Photosynth Res (2007) 91:133–324 DOI 10.1007/s11120-007-9197-6

© Springer Science+Business Media B.V. 2007

14th Photosynthesis Congress - PS07

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PL.1 Photosynthesis: A Blueprint for Powering the Planet

PL.1 Oxygen Evolution

D Nocera (Massachusetts Institute of Technology)

R Debus (University of California)

The supply of secure, clean, sustainable energy is arguably the most important scientific and technical challenge facing humanity in the 21st century. Rising living standards of a growing world population will cause global energy consumption to increase dramatically over the next half century. Within our lifetimes, energy consumption will increase at least two-fold, from our current burn rate of 12.8 TW to 28 – 35 TW by 2050 (TW = 1012 watts). This additional energy needed, over the current 12.8 TW energy base, is simply not attainable from long discussed sources – these include nuclear, biomass, wind, geothermal and hydroelectric. The global appetite for energy is simply too much. Petroleum-based fuel sources could be increased. However, deleterious consequences resulting from external drivers of economy, the environment, and global security dictate that this energy need be met by renewable and sustainable sources. Of the possible sustainable and renewable carbon-neutral energy sources, sunlight is preeminent. If photosynthesis can be duplicated outside of the leaf – an artificial photosynthesis if you will – then the sun’s energy can be harnessed as a fuel. The combination of water and light from the sun can be used to produce hydrogen and oxygen. The hydrogen can then be combined with the oxygen in a fuel cell to give back water and energy. In the overall cycle, sunlight is converted to useful energy. This talk will place the scale of the global energy issue in perspective and then discuss how an artificial photosynthesis to power our planet might be achieved.

Photosystem II (PSII) uses solar energy to extract electrons and protons from water, liberating molecular oxygen as a by-product. The reaction is exceptionally demanding from a thermodynamic perspective and the reaction mechanism is not yet fully understood. However, the recent availability of X-ray crystallographic structural models for PSII has dramatically increased the power of mutagenesis and spectroscopy to provide mechanistic insight. The new structural models are facilitating the development of both detailed mechanistic hypotheses and experiments designed to describe the roles of specific amino acid residues. Many of these experiments involve Fourier Transform Infrared (FTIR) difference spectroscopy. FTIR difference spectroscopy is an extremely sensitive tool for characterizing the dynamic structural changes that occur during an enzyme’s catalytic cycle, such as changes in molecular interactions, bonding, protonation states, and protein backbone conformations. In PSII, the frequencies of numerous vibrational modes change as the Mn4Ca cluster is oxidized through the catalytic cycle. Many of these modes correspond to amino acid residues that either ligate the Mn4Ca cluster, are coupled to the Mn4Ca cluster through hydrogen bonds, interact electrostatically with the Mn4Ca cluster, or have side chains whose protonation states change as the Mn4Ca cluster is oxidized. This talk will describe isotopic labeling and mutagenesis studies that have been undertaken to identify these modes. The identification of these modes will help describe the structural changes that accompany the oxidations of the Mn4Ca cluster and will provide insight into how the Mn4Ca cluster’s reactivity is regulated by its protein environment to maximize the efficiency of water oxidation and minimize the release of toxic, highly reactive intermediates. This work is supported by the National Institutes of Health (GM76232).

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PL.1 Primary Processes – From isolated complexes to intact plants

PL.1 The plumbing of photosynthesis. Integrating the electron and proton transfer reactions in vivo

A Holzwarth (Max-Planck-Institut)

D Kramer (Washington State University), A Kanazawa (Washington State University), J Cruz (Washington State University), J Cape (Washington State University), T Avenson (Washington State University), I Forquer (Washington State University), K Takizawa (Washington State University), C Sacksteder (Washington State University), M Bowman (Washington State University)

It has been assumed for decades that bacterial reaction centers represent the “role model” for the mechanism of the early electron transfer processes in reaction centers (RCs). More detailed ultrafast spectroscopic studies during the recent years have shown however that the RCs of oxygen-evolving organisms, i.e. photosystems (PS) I and PS II, actually employ different mechanisms of charge separation. The results and relevance of recent studies – mostly based on a combination of ultrafast spectroscopy and RC point mutations - leading to these conclusions will be discussed. Furthermore, the detailed insight gained from ultrafast spectroscopic studies in isolated complexes can now be applied to the understanding and unravelling of time-resolved data of highly complex systems. Thus processes, like e.g. high-light adaptation, non-photochemical quenching etc. can now be studied at the level of entire plants. The progress in this area and its relevance for the understanding of physiological adaptation processes will be highlighted in my talk. 1. Holzwarth, A. R. 2007. Ultrafast In: Ultrafast Processes in Chemistry and Biology, W. Zinth, editor. Springer-Verlag, Heidelberg, Berlin. In print. 2. Holzwarth, A. R., M. G. Müller, J. Niklas, and W. Lubitz. 2006. Biophys. J. 90:552-565. 3. Holzwarth, A. R., M. G. Müller, M. Reus, M. Nowaczyk, J. Sander, and M. Rögner. 2006. Proc. Natl. Acad. Sci. USA 103:6895-6900.

The reactions of photosynthesis must be integrated to balance efficiency in energy conversion with the avoidance of deleterious side reactions. The talk will focus on areas where integration is finely tuned in surprising ways. The first area concerns the oxidation of quinol at the cytochrome b6f and bc1 complexes, where reactive oxygen generation appears to be avoided by destabilization (rather than the typical stabilization) of reactive intermediates of catalysis. Similarly, the proton circuit of photosynthesis balances and regulates photosynthesis by finely incrementally restricting proton flux at the ATP synthase and dissipating transthylakoid electric field. We will emphasize the fact that progress in these areas has been driven by the development and application of in vitro and in vitro spectroscopic approaches.

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PL.1 Sugar and Energy Sensing and Signalling Networks in Plants

PL.1 Harnessing photosynthesis for tomorrow’s world: humans, crop production and poverty alleviation

J Sheen (Harvard University), Y Cho (Harvard University), E Baena González (Harvard University), Q Hall (Harvard University), F Rolland (Harvard University), Y Xiong (Harvard University), S Yoo (Harvard University) Photosynthesis is the fundamental metabolic process that supports all life on Earth. In response to environmental cues, photosynthesis activities modulate cellular sugar and energy levels that also serve as potent signalling molecules. Multiple sensors monitor and respond dynamically to sugar and energy signals and initiate signal transduction pathways to globally control distinct target genes that govern cellular metabolism, physiological and developmental programs in plants. To elucidate the fundamental and complex glucose and energy signalling networks, we have taken a combination of cellular, genetic, genomic, and proteomic approaches. Using Arabidopsis as a genetic model, we have isolated and characterized glucose insensitive (gin) and glucose oversensitive (glo) mutants. Our studies of gin and glo mutants have revealed extensive and intimate connections between glucose and plant hormone signalling pathways. We have also provided compelling evidence for the uncoupling of glucose signalling and glucose metabolism in controlling gene expression and developmental processes. Many genes involved in carbon and nitrogen metabolism and storage, cell cycle, and stresses are similarly regulated by glucose in yeast, animals and plants. The evolutionarily conserved glucose sensor hexokinase and energy sensor protein kinases control the energy budget and resource utilizations through the function of nuclear signalling complexes. The flexible and reversible responses to both low and high glucose signals in plant growth promotion and inhibition, respectively, as well as sugar and energy starvation depend on cell types, developmental state, multiple nutrient status, and environmental conditions. The plasticity of plant developmental programs could therefore be attributed to versatile sugar and energy sensing and signalling activities in the plant signal transduction networks. Cheng WH et al. (2002). Plant Cell, 14: 2723-2743 Leon P & Sheen J (2003). Trends in Plant Science, 8: 110-116 Moore B et al. (2003). Science, 300: 332-336 Yanagisawa S et al. (2003) Nature, 425: 521-525 Rolland F et al. (2006) Annu Rev Plant Biol, 57: 675-709 Cho YH et al. (2006) Cell, 127: 579-589

J Sheehy (International Rice Research Institute) Agriculture is the indispensable base of human society and the nature and productivity of agriculture are determined by water, climate and the products of research. Clearly, photosynthesis is the solar energy–dependent process on which human existence ultimately depends. Nonetheless, each day passes with 854 million people hungry and, for that reason, the United Nations Millennium Declaration committed the world’s nations to ‘eradicate extreme poverty and hunger’. Sixty percent of the world’s population lives in Asia, where each hectare of land used for rice production currently provides food for 27 people, but by 2050 that land will have to support at least 43 people. In 2007, about 250 million tonnes of carbon will be fixed in rice grains; by 2050, fixation will have to rise to about 400 million tonnes. However, the elite rice cultivars, which dominate the food supply of the millions of poor people in Asia, have approached a yield barrier and growth in production is slowing. The role of photosynthesis in solving the problems of tomorrow’s world will be examined. In particular, the possibility of producing a quantum increase in yield, water-use efficiency and nitrogen-use efficiency by developing C4 rice will be discussed.

PL - Plenary 7 PL.1 Chlorophyll biosynthesis: enzymology on a global scale N Hunter (University of Sheffield) Chlorophyll is the cofactor for photosynthetic complexes that harvest sunlight and convert it to photochemical energy. Thus, the biosynthesis of chlorophyll takes place on a massive scale, with billions of tonnes synthesised every year on land and in the oceans. It is important to understand the mechanisms of the individual steps, the kinetic coupling between enzymes and the ways in which flux down this important pathway is controlled. A quantitative analysis of the pathway enzymes requires large quantities of pure protein, now available through heterologous overexpression of chlorophyll biosynthesis genes. This approach has laid the foundations for mechanistic studies on the chlorophyll biosynthetic enzymes magnesium chelatase, magnesium protoporphyrin methyltransferase and protochlorophyllide reductase (POR). POR catalyses the reduction of protochlorophyllide to chlorophyllide. Since the catalytic cycle of POR is triggered by light, this enzyme is interesting, not only from the standpoint of chlorophyll biosynthesis, but also in terms of enzyme catalysis. POR presents a unique opportunity to study enzyme catalysis and reaction dynamics at low temperatures and on ultrafast timescales. For example, studies of POR overcome the near-impossibility of distinguishing between enzyme conformational changes and the actual catalytic steps, since conformational and catalytic events are triggered separately by light. Remarkably, memory of the initial light-activation step persists on a long timescale, which reduces the frequency of ‘hits’ by photons needed to drive the subsequent catalytic step of chlorophyll biosynthesis and which allows photosynthetic organisms to make chlorophyll and grow at low light intensities.

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PL.1 Assembly of photosynthesis proteins in the cholroplast: do it yourself provided the nucleus sets the rhythm

PL.1 Measuring the Atmosphere to Monitor the Biosphere

F Wollman (Institute de Biologie Physico-Chimique), Y Choquet (Institut de Biologie Physico-Chimique), D Drapier (Institut de Biologie Physico-Chimique), J Girard-Bascou (Institut de Biologie Physico-Chimique), S Eberhard (Institut de Biologie Physico-Chimique), R Kuras (Institut de Biologie Physico-Chimique), L Lezhneva (Institut de Biologie Physico-Chimique), C Loiselay (Institut de Biology Physico-Chimique), C Raynaud (Institut de Biologie Physico-Chimique), D Saint Marcoux (Institute de Biologie Physico-Chimique) Photosynthetic eukaryotes convert light energy into chemical free energy using a limited number of membrane-embedded oligomeric proteins of dual genetic origin. Nucleus-encoded subunits assemble with chloroplast-encoded subunits in a series of active enzymes in the thylakoid membranes that perform a light-driven electron flow from water to ferredoxin that is coupled to ATP synthesis. The biogenesis of these protein complexes raises a number of fundamental questions, three of which will be addressed in this presentation. 1) How does a plant or algal cell balance the expression levels of the various subunits of a photosynthetic protein, given the fact that the gene copy number for chloroplast-encoded subunits exceeds by several order of magnitude the gene copy number for nucleus-encoded subunits? 2) Are these subunits produced in the stoichiometric ratio required for their functional assembly in photosynthesis proteins? 3) What do we know of the protein toolbox used in the assembly of redox cofactors with their apoprotein targets? The cytochrome b6f complex from the unicellular green alga Chlamydomonas reinhardtii, will be used as a paradigm for these issues in chloroplast protein biogenesis. We will examine how our major observations apply to organellar protein complexes from other sources.

J Berry (Carnegie Institution, Department of Global Ecology) Precise measurements of CO2 concentration (to a common reference) are now made at many locations around the world. A picture of seasonal and spatially resolved patterns of CO2 concentration change is emerging from these data. These measurements provide a window on the activity of the biosphere at various scales and can provide important information on the response of the biosphere to climate change. A quantitative understanding of these CO2 changes in the atmosphere requires a great deal of information on how the activity of the biosphere is distributed across the globe; how these activities affect the adjacent atmosphere and how these local changes mix through-out the whole atmosphere. This draws heavily on the meteorological infrastructure that has been developed to forecast weather, but the improved understanding of the biosphere that emerges also promises to improve forecasts. This synergistic relationship is driving the development of a powerful infrastructure to monitor the activity of the biosphere. Expanded monitoring of CO2 and other trace gases, satellite measurements, and new modeling and data analysis procedures are parts of the system, but progress is also limited by our ability to obtain information from remote sensing measurements and our understanding of important physiological and ecological processes linked to photosynthesis.

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PL.1 The evolutionary transition from anoxygenic to oxygenic photosynthesis

PL.1 How the evolution of oceanic photosynthesis shaped the biogeochemistry of Earth

R Blankenship (Washington University), W Swingley (Hokkaido University), M Hohmann-Marriott (Institute of Biomedical Imaging and Bioengineering), J Raymond (Lawrence Livermore National Laboratory)

P Falkowski (Rutgers University)

Photosynthesis is a central biological process that has a long and complex evolutionary history. The photosynthetic machineries found in the existing groups of phototrophs have both common and divergent characters, suggesting an evolutionary process that combines de novo gene appearance, gene duplication, gene and pathway recruitment and loss, with both vertical and horizontal genetic transfer. The earliest phototrophs were almost certainly anoxygenic (non-oxygen-evolving) and were in existence on Earth by at least 3.4 billion years ago and possibly somewhat earlier. Oxygenic (oxygen-evolving) phototrophs were undoubtedly in existence by 2.4 billion years ago when free molecular oxygen, the waste product of oxygenic photosynthesis, began to accumulate in the atmosphere. These organisms may have been present up to several hundred million years before that time. Several lines of evidence, including molecular evolution analysis, structural comparisons, and biochemical and biophysical data, suggest that all modern photosynthetic reaction centers are derived from a single ancient common ancestor and that the anoxygenic phototrophs preceded oxygenic ones. The transition from anoxygenic to oxygenic photosynthesis was accompanied by a number of evolutionary innovations, including multiple gene duplication and divergence events, modification of the pigment biosynthesis pathways, invention of the oxygen evolution center and a dramatic increase in the reaction center protein subunit complexity.

The oxidation state of the atmosphere and ocean was altered approximately 2.3 billion years ago(Ga), when the signal of mass independent isotopic fractionation of sulfur essentially disappeared. This signal is thought to reflect the production of ozone in the stratosphere; a phenomenon that requires the production of molecular oxygen. However, molecular fossil evidence suggests that oxygenic photosynthetic organisms (cyanobacteria) evolved at least 500 million years earlier. Hence, there appears to have been a very long lag between the evolution of oxygenic photosynthesis and the rise of oxygen in Earth’s atmosphere. I will discuss what processes contributed to the lag, focusing on the co-evolution of two major metabolic pathways, namely oxygenic photosynthesis and nitrogen fixation, and how these pathways came to form global biogeochemical cycles. I will also highlight the importance of tectonic processes in determining the burial efficiency of photosynthetically derived organic matter in the lithosphere, without which there would oxygen would not have accumulated in Earth’s atmosphere. I will conclude with an analysis of the subsequent changes in oxygen and carbon dioxide over the past 500 million years of Earth’s history and how the concentration of these two gases will change in the future.

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PS1 - Bioenergy PS1.1 Achieving environmentally and economically viable biofuel feedstock crops. Why improvement of photosynthetic efficiency is critical and attainable. S Long (University of Illinois), F Dohleman (University of Illinois), X Zhu (University of Illinois) Emergence of economically and environmentally viable energy industries around cellulosic biofuels depend on maximizing dry matter yields per unit land area while minimal inputs. This requires a very different plant design to our major food crops, and very different selection criteria, where photosynthetic efficiency is central. This gives a new impetus to basic photosynthetic research feeding into emerging biofuel crops. A theoretical analysis will be presented that quantifies six potential routes to increasing photosynthetic efficiency. These range from canopy architecture to improved regeneration of the acceptor molecule for CO2. While it is uncertain whether some of these changes could ever be realized or would actually result in increased photosynthesis, others could clearly be realized on a short time scale. Collectively these changes could improve efficiency and therefore yield potential by ca. 50%. Since some of these changes could be achieved by transgenic technology, the time by which they could also be realized in material for the production of commercial cultivars will be considerably less than via conventional breeding and potentially within a 10-15 year time horizon. Supercomputers now allow simulation of the complete photosynthetic process, including all intermediate pools and proteins in silico. Application of evolutionary algorithms suggest manipulations that could result in substantial increases in photosynthtic efficiency.

