D e s c o l a s - G r o s L a b o r a t o i r e d ' H y d r o b i o l o g i e Marine, U n l v e r s i t ~ M o n t p e l l i e r II, U R A CNRS 1355, CC 093, 34095 M o n t p e l l i e r C e d e x 5, F r a n c e V a r i a t i o n s of p r i m a r y p r o d u c t i o n m e a s u r e m e n t s c a l c u l a t e d with two d i s t i n c t m e t h o d s were studied, d u r i n g one year 93-94, in a c o a s t a l l a g o o n with an important oyster production. Each month, three areas were s a m p l e d ; i n the oyster cultures (ZA), outside (ROQ), and a offshore reference area (SEA). For e a c h sampling, w i t h e n v i r o n e m e n t a l measurements, two m e t h o d s were applicated: the r a d i o c a r b o n (14C) m e t h o d (Steemann Nielsen, 1952, J. Cons. Inst. Explor. Met, 18 117-140) and e n z y m a t i c e s t i m a t e s of carbon i n o r g a n i c a s s i m i l a t i o n (carboxylases, D e s c o l a s - G r o s & Fontugne, 1985, Mar. Biol., 87 1-6). The results show a linear relationship b e t w e e n the 14C m e t h o d and R u b i s c o a c t i v i t y e s t i m a t e in ZA only. In ROQ and SEA area, some periods present a high level of 14C assimilation, p a r t i a l l y e x p l a i n e d by h e t e r o t r o p h i c m e t a b o l i s m (~carboxylation) of p h y t o p l a n k t o n ' s CO 2 assimilation. The s i m u l t a n e o u s use of two m e t h o d s reveal v a r i a t i o n s in m e t a b o l i c p a t h w a y s u s e d for CO 2 assimilation. First, if most of w i n t e r p r i m a r y p r o d u c t i o n can be a c c o u n t e d b y Rubisco activity, in sun,her this a c t i v i t y was lower. Secondly, this a s s o c i a t i o n allows to d i s t i n g u i s h p h y t o p l a n c t o n i c m e t a b o l i s m in the oyster's area f r o m others areas (ostreiculture impact ?).

W.J. Henley I & K.H. Dunton2 1 Botany Dept., Oklahoma State Univ., Stillwater, OK 74078-0293, USA; 2 Marine Science Institute, Univ. of Texas at Austin, Port Aransas, TX 78373-1267, USA Field-collected Laminaria solidungula thalli were maintained at 0 + 1.5°C for seven months in factorial experiments: two light regimes (continuous darkness or -25 ~tmolphotonsm-2 s-1 on a 12:12h L:D cycle) x two nutrient regimes C f' nutrients + nitrogen). Photosynthetic properties and pigments, measured at the end of the experiment, responded to the light and nitrogen treatments. Thaili held in darkness (as in arctic winter) retained full photosynthetic capacity and maximum quantum yield, although they grew minimally compared to winter m situ growth rates, probably because of negligible initial carbohydrate reserves. L. solidungula appears well adapted to survive prolonged winter darkness under turbid ice cover yet can quickly benefit from any available light.

P-28-028 COz FIXATION SYSTEM BY CHLORELLA ON A FLUTER MEMBRANE M. Tsutsumi 1, K. Kawachi 1, M. Kaneko 1, & M. Tsuzuki z 1Toshiba Corporation, Fuchu, Tokyo 183, and 2School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-03, Japan. Because of the increase in the atmospheric CO2 concentration, it is important to develop technology of CO2 fixation. Microalgae and cyanobacteria have been considered as possible biological materials for the CO?. fixation system, though there are several problems still to be solved. In a large scale of mass culture, a large amount of water and nutrients are required. We examined a possibility of CO?. fixation by Chlorella on a filter membrane. The rate of CO2 fixation by Chlorella on the membrane was almost the same as that in liquid medium and reached 5g CO?.'m-Z'h-1 at 10% CO?-. The ability of CO;, fixation lasted for at least a week when the cells were moistured occasionaly. Therefore, we concluded that CO2 fixation by Chlorella on a membrane is applicable to technology.

P-28-029 THE EFFICIENCY OF PHOTOSYSTEM II ELECTRON FLOW AND CARBON ASSIMILATION DURING PHYTOPLANKTON DEVELOPMENT IN A MESOCOSM. C. Geel 1, M. Steendijk 2, J.C.H. Peeters 2 and J.F.H. Snel1. 'Dept of Plant Physiology, Wageningen Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands; 2National Institute for Coastal and Marine Management, P.O. Box 8039, 4330 EA Middelburg, The Netherlands. The effect of eutrophication on primary production of a marine ecosystem in a mesocosm was investigated. The efficiency of photosystem (PS) II electron flow was monitored with a chlorophyll fluorescence system which was based on a previously developed Xe-PAM fluorometer. The efficiency of PSII electron flow shows diurnal variations from about 0.7 during the night an early in the morning to values of about 0.3 at noon. Light-response curves of ~4CO~ assimilation and PSII electron flow were measured in the laboratory on mesocosm samples to determine the relation between the efficiency of PSII electron flow and 14CO2 fixation. Under these conditions the correlation between PSII electron flow and carbon assimilation is good. This allows estimation of total '4CO2 fixation in the mesocosm from PSII electron flow and light climate measurements.

233

Poster

P-28-030 SPATIAL ACTIVITY

AND OF

DIURNAL VARIATIONS THE TROPICAL SEAGRASS

~N THE PHOTOSYNTHETIC THALASSIA TESTUDINUM

M. Merino I, S. Enrlquez 2, R. J. Strasser 3 & R. Iglesias-Prieto 4 IInstituto de Ciencias del Mar y Limnologia, Universidad N a c i o n a l A u t 6 n o m a de M4xico, 04510 M e x i c o D. F. M~xicg; 2Freshwater Biological Laboratory, Helsing~rsgade 51, 3400 Hiller~d Denmark; 3Laboratoire de Bio4nergetique, Universit4 de Geneve, Station Botanique, CH-1254 Lullier~Gen~ve Switzerland: 4Departamento de Ecologla, Centre de Investigaci6n Cientlfica y Educaci6n Superior de Ensenada, Km 107 Carretera Tijuana-Ensenada, Ensenada B.C. 22860 M6xico We have analyzed the photosynthetic responses of the seagrass T. testudinumalong a transect perpendicular to the coast line. The results reveal a correlation between the sediments size distribution and organic material content with the parameters of the fluorescence induction curves of the leaves, as well as with several demographic properties of the intact plants. We have also analysed the diurnal variations in the fluorescence induction curves of a population oE seagrasses exposed to very pronounced internal irradiance gradient. Comparative analyses of the induction curves taken at the apical and basal portions of the leaves, indicate that the photoprotective mechanisms were not sufficient to prevent photodamage of the apical parts.

P-28-033 SEASONAL TRENDS IN GAS EXCHANGE IN SUNFLOWER AS AFFECTED BY TILLAGE TECHNIQUES L. G. Angelini, L. Ceccarini, P. Belloni & E. Bonari Agronomy Department, Pisa University, Via S. Micbele degli Sealzi n. 2, 56124 Pisa, Italy The influence of tillage management on gas exchange and dry matter accumulation on sunflower (cv. Stromboli) was analyzed in Central Italy on a very clayey soil where the techniques of minimum tillage (10-15 em deep disk harrowing -MT) and conventional tillage (ploughing to a depth of 50 cm - CT) have been tested since 19981. Gas exchange measurements were taken over a period of increasing water deficit from the vegetative stage to beginning of maturity. In parallel with this, crop growth, leaf water potential and changes in soil water content were examined. Seasonal values of LAI, biomass and plant height were higher under CT in comparison with MT. CO2 exchange rates were significantly lower under MT compared to CT on 7 measurement dates during a dry period. Plants under ploughed soil showed higher transpiration rates and higher water use efficiency. When water stress oceured the largest treatment differences in seasonal net photosynthesis values were observed during bud flower development and full flowering. The low photosynthetic rates under MT as compared with CT have been related to water availability, in particular under conditions of high evaporative demand and low precipitation as usually occur in the Mediterranean climate.