PS1.2 Photosynthetic hydrogen production: genes, proteins and effects T Melis (University of California, Berkeley) 2-production is observed. Recent advances were based on the cloning and application of genes for the chloroplast sulfate permease, genetically attenuating the sulfate uptake capacity of the green algae. Such modification limited the capacity of photosynthesis, permitting cellular respiration to consume photosynthetic oxygen and eliciting a spontaneous and sustained H2-production. Green algae under bright sunlight have low solar conversion efficiencies. Reason is the large arrays of light absorbing Chl antenna molecules in the photosystems, where the rate of photon absorption far exceeds the rate at which photosynthesis can utilize them, resulting in dissipation and loss of the excess energy as fluorescence or heat. Up to 80% of absorbed photons could thus be wasted, lowering solar conversion efficiencies and cellular productivity. The work identified genes that regulate the Chl antenna size of photosynthesis, and described manipulation of their expression to attain a “truncated Chl antenna size”. The latter helps to diminish over-absorption and wasteful dissipation of excitation energy by individual cells, while permitting for greater transmittance of light deeper into the mass culture. Such altered optical properties of the cells resulted in greater photosynthetic productivity and better solar conversion efficiency in a mass culture. The above-described advances are based on genetic modifications that compromise cell fitness in green algae but promote conditions for photosynthetic H2 production.

PS1.3 The Solar Bio-fuels consortium: Developing advanced bio-fuel production systems B Hankamer (University of Queensland), P Schenk (University of Queensland), U Marx (University of Queensland), C Posten (University of Karlsruhe (TH)), O Kruse (University of Bielefeld) The recent Stern Review (The Economics of Climate Change) [1] and the 2007 reports by the Intergovernmental Panel on Climate Change have left little doubt that rapid action is needed to develop CO2 free energy technologies. Indeed to stay below atmospheric CO2 levels of 450ppm, which is widely seen as the highest safe CO2 option, requires the installation of ~11TW-yr CO2 free energy capacity by 2025 (Compared with a total global energy demand of ~13 TW-yr). Currently fuels make up ~67% of the global energy market (Total global energy market =13 TW-yr) [2]. In contrast global electricity demand accounts for only 33%. Yet despite the importance of fuels, almost all CO2 free energy production systems under development are designed to drive electricity generation (e.g. clean-coal technology, nuclear, photovoltaic & wind). In contrast, and indeed almost uniquely, bio-fuels also target the much larger fuel market and so in the future, will play an increasingly important role. Photosynthesis is central to all biofuel production (bio-diesel, bio-ethanol, bio-H2, bio-methane, biomass-to liquid (BTL). Here the advances made by the Solar-Biofuels Consortium at the level of light harvesting antenna engineering, metabonomics, the development of alternative fuels streams, enhanced bio-H2 production and the development of large bioreactors will be presented. 1. Stern, N., Stern Review on the Economics of Climate Change. 2006, HM Treasury: London. p. 576. 2. Hoffert, M., et al., Energy implications of future stabilization of atmospheric CO2 content. NATURE, 1998. 395(6705): p. 881-884.

PS1.4 Studying Structure and Function of Hydrogenase: Basis for a Biological Hydrogen Production W Lubitz (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany) Hydrogenases catalyze the reversible oxidation of molecular hydrogen and play an important role in the metabolism of many anaerobic micro-organisms, including photosynthetic systems. An understanding of these enzymes is of great interest for a future hydrogen economy based on genetically modified organisms or biomimetic catalysts1. In this contribution the structure and function of different hydrogenases are introduced. [NiFe] and [FeFe] hydrogenases are the two main classes of this type of enzyme. They contain bridged binuclear transition metal cores in their active sites which are tuned by a special ligand environment to fulfil the task of efficient hydrogen conversion to protons and electrons – or vice versa – via a heterolytic H2 splitting mechanism. The ligand sphere is characterized by thiolate ligands as well as an unusual coordination of the iron by small diatomic molecules (CN- and CO). The crystallization of representatives of both types of enzymes has provided the spatial structure of the [NiFe] and [FeFe] hydrogenase for the oxidized and reduced forms. The various intermediate states in the catalytic cycle are studied by spectroscopic techniques, where FTIR and EPR play a central role. The activation of the enzyme, the inhibition by CO, the sensitivity

137 14th Photosynthesis Congress - PS07 to molecular oxygen as well as substrate binding and product formation have been investigated. This led to proposals for the catalytic cycles of the [NiFe] and the [FeFe] hydrogenases.

PS1.7 The TL29 protein a proposed ascorbate regulator in the thylakoids lumen of Arabidopsis thaliana

1W. Lubitz and W. Tumas (eds.) “Hydrogen” Chem. Rev. thematic issue (2007), in press.

I Granlund (Biological Chemistry, Umeå University), M Schubert (Biological Chemistry, Umeå University), W Schröder (Biological Chemistry, Umeå University)

PS1.5 The European solar to fuel initiative

The TL29 protein is one of the more abundant proteins found in the chloroplast lumen in various plants. Based on the sequence homology to ascorbate peroxidases, TL29 (recently referred to as APX4) has been implicated to form part of the plant defense system against reactive oxygen species, but no supporting biochemical evidence has been presented, to date. To characterize the TL29 protein we investigated the distribution and analyzed the pattern of appearance within the thylakoids membrane in Arabidopsis thaliana. Part of the protein pool was found to be associated with the thylakoids membrane and the major part was located to the grana region of the thylakoids. The expression analysis showed that the levels of TL29 protein was induced by light and increased upon high light illumination. To investigate the putative peroxidase function for the TL29 protein we analyzed the peroxidase activity in the thylakoids lumen from wild type Arabidopsis plant using 2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid). These measurement showed a clear peroxidase activity, which was unaffected by protease treatment of the intact thylakoids before obtaining the lumen fraction. However, when an ascorbate dependent assay was used, no lumen associated peroxides activity could be detected. Lumen fractions isolated from TL29 knockout plants show the same degree of peroxidase activity as in the wild type lumen showing that the detected lumen peroxidase activity is not associated with the TL29 protein. Interestingly, ascorbate-binding assays using recombinant TL29 protein indicated that the protein indeed could bind ascorbate.

H de Groot (Leiden University), E Aro (Turku University), D Bassani (Bordeaux University), R Cogdell (Glasgow University), R van Grondelle (VU Amsterdam), L Hammarstrøm (Uppsala University), A Holzwarth (MPI Bioanorganische Chemie), O Kruse (University of Bielefeld), V Sundström (University Lund) The world’s energy requirements are predicted to double by 2050 due to a growing population with unfulfilled needs and rising expectations. If we fail to fill the energy gap at an affordable price without disruptions of the supply, our societies will face conflict and insecurity. In parallel, the use of energy by our economies leads to a continued threat of severe and irreversible environmental damage. Presently 70% of all energy use is in fuels, mainly for heating and transportation and this secondary energy mix is unlikely to change fundamentally over a short time. In a recent white paper a European task force has assessed the research needs required to arrive over a period of 1-2 decades at the production of environmentally clean solar fuels, starting from recent scientific progress in basic research in photosynthesis. The results of the study group are available to the scientific community and European scientists want to capitalize on the recent excellent scientific progress to learn from Nature how to harness solar energy for sustainable production of primary energy carriers like hydrogen from water or carbon based fuels from CO2 at an affordable cost and at much higher efficiency than is presently possible with biomass. The goal of this presentation is to outline an implementation strategy for European science to arrive at carbon neutral solar fuels production. PS1.6 Antenna activity of Metal-substituted Chl a bound to the Chl a-binding sites in PS1 RC complexes I Ikegami (Faculty of Pharmaceutical Sciences, Teikyo University), S Ohtake (Faculty of Pharmaceutical Sciences, Teikyo University) Most Chl a in the PS1RC complexes was removed without any loss of P700 by ether treatment, yielding the antenna-depleted PS1 RC complexes with a Chl a/P700 ratio of 12. On addition of about 60 Chl a (/P700) with phosphatidylglycerol, about 20 Chl a (/P700) were bound to the complexes, and they showed the same antenna activity as Chl a remaining in the complexes. The ratio of the bound Chl a to the added Chl a was about one-third, irrespective of the amount of Chl a added. Zn- and Cd-Chl a bound to the complexes with the same binding affinity and with the same antenna activity as Chl a. The results suggest that they almost bind to the Chl a-binding sites locating in the core region. Hg-Chl a bound to the Chl a-binding sites with the same binding affinity as Chl a, but without any antenna activity. The results could be explained by extremely low fluorescence yield of Hg-Chl a, indicating the lifetime of its excited state too short to transfer its excitation energy to the neighboring Chl. Cu-Chl a bound to the complex, but with the formation of aggregated forms of the bound Cu-Chl a and without any antenna activities. The results suggest that Cu-Chl a could bind not to the Chl a-binding sites, but probably on the protein surface. The same results as Cu-Chl a were obtained on reconstitution with Phe a, which bound to make aggregated forms without any antenna activity.

PS1.8 A thylakoid prokaryotic potassium channel is involved in the regulation of photosynthesis in cyanobacteria M Zanetti (University of Padova), E Bergantino (University of Padova), N la Rocca (University of Padova), E Teardo (University of Padova), G Giacometti (University of Padova), I Szabò (University of Padova) A putative Shaker-like potassium channel has been identified in the genome of Synechocystis sp. PCC6803. This channel, named SynKCh, was cloned from Synechocystis and expressed in eukaryotic cells. Electrophysiological analysis demonstrated that it works as a voltage-gated outwardly rectifying, potassium selective channel. In order to investigated the physiological role of this channel, SynKCh-deficient Synechocystis mutant was obtained. This mutant did not grow in normal conditions; however growth could be restored in the presence of Na2S2O3 (an electron donor in cyanobacteria), under high-intensity light illumination and in dark. An antibody raised against a recombinant SynKCh revealed that the expression of the channel was up-regulated by exposing cyanobacteria to light and down-regulated in presence of photosynthesis inhibitors. Immunogold electron microscopy revealed that the channel is localized in thylakoid membranes in cyanobacteria. PAM fluorimeter experiments showed that the mutant cyanobacteria are unable to build up proton gradient across thylakoid membranes during photosynthesis. These data suggest that the channel may be involved in the regulation of photosynthesis. PS1.9 Partitioning of carbon into non-structural carbohydrates and cell wall polysaccharides using 13C label in Switchgrass M Soundararajan (University of Nebraska-Lincoln), B Nemeck (University of Nebraska-Lincoln)

138 14th Photosynthesis Congress - PS07 Switchgrass (Panicum virgatum L.).has drawn a considerable research effort in the area of renewable energy production due to its biomass. Results from a previous study amongst switchgrass populations differing in in vitro dry matter digestibility showed only modest varations in cell wall hemicellulose and cellulose concentration but a significant change in the lignin concentrations in the cell wall. Carbon isotope ratio (CIR, ∂13C, ‰) analyses of stover tissue from both the lowland (KANLOW) and the upland (SUMMER) cultivars of switchgrass indicated that CIR of Kanlow was less negative (-12.7‰) than the upland variety Summer (-13.1). Preliminary observations on the CIR of cellulose from switchgrass showed a less negative ∂13C value (-12.6) than the leaf CIR (a surrogate for non-structural carbohydrates) which showed a ∂13C value of -13.1. The purpose of this study is to follow carbon partitioning into the cell wall structural carbohydrates of Switchgrass plants at various stages of their growth. Preliminary results from 13CO2 pulse labeling of greenhouse grown Switchgrass plants indicated an active partitioning of carbon in their stem tissue over a period of 6 weeks than either in the leaf or root tissues. Results from further analyses of carbon partitioning into hemicelluloses, celluloses and lignins from the stem tissues from both Kanlow and Summer cultivars of switchgrass plants exposed to labeled 13CO2 in the greenhouse, will be presented. PS1.10 Entry into the isomerization photocycle of Proteorhodopsin studied by visible and midIR transient femtosecond spectroscopy A Rupenyan (Vrije Universiteit Amsterdam), I H.M. van Stokkum (Vrije Universiteit Amsterdam), R van Grondelle (Vrije Universiteit Amsterdam), K Hellingwerf (Swammerdam Institute for Life Sciences), M Groot (Vrije Universiteit Amsterdam) Proteorhodopsin (pR) is a proton pump protein of the microbial rhodopsin family found in marine γ-proteobacteria. Upon light absorption it undergoes a photocycle, completed in 20ms after a proton translocation from the cytoplasmic to the extracellular side and reisomerization of the chromophore. The absorption maximum of pR exhibits a blue shift upon increasing the pH of the surroundings, due to the change of the protonation state of the primary proton acceptor Asp97. We study the mechanism of photoisomerization of the retinal chromophore at alkaline and acidic pH by femtosecond time-resolved spectroscopy probing in the visible and in the IR spectral regions. We observe biphasic decay of the excited state with time constants of 0.7ps and 18ps for pR at pH 6.5 and 0.4ps and 20ps at pH 9.5. The IR spectra of PR at pH 6.5 where Asp97 is protonated and at pH 9.5 are clearly different, showing changes in the amide II band of the protein (1550cm-1) during the photoisomerization. The results indicate that in addition to the different charge distribution around the chromophore, the conformational state of the protein influenced by the change of pH is also important. From our modelling we conclude that the isomerization occurs directly from the ground state, and isomerization trough relaxed excited state can be excluded. PS1.11 Hydrogen photoproduction in Chlamydomonas reinhardtii: electron transfer pathways and improvement G Peltier (CEA CNRS Cadarache), C Desplats (CEA CNRS Cadarache), B Ghysels (CEA CNRS Cadarache), L Constans (CEA CNRS Cadarache), V Chochois (CEA CNRS Cadarache), A Beyly (CEA CNRS Cadarache), P Carrier (CEA CNRS Cadarache), S Cuiné (CEA CNRS Cadarache), L Cournac (CEA CNRS Cadarache) Some unicellular green algae such as Chlamydomonas reinhardtii can produce hydrogen in the light thanks to the presence of an hydrogenase coupled to the photosynthetic electron transport chain. Although producing hydrogen from solar energy and water is of considerable

interest, biotechnological applications are limited by the oxygen-sensitivity of the hydrogenase. A time-based separation of hydrogen and oxygen production phases can been used to circumvent this limitation. During the oxygenic phase, carbohydrate compounds (mainly starch) accumulate, and are converted to hydrogen during the anaerobic phase through a PSI-dependent process. Reactions involved in the anaerobic conversion, including reduction of PQs from stromal donors, which constitute the limiting part of the process, remain to be characterized. In contrast to higher plants, C. reinhardtii chloroplasts do not contain a NDH complex analogous to complex I, catalyzing non-photochemical reduction of PQs. It was previously proposed that a type II single subunit NADH dehydrogenase may fulfil this function in C. reinhardtii, and different ORFs have been identified in the nuclear genome of this alga. Based on immunodetection and MALDI-TOF data, we show that one of these ORFs encodes a type II NADH dehydrogenase containing FMN as a cofactor and associated to C. reinhardtii thylakoids. Moreover, expression and targeting to chloroplasts of a bacterial type 2 NADH dehydrogenase is shown to significantly increase rates of PS I-mediated starch to hydrogen conversion. The potential of using genetic approaches to optimize hydrogen photoproduction in algae will be discussed.

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PS2 - Reaction Centres: the powerhouses of photosynthesis PS2.1 Directionality of electron transfer within Photosystem I K Redding (University of Alabama), A Jasaitis (University of Alabama), R Luthra (University of Alabama), M Byrdin (Institut de Biologie Physico-Chimique), C Slavov (Max-Planck-Institut), M Müller (Max-Planck-Institut), B Bullock (University of Alabama), A Holzwarth (Max-Planck-Institut), F Rappaport (Institut de Biologie Physico-Chimique) Like all photosynthetic reaction centers (RC), the core of Photosystem I (PSI) is composed of two related membrane polypeptides that sequester electron transfer (ET) cofactors into two pseudo-symmetrical branches, each consisting of two chlorophylls and a quinone. An open question in the evolution of photosynthesis is how this symmetrical structure has been modified in type 2 RCs (e.g. Photosystem II and proteobacterial reaction centers), in which ET proceeds exclusively down one branch. We have used a combination of site-directed mutagenesis and kinetic spectroscopic techniques to prove the factors controlling the use of each branch in PSI. We have shown that directionality can be altered by breaking H-bonds to the ec3 chlorophyll, as visualized by the amplitudes of electron transfer from the two phylloquinones (PhQA and PhQB). Recent ultra-fast analyses have further found that these mutations affect the primary charge separation step, consistent with recent data on P700 mutants (Holzwarth et al., 2006, Biophys. J. 90:552-65) and providing further supporting for the idea that this represents RC* ® ec2+ ec3–. Results on new mutants designed to disrupt coupling within the ec2A/ec3A and ec2B/ec3B pairs will also be presented. Further evidence for bi-directional ET is provided by mutants that drastically slow ET from PhQA; in these, a new kinetic phase (with ~150-ns decay time) appears, which we have assigned to the FX ® FA step and which requires prior electron transfer from PhQB. We are testing this hypothesis by combining these mutations with those that affect directionality and/or the FA cluster.

PS2.2 Direct Reduction of QB by B-branch Electron Transfer in Bacterial Reaction Centers M Okamura (University of California), M Paddock (University of California) Reaction centers (RC) from purple bacteria contain two pseudo-symmetrical chains of cofactors spanning the membrane; an active A-branch and an inactive B-branch. The primary light reactions involve fast photochemical electron transfer along the A-branch from the primary electron donor, the bacteriochlorophyll dimer, through bacteriochlorophyll and bacteriopheophytin to the tightly bound primary quinone QA (t ~ 10-10 s). This is followed by slower (t ~10-4-10-3 s) thermally activated, proton-coupled electron transfer to a secondary quinone QB. The mechanism of QB reduction is more complex than the electron transfer steps in QA reduction and involves proton transfer reactions and protein dynamics. In order to investigate the QB reduction mechanism we have studied the direct reduction of QB by electron transfer through the B-branch to utilize the fast rate and high driving force for electron transfer to QB. Following the work of other groups, we used mutations in RCs from Rb. sphaeroides that block QA binding and increase the electron transfer efficiency through the B-branch, Using these modified RCs we studied the efficiency of QB reduction at room temperature and at cryogenic temperature, the recombination kinetics

from the P+QB- state and the EPR and ENDOR spectra of QB-∙ frozen in the light and frozen in the dark. The results of these studies indicate heterogeneity in the state of QB that is not observed for QA. This heterogeneity is attributed to conformational flexibility at the QB site necessary for proper function in proton coupled electron transfer and turnover.

PS2.3 Unidirectional Electron Transfer inside the Chlorophyll d-containing Photosystem I Reaction Center Complex of Acaryochloris marina S Itoh (Division of Material Science (Physics), Graduate School of Science, Nagoya University), T Uzumaki (Division of Material Science (Physics), Graduate School of Science, Nagoya University), S Takaichi (Nihon Medical Univ.), M Iwaki (School of Science, Nagoya University, Nagoya, 456-0862 Japan, 2Tokyo Medical College, Tokyo, 3Department of Chemistry, University College, London University), S Kumazaki (Department of Chemistry, Kyoto University), K Itoh (Division of Material Science (Physics) Photosystem I (PS I) reaction center complex was isolated from a cyanobacterium Acaryochloris marina that undergoes oxygenic photosynthesis with chlorophyll (Chl) d, which absorbs light of 700-740 nm, as the major pigment. The complex contained 88 Chl d: 1.1 Chl a: 2.0 phylloquinone: 19 carotenoids and showed peaks of absorption and fluorescence bands at 710 and 718 nm, respectively. Laser excitation induced the 10-ps bleach and the 50-ps recovery of the absorption change at 680nm suggesting the chemical identity of the electron acceptor A0 as Chl a in parallel with the biphasic 8- and 50-ps bleachings of the electron donor Chl d-dimer P740. ESR signals of P740+, iron sulfur centers (FX-, FA-, FB-), A1- (phyllosemiquinone) and P740 triplet state (P740T) and the spin-polarized P740+ and A1radical pair state were identified to be similar to those in other PS I. Amino acid sequences of PsaA, PsaB and PsaC polypeptides were somewhat different from those in other cyanobacteria and plants. The ligands for A0 was Met in PsaA while the one for A0' was not Met but Leu in PsaB. The latter differs from all the other PS I. The results indicate the unidirectional asymmetric electron transfer from a dimer of Chl d (P740) to A0 (Chl a-680), A1 (phylloquinone) on the PsaA branch, and then, to the FeS clusters in the PS I of A. marina.

PS2.4 The Heliobacterial Reaction Center J Golbeck (The Pennsylvania State University) Heliobacteria (Hb) contain a homodimeric photosynthetic reaction center (RC), but unlike green sulfur bacteria, they do not contain an extended antenna system. Given their relative simplicity, Hb should be ideal for study of a prototypical Type I RC. However, there exist large gaps in our knowledge, particularly with regard to the nature of the secondary and tertiary acceptors. This situation, however, is changing rapidly. First, the low molecular mass polypeptide that contains the FA and FB iron-sulfur clusters has been identified. The change in the lifetime of the flash-induced kinetics from 75 ms to 15 ms on its removal shows that the former arises from the P798+ [FA/FB]– recombination, and the latter from the P798+ FX– recombination. Second, FX has been identified in HbRC cores by EPR and Mössbauer spectroscopy, and shown to be a [4Fe-4S]1+,2+ cluster with a ground spin state of S = 3/2. Because all of

140 14th Photosynthesis Congress - PS07 the iron in HbRC cores is in the FX cluster, a ratio of ~22 Bchl g/P798 is calculated from chemical assays of non-heme iron and Bchl g. Third, the N-terminal amino acid sequence of the FA/FB-containing polypeptide has led to the identification and cloning of its gene. The expressed protein can be rebound to HbRC cores, thereby regaining both the 75 ms kinetic phase resulting from P798+ [FA/FB]– recombination and the light-induced EPR resonances of FA–/FB–. The gene was named ‘pshB’ and the protein ‘PshB’ in keeping with the accepted nomenclature for Type I RCs. Funded by DOE Energy Biosciences. PS2.5 The Heme-containing Portion of Cytochrome Chlorobium tepidum: Its Over-expression in

cz

from

H Oh-oka (Osaka University), M Higuchi (Ibaraki University), T Kondo (Nagoya University), H Mino (Nagoya University), S Itoh (Nagoya University), Z Wang (Ibaraki University) Green sulfur bacteria has the type 1 reaction center (RC) similar to photosystem I in cyanobacteria and higher plants. Cytochrome cz (cyt cz) contains a monoheme and serves as a direct electron donor to the P840. It is anchored to membranes with its N-terminal hydrophobic helices and holds a heme in its C-terminal hydrophilic domain. As the electron transfer rate strongly depends to the viscosity of reaction media, the heme-containing moiety is considered to fluctuate at any given time while searching for the appropriate reaction surface on the RC complex. We over-expressed the C-terminal heme-containing portion (C-cyt cz) in E. coli in order to study its reaction characteristic in details. The DNA fragment encoding the C-terminal half of cytochrome cz was amplified with PCR and cloned into the expression vector of pET12a. We constructed the co-expression system with the help of pEC86 that carried the cytochrome c maturation genes cluster derived from E. coli. The C-cyt cz could be expressed in a periplasm in a E. coli strain C41 and purified with an ion-exchange chromatography followed by a gel permeation one. The C-cyt cz showed a typical absorption spectrum of c-type cytochrome with an a-peak wavelength of 550.5 nm, which was slightly shorter than the intrinsic one in vivo. The ESR spectra indicated the presence of both low- (S=1/2) and high-spin (S=5/2) hemes in a single preparation, although the latter contribution seemed to be relatively lower. PS2.6 Structure-function relations of the Quinone Electron Acceptor (A1)-site in cyanobacterial Photosystem I D Stehlik (Freie Universität Berlin), I Karyagina (Freie Universität Berlin), J Golbeck (Freie Universität Berlin) The quinone (A1) binding site in Photosystem I has unique properties in comparison to other well characterized quinone binding sites in photosynthetic reaction centers and other membrane bound cofactor-protein complexes with functional quinone cofactors: (i) it has to acquire the most negative redox potential for one electron reduction among all known quinone binding sites in order to be able to perform its function in NADPH reduction; (ii) a single H-bond formed from the protein environment to one carbonyl group only both, in the ground-state X-ray structural model and in the first stabilized charge-separated radical-ion pair P700●+ A1●- state. New results are presented with selectively 13C labeled high spin density ring positions of suitable 1,4-naphthoquinone (NQ) derivates placed into the intact A1 binding site. In addition, recent results on various A0 mutants are presented. They confirm and detail previous conclusions. Since electron transfer under physiological conditions proceeds predominantly along the PsaA-branch

in cyanobacterial PS I, a single binding site A1A can be studied. Due to the single H-bond it is highly asymmetric in the spin density distribution across the quinone ring system providing a critical test for DFT calculations and hints for possible significance in biological function. The negative redox potential is not due to large individual contributors but composed by a large sum of small contributions which have been only partially identified so far. Extensive comparison is made with other quinone binding sites, in particular the recently well characterized QA-site in reaction centers of purple bacteria.

PS2.7 Energetics of Photosystem II charge recombination in Acaryochloris marina studied by thermoluminescence and flash induced chlorophyll fluorescence measurements I Vass (Institute of Plant Biology, Biological Research Center, Szeged, Hungary), K Cser (Institute of Plant Biology, Biological Research Center, Szeged, Hungary), A Telfer (Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK), Z Deak (Institute of Plant Biology, Biological Research Center, Szeged, Hungary) We have studied charge recombination characteristics of Photosystem II (PSII) redox components in whole cells of the chlorophyll d-dominated cyanobacterium, Acaryochloris marina, by flash-induced chlorophyll fluorescence and thermoluminescence measurements. Our data show that the thermoluminescence intensity is enhanced significantly (2.5 fold), on the basis of equal amounts of PSII, in Acaryochloris as compared to the chlorophyll a containing cyanobacterium, Synechocystis 6803. Flash induced chlorophyll fluorescence was slowed down both in the presence and absence of the QB site inhibitor, DCMU, in Acaryochloris relative to that in Synechocystis. In addition, chemical reduction of the PQ pool retarded fluorescence relaxation in the absence DCMU in Acaryochloris to a significantly larger extent than in Synechocystis. These data indicate that the energetics of charge recombination are modified in Acaryochloris marina with changes at the level of pheophytin, QB and PQ, on the acceptor side, and of Tyr-Z and the Mn cluster, on the donor side of PSII. The significance of the difference in energetics between chlorophyll d and chlorophyll a oxygenic organisms will be discussed. PS2.8 Regulation of photoprotection by non-radiative charge recombination in Photosystem II K Cser (Institute of Plant Biology), I Vass (Institute of Plant Biology) We studied the influence of charge recombination reactions in Photosystem II on photodamage of Synechocystis 6803 cells, in which the redox properties of the primary pheophytin acceptor, Phe, and of the first quinone acceptor, QA, were modified. Replacement of the D1-Gln130 residue by Glu and Leu shifts Em(Phe/Phe-) by about + 30 and -50 mV, respectively. In the D1Q130E mutant the overall charge recombination from the S2QA- state is accelerated, and the contribution of the non-radiative recombination pathway via the P680+Phe- primary radical pair is enhanced relative to the WT. The opposite effect, i.e. strong stabilization of charge recombination from S2QA-, and decreased contribution of the non-radiative recombination pathway was observed in the D1Q130L mutant. Photodamage of PSII was decreased in D1Q130E relative to the WT and the opposite effect was observed in D1Q130L. In the presence of the electron transport inhibitor bromoxynil, which decreases Em(QA/QA-), overall charge recombination from S2QAwas accelerated, relative that observed in the presence of DCMU, which

141 14th Photosynthesis Congress - PS07 increases Em(QA/QA-). In addition, photodamage was accelerated by bromoxynil and retarded by DCMU in all the three strains. We conclude that the extent of photodamage is determined by the yield of 3Chl production via non-radiative charge recombination through the P680+Pheradical pair, which is influenced by the free energy gap between Phe and QA, as well as between P680+Phe- and the ground state of relaxed P680. The D1-Q130E amino acid exchange, which occurs in many cyanobacteria during light adaptation appears to be an important mechanism of regulating phototolerance. PS2.9 Involvement of phosphatidylglycerol in the assembly and function of photosystem II Z Gombos (Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, H–6701 Szeged, Hungary), S Toth (Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, H–6701 Szeged, Hungary), H Laczko-Dobos (Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, H–6701 Szeged, Hungary), B Ughy (Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, H–6701 Sze Phosphatidylglycerol (PG) is a ubiquitous constituent of thylakoid membranes in plants and cyanobacteria. Our earlier results (Hagio et al 2000, Plant Physiol 124: 795-804; Gombos et al 2002, Biochemistry 41: 3796-3802) suggested that PG is indispensable for photosynthesis. In the present study, we used a Synechocystis mutant, called PAL, which is devoid of the light-harvesting phycobilisomes (Ajlani et al 1998, Plant Mol Biol 37: 577-580). The photosystem II to I ratio is 1 in PAL, whereas normally a ratio of 1/3 is found in wild-type Synechocystis. Using PAL, we isolated a new deletion mutant in which the cdsA gene, encoding an enzyme involved in PG biosynthesis, was inactivated. The PAL?cdsA mutant was initially grown in the presence of PG and then cells were transferred to PG-depleted medium. Chl a fluorescence was recorded during PG starvation. In the PAL mutant, the Fv/Fm value was relatively high (~ 0.7) and chl a fluorescence (OJIP) kinetics were evocative of those of higher plants. PG depletion resulted in a 30 % increase of Fo that can be explained by the detachment of CP43 and CP47 from the photosystem II reaction centers. Chl a fluorescence induced by short repetitive pulses (twenty 300 or 500 µs pulses, spaced 2.3 ms apart) revealed that the electron transfer between QA and QB became slower in the absence of PG. We conclude that PG facilitates the QA-QB electron transfer and the assembly of photosystem II core complexes, whereby PG is an indispensable component of the photosystem II core complex. PS2.10 Theoretical modelling of the optical properties and the exciton dynamics of the isolated PSII reaction centre. R Pishchalnikov (Max Planck Institute for Bioinorganic Chemistry), M Mueller (Max Planck Institute for Bioinorganic Chemistry), A Holzwarth (Max Planck Institute for Bioinorganic Chemistry) There is still a considerable debate [1] over the mechanism of charge separation in the PSII reaction center and the understanding of its spectral properties. In this investigation the femtosecond pump-probe spectra, exciton dynamics and charge separation kinetics of the isolated PSII Reaction center were modeled using modified Redfield theory. This theory implies the doorway-window representation derived using projection operator techniques. We consider the spectral density function using 48 vibronic modes to describe the absorption properties of a monomer chlorophyll molecule and to calculate the exciton transfer rates. The radical-pair states pump-probe spectra were extracted from the

experimental data. For fitting we used the differential evolution algorithm. The choice of the proper strategy and tuning parameters allows us to get a reasonable rate of convergence. By fitting the model to the femtosecond data we explore the recently proposed mechanism of charge separation with incorporated the accessory Chl D1 as the primary electron donor [2,3]. [1] Renger, G. Holzwarth, AR. 2005, Photosystem II: The Light-Driven Water: Plastoquinone Oxidoreductase 139-175 [2] Holzwarth, AR. Müller, MG. Reus, M. Nowaczyk, M. Sander, J. Rögner, M. PNAS, 2006, 103, 18, 6895-6900 [3] Groot, M.-L. Pawlowicz, N.P. van der Wilderen, L.J.G.W. Breton, J. Van Stokkum, I.H.M. van Grondelle, R. 2005, PNAS, 2005, 102, 37, 13087-13092 PS2.11 Influence of the protein environment on the regulation of the Photosystem II activity – a time-resolved fluorescence study M Szczepaniak (Max Planck Institute for Bioinorganic Chemistry, Mülheim, Germany), J Sander (Plant Biochemistry Department, Ruhr-University, Bochum, Germany), M Nowaczyk (Plant Biochemistry Department, Ruhr-University, Bochum, Germany), M Müller (Max Planck Institute for Bioinorganic Chemistry, Mülheim, Germany), M Rögner (Plant Biochemistry Department, Ruhr-University, Bochum, Germany), A Holzwarth (Max Planck Institute for Bioinorganic Chemistry, Mülheim, Germany ) Photosystem II (PS II) is a complicated machinery that converts light energy to chemical energy by splitting water in the most important biological process on earth. The early photosynthetic processes, charge separation and electron transfer, can be regulated in many ways, amongst which the intrinsic properties and the internal dynamics of particular cofactors in the electron transfer chain as well as their interactions with the surrounding proteins play a significant role. In this study we are also particularly interested in the protein dynamics triggered by the primary photosynthetic events. We compare the time-resolved fluorescence of the cyanobacterial (T. elongatus) intact dimeric Photosystem II core particles isolated from WT and RCs containing the psbA3 gene product. The D1 protein of the PS II RC is present in three different copies in cyanobacteria: psbA1, psbA2 and psbA3. Under physiological light conditions only two copies, psbA1 and psbA2 are expressed. In the investigated “mutant” the psbA1 and psbA2 genes were knocked-out, resulting in the overexpression of the psbA3 gene. The analyzed psbA3-containing PSII preparation differs from WT in over ten amino acid residues, some positioned close to the early electron transfer chain cofactors. Thus, the influence of this amino acid alteration, mainly the interaction between active pheophytin and the D1-Q130 residue (D1-E130 in the case of the psbA3 preparation), on the primary events was studied in PSII particles with reaction centers (RC) in both the open and the closed states. Time-resolved fluorescence spectroscopy was performed using time-correlated single photon timing at room temperature. The WT and the psbA3 “mutant” fluorescence kinetics are compared in terms of the rate constants of the early processes and the changes in the free energy differences DG between the radical pair intermediates. PS2.12 The role of AspL213 for the binding of coenzyme Q to the photosynthetic reaction center E. -M. Krammer (Structural Biology/Bioinformatics Group, University of Bayreuth), A. R. Klingen (Structural Biology/Bioinformatics Group, University of Bayreuth), G. M. Ullmann (Structural Biology/Bioinformatics Group, University of Bayreuth) The photosynthetic reaction center is a transmembane protein, that

142 14th Photosynthesis Congress - PS07 converts light energy into the chemical energy of reduced coenzyme Q bound in the QB site of the protein. Two positions of coenzyme Q in the QB site (distal or proximal to the non-heme iron) have been observed in crystal structures. We have investigated the binding behavior of the educt (Q) and the first intermediate (Q.-) of coenzyme Q reduction to its two positions in the QB site of the photosynthetic reaction center. The protein and its environment are described by a continuum electrostatic model for which the Poisson-Bolzmann equation can be solved. Resulting protonation and binding state energies have been sampled by the Metropolis Monte Carlo algorithm. We thereby obtained binding probabilities in dependence of pH and coenzyme Q concentration. A central aspect of our study is to identify residues which are involved in the binding of coenzyme Q. Coupling of the binding process and of the (de)protonation of certain protonatable residues was therefore evaluated based on a correlation quantity. It is known, that AspL213, a residue located in the QB binding site, plays a dual role in the light-induced reaction. AspL213 is involved in the proton transfer to coenzyme Q and stabilizes a proton in the environment of coenzyme Q. Based on our calculations, we suggest a third role for AspL213: protonation of AspL213 is a prerequisite for the binding of the first reaction intermediate. PS2.13 Structural aspects of interactions between the primary donor and cytochrome in heterodimer mutant reaction centers of Blastochloris viridis

K Saito (Univ. of Tsukuba, Japan), M Koichiro (Univ. of Tsukuba, Japan), H Sumi (Univ. of Tsukuba, Japan) H- on the primary electron donor P and pheophytin H. Normally charges are further separated by electron transfer from H– to quinone Q. Occasionally, however, the electron on H– and the hole on P+ recombine to produce a spin triplet state (3B*) on the so-called accessory chlorophyll B located between P and H. This is one of the specificities of PS II from other photosystems, since the triplet state is formed not on B but on P in the purple-bacterial photosystem. In this study, we clarify theoretically the pathway and the mechanism of the triplet-state formation in PS II. In this formation, we can consider three pathways with different intermediate states: 1) the hole-electron type (P+BH- ? PB+H- ? P3B*H), 2) the electron-hole type (P+BH- ? P+B-H ? P3B*H), and 3) the excitation-transfer type (P+BH- ? P+B-H ? 3P*BH ? P3B*H). Since these pathways are mediated by at least one intermediate state, the mediation is classified between mutually-opposite limits of stepwise (ordinary-sequential) and unistep (superexchange). A mechanism halfway between the two limits is called the hot one, where the final transition occurs during thermalization of phonons in the intermediate state. Which pathway participates? and how classified is the mediation mechanism? The answers depend on the magnitude of the hole transfer integral Jh relative to the electron one Je. When Jh << Je, it occurs by the hot mechanism in 3), otherwise by the stepwise limit in 1). +

N Ponomarenko (University of Chicago), O Poluektov (Argonne National Laboratory), L Li (University of Chicago), E Bylina (KAIROS Scientific), R Ismagilov (University of Chicago), J Norris, Jr. (University of Chicago)

PS2.15 Structure for thermostability of photosynthetic reaction center from thermophilic purple sulfur bacterium, Thermochromatium tepidum.