P-28-031 THE RELATIONSHIP BETWEEN ELECTRON TRANSPORT RATE THROUGH PSII AND CO 2 GASEXCHANGE IN ANTARCTIC CRYPTOGAMS.

P-28-034 GAS E X C H A N G E S IN TWO INBRED LINES OF S U N F L O W E R WITH DIFFERENT LEAF CHARACTERISTICS.

B. $Ghroeter 1 , ]V.G.A. Green 2 , L. Kappen 1 , R.D. Seppelt3 {~nd K. Maseyk = ,Botanisches Institut, Universitfit Kiel, D-24098 Kiel, Germany; =Biologic~J Sciences, University of Waikato, Hamilton, N e w Zealand; Australian Antarctic Division, Kingston, Tasmania, Australia

L. 0. Angelin~t , P. Betloni t &A. Merrien2 1A~ronomv Department Pisa University, Via S. Michele degli Scalzi n. 2, - - Pisa, - Italy; 2Ddpartement ' 56124 Etudes et Reeherehes Centre de Biologic Appliqu6e, Rue de Lagny, 77178 Saint-Pathus, France.

Carbon dioxide exchange and chlorophyll a fluorescence were measured simultaneously in the lichen Umbilicaria aprina and the moss Bryum argenteum in the field in continental Antarctica over a range of temperatures. The relationships between carbon dioxide exchange and chlorophyll a fluorescence parameters differed considerably between the moss and the lichen. Electron transport rate (ETR) was a valid measure for gross photosynthesis only for the moss, but gave no indication of overall carbon balance. The situation for the lichen was complex with the relationship between CO 2 exchange and ETR often being nonlinear and very temperature-dependent

P-28-032 FLASHING LIGHT EFFECTS AND ENERGY ECONOMY IN ALGAL

CULTURE (CHLORELLA PYRENOIDOSA): APPLICATION OF LIGHT EMITTING DIODES IN BIOREACTORS. H.C.P. Matthiis x, H. Balke t, U.M. van Hes l, B.M.A. Kroon l, L.R. Mur 1, P. Paul 2 and R.A. Binot3 tDepartment of Microbiology, University of Amsterdam; 2Comprimo consulting services, Amsterdam; 3ESA/Estec, European Space Research and Technology Centre, Noordwijk, the Netherlands. A device for continuous culture of phototxophic organisms with light emitting diodes as sole light source is described. The diodes applied show a peak emission at 660 nm with a half power bandwidth of 30 nm. No need for additional supply of blue light was found. Using flash modulation permitted application of a higher voltage which yielded stronger light output during flashes. Extension of the dark period between the 5 t~s flashes to 45 ps (10 % duty cycle) still permitted the maximum growth rate to be reached. A modulation domain rendering relatively high efficiency of light energy conversion was identified. We conclude that application of flashes provides a major gain in energy economy, making LED illumination favorable in comparison to ordinary luminiscent light sources.

Gas exchanges in two self-pollinated sunflower lines with the same leaf area but with different leaf size (SL small leaves and LL large leaves), growing in different row arrangements (80, 50, 30 em),were examined. Field measurements of CO2 and water vapour exchange were made at flowering (F2-F3) and during early maturity (M0-M1). Range of leaves in various positions on plants was studied in order to better understand the relationships between photosynthesis and leaf position on the stem. Differences in net photosynthesis, transpiration and water use efficiency between the two genotypes and between different row widths were observed.Tbe SL line showed higher rates in photosynthetic activity per unit leaf area and greater water use efficiency. Different row widths affected the photosynthetic rate and a gradual reduction of photosynthetic activity was observed as row width became smaller.The different leaf architecture of the two genotypes affected the photosynthetic activity of the upper and middle portion leaves as row width changed.Thus a significant drop in CO2 net assimilation values and water use efficiency was observed from 80 to 50 em m the LL type, characterized by larger leaves which were immediately affected by shadow.

P-28-035 PHOTOSYNTHETIC O~ EVOLUTION AND QUANTUM YIELD OF SOME WHEAT GENOTYPES DURING LEAF SENESCENCE _Paiev~6, S. 1, Krstid, B.1, Plesni6~r, M.2, Stankovi¢5, ~.1, Den6i¢5, 5. 2 llnstitute of Biology, Faculty of Sciences,21000 Novi Sad, Yugoslavia 21nsfituteof Field and Vegetable crops, 21000 Novi Sad, Yugoslavia The relationshipsbetween net photosynthetic 02 evolution, quantum yield, dark respiration rate, pigment content, genotyp and leaf age were investigated in a field experiment during two years, lnvesligafionsincluded 15 genotypes of winter wheat important in selection. Photosynthesis and other parameters were measured at flag and second leaveS at the four stageS during postanthesis period.Net photosyntheSis rate and dark respiration rate varied significantly according to genotyp and leaf age. The quantum yield, giving information for the definition of m a x i m u m genetic potential capacity of photosynthesis since it determines the activityof PSI[, also varied in relationto genotype, but variations were evident at the last period of vegetalion. Significant decrease of quantum yield also occurred between fleg and second leaves in most of investigated 9enotypes. During seneScence differentgenotypes showed differentdegradation degree of P 5 ll.Concenirafion of photosynthetic pigments also varied, but there were not always significant correlation between net photosynthetic rate and

pigment content.

234

Poster.

P-29-003 THE THERMAL SIGNAL DURING LIGHT-INDUCED INDUCTION KINETICS OF PHOTOSYNTHESIS AND ITS DARK-RECOVERY Poster

session

C. Buschmann Botanical Institute II, Univ. of Karlsruhe, D - 76128 Karlsruhe, Germany

29

Energy of light absorbed in a leaf is converted only into (a) photosynthetic activity, (b) chlorophyll fluorescence, and (c) heat. Therefore the increase of one component must lead to the decrease of at least one of the other two components. Photosynthetic activity is often deduced from fluorescence assuming an antiparallel behaviour. However, this can only be true if heat production is very low, constant or parallel to one of the other two types of energy. Without heat detection the low fluorescence, e.g. in non-photochemical quenching or photoinhibition, is often explained as "increase of thermal dissipation". Here simultaneous measurements of the thermal photoacoustic signal and of fluorescence of leaves during the light-induced induction kinetics of photosynthesis and its dark-recovery are presented. They show that heat dissipation is neither low, constant nor parallel to fluorescence. Therefore the thermal signal should always be measured in order to fully understand the way leaves convert the energy of absorbed light.

Photosynthesis: global aspects P-29-001

-

P-29-036

P-29-004

P-29-001 DAILY IONIC OSCILLATION IN LEAF-CELL SAP OF TROPICAL CAM PLANTS UNDER FIELD CONDITIONS.

E F F E C t OF O Z O N E ON PHOTOSYNTHESIS AND G R O W T H OF EGYPTIAN CI.~TIVAR OF C O T T O N

E. Ollvares & G. Agular Centro de Ecolog£a IVIC, Apdo. 21827. Caracas 1020A. Venezuela. Parallel to typical oscillation of titratable acldlty of CAM plants, a delta in the concentration of ions in dawn-dusk leafcell sap was observed i n Fourcroya huraboldtiana (Agavaceae), sampled in the succeselon of a cloud forest in Venezuela, in C~us~a ~osea (C1uslaceae) in a dry forest growing on calcareous soils and on asphalt in a natural oll sp~ll area, where also Clusla sp., TilZandsia f~exuosa (Bromeliaceae) and ~ubpiloce~eous ottonis (Cactaceae) were tested. Anions were anallzed by ionic cromatography and cations by atomic absorption spectrometry. The sulphate concentration is related to the pN of the cell sap. The leaf-cell sap of F. ~ m b o ~ G dusk samples were tltrated with mallc acid to obtain the low dawn pH in the field and the sulphate concentration was compared. An increase of the initial values was observed, as occurred in the field dawn samples.

P-29-002 EFYECTS OF EL]CVATEDCO= ~ l a N ON ~ C(IWOBIUATIONOF 'IlffIAI~ID MUmgA..'~B AND ON STAI~I-I AC(XMJLATION IN (]-HJI~0PL&.qI~ l l J , V ~ OF D l g F i ~ q ~ Clil3PS BOo-Yu Zu¢~ Gel-Zhan Jlang & Tlng-Yun Kuan~ Dept. of Photosynthesis, I n s t i t u t e of Botany, Academia Slnic& Boljlng 100044, China e f f e c t s of elevated CO= concantratian on the chloroplast u l t r a s t r u c t a r e were studied In leaves of C. plants 21~dle~,o u t l v a and Glyvl/Je ~ . . and C. plant ..qwtau'la If.miles. ~ raspemm to elevated 13~ coneantrattan (660-700 pIzd was characterized by drsmatlc ~k.n~es in thylakoid mmbrass of masophyll c e l l chloroplasts, and by d r a s t i c changes in starch accumulation in chloroplasts of mesophyll and bundle-sheath c e l i a The grana thylakotda, asp. starch grains in the sponge sad bundle-sheath c e l l s In the Ca plants were more than those of the control. These canformati0aal changes of thylakoid membranes consisted with our functional results, viL the capacity of chloroplasts for l i g h t abuorptiun sad efficiency of PSII were increased (Lu et el, in press). However, I/ran the starch accumulated too much, the grant esF the stratus tbylskoids eunld be recognized especially in the C. plant. This might c a s i s t a c o r r e l a t i o n between the emformatlun of s t r a t a thylakoid= and starch accumulation, bacause eunversion and transfer of starch need energy f r ~ ATP, and EF for ATP formation were d i s t r i b u t e d mainly un PSu face of stream thylakoid membranes, and the end and margin mel~ranes of grmm thylakoida.

Hassan, I.A.:, Asl'maore, M.R. ~, Bell, LN.B. "~& Friga, G. ~ i Dep~'ment of Botany & Microbiology, Faculty of Science, Alexandria University, 21526 E1 Shatby, Alexandria, Egypt; ~Department of Biology., Imperial College at Silwood Park, Ascot, Barks, SL5 7PY. U.K.

Over recent years convincing evidence has emerged ofbo~ a decrease in sl~ospheac ozone (O~)and an increase in tropospheric O, concemration, rh~e ,, an inlilnate relationship between O~ and photosynthesis (A) which is ultimately linked to plant growth and yield. Egyptian culUvar of cotton (Go&~,piumhirsutum L.) was exposed to 701111-:O~. Photosynthetic rate was reduced by 18%, and chlorophyll content and growth of the plant were also reduced by O~ exposure. O~-indu~d reduaiom in A were rela~edto declines in growtl~ Scince exposure to O~ concenUation typical of level of the pollutant observed in Egypt reduced A and growfiaof the species tested, reductions in A may be occurring in othertu~,ortant crops in Egypt,

P-29-005 P H O T O S Y N T H E T I C A C C L I M A T I O N T O G R O W T H IN E L E V A T E D C O 2 IN N A T I V E U K H E R B A C E O U S S P E C I E S Phillip Davey, Peter Farage & Steve Long. Department o f Biology, University o f Essex, Colchester, CO4 3SQ, UK. The acclimation o f photosynthesis to elevated CO2 and interactions with nitrogen was investigated for a range o f U K native species. Bellis perennis, Chamaenerion angustifolium, Dactylis glomerata, P o t w m u a and Rumex obtusifolius were g r o w n at a current ([CO2]amb.) and elevated ([CO2]dev.) CO 2 concentrations with high and low nitrogen application regimes. Gas exchange analyses were used to calculate in vivo carboxylation capacity (Vc,max) o f Rubisco and the maximum capacity for the regeneration o f RubP. These species showed contrasting degrees and directions o f photosynthetic acclimation. Growth o f D.glomerata and C.angustifolium at [CO2]elev" lead to an acclimation response at low nitrogen supply, the decreases in Vc.max suggested a reallocation o f nitrogen away from Rubisco. B. perennis, P. annua and R.obmsifolins displayed no acclimatory interaction with [CO2] and nitrogen, which stimulated CO 2 uptake at [CO2]elev ' regardless o f nitrogen supply. Investigating these responses are critical for parameterising mechanistically based models o f natural vegetation responses to climate change.

235

Poster

P-29-006 LIGHT A C C L I M A T I O N AND O Z O N E T O L E R A N C E OF W H I T E PINE

Mare Cherland, John. W. Malcolm and Roger M. Cox, Natural Resources Canada, Canadian Forest Service - Maritimes, P.O.B.4000, Fredericton, New Brunswick, Canada, E3B 5P7 ~ a n forested areas have to cope with increasing loads of air pollutants. Little is known about the response of sun and shade parts of a tree to O3. Trees from 2 eastern white pine populations, that are arising from 2 zones of pollution, were acclimated to either full Sun or 60% neutral shade. A branchchamber fumigation system delivered 3 O3 treatments (rnirnicking natural events but peaking at different concentrations) to branches of the same tree for 110 consecutive days. The oldest Son-adapted needles showed the most visible damages. High 03 levels (200-ppb peak treatment) had detrimental effects on both CO 2 f'ucation and stomatal conductance over the season, particularly on shade adapted trees. An initial stimulation of net CO 2 f'ucation was observed for 80-ppb peak treated branches, before it declined to its initiaJ level. New needles seemed to be more tolerant than old ones to 03 treatmenL We can conclude that light environment seems to affect plant responses to ozone. From those data, a model is constructed to take into account the difference in response to ozone of differentially light acclimated parts of the U-ee.

P-29-007 SLOW CHLOROPHYLL FLUORESCENCE, NET CO2 ASSIMILATION AND CARBOHYDRATE RESPONSES IN POLYTRICHUM FORMOSUM TO ELEVATED CO2 CONCENTRATIONS

Zsolt Csintalan 1, ZoltSn Tuba 1 and Eric Laitat 2 1Dept of Botany and Plant Physiology, Agricultural University of G0d0110, P~iter K. u. 1., H-2103 GODOLLO, Hungary; 2Dept of Plant Biology, Agricultural Faculty of Gembloux, Passage des d~port4s, 2, B-5030 GEMBLOUX, Balgique

P-29-009 HEAVY METALS DETECTION USING THYLAKOIDS ENTRAPPED IN POLYVINYLALCOHOL-SBQ

R. Rouillon 1, Y. Gingras 2, R. Carpentier 2 and J-L. Marty 1 1 : Universit6 de Perpignan, Centre de Phytopharmacie, GERAP URA CNRS n ° 461, 52 Av. De Villeneuve 66860 PERPIGNAN FRANCE 2 : Centre de Recherche en Photobiophysique, Universit6 du Qu6bec a TroisRivi~re, C.P. 500 Trois-Rivi~res, QUEBEC, CANADA, G9A 5H7 A new immobilization method based on entrapment of thylakoids in a photo-crosslinkable polyvinylalcohol bearing styrylpyridinium groups was developed. The immobilizing process was simple and led to a membrane which allowed a good storage stability in a dry state (25% activity after one year at -18°C). The photosynthetic activity was electrochemically determined in a special compartment cell using a three-electrodes system (platinum working and counter electrodes and an Ag/AgCI electrode as the reference). The efficiency of this technique for the detection of the herbicides diuron and atrazine which inhibit photosynthetic electron transfer at the level of the photosystem I1 plastoquinone binding protein has been shown (R. Rouillon, M. So16, R. Carpentier and J-L. Marty, 1995, Sensors and Actuators B, in press). In this work, we have studied the detection of various heavy metals such as mercury, copper, cadmium, zinc and nickel which have an inhibitory effect on photosynthetic electron transport.