The influence of interactions among cofactors involved in re-reduction of the photooxidized primary electron donor in the reaction center of Blastochloris viridis with a bound cytochrome subunit was studied in heterodimer mutant. In this H(M200)L mutant, one of two bacteriochlorophyll molecules of primary donor is replaced by a bacteriopheophytin due to substitution of coordinating histidine by leucine in M subunit. EPR characterization of this heterodimer mutant reaction centers reveals the presence of high spin iron in addition to the low spin iron typical of cytochrome hemes in the wild type organism. These observations could be indicative of ligand field symmetry lowering for one or more hemes, as the spin-state of iron (III) porphyrins is dependent on the strength of the axial ligations. To clarify the origin of the modified EPR signal of the heme, the structural details of the altered protein complex were analyzed. Reaction centers with the H(M200)L mutation of the bacteriochlorophyll binding site of the primary donor were crystallized using microfluidic technology. The structure has been refined to a resolution limit of 2.5 Å. Electron density maps confirm the conversion of bacteriochlorophyll of the M site into bacteriopheophytin due to substitution of histidine by leucine. Concomitant structural changes, including positioning and orientation of two tetrapyrrole rings forming the primary donor appear to be relatively minor. Small structural modifications of the protein matrix around the bacteriochlorophyll were detected. The electrostatic interaction between the heterodimer and its nearest redox-active heme c559 is being investigated as the source of the change from low spin to high spin iron as revealed by EPR. Ultimately, determination of the modified primary donor potential is required to define the origin of the altered iron spin state.

M KOBAYASHI (Department of Chemical Science and Engineering, Ariake National College of Technology, Omuta, Japan), Y Shimada (Department of Biomolecular Engineering, Tohoku University, Sendai, Japan), Y FUJIOKA (Department of Biomolecular Engineering, Tohoku University, Sendai, Japan), H SUZUKI (Fuculty of Science, Ibaraki University, Mito, Japan), Z WANG (Fuculty of Science, Ibaraki University, Mito, Japan), T NOZAWA (National Institution for Academic Degree and University Evaluation, Tokyo)

PS2.14 Mechanism of spin-triplet-state formation on the accessory chlorophyll in the reaction center of photosystem II

We have been reported the three dimensional structure of photosynthetic reaction center (RC) from thermophilic purple sulfur bacterium, Thermochromatium tepidum, which indicated that the RC have three specific arginine residues with positive charge on the membrane surface of the periplasmic side [T.Nogi et al., PNAS, 97, 13561(2000)]. We investigated the thermal stability of the RCs in the intracytoplasmic membrane (ICM) and the reconstituted liposome by lipids and isolated LH1-RC or RC from Tch.tepidum by measurement for their absorption and circular dichroism spectra. The comparisons of the stability of the RC in those showed that the thermostability of the RC could be raised by the existence of LH1 surrounding RC. Analysis of the primary structure of the a and ß polypeptides of Tch.tepidum LH1 showed deletion of an asparic acid residue in a polypeptide at the membrane surface of the periplasmic side. This deletion leads negative charge on the membrane surface on the same side where the above-mentioned arginine residue of RC exist. Since extra-ionic interactions between these ions of RC and LH1 for Tch.tepidum might contribute to the tight association between them, this might be an important factor in the thermostability of the RC in Tch.tepidum. The thermal stability of RC was also affected by its subunit constitution: the RC containing a cytochrome subunit was more thermostable than the cytochrome-detached RC. This suggests that the cytochrome subunit contributes not only to the transport of electrons to the photo-oxidized special pair cation, but also to the stability of the special pair pigments from denaturing.

143 14th Photosynthesis Congress - PS07

PS2.16 Detection of quinone function in the homodimeric type-I reaction center of Heliobacterium modesticuldum T Kondo (Nagoya University), H Mino (Nagoya University), M Matsuoka (Osaka University), C Azai (Osaka University), H Ohoka (Osaka University), S Itoh (Nagoya University) Heliobacteria have the type I reaction center (RC) complex that is homodimeric in contrast to the heterodimeric PS I and type II RCs. The function of quinone in the heliobacterial RCs are not fully identified yet. We studied the flash-induced ESR signal in the isolated RC core complex of Heliobacterium modesticaldum [1], which is depleted of iron sulfur centers FA/FB and show turnover of FX [2]. We detected the E/A pattern spin polarized ESR signal with a decay time constant of 4 ms and assigned it to originate from the P800+FX- radical pair as reported in the membrane preparations [3]. The RC also showed another new E/A/E pattern signal that has the decay time constant of 70 μs. We assigned this signal to be originated from the P800+A1- radical pair in comparison with the P700+A1- state signal in PS I RCs. The result suggests the function of quinone in this RC. The E/A/E ESR spectral pattern was ascribed to the different orientation of menaquinone in this RC compared to that of phylloquinone in PS I. The distances and the electron transfer rates between A0, A1, and FX in the heliobacterial RC might be interpreted by the unique orientation of A1-menaquinone. [1] H.Oh-oka. (2007) Photochem. Photobiol., 83 177-186. [2] R.Miyamoto et al. (2006) Biochemistry, 45 6306-6316 [3] A. van der Est et al. (1998) Biochem. Biophys. Acta, 1409 87-98 PS2.17 Electron transfer reactions in photosystem I and II of the chlorophyll d containing cyanobacterium, Acaryochloris mari A Telfer (Imperial College London), A Pascal (CEA Saclay), J Barber (Imperial College London), M Schenderlein (Technische Universtät Berlin), E Schlodder (Technische Universtät Berlin), M Çetin (Technische Universtät Berlin) The redox properties of photosystem I (PS I) of the chlorophyll d dominated cyanobacterium, Acaryochloris marina, have been reinvestigated. We find that the redox potential of the primary donor, P740, is higher than that originally reported by Hu et al. (PNAS [1998] 95, 13319-13323) and is, indeed, very similar to that of P700 in chlorophyll a dominated organisms. This has been confirmed by Bailleul et al. (these proceedings) and explains the apparently normal rates of electron transfer between PS II and PS I that these authors demonstrate in this organism. Low temperature transient absorption spectroscopic properties of PS I and P740 will be presented. Secondary electron transfer reactions in non-oxygen evolving PS II of A. marina have also been investigated and the operation of an electron cycle around PS II, via alpha-carotene, similar to the beta-carotene cycle seen in chlorophyll a organisms, will also be described. PS2.18 EPR and ENDOR Studies of the Protonated Rhodosemiquinone in the QB Site in Bacterial Reaction Centers Lacking QA. M Okamura (University of California, San Diego), M Paddock (University of California, San Diego), R Isaacson (University of California, San Diego), J Shepherd (Gonzaga University) Rhodobacter sphaeroides, the proton coupled electron transfer reaction

leading to the reduction of the quinone QB has been shown to proceed through a transient protonated semiquinone intermediate state. eH+ eH+ QB → QB- · <=> QBH· → QBH- → QBH2 Although the protonated state of the native ubisemiquinone has not been observed, the protonated semiquinone state of the amino-quinone, rhodoquinone, which has a higher pKa, has been detected by optical kinetic experiments (Graige et. al. Biochemistry 38, 11465-73 (1999)). In order to study the properties of the protonated semiquinone we have investigated the EPR and ENDOR spectra of the semiquinone of rhodoquinone-3 bound in the QB site of RCs. These studies were been done using mutant RCs which have been modified to eliminate the binding of QA, which can also form a semiquinone that interferes with the measurements on QB- ·. In addition, the RCs contained mutations which allow direct reduction of QB by B-branch electron transfer from BChl2 to BChl to BPh then directly to QB (Paddock et. al. Biochemistry 44, 6920-8 (2005)). An EPR signal (35 GHZ, 80K) characteristic of the semiquinone was observed upon illumination of mutant RCs containing rhodoquinone. EPR and ENDOR measurements made at pH 9.5 and 4.5 showed spectral changes that were attributed to changes in H-bonding upon protonation. PS2.19 Detection of Ultra-Fast Process in the Energy and Electron Transfer Dynamics in Photosystem I Reaction Center Y Shibata (Nagoya University), S Kawamoto (Nagoya University), Y Satoh (Nagoya University), S Itoh (Nagoya University) Primary energy and electron transfer dynamics in photosystem I (PSI) have been extensively studied so far. We measured the time-resolved fluorescence dynamics of spinach PSI (in the whole spectral region) at 660-730 nm over a wide time range from 0.1 ps to 2 ns at room temperature. The sensitivity-corrected time-resolved fluorescence spectra were calculated by combining the data from the fluorescence up-conversion technique at 0.1 to 50 ps and that from the streak camera at 10 ps to 2 ns after the excitation with a 0.1 ps laser pulse at 430 nm. This excitation wavelength is expected to excite all the antenna chlorophylls almost uniformly (non-selectively). The global decay-fitting analysis revealed 5 components with time constants of 0.17 ps, 6.9 ps, 19 ps, 96 ps, and 420 ps. The shortest 0.17 ps time constant is significantly shorter than the 1.5-ps one reported previously [Holzwarth et al. J. Phys. Chem. B 109 (‘05)]. In the kinetic model proposed by Holzwarth et al., the 1.5 ps component has been assigned to the fastest trapping process. The detection of the 0.17-ps component instead of the 1.5-ps one in the present study offers the new information for the estimation of the trapping time constant in PSI. PS2.20 The primary electron acceptor of Photosystem II is weakly coupled to the accessory chlorophyll and the plastoquinone QA N Cox (Australian National University), J Hughes (Australian National University), R Steffen (Australian National University), P Smith (Australian National University), A Rutherford (CEA Saclay), E Krausz (Australian National University), R Pace (Australian National University) An analysis of optical and EPR spectra of chemically reduced PSII core complexes isolated from spinach is presented. In these samples, QA is pre-reduced in darkness, allowing the photo-accumulation of its electron transfer pathway precursor, PheoD1. We report low-temperature (2-200K) spectral changes in CD and absorption spectra associated with

144 14th Photosynthesis Congress - PS07 PheoD1 photo-reduction. The area of the narrow (2nm FWHM) bleach at 683.8nm is fully commensurate with that of an isolated pheophytina, indicating a weak coupling to its neighbouring pigment, the accessory chlorophyll ChlD1. Also, a highly structured second derivative pattern is seen in the change in the CD at 683.8 nm upon photoreduction. This is also interpreted as indicative of a weak PheoD1-ChlD1interaction. Corresponding EPR measurements of the trapped PheoD1- - which give rise to a “split signal” centered about g~2.0 - agree quantitatively with optical results. A theoretical model of the split signal interaction was developed using spin Hamiltonian formalism, in which the reduced pheophytin interacts with the pseudo spin ½ quinone-iron coupled complex. It establishes that the magnitude of this coupling is minimal with J~|50G|. The EPR analysis requires the presence of the two lowest doublets of the quinone-iron system to simulate the observed spectrum, implying the signal is a composite of two distinct spectral patterns. Consequently, the PheoD1 can be considered electronically isolated from QA and only weakly coupled to ChlD1.

PS2.21 Purification and biochemical characterization of PSI-LHCI supercomplex in Chlamydomonas reinhardtii M Sumimoto (Okayama University), T Onishi (Okayama University), J Shen (Okayama University), Y Takahashi (Okayama University) The unicellular green alga Chlamydomonas reinhardtii contains nine distinctive light-harvesting complexes I (LHCI complexes) designated Lhca1-9 (1) as well as fourteen photosystem I (PSI) polypeptides (PsaA-L, N, and O) (2). It appears that the PSI-LHCI supercomplex in C. reinhardtii is larger than that in higher plants that contain only four distinctive Lhca polypeptides (3). Since it is of interest to characterize the structure of the PSI-LHCI supercomplex in C. reinhardtii, we have improved a method to purify the PSI-LHCI supercomplex on a larger scale in order to carry out structural analysis. Thylakoid membranes were purified, washed with NaBr, and subsequently solubilized with n-dodecyl-b-D-maltoside. The resulting extracts were fractionated on sucrose density gradient. We optimized sucrose density gradient for ultracentrifugation for a larger sample scale and for a better separation of PSI and PSII complexes. The resulting PSI-LHCI enriched fractions were subsequently applied onto DEAE column. The conditions for elution buffer were optimized to eliminate minor contamination of other polypeptides. A highly purified PSI-LHCI supercomplex retaining PSI activity was obtained. The preparation contained most constituent PSI polypeptides except two peripheral subunits such as PsaN and PsaO. We found that PsaG is rather unstable in PSI complex and is easily dissociated from PSI complex. We also found that PsaG is highly susceptive to protease digestion. 1. Takahashi, Y. et al. Biochemistry 43 (2004) 7816-7823 2. Hippler, M. et al. Protist 153 (2002) 197-220 3. Ben-Shem, A. et al. Nature 426 (2003) 603-635 PS2.22 Spectroscopic investigation of electron and proton transfer in the photosynthetic reaction center of Rhodobacter sphaeroides D Onidas (Department of Biophysics, Ruhr-University Bochum), S Hermes (Department of Biophysics, Ruhr-University Bochum), J Stachnik (Department of Biophysics, Ruhr-University Bochum), K Gerwert (Department of Biophysics, Ruhr-University Bochum) The reaction center (RC) from the purple nonsulfur bacterium Rhodobacter sphaeroides uses light energy to reduce QA, the primary electron acceptor, and then, QB, the secondary electron acceptor. Previous Fourier-transform infrared (FTIR) measurements led to the

suggestion of an electron transfer mechanism involving an intermediary electron donor [1]. Fe2+ has been excluded as an intermediary electron donor by time-resolved X-ray absorption on the Fe-K-edge [2]. We study RC mutants by FTIR spectroscopy to search for the intermediary electron donor and to better understand molecular mechanisms of the coupled electron and proton transfer reactions. We showed that after reduction of QA, protons are taken up via Asp210 and transported in a Grotthuss mechanism via a protonated water chain to QB. Mutation of Asp210 to Asn leads to a deceleration of oxidation of QA-. Using time-resolved FTIR spectroscopy we characterized molecular reaction mechanisms of this mutant and found that QB- formation precedes QA- oxidation even more pronounced than in the wild type RC [3]. We proposed that specific bands belong to the intermediary electron donor. Time-resolved continuum bands were recorded and indicate protonation state changes of a protonated water cluster [4]. [1] A. Remy, K. Gerwert, Nat. Struct. Biol., 2003, 10(8), 637-644. [2] S. Hermes, O. Bremm, F. Garczarek, V. Derrien, P. Liebisch, P. Loja, P. Sebban, K. Gerwert, M. Haumann, Biochemistry, 2006, 45, 353-359. [3] S. Hermes, J. M. Stachnik, D. Onidas, A. Remy, E. Hofmann, K. Gerwert, Biochemistry, 2006, 45(46), 13741-13749. [4] F. Garczarek, K. Gerwert, Nature., 2006, 439(7072), 109-112. PS2.23 What is the origin of the highly dispersive quantum efficiencies for secondary donor oxidation at low temperature in Photosystem II? J Hughes (Australian National University), R Steffen (Australian National University), N Cox (Australian National University), P Smith (Australian National University), R Pace (Australian National University), A Rutherford (CNRS, CEA, Saclay), E Krausz (Australian National University) reduction following illumination at low temperature using green light is highly dispersive [1]. The data can be described [1] by three nearly equal populations that each differs in QE by approximately an order of magnitude from ~100 to ~10-3. With direct excitation of the weakly absorbing lowest energy optically accessible absorption band of PSII (700-730 nm), the QE decreases by up to ~104 [1]. We have extended our studies to Thermosynechococcus elongatus PSII core complexes. Our aim is to determine the origin of the dramatic 100-104 decrease in QE of QA reduction that follows primary charge separation induced by direct excitation of the weak long wavelength (700-730 nm) absorption band of PSII. We have used optical and EPR spectroscopy at low temperatures (<20 K) of both spinach and Thermosynechococcus elongatus PSII core complexes to quantify the secondary electron donors that are oxidised following either green or deep red (700-730 nm) illumination. Distinctly different electrochromic patterns were obtained for deep red vs. green illumination. We address the question of whether a particular secondary donor can be associated with a subset of PSII exhibiting a particular range of QEs, and address the origin of the dramatic decrease in QEs that we observe. The mechanism of primary charge separation is discussed in light of these results. A