P-29-010 T E M P E R A T U R E A N D LIGHT D E P E N D E N C E S OF C H L O R O P H Y L L F L U O R E S C E N C E in vivo FOR NORWAY S P R U C E S E E D L I N G S K. Roha~ek & P. ,~iffel

Institute of Plant Molecular Biology, Academy of Sciences, Brani~ovskd 31, CZ-370 05 Cesk4 Bud~jowce, Czech Republic

Moss cushions of PolytWchum formosum ware placed into open top chambers maintained at air CO2 concentrations of 350, 467, 683 and 700 pmol mol "1 in Vielsalm, Belgium in August, 1992. Photochemical activity in the high CO2 mosses either did not differ (467pmol mol "1 CO2) or were lower (583 and 700 pmol mo1-1 CO2) than in the ambient ones after 11 months of exposure. The fluorescence ratio F690/F735 (both at maximum and steady-state fluorescence levels) indicated an increasing decrease in chlorophyll a + b content along the CO2 gradient. Net CO2 assimilation at optimum water content was lower at all experimental elevated CO2 concentrations than in the ambient. The initial slope of the curve of net CO2 assimilation as a function of 'internal' CO2 concentration was lower in all high CO2 treatments than in the ambient one. The indicated limitation of Rubisco capacity was highest in the 583 and 700 .umol mo1-1 CO2 treatments. Starch content increased with increasing CO2 concentrations. It appeared that the acclimation of photosynthesis was of downward type in the mosses (plants without stomata) and that acclimation had an effect on photochemical processes of photosynthesis also.

Dependence of chlorophyll fluorescence in vivo on ambient temperature and/or photosynthetic photon flux density (PPFD) was determined for the Norway spruce seedlings. The highest values of potential yield of PS2 photochemical reactions ( ~ ) , efficiency of PS2 electron transport (~o) and quantum yield of PS2 electron transport (d~j) were observed between 20 and 24 "C - the temperature optimum. From 20 to 4 oC, the photochemical quenching (qp), ~p, d)o and d~, declined. This indicates that low temperatures reduce the rate of a linear electron transport. A strong increase of nonphotochemical quenching (q,) and relative changes of a ground fluorescence (qo) occurring from 27 to 34°C are likely related to thermal dissipation within thylakoid membrane. No significant effect of the PPFD on all six fluorescence parameters was observed between 150 and 700 pmol.m-2.s -1. With PPFD decreasing below 150 IJmol.m-2.s -~ a pronounced increase of qN and qo occurred. It is likely related to the RuBPCO inactivation.

P-29-008 DO SHADE AND ELEVATED CO t CONCENTRATION HAVE AN I N T E R C T I V E EFFECT ON PHOTOSYNTHESIS? ANALYSIS USING W H E A T G R O W N UNDER FREE-AIR CO t E N R I C H M E N T (FACE).

P-29-011 THE EFFECTS OF CO2 ENRIDHMENTAND ELEVATED TEMPERATURE ON RUBIaOO ~CTIVITY AND CONTENT OF WHEAT (TR/TICUM AESTZVUI4 L. VAR POLKI~) FLAG LF~VES

C.P. Osborne l, S.P. Long2,1, R.L. Garcia 3,4, G.W. WalP, B.A. Kimball3, P.J. Pinter 3, Jr., R.L. LaMorte~ and G.R. Hendrey2. tDept, of Biology, University of Essex, Colchester CO3 3SQ, UK; 2Applied Science, Brookhaven National Laboratory, USA; 3USDA, Agricultural Research Service, USA; 4LI-COR, inc., USA.

E-M. Tuhkanen, K. Hakala, T. M e l a Agric. Res. C e n t r e of Finland. Inst. of Crop and Soil sci. 3 1 6 0 0 J o k i o i n e n . F i n l a n d

Leaf photosynthetic carboxylation capacity ( V ~ , ) was estimated in vivo for a spring wheat crop grown at an elevated atmospheric CO2 concentration (c~) using FACE. At current ambient c v Ve,maxdeclined with increased leaf depth in the canopy. The decline was significantly greater under elevated ca, indicating a synergistic interaction. However, despite losses in Vc.m~ leaf CO2-uptake measured in situ was higher in elevated c a grown plants than in controls over an entire diurnal time-course. The relative stimulation of dally net photosynthesis by elevated ca was greater with increased leaf depth in the canopy. Results support the hypothesis that shaded leaves within the canopy experience greater loss of active Rubisco at elevated c.. In spite of this loss, there was still a net increase in the photosynthesis of these shaded leaves under elevated c=.

236

F i e l d - s o w n s p r i n g w h e a t w a s g r o w n in open top c h a m b e r s at a m b i e n t a n d e l e v a t e d t e m p e r a t u r e (+3°C), and at a m b i e n t or e l e v a t e d CO 2 (700 ppm) at b o t h t e m p e r a t u r e s . D u r i n g the e x p e r i m e n t a l y e a r s 1992-94 t h e r e w a s no c o n s i s t e n t c h a n g e of R u b i s c o a c t i v i t y or c o n t e n t of t h e f l a g leaves in the d i f f e r e n t treatments. R u b i s c o a c t i v a t i o n s t a t e w a s lower in e n r i c h e d p l a n t s t h a n in c o n t r o l p l a n t s o n l y at e l e v a t e d t e m p e r a t u r e in 1994. R u b i s c o a c t i v i t y on leaf area b a s i s was h i g h e r in c o n t r o l p l a n t s at a m b i e n t t e m p e r a t u r e in 1994. T h e s p e s i f i c a c t i v i t y of R u b i s c o was lower in c o n t r o l p l a n t s at a m b i e n t t e m p e r a t u r e in 1992. S o l u b l e p r o t e i n per u n i t leaf area w a s h i g h e r in control p l a n t s at both temperatures in 1994. Chl a/b ratio was s i g n i f i c a n t l y lower in e n r i c h e d p l a n t s at e l e v a t e d t e m p e r a t u r e in 1993.

Poster

P-29-015

P-29-012 THE

EFFECTS

(Triticum

OF

CLIMATIC

aestivum

L.)

CHANGE ON SPRING WHEAT PHOTOSYNTHESIS AND GROWTH

K. H a k a l a , E - M . T u h k a n e n a n d T. M e l a Agricultural research centre of Finland, Institute of crop and soil science, FIN-31600 Jokioinen, Finland According to current predictions, the climate will be w a r m e r a n d t h e CO= l e v e l s h i g h e r in t h e f u t u r e . I n c r e a s e s in g r o w i n g s e a s o n t e m p e r a t u r e s a n d C O 2levels are potentially beneficial f o r w h e a t , a Csp l a n t c o m m o n l y c u l t i v a t e d in F i n l a n d . T h e e f f e c t s o f elevated growing season temperatures (3*C a b o v e a m b i e n t ) a n d d o u b l e d CO= l e v e l s o n t h e p h o t o s y n t h e s i s a n d g r o w t h o f f i e l d - s o w n s p r i n g w h e a t w e r e s t u d i e d in Finland, Jokioinen (60°49'N, 23*30'E) in o p e n - t o p chambers in the field (ambient temperatures) and in a special greenhouse (elevated temperatures). Even if the years 1992-1994 were climatically different, a 20-80 % increase in photosynthetic activity in e l e v a t e d C02 w a s f o u n d i n b o t h a m b i e n t a n d e l e v a t e d temperatures. The growth and yield of wheat, again, varied through the experimental years. The role of photosynthesis in w h e a t g r o w t h a n d y i e l d p r o d u c t i o n an t h e c h a n g i n g c l i m a t e is d i s c u s s e d .