(1) J.L. Hughes, P. Smith, R. Pace, E. Krausz, Biochim. Biophys. Acta, 1757 (2006) 841-51 PS2.24 Triplet States in Photosystem II Reaction Centers Studied

145 14th Photosynthesis Congress - PS07 with EPR and ENDOR Spectroscopy S Prakash (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany), J Niklas (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany), M van Gastel (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany), W Lubitz (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany) The chlorophyll triplet state 3P680 in photosystem II (PS II) is formed by recombination of the primary radical pair. In D1/D2 complexes of PS II from spinach this state was investigated using time-resolved EPR and ENDOR spectroscopy. The zero field splitting (zfs) parameters were determined over a large temperature range (10 250 K) with time-resolved EPR (pulse and transient) spectroscopy. The results showed that the triplet exciton is localized on a single chlorophyll at low temperature and delocalized over several (≥ 2) pigments at higher temperature. The spin density distribution has been determined for the triplet chlorophyll from a comprehensive set of the 1H hyperfine coupling constants (hfc) which could be measured by orientation selection pulse ENDOR spectroscopy at Q band. These results agree well with previous observations in frozen solution at X band1. Due to improved sensitivity and resolution more hyperfine couplings are resolved. The analysis supports the localization of the triplet on a single chlorophyll. DFT calculations using the ORCA program2 are presented for the triplet states for various chlorophylls in the PS II reaction center yielding both the zfs and hf parameters. The agreement between experimental and theoretical values allows for the determination of the details of the electronic structure of pigment triplet states in photosynthesis. References: Lendzian, et al Biochim. Biophys. Acta, Bioenerg 2003, 1605, (1-3), 35-46. 2 ORCA- An ab intio, DFT and semiempirical electronic structure package, Version 2.6, F. Neese, Universität Bonn (2007). 1

PS2.25 A model for temperature-dependent peak shift of the bacterial reaction-center absorption K Mukai (Tsukuba University), K Saito (Tsukuba University), H Sumi (Tsukuba University) Absorption spectra of the purple-bacterial reaction center have three bands in the near-IR region. They are ascribed to the Qy electronic transition of bacteriochlorophyll-a (Bchl) and bacteriopheophytine-a molecules. The lowest one of them, assigned to the lower excited state of the special pair of Bchls, exhibits a striking temperature dependence, decreasing markedly its energy with lowering temperature. The other two bands do not show such a temperature dependence. A model for this temperature dependence shift of the special-pair absorption is given, by constructing potential energy surfaces for the electronic excited and the ground state of the special pair. These surfaces are composed of the interaction with the protein vibration and the electronic energy. The excited state of the special pair is the exciton state, whose energy is sensitive to the intra-pair distance and the mutual angle. Their variations make the excited-state energy surface markedly anharmonic. Employing these energy surfaces, the absorption band was calculated as a function of temperature. The large peak shift with temperature could successfully be reproduced. This mechanism does not rely on the thermal expansion which has been assumed so far to be effective in the peak shift without detailed calculation. The peak shift is due to the

density-of-states difference between the energy surfaces. PS2.26 Effect of capsaicin on the photosynthetic performance of Scenedesmus obliquus cultures in vivo K Loulakakis (Department of Floriculture and Greenhouse Crops, School of Agricultural Technology, Technological Education Institute of Crete, P.O. Box 140, 710 04 Heraklion, Crete, GREECE), E Navakoudis (Department of Floriculture and Greenhouse Crops, School of Agricultural Technology, Technological Education Institute of Crete, P.O. Box 140, 710 04 Heraklion, Crete, GREECE), K Kotzabasis (Department of Biology, University of Crete, GREECE) Capsaicinoids are the principle pungent of capsicum fruits with capsaicin and dihydrocapsaicin to be responsible for more than 90% of the pungency. Capsaicin has been reported to act as a competitive inhibitor in vitro in both, plant PSII and bacterial reaction center. Due to this inhibitory action on the photosynthetic apparatus and the structural similarity with known herbicides, capsaicinoids could be exploited as herbicides or as leads for synthetic herbicide designing. The purpose of this work is to further investigate the action of capsaicin in the photosynthetic apparatus and to test its in vivo activity in cultures of the green alga Scenedesmus obliquus. In this direction, chlorophyll fluorescence was used to screen the photosynthetic performance of the cell cultures of S. obliquus, following capsaicin supplementation. Increasing concentrations of capsaicin showed a reduction of the photosynthetic efficiency (Fv/Fm) of the cultures, indicating that capsaicin exhibits in vivo activity. Further processing of the fluorescence induction kinetics by JIP-test analysis provided significant information on the alterations induced in the structure and function of the photosynthetic apparatus. These results not only confirm that the capsaicin inhibitory action is a common trait among photosynthetic species, but also prove that QB is the binding site for capsaicin in vivo, inducing the closure of the PSII reaction centers. Further work is in progress in order to investigate the possible phytotoxical action of capsaicinoids on intact higher plants. This work was supported by an Archimedes Project, co-funded by the European Social Fund and National Resources. PS2.27 In vitro reconstitution of the cytochrome b559 from higher plants R Picorel (EEAD-CSIC) Maria A. LUJAN, Miguel ALFONSO, Sara LOPEZ, Inmaculada YRUELA and Rafael PICOREL (Aula Dei Experimental Station, Consejo Superior de Investigaciones Científicas (CSIC) The cytochrome (Cyt) b559 is a membrane-protein component of the photosystem II reaction center but its function still remains unclear. This Cyt consists of two polypeptide subunits, a and ß. The a-subunit (9 kDa) is encoded by the psbE gene and the ß-subunit (4.5 kDa) by the psbF gene. Both genes are of chloroplastic origin and each polypeptide spans the thylakoid membrane with a single a-helix. The two polypeptides are bound by a heme group coordinated by two histidines, one from each subunit. Our work is aiming to obtain in vitro reconstitution of the plant Cyt b559 for future structural and functional studies. To that end we have cloned and overexpressed each subunit from sugar beet in E. coli as a fusion protein using the expression vector pMALc2x. When the fusion protein expression was induced at standard conditions, most of that protein appeared as inclusion bodies. This fraction was purified by washing with Triton X-100 and denatured with 7 M urea. For reconstitution, we mixed the two fusion proteins with equimolar amounts of heme. The reconstitution was carried out by dyalisis against

146 14th Photosynthesis Congress - PS07 gradually decreasing urea concentrations, and, finally the free heme was removed by chromatography to avoid spectral interferences. The reconstitution was analyzed by UV/Vis spectroscopy; i.e., the reconstituted Cyt b559 redox difference spectrum was very similar to that of the native Cyt b559 from higher plants, with a typical maximum of the a-band and b-band at 559.5 nm and 530 nm, respectively. PS2.28 The Electron Acceptor Quinone A1 of Photosystem I Investigated by Pulse EPR and ENDOR Spectroscopy J Niklas (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany), B Epel (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany), M Antonkine (1) Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany 2) Institut für Experimentalphysik, Freie Universität Berlin, Berlin, Germany), S Sinnecker (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany), M Pandelia (Max Planck Institute for Bioinorganic C The quinone vitamin K1 (VK1) is part of the electron transfer chain in photosystem I (PS I). After light-induced charge separation the spin polarized radical pair P700∙+A1∙– (protein bound VK1 is denoted A1) is formed. The protein bound quinone exhibits physical properties different from those of VK1 in vitro. The X-ray structure of PS I indicates that VK1 in the A1 binding site is involved in asymmetric hydrogen bonding with the protein. EPR and ENDOR spectroscopy were used to investigate the light-induced radical pair P700∙+A1∙– and the stationary radical anion A1∙– and were compared with VK1∙– in vitro.1 One- and two-dimensional TRIPLE spectroscopy was used to determine relative orientations and signs of the hyperfine (hf) coupling tensors. Variable mixing time (VMT) ENDOR2 was employed to determine the absolute signs of the hf coupling constants in the radical pair P700∙+A1∙–. We exchanged the quinone in the A1 binding site of menB mutant PS I with fully deuterated vitamin K3. This allowed us to differentiate between proton hyperfine couplings from the quinone and from the protein environment. Furthermore, DFT calculations were performed on a model of the A1 binding site, which yielded hf tensors in very good agreement with the experiment. The experimental and theoretical results clearly show that the single-sided H-bond to A1∙– is indeed the crucial factor that determines the particular electronic structure of this radical. 1 2

Epel et al., J. Phys. Chem. B, 2006, 110, 11549-11560 Epel et al., Appl. Magn. Reson., 2006 , 30, 311-327

PS2.29 Redox Reactions of the Non-Heme Iron of Photosystem II: An EPR Spectroscopic Study

as a single, one-electron acceptor site: EPR quantitation of both the oxidized donor yield and the Fe2+ QA– yield found no evidence for the formation of more than one oxidized secondary electron donor per PSII. The photooxidized chlorophyll and carotenoid secondary electron donors were shown, for the first time, to be capable of charge recombination with photoreduced Fe2+, oxidizing the iron in a temperature-dependent fashion below 300 K. Two redox populations of Fe3+ were revealed at low temperatures. One population was photoreduced at the lowest attainable temperatures, while the other was fully photoreduced only at temperatures above ca. 140 K, having an apparent reduction potential below that of the QA / QA– couple (–80 mV) at lower temperatures. It is hypothesized that the redox activity of the non-heme iron depends upon the existence of a facile proton-transfer pathway linking the site to the stromal surface of the protein, and that the redox activity of the non-heme iron may probe redox-coupled proton-transfer reactions around the QB site. Supported by the DOE, Office of Basic Energy Sciences, Division of Chemical Sciences DE-FG02-05ER15646. PS2.30 An investigation into thermodynamics of the thermal denaturation of the reaction centre of Rhodobacter sphaeroides in vitro and reconstituted into liposomes. F Bohles (Queen Mary, University of London), S Santabarbara (Queen Mary, University of London), P Heathcote (Queen Mary, University of London), M Jones (University of Bristol) The thermodynamics of thermal stability of the reaction centre (RC) of Rhodobacter sphaeroides isolated in detergent micelles and reconstituted into liposomes has been investigated. The temperature dependence of the absorbance spectra and circular dichroism spectra were investigated in the ultra-violet region and the visible region to investigate the thermal stability of the protein component of the RC, and the co-factors in their native binding pockets, respectively. The isolated RC in detergent micelles shows three melting transitions at 8 °C, 45 °C and 113 °C. For these three transitions the melting entropies (ΔHm) are 210 kJ mol-1 320 kJ mol-1 and 412 kJ mol-1 and specific heat capacities (ΔCp) are 0.68 kJ K-1 mol-1, 0.5 kJ K-1 mol-1 and 0.2 kJ K-1 mol-1, respectively The transition at 8 °C is attributed to a conformational change affecting the carotenoid binding site. The 45°C melting point was observed when monitoring both protein and co-factor absorption, while the transition at 113 °C was monitored in the UV region only. In reconstituted RCs only one melting transition was observed at 83 °C in the visible region of the absorption spectrum. Moreover, we observed a lower ΔHm (140 kJ mol-1) and a substantial increase of ΔCp (2.2 kJ K-1 mol-1) in the liposome-reconstituted RC. The balance between entropic and enthalpic contribution to the temperature dependent free energy of melting provide a microscopic description of the temperature stabilisation of the RC conferred by a membrane-mimicking environment.

PS2.31 Electronic structure of axial histidines in photosynthetic reaction centres of Rhodobacter sphaeroides

J McEvoy (Regis University), G Brudvig (Regis University) We have used EPR spectroscopy to investigate the redox chemistry of the non-heme iron of photosystem II, focusing on the cryogenic electron-transfer reactions of iron, QA and a variety of secondary electron donors, namely chlorophylls, carotenoids and tyrosine D. Glycolate coordination to the iron was used in combination with potassium ferricyanide to preoxidize the non-heme iron completely and prime it for photoreduction. The oxidized Fe3+ and QA centers operated

A Alia (Leiden Institute of Chemistry), P Wawrzyniak (Leiden Institute of Chemistry), F Buda (Leiden Institute of Chemistry), J Matysik (Leiden Institute of Chemistry), H de Groot (Leiden Institute of Chemistry) Histidine residues are the axial ligands to primary electron donor chlorophylls in all known photosynthetic reaction centres (RC). In bacterial reaction centres, mutation of the axial histidines has been demonstrated to affect kinetics of electron transfer and symmetry of the

147 14th Photosynthesis Congress - PS07 electron spin distribution. In the present study, different Magic angle spinning (MAS) NMR techniques are applied in order to explore the electronic structure of the histidines co-ordinating to Mg2+ in bacterial reaction centres. 15N MAS NMR spectra of [13C6,15N3]-histidine labeled bacterial reaction centers clearly show two different classes of axial histidines in RC. Out of four magnesium bound histidines, one show very different electronic structure in which magnesium bound nitrogen resonates 6 ppm downfield compared to rest of the three histidines. This data was further confirmed by 2D heteronuclear (1H-13C) dipolar correlation spectrum that shows clear separation of axial histidines into two catogories based on the chemical shifts of d carbon and d proton of axial histidines. The DFT calculations corroborate that one of the axial histidine in reaction centre may be carrying partial negative charge in ground state due to partial displacement of the proton of pros nitrogen of the imidazole ring. We propose that this could be the axial histidine ligated to one of the special pair bacteriochlorophyll in the active branch of bacterial reaction centres and might be involved in tuning the properties of the active branch. PS2.32 Proton uptake in the reaction center mutant L210DN from Rhodobacter sphaeroides via protonated water molecules J Stachnik (Ruhr-University Bochum), S Hermes (Ruhr-University Bochum), D Onidas (Ruhr-University Bochum), E Hofmann (Ruhr-University Bochum), K Gerwert (Ruhr-University Bochum) The reaction center (RC) of the purple nonsulfur bacterium Rhodobacter sphaeroides uses light energy to reduce and protonate a quinone molecule, QB (the secondary electron acceptor), to form quinol, QBH2, which is released from the RC. Asp210 in the L-subunit was shown to be a catalytic residue in this process. In the L210DN mutant the proton uptake pathway is disturbed, which slows down the proton transfer to QB and leads to a deceleration of the re-oxidation of QA- in the QA-QB à QAQB- transition. Previous Fourier-transform infrared (FTIR) measurements led to the suggestion of a mechanism of electron transfer involving a intermediary electron donor X [1]. QB- formation in the L210DN mutant precedes QA- oxidation even more pronounced than in the wild type RC [2]. The structure of the L210DN mutant was solved to 2.5 Å. There are no major structural differences as compared to the wild type protein. We found QB in the distal position and a chain of water molecules between Asn210 and QB, which is highly conserved in wild type RC structures. Continuum absorbance changes in FTIR-spectra indicate deprotonation of a protonated water cluster, most likely of the water chain between Asn210 and QB. In addition an H/D exchange spectrum of the L210DN mutant in the ground state evidences a water molecule with a dangling hydroxyl group. References: [1] A. Remy, K. Gerwert, Nat Struct Biol, 2003, 10(8), 637-644. [2] S. Hermes, J.M. Stachnik, D. Onidas, A. Remy, E. Hofmann, K. Gerwert, Biochemistry, 2006, 45(46), 13741-13749. PS2.33 Secondary Donors in Low-Temperature Optical Spectroscopy of Photosystem II R Steffen (Australian National University), J Hughes (Australian National University), N Cox (Australian National University), P Smith (Australian National University), R Pace (Australian National University), A Rutherford (Institut de biologie et biotechnologies de Scalay), E Krausz (Australian National University)

At low temperature (5K) light-induced charge separation in photosystem II is followed by charge stabilisation due to reduction of P680+ by secondary donors such as carotenoid (Car) or chlorophyll (Chl) which compete with P680+ QA- charge recombination. In order to study charge stabilisation in photosystem II at low temperature we utilised a newly developed CCD-based absorption spectrometer providing minimal actinic fluence and reasonable time (100 ms) and spectral (0.5 nm) resolution while covering a large spectral window from 470 – 1100 nm. This enabled us to follow the electron transfer reactions in photosystem II by monitoring the specific signals for QA-, Chl+ and Car+ simultaneously in the wavelength and time domain. We show that, as a result of inducing multiple turnovers in photosystem II during the course of illumination with either continuous green light or saturating laser flashes, different donors are generated. These include two different carotenoids, chlorophyll and a third as not yet identified donor. The relative amounts of these donors change with the number of photons absorbed per reaction centre. In a prolonged illumination Car+ is largely replaced by the other more stable donors, resulting in slower recombination kinetics in the dark after illumination. In addition the kinetic separation of different donor species enables us to identify spectral features to either Car or Chl oxidation processes. PS2.34 The lipid environment influence on spectral and kinetic properties of semiquinones in bacterial photosynthetic reaction centres. F Milano (CNR – Istituto per i Processi Chimico Fisici), A Agostiano (Dipartimento di Chimica Università di Bari), E Altamura (Dipartimento di Chimica Università di Bari), L Giotta (Dipartimento di Scienza dei Materiali - Università di Lecce), L Nagy (Department of Biophysics – University of Szeged – Szeged (Hungary)), P Maroti (Department of Biophysics – University of Szeged – Szeged (Hungary)), M Trotta (CNR – Istituto per i Processi Chimico Fisici) Quinones perform a key role in the energy conversion of biological systems, coupling oxydation/reduction reactions to proton uptake or release across membranes. In bacterial photosyntesis a cyclic electron flow occurs between the Reaction Centrer (RC), the bc1 complex and the cytochrome c2; this movement of electrons is accompanied by a net flow of protons from the periplasmic to the cytoplasmic space. Electrons are shuttled beteween the membrane-spanning RC and bc1 complex by the hydrophobic ubiquinone-10 (UQ10). In the RC crystal structure two molecules of UQ10 are found in distinct binding sites, termed QA and QB. The quinone in the QA pocket acts as a single electron carrier and is firmly associated to the protein, while the one in the QB pocket can undergo to double reduction and protonation and is loosely bound. When isolated RC are hit by a light flash, the charge separated state D+QB− (or D+QA− in the presence of QB inhibitors) is produced. Here D is the primary donor, a bacteriochlorophyll dimer. In the presence of a suitable electron donor to D, a relatively stable semiquinone is formed. The properties of the two semiquinones are mainly modulated by the protein environment, but fine adjustments are achieved by the interaction with the protein surrounding, namely the lipidic bilayer. We have reconstituted the RC in liposomes made by different and physiologically important phospholipids and recorded the spectra of the semiquinones with the aim of study protein-lipid interactions that are involved in the fine tuning of these key intermediates. PS2.35 Influence of bilayer thickness on photosynthetic Reaction Centres function.