T S E I N T E R A C T I V S E F F E C T S OF E L E V A T E D PROTOSYNTHSSIS IN S P R I N G W H E A T

C O 2 a n d 03 C O N C E N T R A T I O N

I*~. McKe~, P.K. Farage & S.P. Long Department of Biology, University of Essex, Wivenhoe Park I Colchester, Essex, CO4 3SQ, O.K. This study investigated the interacting effects of carbon dioxide and ozone on photosynthetic physiology in the flag leaves of spring wheat (Triticum aestivum L. cv. Wembley), at three stages of development. Plants were exposed throughout their development to reciprocal combinations of two carbon dioxide and two ozone treatments: [C02] at 350 or 700 mmol mo1-1, [03] at <5 or 60 nmol mo1-1. Gas exchange analysis, coupled spectrophotometric assay for ribulose-l,5-bisphosphate carboxylase/oxyqenase (RuBisCO) activity, and gel electrophoresis, were used to examine the relative importance of pollutant effects on i) stomatal conductance , ii) quantum yield, and iii) RuBisCO activity, activation, and concentration. Independently, both elevated [CO2] and elevated [03] caused a loss of RuBisCO protein and activity. In combination, elevated [C02] partially protected against the deleterious effects of ozone. It did this partly by reducing stomatal conductance, and thereby reducing the effective ozone dose. Elevated [03] caused stomatal closure largely via its effect on photoassimilation.

P-29-016

P-29-013 RELATIONSHIP BETWEEN PHOTOSYNTHESIS AND ~PINENE EMISSION FROM Quercus//exLEAVES UNDER DIIqqfRENT ENVIRONMENTAL CONDITIONS F. Loreto l, P. Ciccioli2, A. Cecinam 2 , E. Brancaleoni: & M. FITdttolti2. IC.N.R.- Istituto diBiochimica ed Eenfislologia Vegetali; 2C.N.R. - Istituto per l'lnquinainento Atmosfedco,via Salaria km 29, 00016 Monteretondo Scalo, Italy

Quercus ilex is one of the most common evergrean tre¢ growing in the Mediterranean area. Contrary to many other oak species, Q. ilex does not emit isoprene. However, our laboratory experiments indicated a detectable emission ofmonoterpenes and particularly of a-pinene from Q. ilex leaves. The emission was temperature, light and CO2 dependent. Temperature dependency was similar to that previously shown for isoprene in Q. rubra, et-pinene emission and photosynthesis were both light dependent. However, light induction of ce-pinene was slower than induction of photosynthesis. When light was turned off photosynthesis dropped suddenly but a-pinene emission decreased gradually. C02 removal caused a gradual reduction of ce-pinene emission. One hour after CO2 removal a 70% reduction of tx-pinene emission was observed. The emission recovered when CO2 supply was restored. Recovery was complete after one hour. CO: enfichraatt to a concentration of 1500 ppm stiraulated photosynthesis and caused stomatal closure, c¢-pinane emission was slightly stimulated by CO2 enrichment These observations indicate that Q.ilex leaves have a small pool of ct-pinene that, contrary to pools stored in specialized organs, responds rapidly to ¢avirenraantal changes, is not dependent on stomatal conductance and is depleted upon carbonremoval. Our results suggest that u-pinene biogenesis and emission in Q. ilex leaves may be similar that of isoprene in other oak species.

P-29-014 PZ21ETRATION OF LIGHT IN PHOTOSYNTHETIC OF SPHERICAL SYMMETRY.

SEE%~MS

J. Gapi~ski I, G. Paillotin 2, A. Dobek I, W. Leibl 3, K. Gibasiewicz I, J. Breton 3, H.-W. Trissl 4 IInstitute of Physics, UAM, Umultowska 85, 61-614 Poznad, Poland; 2INRA, 147 r. de l'Universit~, 75338 Paris, Cedex 07, France; 3Section de Bio~nerg~tique, DBCM, CEN Saclay, F-91191 Gif-sur-Yvette Cedex, France; 4Abt. Biophysik, Universit~t OsnabrUck, Barbarastr. ii, D-4500 Osnabr~ck, Germany In our recent publications we presented a theory that explained quantitatively the origin, polarity, and amplitude of the photovoltage induced by short light flashes in suspension of chloroplasts and oriented membranes. Here we demonstrate the light distribution in photosynthetic systems like chromatophores, some photosynthetic bacteria, EDTA-treated or inside-out vesicles from chloroplasts, all exhibiting spherical symmetry. An analytical solution of light intensity distribution and induced dipole moment value was found for the case of a single spherical layer with high coefficient of absorption (photosynthetic membrane) immersed in non-absorbing medium (water). We present numerical results obtained for different wavelengths and different pigments and we compare them with experimental data of photovoltage induced by picosecond light flashes.

ON

EFFECTS OF ELEVATED TEMPERATURE AND CO2ON PHOTOSYNTHESIS, RUBISCO ACTIVITY AND AMOUNT IN MEADOW FESCUE LEAVES Riitta Heli6, Kaija Hakala, Timo Mela Agricultural Research Centre of Finland, Institute of Crop and Soil Science, 31600 Jokioinen, Finland Meadow fescue (Festuca pratensis) was field sown and grown under four different treatments: (1) elsvated CO2 (700 ppm) and elsvated temperature (+3°C above ambient), (2) ambient CO2 and elsvated tsmperature, (3) elevated CO2 and ambient temperature, (4) ambient CO2 and ambient temperature. The photosynthetic rate was 60% greater in the elevated CO2 and elevated temperature compared to the ambient treatment. Solely elevation of CO2 level increased the photosynthesis 40%, where as elevated temperature raised photosynthesis 10%. Rubisco activity in the meadow rescue leaves was very similar in all four treatments. How ever the amounts of rubisco as well as total leaflet soluble protein and chlorophyll decreased in elevated temperature, which indicates a higher spesific activity of rubisco. Elevation of CO2 level did not effect the amounts of rubisco, total leaflet soluble protein or chlorophyll.

P-29-017 THE INCREASE OF WATER USE EFFICIENCY OF MAIZE PLANT GROWN AT ELEVATED ATMOSPHERIC CO2 IS EXPLAINED BY LEAVES GAS EXCHANGES.

Olivier Bethenod. Fran~;oise Puget, Laurette Combe, Unit6 de Bioclimatologie, INRA, 78850 ThivervaI-Grignon, France Maize plants (Zea m_a~s,cv DEA) were exposed to normal ambient or doubling of CO2 (700 /.tl 1- ) throughout 4 growing seasons in a ventilated glasshouse. At the time of harvest, water use (cumulated transpiration) of maize plants in elevated CO2 decreased by 15% and water use efficiency (plant dry weight/cumulated transpiration) increased by 23.7%. At flowering time, similar results were obtained. These results were explained by higher stomatal conductances in the greenhouse with normal CO2 concentration. In terms of both maximal net CO2 assimilation and mesophyll conductance, measured using an open gas chamber, no difference was noticable at leaf level. In both treatments net CO2 assimilations and stomatal conductances were strongly linked. Based on the two curves of net CO2 assimilation versus stomatal conductance obtained at 350 lal 1"1 or 700 lal 1-1 respectively, no photosynthetic acclimation to high CO2 was observed.

237

Poster i

P-29-018 M O D I F I C A T I O N OF THE PNOTOSYNTHITIC RESPONSE TO ELEVATED CO2 BY GROWTH TEMPERATURE AND DEPOLIATION TREATMENTS.

C.M. Stirlinu I and Davey P.A. 2 IInstitute of Terrestrial Ecology, University of Wales, Deiniol Rd., Bangor, Wales LL57 2UP; 2Dept. of Biology, University of Essex, Wivenhoe Park, Colchester, Essex, U.K. CO4 3SQ. Species were grown in a suite of Solardome glasshouses programmed to provide a factorial combination of two levels of CO 2 concentration, [C02] (ambient/ambient + 340ppm) and two levels of temperature (ambient/ambient + 3°C). A/Ci curves were constructed for the first fully expanded leaf between 75 and i00 days. Acclimation of photosynthesis (A) to elevated [CO 2] varied widely between species; significant upward adjustment of the A/Ci response was observed for the slow-growing species H.nummularium, whilst the reverse was true for the fast growing species C.ancustifolium and D.alomerata. Recovery in the carboxylation efficiency of plants grown at elevated [CO2] following partial defoliation is consistent with the view that A at elevated [C02] is largely mediated by sink limitations. Relaxation of sink limitations to A may also explain the observed stimulation of photosynthesis at elevated [CO 2} in four of the seven species grown at ambient + 3 °C.

P-29-021 PHOTOSYNTHESIS, GROWTH AND NUTRIENT ASSIMILATION IN NONNODULATED PHASEOLUS VULGARIS L. EXPOSEO TO ELEVATED CARBON DIOXIDE J M. Mtwara & C.E.J. Boths

SchOnland Botanical Laboratories, Botany Department, Rhodes University, P.O. Box 94, Grahamstown, 6140, Republic of South Africa. The response of Phaseolus vul.~aris L. cv. Contender grown at either ambient or elevated (360 and 700 p.mol mol", respectively) concentrations of atmospheric CO=, in controlled environment chambers, was monitored from 10 days after germination (DAG) until senescence. Elevated CO~ significantly increased total plant height (TPH), leaf area (LA) and dry weight (DW) whilst specific leaf area (SLA) was signilicantly reduced. While the trends in growth parameters were different between [CO2], some physiological processes appeared transient. In particular, net assimilation rate (NAR), foliar nutrient Concentrations of N, Mg and Cu for example, CO= enrichment significantly increesed NAR but from 20 DAG, a steady decline to almost similar levels to those measured in plants grown under ambient CO= occurred. A simiier trend was observed for leaf N where the loss of leaf nitrogen in elevated Cos-grown plants, was greater than that observed for ambient CO= plants. Under enhanced CO2, the foliar concentrations of K and Mn ware increased significantly whilst P, Ca, Fe and Zn were reduced significantly. Changes in Mg, and Cu concentrations were insignificant. In addition high CO= grown plants exhibited a pronounced leaf discoloration or chlomsis, coupled with a significant reduction in leaf longevity.

P-29-019 ~JESP~a~ OF ~ A~SPHRRXC ~

PHOTOSk~INSZS TO a N O R T - T E ~

TLUCTUATIONE IH

P-29-022 ESTIMATING CO2 FLUXES WITH STABLE ISOTOPES

DZG~EIDZ

Xue-Feng Wang & Dan Yakir Dept of Environ Sci Weizmann Institute of Sci Rehovot 76100, Israel

G.R. Hendrey I, S.P. Long 2"1, N.R. Baker 2 & I.F. McKee 2 IBiosystems and Process Sciences Division, Brookhaven Laboratory, Upton, NY 11973, USA; 2Department of University of Essex, Colchester, C04 3SQ, Essex, UK

National Biology,

In their normal habitats and in experiments plants are exposed to concentrations of CO2 (Ca) that may undergo short-term variations over periods of a few seconds to minutes. In nature such variation is in the range of about 5% from the global average, or 350 ~ 20 tunol mol "I, while in well-run CO2 enrichment experiments, c. may vary by ~I0--20% of the set point concentration. The effects of this variability on photosynthesis must depend on both the extent and duration of the change in ca. In the present studies, photosynthetic fluorescence (Ft) was monitored for wheat leaves (Triticum aestivum cv. Hereward) exposed to CO2 oscillating symmetrically by 225 ~Lmol mcl -I about a ca set point concentration of 575 or 650 pmol mol -I. No response was detected to half-cycle step changes in ca lasting less than two seconds, but at half-cycles of two seconds or longer, the response of Ft was pronounced.

Poorly known global distribution of photosynthetic activity in the terrestrial biota impede better understanding of the atmospheric CO2 budget. A significant development in this respect was the suggestion that local and global scale gradients of laO in atmospheric CO2 are influenced by CO2 exchange associated with plant photosynthesis and respiration. We tested these ideas at the landscape scale by measuring gradients of 13C in CO2 and of t80 in CO2 and water vapor in the atmosphere above fields of crop plants, together with measurements of wind speed and the isotopic composition of the plants. The data were then used to estimate photosynthetic CO2 uptake (gross primary productivity, GPP) and evapotranspiration (El') fluxes. CO2 fluxes derived from tsO gradients compared favorably with those derived from gradients in ~3C and both were in agreement with direct concentration measurements. Obtaining the intercept in a plot of the isotopic ratios as a function of 11[CO2] in the boundary layer (for either t3C or t80), provided additional means to test some of the assumptions involved in the application of the isotopic approach. The results provided a useful link between laboratory and global atmospheric studies directed at increasing our ability to identify sinks and sources of CO2 in the terrestrial biosphere.

P-29-023

P-29-020 ELEVATED CO 2 AND T E M P E R A T U R E E F F E C T S O N E N Z Y M E S OF SUCROSE AND STARCH S Y N T H E S I S I N SOYBEAN Aria H Pennanen l, Joseph CV Vu 2, L Hartwell Allen Jr 2, & George Bowes3. 1ll)ept Plant Biology, Univ Helsinki, 00014 Helsinki, Finland; 2USDA-ARS & Agronomy Dept; °Dept Botany, Univ Florida, Galnesville, FL 32611, USA Soybean (cv. Bragg) was grown in sunlight under four temperature regimes (28/18 to 40/30°C) and 350 or 700 ~ CO2 L -l. At a growth temperature of 280C, elevated [CO2] had little effect on the activities of sucrose syuthase (SS), sucrose phosphate syuthase (SPS) and ADP-glucose pyropbosphorylase (ADPGPP), whereas Q-enzyme (Q-E) activity was reduced. Under the high [CO2], SS, SPS and ADPG-PP all increased in activity with higher growth temperatures. At ambient [CO2] the situation differed with respect to SS and SPS, as activity actually decreased with increasing temperature, although this was dependent on the time of day, especially for SS. Both ambient- and high-[CO2] grown plants showed a loss of Q-E activity with higher growth temperatures, but the decline was not as great at high [CO2]. For soybean, growth at elevated [CO2] appeared to moderate the potentially adverse effe~s of high growth temperatures on the activity of carbohydrate enzymes. Furthermore, except for Q-E, no down-regulation of activity was observed at high [CO2]; this included the major regulatory enzymes, ADPG-PP and SPS.

238

ENHANCEMENT IN GROWTH AND P R I M A R Y PHOTOCHEMISTRY OF ANABAENA DOLIOLUMTREATED WITH INDUSTRIAL EFFLUENT

M.Panda & B.Biswal S c h o o l of L i f e S c i e n c e s , S a m b a l p u r Jyotivihar-768019,Orissa,India

University,

Cyanobacteria a r e u s e d in a b a t e m e n t of i n d u s t r i a l effluent and production of biomass from the waste water. Since paper mill wastes contain important ions essential for different physiological processes including photosynthesis,an a t t e m p t is m a d e in t h e p r e s e n t w o r k to e x a m i n e i t s e f f e c t s o n g r o w t h a n d p r i mary photochemistry of thylakoid membranes of Anabaena doliolum.The effluent significantly stimulates g r o w t h in t e r m s of c h l o r o p h y l l and biomass production. T h e h i g h g r o w t h r a t e of t h e o r g a n i s m is a t t r i b u t e d to effluent induced enhancement in p r i m a r y p h o t o c h e m i c a l reactions associated both with PSI and PSII of thylakoids.Photosynthetically essential ions like magnesium,calcium and chloride,major components of efflue n t in a d d i t i o n t o its e n r i c h m e n t with nitrogen,may possibly modulate photosynthetic efficiency of t h e organism during the treatment.