148 14th Photosynthesis Congress - PS07 M Trotta (CNR – Istituto per i Processi Chimico Fisici), F Milano (CNR – Istituto per i Processi Chimico Fisici), N De Nicolò (Dipartimento di Chimica Università di Bari), L Giotta (Dipartimento di Scienza dei Materiali - Università di Lecce), L Nagy (Department of Biophysics – University of Szeged – Szeged (Hungary), P Maroti (Department of Biophysics – University of Szeged – Szeged (Hungary), A Agostiano (Dipartimento di Chimica Università di Bari) The photosynthetic Reaction Centre (RC) is a transmembrane pigment-protein complex where the energy associated to the electromagnetic solar radiation is converted into chemical energy in the form of a charge separated state. Following absorption of a photon the bacteriochlorophyll dimer (D) reaches its singlet excited state (D*) and then transfers an electron through a chain of cofactors to the final electronic acceptor QB. In absence of electron donors to D, a charge recombination reaction occurs with a lifetime in the seconds timescale, strongly influenced by the protein environment. The membrane-spanning portion of the RC is about 35 Å and is normally embedded in the intracitoplasmic membrane where the most representative phospholipid is palmitoyl-oleoyl- phosphatidylcholine (POPC) with side chains of 16 (saturated) and 18 (monounsaturated) carbon atoms (nc) forming a 44 Å thick bilayer. To assess the effect of a complete or partial covering of the transmembrane portion, RC have been reconstituted in liposomes made by 1,2 diacyl – phosphatidylcholine with nc spanning from 9 to 14, ensuring that the bilayers formed are in the liquid-crystalline phase. RC embedded in liposomes made by POPC, showing a charge recombination lifetime of 2.2 s, was taken as a reference system. A discontinuity in the charge recombination reaction lifetime was found between nc = 12 and nc = 14 corresponding to a bilayer thickness of 32 and 37 Å respectively. PS2.36 The Distant Protein Environment influences the Re-dox Potentials of the Quinone Acceptor A1 and the FX Iron-Sulphur Cluster in Photosystem I: TR EPR Study of Mutants of the D575< I Karyagina (FU Berlin) Multifrequency TR EPR is applied to measure the kinetics of electron transfer (ET) from A1 to FX in site-directed variants of the amino acids D575PsaB and D566PsaB in Photosystem I. Electrostatic calculations predict that the partial negative charges associated with these Asp residues play a significant role in modulating the Red-Ox potentials of A1 and FX. To test this prediction, the side chains of residues 575PsaB and 566PsaB were changed from negatively charged (Asp) to neutral (Ala) and to positively charged (Lys). The rate of ET from A1A to FX was found to decrease slightly in the D575PsaB but increase in the D566PsaB variants from wild type in the sequence D/A/K. These results are consistent with the expectation that changing the partly negatively charged Asp residue will shift the Red-Ox potential of nearest cofactors to more positive values and this shift will depend on the distance between the cofactor and the Asp residue position. According to the X-ray structure model (1JB0), the Red-Ox potentials of A1A should experience a larger shift than A1B and even larger than FX in the D575PsaB variants. But FX should experience a larger shift than either A1A and A1B in the D566PsaB variants. As a consequence, the driving energy (the Red-Ox potential difference) and the ET rate from A1A to FX will decrease in the former and increase in the latter case. The experimental findings agree to a large extent with the predictions from electrostatic calculations as will be discussed in detail. PS2.37 Bacterial reaction centers with bound Mn are capable of

light-controlled enzymatic activity J Allen (Arizona State University), W Lee (Arizona State University), A Tufts (Arizona State University), J Williams (Arizona State University) The reaction center from Rhodobacter sphaeroides has long been used as a model for photosystem II, and our group has incorporated features of photosystem II such as a highly oxidizing primary donor that is capable of oxidizing tyrosine residues. We have also introduced a Mn cofactor bound at a location analogous to that of the Mn cluster of photosystem II. In addition to characterizing the new light-driven electron transfer reactions involving Mn as a secondary donor to the oxidized bacteriochlorophyll dimer, we are examining the modified reaction centers for the types of functional properties found in other Mn-binding enzymes. For example, superoxide dismutase catalyzes the conversion of superoxide anions in a critical antioxidative reaction. An optical assay used to quantify the amount of superoxide dismutase activity by measuring the concentration of superoxide anion was employed to test for such activity in reaction centers. The modified reaction centers with bound Mn were found to be capable of reacting with superoxide. This enzymatic activity was present only when the reaction centers were exposed to light and only when the bacteriochlorophyll dimer was highly oxidizing. These results indicate that the superoxide conversion proceeds from the oxidized state of the Mn cofactor. The modifications of the reaction center provide a pathway by which a phototroph capable of enzymatic activity could have been an intermediate in the evolutionary development of organisms containing oxygen-evolving complexes. The ability to regulate the enzymatic activity using light provides the possibility of developing new light-controlled enzymes. PS2.38 Mutational analysis of the EC2-EC3 environment in PS1 R Luthra (University of Alabama), K Narasimhulu (University of Alabama), A Jasaitis (University of Alabama), K Redding (University of Alabama), A Ozarowski (National High Magnetic Field Lab, Tallahassee, FL), J von Tol (National High Magnetic Field Lab, Tallahassee, FL), F Rappaport (Institut de Biologie Physico-Chimique, Paris), M Müller (Max-Planck-Institut fur Bioanorganische Chemie), C Slavov (Max-Planck-Institut fur Bioanorganische Chemie), A Holzwarth (Max-Planck-Institut fur Bioanorganische Chemie) Photosystem I (PS1) has two branches of cofactors (A and B), which are potential electron transfer pathways to the FeS cluster FX. Each branch consists of two chlorophylls (ec2 and ec3) and a phylloquinone (PhQ). P700 has been thought to be the site of primary charge separation, but mutations to the H-bond donor near P700 seem to have no effect on directionality (Li et al., 2004, Biochemistry 43:12634-47) or on the rate of primary charge separation (Holzwarth et al., 2006, Biophys. J. 90:552-65). Mutation of the H-bond donor to ec3A (PsaA-Tyr696) or ec3B (PsaB-Tyr676) had a marked effect on directionality. The PsaA-Y696F mutation increased the amplitude of the faster kinetic component (assigned to electron transfer from PhQB) and decreased the amplitude of the slower kinetic component (assigned to electron transfer from PhQA), while the PsaB-Y676F mutation had the opposite effect. Recent ultrafast data shows that these mutations slow primary charge separation, implicating ec3 as one of the species involved in this step. We have also been able to obtain high-field EPR (413 GHz) spectra of photo-accumulated “A0-” with clear resolution of the g-tensor for WT and both mutants. We are examining other mutations to the environment of ec2A and ec2B, specifically an alanine close to the pair (PsaA-Ala684 and PsaB-Ala664) and the H-bond partner to the axial ligand H2O (PsaB-Asn591 and PsaA-Asn604). Like PsaA-Y696F, the PsaA-A680D mutant shows a similar increase in the faster component, indicating redirection of electrons down the B branch.

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PS2.39 Triplet photoprotection by carotenoid in intact photosystem II cores A Holzwarth (Max-Planck-Institut), V Martinez-Junza (Max-Planck-Institut), M Szczepaniak (Max-Planck-Institut), S Braslavsky (Max-Planck-Institut), J Sander (Ruhr-Universität Bochum), M Rögner (Ruhr-Universität Bochum) Two ß-carotenes are located in the D1-D2 reaction center (RC) of photosystem II (PSII) in the X-ray structure.1 Despite the presence of two carotenes and a high yield of triplet chlorophyll production, no triplet carotene quenching has been observed so far in the RC of PS II. We have used nanosecond transient spectroscopy to study the triplet kinetics and photoprotection in intact PS II cores from T. elongatus. We report for the first time efficient photoprotection by 3Chl-to-3Car transfer. We assign the 3Car to the D2 carotene, assigning a photoprotective role to the otherwise inactive branch of the RC. [1] Loll, B., Kern, J., Saenger, W., Zouni, A., Biesiadka, J. Nature 438, 1040-1044 (2005). [2] Telfer, A. Photochem. Photobiol. Sci. 4, 950-956 (2005). PS2.40 The dual-branched electron transfer in photosystem I A Holzwarth (Max-Planck-Institut), M Müller (Max-Planck-Institut), C Slavov (Max-Planck-Institut), R Luthra (University of Alabama), K Redding (University of Alabama) There exists clear evidence - based on the bi-phasic reduction of the iron-sulphur cluster Fx - that in PS I electron transfer (ET) occurs in both branches. However, the relative efficiencies and in particular the mechanism and the rates of the early ET steps have not been determined. We present here ultrafast transient absorption measurements on two mutants (A-Y696F, B-Y676F) from C. reinhardtii We show that primary CS slows down in both mutants relative to the w.t. We were able to separate the kinetics in both branches and determined all the rates and yields of the early ET steps. The data provide clear evidence for efficient ET along both branches. PS2.41 Charge separation and energy transfer in the Photosystem II core complex studied by femtosecond mid-infrared spectroscopy. N Pawlowicz (Vrije Universiteit Amsterdam), M Groot (Vrije Universiteit Amsterdam), I van Stokkum (Vrije Universiteit Amsterdam), J Breton (Service de Bioénergétique, CEA-Saclay), R van Grondelle (Vrije Universiteit Amsterdam) The core of photosystem II (PSII) of green plants contains the reaction center (RC) proteins D1D2-cytb559 and two core antennas CP43 and CP47. We have used time-resolved visible pump/mid-infrared probe spectroscopy in the region between 1600 and 1800 cm-1 to study the energy transfer and charge separation events within PSII cores. The absorption difference spectra in the region of the keto and ester chlorophyll modes show spectral evolution with time constants of 3 ps, 27 ps, 200 ps and 2 ns. Comparison of IR difference spectra obtained for the isolated antennas CP43 [1], CP47 [2] and the D1D2-RC [3] with those measured for the PSII core allowed us to identify the features specific for each of the PSII core components. From the presence of the CP43 and CP47 specific features in the spectra up to time delays of 20-30 ps, we conclude that the main part of the energy transfer from the antenna’s to the RC occurs on this time scale. Direct excitation of the

pigments in the RC leads to radical pair formation of PD1+PheoD1¯ on the same time scale as multi-excitation annihilation and excited state equilibration within the antennas CP43 and CP47, which occur within ~1-3 ps. The formation of the earlier radical pair ChlD1+PheoD1¯, as identified in isolated D1D2 complexes with time-resolved mid-IR spectroscopy [3] is not observed in the current data, probably because of its relatively low concentration. Relaxation of the state PD1+PheoD1¯, caused by a drop in free energy, occurs in 200 ps in closed cores. We conclude that the kinetic model proposed earlier for the energy and electron transfer dynamics within the D1D2-RC [3], plus two slowly energy transferring antennas C43 and CP47 explains the complex excited state and charge separation dynamics in the PSII core very well. We further show that the time resolved IR-difference spectrum of PD1+PheoD1¯ as observed in PSII cores is virtually identical to that observed in the isolated D1D2-RC complex of PSII, demonstrating that the local structure of the primary reactants has remained intact in the isolated D1D2 complex. [1] Di Donato, M., van Grondelle, R., Groot, M. L., submitted to J. Phys. Chem. [2] Groot, M. L., Breton, J., van Wilderen, L. J. G. W., Dekker, J. P., van Grondelle, R. (2004) J. Phys. Chem. B. 108, 8001-8006. [3] Groot, M. L., Pawlowicz, N. P., van Wilderen, L. J. G. W., Breton, J., van Stokkum, I. H. M., van Grondelle, R. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 13087-13092. PS2.42 (A1--A1) FTIR Difference Spectra Obtained using Photosystem I Particles With 18O and Specifically Occupying the A1

13

C Labeled Quinones

G Hastings (Georgia State University), P Banderanayake (Georgia State University) Time-resolved step-scan Fourier transform infrared difference spectroscopy (TRSS FTIR DS) has been used to produce (A1¯-A1) FTIR difference spectra for PS I particles from S. 6803 that have a 2-methyl naphthaquinone (2MNQ) occupying the A1 binding site. 2MNQ is the same as phylloquinone except that the phytyl chain is replaced with a hydrogen atom. We have also obtained spectra for PS I particles containing 2MNQ that is 18O labeled, and specifically 13C1 or 13C4 labeled. These spectra allow a clear demarcation between bands associated with the quinone and bands associated with other molecular groups in the binding site. To aid in the interpretation of the (A1--A1) FTIR difference spectra we have obtained FTIR absorption spectra for all labeled and unlabeled quinones studied, and have used density functional methods to calculate the FTIR absorbance and “anion minus neutral” absorbance difference spectra for all of the quinones studied. Finally, we have used QM/MM methods (ONIOM method) to calculate the vibrational properties of quinones in the A1 binding site.

PS2.43 Calculated Electronic Spectra of Chlorophylls in Solution S Parameswaran (Department of Physics and Astronomy, Georgia State University), G Hastings (Department of Physics and Astronomy, Georgia State University) Chlorophylls and bacteriochlorophylls play a fundamental role in photosynthesis. They are used to capture and funnel solar energy to the reaction center. They are also the primary units used to convert excitation energy into chemical products. Knowledge of their function in various environments, from solution, to photosynthetic light harvesting

150 14th Photosynthesis Congress - PS07 complexes, to reaction center protein complexes, is therefore of considerable importance. As a first step in developing a quantitative model of chlorophylls in various environments we have been using time dependent density functional theory to calculate the electronic properties of several chlorophyll structures in various solvents. Up until now most studies have been limited to isolated pigments in the gase phase. In particular, we have used time dependant density functional methods [B3LYP/6-31G(d)] in conjunction with the polarizable continuum model, to obtain fully optimized structures of chlorophylls a, b, c1, c2, d and bacteriochlorophylls a, b, c, d, e and g, in both polar and non-polar solvents. The calculated wavelengths for the Qy, Qx and soret bands of the different bacteriochlorophylls and chlorophylls were found to agree well with experimental spectra. We have also used QM/MM methods to calculate the electronic properties of chlorophyll-a in the presence of 40 explicitly added solvent molecules. Solvent induced electronic band shifts calculated using this explicit solvent model are compared to the results obtained using the polarizable continuum model. In this way the applicability of the polarizable continuum model is assessed. PS2.44 Calculation of the Vibrational Properties of Chlorophyll-a R Wang (Department of Physics and Astronomy, Georgia State University, Atlanta, GA 30303), G Hastings (Department of Physics and Astronomy, Georgia State University, Atlanta, GA 30303) Chlorophylls are important pigments in photosynthetic processes, and they have been widely studied using vibrational spectroscopies. In spite of this, little work has been undertaken to quantitatively assess the vibrational structure of chlorophyll. With this in mind we have used density functional theory (DFT) to calculate the vibrational properties of chlorophyll-a (Chl-a) in the gas phase and in the presence of solvents. Gas phase calculations do not accurately simulate the vibrational properties of the carbonyl groups of either neutral or cationic Chl-a. That is, the calculated results do not agree with experimental observations. Here we undertake calculations to investigate how different factors impact the carbonyl vibrational modes of Chl-a. Firstly, we use DFT in combination with the polarizable continuum model to investigate how different solvents impact the vibrational properties of Chl-a. Secondly, how additional peripheral groups modify the vibrational properties of Chl-a and Chl-a’ are investigated. Thirdly, the effects of hydrogen-bonding to the carbonyl groups are investigated. Our calculations show that solvent effects, as described by the polarizable continuum model, cannot explain the differences between theory and experiment. Additional peripheral groups cannot explain the differences either. Hydrogen bonding to the carbonyl groups of Chl-a appear to be the only way to reconcile calculations with experimental spectra. We therefore propose that in all previously determined experimental IR spectra of Chl-a in solvent, hydrogen bonding effects predominate, possibly because water has never been completely removed in any of these previous experiments. PS2.45 Photo-CIDNP MAS NMR on photosynthetic reaction centres J Matysik (Leiden University) The first step of photosynthesis, the charge separation upon light-induced electron transfer, is of unsurpassed efficiency having quantum yields close to unity. In all six RCs which have been tried, originating of various branches of the evolutionary tree, photo-CIDNP has been observed, despite the window of the conditions for the occurrence of this phenomenon is rather narrow. Hence, we suppose that the conditions allowing for the production of photo-CIDNP are

conserved in evolution. On the other hand, despite of lots of efforts until now no photo-CIDNP has been reported from any artificial RC system, having also low quantum yield. Therefore, there may be a link between the fundamental conditions, allowing for the production of photo-CIDNP, and the efficiency of light induced electron transfer. In that case, understanding of these fundamental conditions could have a large impact on the synthesis of artificial RC systems. Three mechanisms producing photo-CIDNP in RCs are proposed and in good agreement with experiments. In these experiments, NMR enhancement factors of above 10000 have been observed, making photo-CIDNP to a hot candidate to overcome the intrinsically low sensitivity and selectivity in solid-state NMR. Currently, the method is used to study the photochemical machinery of various RC systems at the atomic resolution. To obtain detailed insight into the mechanisms, the build-up of photo-CIDNP in RCs is studied with nanosecond-flash experiments, allowing for observation of the evolution of electron spin density during the birth of the radical pair.

PS2.46 The origin of the high redox force of photosystem II: A photo-CIDNP MAS NMR analysis A Diller (University of Leiden), A Alia (University of Leiden), E Roy (University of Leiden), P Gast (University of Leiden), H de Groot (University of Leiden), H van Gorkom (University of Leiden), G Jeschke (University of Konstanz), C Glaubitz (University of Frankfurt), J Matysik (University of Leiden) Photochemically induced dynamic nuclear polarisation (photo-CIDNP) enhances the intensity of a NMR line by inducing a non-Boltzmann distribution of the nuclear spin states. The observation of photo-CIDNP by magic angle spinning (MAS) NMR in photosynthetic reaction centers (RCs) allows for a spectacular increase of NMR signals up to factor 10000, providing significant increase of sensitivity and selectivity of signals originating from cofactors involved into electron transport. Hence, photo-CIDNP MAS NMR allows analysing the photochemical machinery of RCs at the atomic and molecular level. Of particular interest is electron donor of photosystem II of plants, having the highest redox potential in living nature. Here, we present both 13C and 15N photo-CIDNP MAS NMR spectra of photosystem II. Combined with existing data from photosystem I, a direct comparison of the donors of both plant photosystems can be made. The comparison clearly demonstrates an inversion of the electron spin density distribution in the donor of photosystem II compared to photosystem I; latter resembles the spin density of an undisturbed chlorophyll molecule. Furthermore, at the donor of photosystem II, electron spin density is observed on a deprotonated histidine. A model, in which a tilted axial histidine affects the electron density distribution in the chlorophyll macrocycle, is proposed. PS2.47 Time-Resolved High-Field EPR Spectroscopy of Natural Photosynthesis: Photoinduced Electron Transfer Pathways in Photosystem I. O Poluektov (Argonne National Laboratory, Argonne, USA), L Utschig (Argonne National Laboratory, Argonne, USA), S Paschenko (Argonne National Laboratory, Argonne, USA), K Lakshmi (Rensselaer Polytechnic Institute, Troy, USA), D Tiede (Argonne national Laboratory, Argonne, USA) 0, a chlorophyll molecule, and A1, a phylloquinone. From A1- the electron is transferred to the [4Fe–4S] cluster FX, and further to FA and FB, two iron-sulfur clusters held within an extrinsic protein subunit Thus, unlike Type II reaction center (RC) proteins, ET in PSI does not

151 14th Photosynthesis Congress - PS07 terminate at two functionally distinct quinines. Is ET in Type I RCs likewise functionally asymmetric? Resolution of this basic, yet important, issue of ET directionality (uni vs. bi) in PSI has remained an experimental challenge. Using high-field (HF) pulsed EPR technique we have resolved two distinct transient spectra of the P+A1- radical pair from PSI RC proteins of the cyanobacterium Synechococcus lividus and correlate their structures with kinetic data and the X-ray crystal structures of PSI. We demonstrate that the geometries of the two distinct donor/acceptor pairs correspond to the charge separated states along the A and B branches, and that our assignments of radical pair geometries are in excellent agreement with the X-ray crystal structure of PSI. Together with previously reported data, the concomitant structural and kinetic information obtained with HF EPR provide unambiguous evidence of bidirectional ET in PSI.

progressive improvement of purification procedures and optimization of crystal quality altered significantly the diffracting properties of crystals, enabling the recent determination of the X-ray crystal structure at 3.4 Å resolution (Amunts, A., Drory, O. and Nelson, N. The structure of a plant Photosystem I supercomplex at 3.4 Å resolution. Nature, 2007). The current crystal structure provides a picture at near atomic detail of 16 out of 17 protein subunits with an additional subunit (PsaN) being identified for the first time on the luminal side of the supercomplex. Positions of 3038 out of 3443 predicted amino acids were assigned as were those of 168 chlorophylls (65 revelaing the orientation of the Qx/Qy transition dipolar moments), 2 phyloquinones, 3 Fe4S4 clusters and 5 carotenoids. In addition the unique interactions between the LHCI and the RC were revealed. The structural information on proteins, co-factors and interactions between them provides a first glimpse at the fine architecture of nature’s most efficient photochemical nano-machine.

PS2.48 Photochemically induced dynamic nuclear polarization studies on photosynthetic reaction centers from diverse organisms

PS2.50 Origin and function of the long-wavelength chlorophylls of PSI in the cyanobacterium A. platensis.

E Roy (Leiden University), A Alia (Leiden University), P Gast (Leiden university), H de Groot (Leiden University), G Jeschke (University of Konstanz), H van Gorkom (Leiden university), J Matysik (Leiden University) Photochemically induced dynamic nuclear polarisation (photo-CIDNP) MAS NMR is a technique in studying the electronic structures of the cofactors involved in the electronic transfer in the RCs. Previously photo-CIDNP was observed in RCs from Rhodobacter sphaeroides (WT, R26) and PSII (spinach) which are type II RCs having quinone as the terminal electron acceptor. Here we present RCs from diverse photosynthetic organisms containing type I RCs that have iron sulphur clusters as terminal electron acceptors, in which photo-CIDNP has been observed by 13C MAS NMR. These RCs range from photosystem I (spinach) [1], green sulphur bacteria (Chlorobium tepidum) [2] and isolated membrane fragments from heliobacteria (Heliobacillus mobilis) [3]. The 13C photo-CIDNP spectral pattern in Rhodobacter sphaeroides WT, PSI and Chlorobium tepidum comprises of emissive (negative) signals, while both emissive and absorptive (positive) signals are observed in Rhodobacter shaeroides R26, PSII and Heliobacillus mobilis. Photo-CIDNP thus appears to be an inherent property of natural RCs. Hence, there may be a link between the fundamental conditions which result in photo-CIDNP and the efficiency of RCs in light induced electron transfer. [1] Alia, E. Roy, P. Gast, H. J. van Gorkom, H. J. M. de Groot, G. Jeschke, J. Matysik (2004) J. Am. Chem. Soc. 126, 12819-26. [2] E. Roy, Alia, P. Gast, H. van Gorkom, H.J.M. de Groot, G. Jeschke, J. Matysik (2007) Biochem. Biophys. Acta, in press. [3] E. Roy et al., to be published.

PS2.49 The structure of plant photosystem I at 3.4 Å resolution A Amunts (Tel Aviv University), N Nelson (Tel Aviv University), O Drory (Tel Aviv University) A plant Photosystem I (PSI) is exquisitely organised, intricate, multi-subunit membrane supercomplex of protein and non-protein components that drive the photosynthesis process. Previously we determined the structure of a plant PSI, which provided the first a-carbon structural model of the supercomplex, containing the reaction center complex (RC) and the peripheral antenna (LHCI). Continuous and

N Karapetyan (A.N. Bakh Institute of Biochemistry RAS) PSI core antenna of cyanobacteria is highly enriched with the long-wavelength (or red) chlorophylls (Chl); the relative content of the longwave Chls in the cyanobacterium A. platensis is of about 10%. The red-most Chls in PSI complex of that cyanobacteria absorb at 740 nm and emit at 760 nm. The longwave bands in 77 K CD spectra of PSI complexes of A. platensis at 711 and 736 nm are indicative on the excitonic origin of red Chls. According to 77 K LD spectra, the transition dipole moments of the red-most states are oriented parallel to the membrane plane. Light-induced difference CD spectra of PSI complexes A. platensis give strong evidence for the delocalization of the excited singlet states in the reaction centre. Therefore P700 cannot be considered as a dimer but should be regarded as a multimer of the six nearly equally coupled reaction centre Chls. Energy absorbed by red Chls migrates uphill with high efficiency to P700 causing its oxidation. Fluorescence at 760 nm of the red-most Chl in PSI of A. platensis is quenched not only by P700 cation radical but also by P700 in triplet state thus indicating the protective role of red Chls. PS2.51 Low quantum yield electron transfer pathways in PS II M Schenderlein (Max-Volmer-Laboratorium für Biophysikalische Chemie, TU Berlin, Straße des 17 Juni 135, D-10623 Berlin, Germany,), M Mroginski (Max-Volmer-Laboratorium für Biophysikalische Chemie, TU Berlin, Straße des 17 Juni 135, D-10623 Berlin, Germany), T Renger ( Institut für Chemie (Kristallographie), Freie Universität Berlin, Takustr. 6, D-14195 Berlin, Germany ), E Schlodder (Max-Volmer-Laboratorium für Biophysikalische Chemie, TU Berlin, Straße des 17 Juni 135, D-10623 Berlin, Germany) At low temperatures (<180 K) the electron transfer in photosystem II (PS II) is inhibited from QA- to QB and from YZ to P680+. Depending on the initial redox state of cytochrome b559 different long-lived states are formed upon illumination (QA-P680Cyt b559, QA-P680Car+, QA-P680ChlZ+). Their formation is interpreted as an electron transfer from alternate electron donors (Cyt b559, Car, ChlZ) to P680+, which occurs with low quantum yield besides charge recombination of QA-P680+. To determine their function in photosynthesis, the formation and decay of these states are investigated by means of Raman, EPR, absorption difference spectroscopy and LD experiments. DFT-calculations on β-carotene and its radical cation have been performed to assist with the assignment of Raman-signals arising in PS II samples after illumination at 77 K. Oxidation of ChlorophyllZ has been analyzed with the focus on

152 14th Photosynthesis Congress - PS07 absorbance changes in the QY and Soret region. All measurements were done using PS II from Thermosynechococcus elongatus as well as PS II from Spinacia Oleracea. Interestingly there are some quite remarkably differences in the formation and decay of the different closed states in these two organisms. PS2.52 Temperature Dependence of the Reduction Kinetics of P680+ in Oxygen-Evolving PS II Complexes throughout the range from 320 K to 80 K E Schlodder (Max-Volmer-Laboratorium für Biophysikalische Chemie, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany) The absorption of light by Photosystem II (PS II) induces a transmembrane charge separation between the primary electron donor P680, which is composed of PD1 and PD2, and the plastoquinone acceptor QA. The photooxidized P680 is re-reduced by the redox active tyrosine TyrZ (D1-Tyr-161) which in turn accepts an electron from the oxygen-evolving complex. Transient absorbance difference spectroscopy has been used to study the reduction kinetics of P680+ after the first flash given to dark-adapted oxygen-evolving PS II complexes from Thermosynechococcus elongatus as a function of temperature between 80 K and 320 K. The half-life of P680+ reduction by TyrZ increases from 20 ns at 300 K to about 4.2 µs at 150 K corresponding to an activation energy of 122 ± 3 meV. Analysis by nonadiabatic electron transfer theory yields edge-to-edge distance of about 9 Å in excellent agreement with the distance between PD1 and TyrZ in the recent structural model of PS II at 3.0 Å resolution [1]. In the range from 260 K to 150 K, the re-reduction of P680+ by TyrZ is increasingly replaced by the charge recombination of P680+ with QA-. It is proposed that reorganization processes which are required for the stabilization of the state P680TyrZox become blocked around 200 K. [1] B. Loll, J. Kern, W. Saenger, A. Zouni, J. Biesiadka (2005) Nature, 438, 1040-104 PS2.53 Electron Transfer between QA- and QB in Rb. sphaeroides Reaction Centers:Recent Advances from FTIR Difference Spectroscopy J Breton (Service de Bioénergétique, CEA-Saclay) Rhodobacter sphaeroides two ubiquinone molecules, QA and QB, play a pivotal role in the coupling of electron transfer to proton uptake. Three distinct observations derived from recent FTIR studies related to the QA–QB?QAQB– reaction will be presented: -- In contrast to native RCs where QB reduction elicits protonation of the single residue Glu L212 in the pH range 4-10, a large pH dependence of the protonation pattern of at least three distinct carboxylic acid residues has been observed in the AspL212/GluL213 swap mutant (Nabedryk et al., 2007, Biochemistry 46, 1176). -- Comparison of the QA–/QA FTIR difference spectra of native and CysM260 mutant RCs demonstrates that the unusually strong hydrogen bond between the carbonyl of QA and His M219 is not essential for efficient electron transfer from QA- to QB. (Breton, J. et al; 2007, Biochemistry 46, in press). -- On the basis of time-resolved FTIR measurements in native RCs, a new and unconventional mechanism has been proposed in which QB– formation precedes QA– oxidation (Remy, A. and Gerwert, K., 2003, Nat. Struct. Biol. 10, 637). The FTIR spectrum of the proposed transient

acceptor (X+/X) has been recently reported in the GlnL210 mutant (Hermes, S. et al., 2006, Biochemistry 45, 13741). The analysis of normalized steady-state QA–/QA and QB–/QB FTIR spectra provides compelling evidence that in both GlnL210 and native RCs the species X+ and X are spectrally indistinguishable from QB and QB–, respectively (Breton, J., 2007, Biochemistry 46, 4459). PS2.54 A0-?A1 electron transfer in Chlamydomonas reinhardtii Photosystem I with replaced A0 axial ligand K Gibasiewicz (Adam Mickiewicz University, Poznan, Poland), V Ramesh (Arizona State University), S Lin (Arizona State University), W Giera (Adam Mickiewicz University, Poznan, Poland), A Dobek (Adam Mickiewicz University, Poznan, Poland), S Bingham (Arizona State University ), A Webber (Arizona State University) Replacement of methionine, the natural axial ligand to the primary electron acceptor (A0) in Photosystem I, with a series of different amino acids results in dramatic increase of the A0- lifetime from ~20 ps in wild type to a few nanoseconds in the mutants in the case of Chlamydomonas reinhardtii [Ramesh et al. (2004) Biochemistry 43:1369–1375; Ramesh et al. (2007) BBA 1767:151-160]. This effect is similar independently if the mutation affects A-side or B-side A0. This observation further confirms an existence of two equivalent primary electron acceptors in both symmetric branches of Photosystem I in Chlamydomonas reinhardtii, which makes this photosystem unusual among other photosystems (from purple bacteria, PS II) which are essentially unidirectional. However, it is still not clear if the bidirectionality of electron transfer in Photosystem I is complete, i.e. if the electron from A0- reaches A1 in both branches or takes another route in the “non-active” branch. In order to solve this issue, in this contribution we will compare kinetics of A0- reoxidation to the kinetics of A1 formation in the case of both A-side and B-side A0 mutants.