Poster P-29-024

THE PRACTICAL USE OF THE POLYPHASIC CHLOROPHYLL A FLUORESCENCE (OJIP) IN B I O T E C H N O L O G Y AND E N V I R O N M E N T A L B I O L O G Y

P-29-027 U S E OF ~ LABSPEC TM ~ I R - 5 1 2 SPECTRORADIaMETER SPECTROSCOPy FROM SINGLE COL0~rIES

F C ~ / N SIT~7

Bruno J. Strasser and R.J. Strasser, Laboratory of Bioenergetics, University of Geneva, CH- 1254, Jussy, Geneva, Switzerland

M. Wiqqli I, R. Ghosh 2, & R. Bachofen 1 iInstitute of Plant Biology and Microbiology, University Zurich, Zollikerstr. 107, CH-8008 Zurich, Switzerland; 2Bioenergetics Laboratory, University of Geneva, CH-1254 Lullier-Geneva, Switzerland

Fluorescence techniques have proven to be useful for the description of the vitality in plants. The conclusion made from fluorescence measurements are always indirect. However reflectance and fluorescence techniques are often the only practical tools to screen a biotype in w v o (from close to remote sensing). Several instruments are commercially available which can be used by a non specialist if a practical manual has been elaborated. Indexes, on the basis of fast fluorescence rise kinetics at one or two emission wave lengths, which characterize the sample are proposed. Only short tests (more than 100 samples measured per hour) can practically and economically be used.

So far screening of mutants of phototrophic bacteria was mainly performed by eye. A new method is presented using a photodiode array detector with fiberoptics to collect absorption spectra from single colonies on petri dishes allowing to n~nitor small deviations of the pign~nt pattern. A good signal to noise ratio is obtained for wavelengths between 400 and 900 run. The strong absorption band around 880 nm is a good ~arget to investigate small shifts. The digital s p e c t r a a l l o w ser~i-automatic processing and n o ~ l i z a t i o n . Accurate aligning as well as a stable light source is a prerequisit for reproducible colony spectra. The spectra obtained show the same features as their counterparts recorded in liquid suspensions. The direct measurement will facilitate the screening process, as small single colonies can be assessed now, elintinating the need of liquid cultures prior recording of absorption spectra.

P-29-025

of

P-29-028

ADAPTATION OF CAMELLIA LEAVES TO VARYING LIGHT CONDITIONS

ELEVATED CO2 CONCENTRATIONS AND VESICULAR ARBUSCULAR MYCORRHIZA INFLUENCE PHOTOSYNTHESIS AND GROWTH OF RAINFOREST SEEDLINGS

M. Tsimillil, G. Krfiger2 and R.J. Strasser 1, 1 Laboratory of Bioenergeties, University of Geneva, 1254-Jussy, Switzerland, 2Department of Soil Sciences, Potchefstroom University, Potehefstroom, 2520, South Africa.

C. E. Lovelock, D. KyUo & K. Winter Smithsonian Tropical Research Institute, PO Box 2072, Balboa, Republic of Panama

Steady states, in Camellia leaves, established under different light conditions, have been studied by the polyphasic Chl a fluorescence rise (OJIP). For each physiological state the multiparameter pattern of functional and structural expressions has been monitored by varying the light intensity for a short l~me (1 see). The shape of the light saturation curve of the leaf, for each derived expression depends strongly on the actually established physiological steady state. This indicates that opt/miration of structure and function in the sample, triggered by the given environmental conditions, takes place.

P-29-026

In tropical forests mycorrhiza have been shown to be important in increasing survival and early growth of seedlings. The aim of this study was to assess whether the influence of mycorrhiza on seedlings was altered by growth under elevated CO2 concentrations. Seedlings of the tree species Beilschmiedia pendula were grown under CO2 concentrations of 800 ppm for 12 weeks. Half the plants were infected with vesicular arbuscular mycorrhiza. Plants infected with mycorrhiza had increased photosynthetic capacities relative to non-mycorrhizal plants, while elevated CO2 concentrations increased photosynthetic rates measured under growth conditions. Both elevated CO2 and mycorrhizal infection increased growth rates by 15 to 20%. Increases in growth due to mycorrhiza or elevated CO2 concentrations were additive when the two treatments were imposed simultaneously.

P-29-029

INTERACTION OF ELEVATED CO 2 AND NITROGEN SUPPLY ON PHOTOSYNTHETIC ACCLIMATION IN WHEAT

PHOTOSYNTHETIC ACCLIMATION OF WINTER WHEATTO ELEVATED CO 2 AND TEMPERATURE

P.K. Fara~,e& S.P. Long, Department of Biology, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ U.K.

D.W. Lawlor, E. Delgado 1, S. Driscoll, V.J. Mitchell, R.A.C. Mitchell, M.A.J Parry.lACR-Rothamsted, Harpandan, Herts, AL5 2JQ, UK; 1 Institute of Advanced Studies, University of the Balearics, Mallorce, Spain

Growth of plants in elevated concentrations of CO2 often results in a reduction of photosynthetic capacity, termed acclimation. This investigation tested the hypothesis that acclimation occurs through a decrease in the quantity of active Rubisco in response to nitrogen limitation. Winter wheat, cv. Hereward, was grown in controlled environments at 350 and 650 l-tmol mo1-1 CO2 with high and low rates of nitrogen supply. Light-saturated rates of net CO2 uptake did not differ significantly between CO2 treatments when measured at the same CO2 concentration provided that the plants received the high rate of nitrogen supply. However, acclimation did occur in forth and subsequent leaves of the elevated CO2 grown plants supplied at the low nitrogen addition rate. Analysis of the A/ci response showed that the maximum in vivo rate of carboxylation was significantlydecreased in plants grown at elevated CO2 with low nitrogen. These results suggest, that under these conditions, acclimation of photosynthesis in wheat to growth in elevated CO2 can be attributed to reduced capacity for carboxylation due to reallocation of nitrogen from Rubisco.

Photosynthetic rate (A) of winter wheat leaves grown in 355 or 700/~mol mor I CO=, combined with ambient or ambient +4oC temperatures, did not change ("acclimate") in capacity for A (from A versus internal CO= Ci - curves under standard conditions) or amounts of soluble or ribulose bis-phosphate carboxylase-oxygenase (Rubisco) protein or chlorophyll (Delgado et al. Plant, Cell and Environment, 17, 1205-1213). However, in another study, although A, measured in growth conditions (but with constant light) increased with elevated CO= in leaf 8, it did not in 10 and decreased in the flag: despite small stomatal conductance, Ci did not limit A. Chlorophyll, soluble protein and Rubisco protein, initial activity and carbamylation state were unaffected by CO= in leaf 8 but decreased in 10 and flag, particularly at ambient + 4°C. I%=t was unaffected. Conclusion: wheat A and composition may acclimate to elevated CO 2 but the causes of differences in response are not yet established.

239

Poster

P-29-030 THE PHOTOSYNTHETIC PERFORMANCE OF OAK (Quercus robur L.) POPULATIONS EXPOSED TO AUTOMOBILE EXHAUST AS AN AIR POLLUTANT L.Makrai 1, S. Dulal 2, E. Lehoczki 1 1: Research Group of the Hungarian Academy of Sciences, Dept. of Botany, JATE University, H-6701 Szeged, POB:657. Hungary 2: Botanical Dept. Eszterhfizy Teachers Training College, H-3301 Eger, POB: 43. Hungary The effects of automobile exhaust in impairing the photosynthetic functions of two populations of oak (Quercus robur L.) were investigated over the past three years between the end of May and August, under natural conditions. The stress effects of automobile exhaust on the photosystem II properties were characterized by using a homebuilt computerized fluorimeter and a dualchannel modulated fluorimeter (Hansatech UK). Significant declines were found in the values of Fv/Fm, Fi, Rfd, and A.p., the fluorescence induction parameters and the photochemical and non-photochemical quenching in the stressed oak population. A more pronounced stress effect was observed on the net photosynthesis measured by CO 2 fixation.

P-29-033 NEW INSTRUMENTS FOR REMOTE SENSING OF PLANT STRESS Patrice BENOIT. Jean-Marc ROTH, Patrick SUMM, Francois TOMASINI ARP, CTT route de Hausbergen 67309 SCHILTIGHEIM Cedex (F) Among the spectroscopic methods developed to caracterize the vegetation, the laser induced fluorescence appears to be a promising tool to evaluate plants physiology under different stress conditions. In the last years, time resolved as well as imaging fluorescence measurements have been applied in laboratory to study plants grown in greenhouse. In order to extend these methods to far-field and outdoor measurements, new instruments are currently developed by ARP in close cooperation with the Groupe d'Optique Appliqute, CRN, Strasbourg. The fast one is a streak camera device having two important characterisitcs : 1) it can be shuttered to decrease the noise due to ambient light, 2) the simultaneous record of the back scattered light and the chlorophyll fluorescence signal allows the numerical fitting of the decay curves even for moving leaves. Results obtained over a distance of 30 m for water stressed tabacco leaves and for maize plants with iron deficiency show the influence of the plant activity on the mean fluorescence lifetime Xm.The second instnmaent concerning the imaging fluorescence is an intensified and gated CCD camera. This camera is compact and allows to image a leaf or a whole plant at different spectral ranges with a high spatial resolution. Some exemples of images obtained for different stress are presented.

P-29-034

P-29-031 EFFECT OF ELEVATED CO2 CONCENTRATION ON THE GAS EXCHANGE OF FOUR XEROPHYTIC SPECIES FROM VENEZUELA

HYDROGEN ISOTOPE RATIOS OF PLANT METABOLITES j. Schleucher 1,2, j. L. Markley 1 & T. D. Sharkey 2

M. D. Fersandez, M. Azkue, A. Pieters & A. Herrera. Centre de Bot.~nica Tropical, Universidad Central de Venezuela. Aptdo. 47577, Caracas 1041-A. Venezuela. The effect of CO2 concentration on gas exchange was assessed in four species from the sersi-add regions of Venezuela (Jatropha .qossypifolia, C3; Iporneea carnea, C3; Altersanthera crucis C4, and Talinum tria~ulare, inducible-CAM). Plants were grown for 46 weeks during the rainy season in two open-top chambers, one at 560/40_ pprs, and the other one at ambient CO2, and in a control plot. No chamber effect on gas exchange was detected. All species increased maximum photosynthetic rate in the §rst 20 weeks, decreasing thereafter to values similar to those of control plants. High CO2 concentration increased carboxylation efficiency in Jatrophe, Altersanthere and Talinurn, whereas it decreased it in Ioornoea. Integrated instantaneous water use efficiency was not affected by CO2 concentration during all of the experiment. Apparent quantum yield increased with high CO2 for all species, whereas apparent dark respiration was enhanced in Altersanthera and Jalropha and decreased in Iporsoea. Partly financed by projects EC CTI1*-CT91-0896 (in cooperation with F.I. Woodward, U. Sheffield) and CONICIT RPVII-270078 (Venezuela)

P-29-032 ACCLIMATION OF PHOTOSYNTHESIS TO RISING CO2 CONCENTRATION IN THE FIELD: A MOLECULAR ANALYSIS. A. Rogers, C.A. Raines, S.P. Long, H. Blum & M. Frehner Department of Biology, University of Essex, Colchester, CO4.3SQ, UKI; Institut ~ r Pflanzenwissenschaflen, ETHZ, CH-8092 Ztirich, Switzedand. Predicting the response of vegetation to rising CO2 concentration is confounded by a loss of photosynthetic capacity with long-term growth in elevated CO2. Understanding the mechanistic basis of this variability is necessary to advance ability to predict the long term responses of communities to rising CO2. Increased soluble carbohydrate levels, as accumulate under elevated CO2, correspond to changed expression of several photosynthetic genes. The replicated Free-Air CO2 Enrichment (FACE) facility in Switzerland is unique in elevating the CO2 concentration around large areas of crops in the field without alteration of microclimate. Western blot analysis of Loliutn perenne grown for two years in FACE at low nitrogen has revealed a decrease in the amount of Rubisco, in relation to total leaf protein. Transcript levels have been determined by Northern blot analysis. The importance of nitrogen availability and source/sink balance on the amount of Rubisco and nitrogen use efficiency are discussed in relation to the results obtained.

240

1 Dept. of Biochemistry, 420 Henry Mall and 2 Dept. of Botany, 132 Birge Hall; University of Wisconsin-Madison, Madison, Wisconsin 53706, USA An understanding of 1H/2H isotope ratios in plants has not been achieved to date because i) the isotopic composition of leaf water is influenced by climate, the plant's water source, and evapotranspirafion and ii) hydrogen from leaf water is incorporated into photosynthate in a number of biosynthetic reactions, each potentially discriminating differently against 2H. However, the internal 1H/2H distribution among diastereotopic sites in metabolites should only be influenced b~ isotope discriminations during biosynthesis. We propose to use internal H/ H distributions to separate physiological and environmental influences on 1H/2H ratios. Data will be presented that demonstrate that the internal 1H/2H distribution in spinach (Spinacia oleracea) glucose is influenced by growth conditions of the plant. Internal 1H/2H distributions of metabolites might become an indicator for plants' physiological status. The findings are also relevant for studies using lI-I/2H ratios to reconstruct prehistoric climates.

P-29-035 OBJECT-DIRECTED APPROACH: A METHOD TO GRAPHIC RENDER OF RUSSIAN TERRITORY PHOTOSYNTHESISCARBON STOCK DATA BASE p. Yu. Voronin 1 , p. V. Konovalov2, V. I. Lukyanovich 3 lK.A.Timiriazev Institute of Plant Physiology, Botanicheskaya35, Moscow ,123276, Russia; 2NovosibirskState Technical University, Novosibirsk, Russia;3Agricultural manufacturefirm "Rial", Iskitim,Novosibirskregion, Russia Graphic image concept of Russian territory photosynthesis carbon stock data base is rendered. Object-directed approach oblige to determine mu]Lotude of arranged in grade objects. These are tied with a map and combined with relative data base. Map's information filling grade can be demonstrated visually also in space and time. Computer map and scale unification support compatibility of different data bases linked with this map standard. According to our estimation the total photosynthetic carbon income to Russia territory is 4.4Gt a year (P.Yu. Voronin et al. 1995. Russian Joamd of Plant Physiology, vol.42, p.334-341.).

Poster P-29-036 PItOTOACOUSTIC MEASUREMENTS OF ENERGY STORAGE IN SCENEDESMUS ARMATUS EXPOSED TO CRUDE OIL

Z. Tukail& J. Szurkowski 2 1Dept. of Plant Physiology, University of Gdatisk, AI. Marszalka Pitsudskiegn 46, 81-378 Gdynia, Poland; 2Inst. of Experimental Physics, University of Gdafisk, Wita Stwosza 57, 80-952 Gdafisk, Poland Spectral response of energy storage (ES) for Scenedesmus armatus (green microalga) untreated and treated by crude oil derived from Baltic Sea was studied using photoacoustic spectroscopy. ES was measured by recording of photoacoustic signal in the absence and presence of background white light and at modulation freguency of 80 Hz. Maximum ES of untreated algae in range of 600-720 nm was observed at 640 urn. At wavelengths longer than 640 nm ES gradually decreased to 680 um and next rapidly dropped reaching the minimum at 720 urn. No inhibition effect of crude oil was observed on ES in Scenedesmus. Moreover, a slight but statistically significant (P>0.95) stimulation effect of crude oil on ES was noted at wavelengths of 680 nm. Time response measurements at 680 um indicate that ES of algae treated was about 5% higher in comparison to control algae regardless the time of exposure. This enhancement may be considered as a result of detoxification mechanism in algae influenced with low concentration of hydrocarbuns in the culture medium.

241