PS2.55 Triplet states in photosynthetic reaction centers of Rb. Sphaeroides A Marchanka (Max-Planck-Institut for Bioanorganic Chemistry, Stiftstr.34-36 45470 Muelheim, Germany), M van Gastel (Max-Planck-Institut for Bioanorganic Chemistry, Stiftstr.34-36 45470 Muelheim, Germany), W Lubitz (Max-Planck-Institut for Bioanorganic Chemistry, Stiftstr.34-36 45470 Muelheim, Germany) The photoexcited triplet state of the primary donor P865 and of the carotenoid in the reaction center of Rb. sphaeroides wild type and mutants were investigated by pulsed EPR spectroscopy. Temperature dependence and time evolution of the triplet states were measured. Different mechanisms of triplet formation were observed: At T=10K, Rb. sphaeroides strain R.26-1 and 2.4.1 (wild type) and the double mutant Gly?Asp(M203)/Ala?Trp(M260)1 form a triplet via the radical pair (RP) mechanism. The electron transport in this species proceeds exclusively via the A-branch of the RC. In the double mutant Leu?His(M214)/Ala?Trp(M260)1 the intersystem crossing (ISC) mechanism is dominant. At T>30K, the radical pair mechanism also contributes to triplet formation. Electron transfer in this species proceeds via the B-branch but with very low triplet quantum yield. This process is temperature dependent. The zero-field splitting parameters of 3P865 are the same for the RP and ISC triplet. The carotenoid takes over the triplet state from 3P865. The amount of carotenoid triplet signal and the transfer rate are temperature dependent. Moreover, the rate of triplet transfer

153 14th Photosynthesis Congress - PS07 differs in Rb. sphaeroides 2.4.1 and mutants. The data confirm that triplet exciton transfer from 3P865 to Car proceeds via the accessory BChl, whereby the 3P865 ?BChla transfer step is rate limiting. Paddock et al. (2005) Biochemistry 44, 6920-6928

1

PS2.56 Quantifying the excitation migration time in Photosystem II. Consequences for primary and secondary charge separation rates and the corresponding drop in free energy. H van Amerongen (Wageningen University), K Broess (Wageningen University), B van Oort (Wageningen University), A van Hoek (Wageningen University), R Croce (University of Groningen) The fluorescence decay kinetics of Photosystem II (PSII) membranes with open reaction centers (RCs), was compared after excitation at 420 and 484 nm. These excitation wavelengths lead to preferential excitation of Chl a and Chl b, respectively, which causes different initial excitation populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 6.6 ± 0.8 ps slower upon 484 nm excitation for preparations that contain on average 2.3 LHCII (light-harvesting complex II) trimers per RC. Using a recently introduced method (Biophysical J 91:3776-3786, 2006) it is concluded that the average migration time of an excitation towards the RC contributes 34 ± 7% to the overall trapping time. This demonstrates that the exciton-radical pair equilibrium (ERPE) model that describes the kinetics of samples without outer antenna [Proc. Natl. Acad. Sci .USA 84, 1987], is not applicable for systems with outer antenna. This prompts us to introduce the MiCS model, which includes appreciable contributions from both the Mi(gration) time and the trapping or C(harge) S(eparation) time to the overall decay. It is conluded that the effective rate of primary charge separation of the entire RC (i.e. not only the primary donor) is (3.7 ± 0.5 ps)-1, the rate of secondary charge separation is (155 ± 42 ps)-1 and the drop in free energy upon primary charge separation is (825±106) cm-1. This large drop in energy occurs faster than generally found for systems without outer antenna.

depend on which anthraquinone has been incorporated. The spin polarization patterns also provide evidence that the rate of electron transfer from A0 to AQ is slow and that significant singlet-triplet mixing occurs in the primary radical pair. Interestingly, the spectra at low temperature from all samples reveal only the fraction of the PSI complexes that still contain plastoquinone. PS2.58 Identification of special pair and ChlZ of Photosystem II in Acaryochloris marina T Tomo (Hall of Global Environmental Research, Kyoto University), T Okubo (Institute of Materials Science, University of Tsukuba), S Akimoto (Molecular Photoscience Research Center, Kobe University), H Miyashita (Hall of Global Environmental Research, Kyoto University), T Tsuchiya (Hall of Global Environmental Research, Kyoto University), T Noguchi (Institute of Materials Science, University of Tsukuba), M Mimuro (Hall of Global Environmental Research, Kyoto University) The special pair and ChlorophyllZ (ChlZ) of the photosystem II (PSII) in the Chl d-dominated cyanobacterium, Acaryochlroris marina MBIC 11017, were studied using FT-IR and electronic absorption difference spectroscopy. We purified photochemically active complexes consisting of a CP47, CP43’, D1, D2, cytochrome b559, PsbI, and a small polypeptide. The pigment composition per two pheophytin (Phe) a molecules was 55 ± 7 Chl d, 3.0 ± 0.4 Chl a, 17 ± 3 a-carotene, and 1.4 ± 0.2 plastoquinone-9. The special pair was detected by a reversible absorption change at 713 nm (P713) together with a cation radical band at 842 nm. FT-IR difference spectra of the specific bands of a 3-formyl group allowed assignment of the special pair. The combined results indicate that the special pair comprises a Chl d homodimer. Two molecules of ChlZ were also identified as Chl d using FT-IR difference spectra and UV/Vis absorption difference spectra. The potential of primary electron acceptor (Phe a) was shifted to a higher value than that in the Chl a/Phe a system. The overall energetics of PSII in the Chl d system are adjusted to changes in the redox potentials, with P713 as the special pair using a lower light energy at 713 nm. Our findings support the idea that changes in photosynthetic pigments combine with a modification of the redox potentials of electron transfer components to give rise to an energetic adjustment of the total reaction system.

PS2.57 Transient EPR Studies of In Vivo Uptake of Substituted Anthraquinones by Photosystem I in Phylloquinone Biosynthetic Pathway Mutants of Synechocystis sp. PCC 6803

PS2.59 Unexpected Difference in the P700 Redox Potential among Oxygenic Photosynthetic Organisms Revealed by Spectroelectrochemistry

A van der Est (Brock University), S Chirico (Brock University), E Drago (Susquehanna University ), W Johnson (Susquehanna University ), J Golbeck (The Pennsylvania State University)

Y Kato (IIS, University of Tokyo), A Nakamura (R&D Dep., Central Japan Railway), T Suzawa (IIS, University of Tokyo), T Watanabe (IIS, University of Tokyo)

Deletion of the menA or menB gene in Synechocystis sp. PCC 6803 results in mutant strains of the cyanobacterium that are unable to synthesize phylloquinone. However, in the absence of phylloquinone, Photosystem I (PS I) incorporates plastoquinone allowing both strains to grow photoautotropically at low light. It has also been shown that the plastoquinone in PS I particles isolated from the mutants can be displaced by incubating the particles with naphthoquinones. Here we show that supplementing the growth medium with various substituted anthraquinones allows the mutants to grow under high light conditions. Chromatographic analysis of PS I particles isolated from cells grown in this way show varying levels of incorporation of AQ. The spin polarized transient EPR (TREPR) signals the samples are remarkably different from both the wild type and the mutants grown without anthraquinone. At room temperature, the spectra reveal that the AQ incorporated into PSI is active in electron transport and the kinetic traces show that electron transfer to the iron sulfur clusters occurs with lifetimes that

The redox potential of the primary donor of photosystem (PS) I, P700, determined over decades mainly by chemical redox titration, exhibits a heavy scatter from +375 to +525 mV vs. SHE (for review, see B. Ke, in Photosynthesis: "Photobiochemistry and Biophysics", Kluwer Academic Pubsihsers, Chap. 28, 2001). Though a part of this scattering would be experimental artifacts, the scattering of the P700 redox potentials values might reflect inherent differences in the properties of P700 among organisms which cannot be expected at a glance from highly conserved amino acid sequences of the PS I reaction center proteins. Thus, we have tried to measure the redox potential of P700 of various oxygenic photosynthetic organisms, such as cyanobacteria, red algae, green algae, and higher plants, precisely by spectroelectrochemical mean using an optically transparent thin-layer cell. Experimental conditions developed by us, which can determine the P700 redox potential with an error range of a few millivolts, revealed significant species-dependence of the P700 redox potential. The results showed that the P700 redox potentials are

154 14th Photosynthesis Congress - PS07 spanning from +398 to +470 mV vs. SHE among different species. Furthermore, the P700 redox potential shifts to positive direction on the order of cyanobacteria, red algae, green algae and higher plants. Possible causes for the species-dependence of the P700 redox potential will be discussed by taking into account the spectroscopic properties of P700 and the natures of the external electron donor proteins, cytochrome c6 and/or plastocyanin.

PS2.60 Fluorescence Lifetime Imaging of crystals of Photosystem I B van (Wageningen University), A Amunts (Tel Aviv University)), N Nelson (Tel Aviv University)), R Croce (University of Groningen)), H van Amerongen (Wageningen University) Photosystem I (PSI) is a multisubunit protein-pigment complex. It possesses Light-Harvesting Complexes (LHCs) that transfer absorbed energy to the core where the excitation energy induces charge separation in the reaction centre (RC). PSI is unique in binding red chlorophylls (Chls), with energy levels lower than that of the RC. These Chls may compete with the RC as an energy sink. The function of these red Chls is not fully known. Many time-resolved optical spectroscopy studies on PSI have revealed very heterogeneous excited state kinetics. Although this at least partly results from the intrinsic properties of PSI , including the presence of the red pigments , it might in principle also be caused by some sample heterogeneity. Here we show that the fluorescence kinetics of (highly homogeneous) crystals of PSI from pea are identical to those of PSI in solution. This justifies the structure-based modelling of spectral properties of PSI . Our new data indicate that the energy flow from LHC to the core proceeds mainly via the red Chls. PS2.61 Site-directed mutagenesis of cytochrome b559 in the cyanobacterium Thermosynechococcus elongatus J Ortega (University of Seville-CSIC) F. Guerrero1,2, M. Roncel1, D. Kirilovsky2, J.M. Ortega1 Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla y CSIC, Américo Vespucio 49, 41092 Sevilla, Spain. 2Laboratoire de Bioénergétique Moléculaire et Photosynthèse, Institut de Biologie et Technologies-Saclay (iBiTec-S), CEA Saclay, 91191 Gif-sur-Yvette, France. Cytochrome b559 (Cyt b559) is an intrinsic and essential component of the photosystem II (PSII) reaction centre in all photosynthetic oxygen-evolving organisms, but its function, although widely investigated, still remains unresolved. Most of the functional hypotheses propose that Cyt b559 may participate in secondary electron transfer pathways protecting PSII against oxidative damage. Mutational studies have not succeeded in demonstrating this redox function of Cyt b559 in PSII because most of the mutants obtained are impaired in the functional assembly of PSII holocomplex. We have constructed a series of site-directed mutants, each carrying a single amino acid substitution, in the thermophilic cyanobacterium Thermosynechococcus elongatus, in order to modify the redox potential of the heme without altering the assembly properties of PSII. We have obtained 19 mutant strains of Cyt b559: 10 in a-subunit (R8I, R8L, I14A, I14S, R18S, W20L, W20Y, I27A, I27T and F31Y) and 9 in ß-subunit (W20I, W20F, W20T, V21T, V28S, V28T, I31A, I31G and F32Y). Efficient liquid cultures of all mutant strains have been obtained. The midpoint redox potential of Cyt b559 in PSII-enriched membranes from 8 of the strains (WT, a-I14A, a-I14S, a-I27A, a-I27T, a-R18S, ß-V28T, ß-F32Y) have been measured. Some of these mutants showed relevant differences in redox properties of Cyt b559 compared with WT. We have also analyzed the PSII functionality of these 8 strains by polarographic and thermoluminescence techniques. The a-R18S mutant strain showed the

most important effects. PS2.62 Primary events in cyanobacterial photosystem I complexes studied using femtosecond selective excitation of antenna and reaction center chlorophylls A Semenov (A.N. Belozersky Institute of Physical-Chemical Biology Moscow State University), I Shelaev (Institute of Chemical Physics), F Gostev (Institute of Chemical Physics), V Nadtochenko (Institute of Chemical Physics), M Mamedov (Moscow State University), O Gopta (Moscow State University), V Shuvalov (Moscow State University), O Sarkisov (Institute of Chemical Physics) The photosystem I (PS I) complex from cyanobacteria contains ~90 light-harvesting antenna chlorophyll (Chl) molecules and the reaction center (RC) electron-transport chain, which includes P700 (Chl dimer), A0 (one or the two Chl monomer molecules), A1 (one or the two molecules of phylloquinone), and the iron–sulfur clusters. The flash-induced kinetics of primary events in PS I complexes from Synechocystis sp. PCC 6803 was studied using femtosecond transient absorption spectroscopy within 400 – 800 nm spectral range at different excitation wavelengths. This approach provided an opportunity to observe the excitation energy transfer between antenna Chl molecules, the formation of excited state P700* and the appearance of ion-radical pair (IP) states in RC. It was shown that 20 fs laser pulses centered at 680 and 700 nm predominantly excite antenna Chl molecules, while the pulse at 720 nm mostly results in selective excitation of the RC primary electron donor P700. The comparison between the flash-induced kinetic spectra showed that: 1) excitation of antenna leads to energy transfer and formation of P700* within ~3-5 ps, while selective excitation of P700 leads to very fast (~50 fs) formation of P700*; 2) generation of the primary IP (most probably P700+A0-) state accomplishes in ~5-8 ps irrespective of selective excitation of either antenna Chls or P700; 3) disappearance of A0- and formation of secondary IP state (P700+A1-) is completed within ~30 ps.

1

PS2.63 The chloroplast encoded PSI-J subunit is required for formation of the plastocyanin binding domain of photosystem I P Jensen (Dept. of Plant Biology, University of Copenhagen), K Amann (Department of Biology, Ludwig-Maximilians-Universität-München), A Zygadlo (Dept. of Plant Biology, University of Copenhagen), A Hansson (Dept. of Plant Biology, University of Copenhagen), J Meurer (Department of Biology, Ludwig-Maximilians-Universität-München ), H Scheller (Dept. of Plant Biology, University of Copenhagen) Photosystem I (PSI) is located in the thylakoid membrane of chloroplasts where it is involved in light-mediated electron transport form plastocyanin on the luminal side to ferredoxin on the stromal side. The plastid encoded psaJ gene encodes a small hydrophobic subunit containing one transmembrane helix. The function of PSI-J in higher plant PSI is so far unknown. Tobacco plants with an inactivated psaJ gene were constructed by chloroplast transformation. The mutant plants were devoid of PSI-J protein and grew photoautotrophically but were slightly smaller and paler than wild-type caused by a 15-20% reduction in the content of PSI indicating that PSI-J is important for assembly or stability of PSI. The functional size of the PSI antenna was not affected suggesting that PSI-J is not involved in binding of LHCI. The specific PSI activity measured as NADP+ photoreduction revealed a 55% reduction in electron transport in the absence of PSI-J. No significant difference in the second order rate constant for electron transfer from

155 14th Photosynthesis Congress - PS07 reduced plastocyanin to oxidized P700 was observed in the absence of PSI-J. Instead, a large fraction of PSI was found to be inactive. Immunoblotting analysis of PSI complexes revealed a secondary loss of the luminal PSI-N subunit. Presumably the absence of PSI-J affects the conformation of PSI-F which in turn affects the binding of PSI-N. This together renders a fraction of the PSI particles inactive. Thus, PSI-J is an important subunit of PSI that together with PSI-F and PSI-N is required for formation of the plastocyanin binding domain of PSI. PS2.64 Structure of Radical Pairs D∙ +QA∙ - in Photosynthetic Reaction Centers Cooled to Cryogenic Temperatures in Neutral and Charge Separated States: A High-Field EPR/PELDO M Flores (Max Planck Institute for Bioinorganic Chemistry, Mülheim an der Ruhr, Germany), A Savitsky (Department of Physics, Free University Berlin, Berlin, Germany), E Abresch (Department of Physics, University of California San Diego, La Jolla, USA), W Lubitz (Max Planck Institute for Bioinorganic Chemistry, Mülheim an der Ruhr, Germany), K Möbius (Department of Physics, Free University Berlin, Berlin, Germany) Photosynthetic reaction centers (RCs) from Rb. sphaeroides, upon excitation by light, undergo electron transfer from a dimeric bacteriochlorophyll donor (D) to a primary ubiquinone acceptor (QA). Structural changes associated with the charge separated state, D∙ +QA∙ -, have been suggested from the difference in electron transfer kinetics in RCs cooled in the dark and under illumination.1 The goal of the present work is to determine the nature of these suggested structural changes. RCs containing only QA, were frozen in the dark and under continuous illumination to trap the DQA and D∙ +QA∙ - states, respectively. The D∙ +QA∙ → DQA recombination kinetics was monitored using time-resolved W-band EPR at 90 K. In RCs cooled prior to illumination, the charge recombination time was 25 ms (fast), in agreement with previous reports.1,2 When the same RCs were warmed to room temperature and recooled under continuous illumination, a stable radical-pair (D∙ +QA∙ -) EPR signal was observed in ~70% of the sample. The charge-recombination time corresponding to the ~30% fraction of the sample was 120 ms (slow). PELDOR experiments on spin-correlated D∙ +QA∙ - radical pairs were performed on the states with “fast” and “slow” kinetics. The results showed similar relative orientations of the pair partners in both states. Since it is not likely that both cofactors move similarly during illumination, it is concluded that the same orientation for QA∙ - is observed in RCs cooled in the dark and in cyclic RCs cooled under illumination. This suggests that the difference in their kinetics is due to a different structural change of the redox cofactors, presumably involving the binding site of the intermediate pheophytin.

-

1 2

Kleinfeld et al., Biochemistry 23, 5780 (1984). Zech et al., Appl. Magn. Reson. 13, 51 (1997).

PS2.65 Study of intersystem electron transfer in the chlorophyll d containing cyanobacterium Acaryochloris marina and a reappraisal of the redox properties of P740 B Benjamin (IBPC), G Finazzi (IBPC), S Benson (Imperial College London), J Barber (Imperial College London), F Rappaport (IBPC), A Telfer (Imperial College London) A far red light driven oxygenic photosynthesis takes place in

Acaryochloris marina, a marine prokaryote whose major pigment is chlorophyll d. The PS1 primary donor, named P740, was identified as being composed of Chl d instead of Chl- a (Hu et al. PNAS, 1998). Its midpoint potential was lower than in Chl a organisms (335mV instead of 420-470mV), apparently compensating for the low quantum energy absorbed (1.68eV instead of 1.77eV). This would leave unmodified the reducing power resulting from charge separation, as argued by Hu et al., but would be expected to affect intersystem electron transfer. Indeed, if the midpoint potential of cytochrome f is similar to that in Chl a organisms (350-370mV), electron transfer would be uphill and might reduce considerably the efficiency of the photosynthetic chain. Hence, we investigated P740 and Cyt f re-reduction kinetics to assess the equilibrium constant between the two complexes. We found a value of ~15 which is consistent with the Cyt f midpoint being ~100mV lower than that of P740. We then directly measured, by equilibrium redox titration, the midpoint potentials of both P740 and Cyt f and found respectively 430mV and 330mV. Both values are similar to those found in Chl a organisms. This indicates that the change in Chl type is not associated with a modification of the midpoint potential of P740, as previously reported. Rather, we propose that the lower reducing power may affect the energetics of electron flow on the acceptor-side of PS1. Kinetic consequences will be discussed.

PS2.66 Primary radical pair P+Bphe– lifetime in Rhodobacter sphaeroides with blocked electron tranfer to QA. Effect of o-phenantrol K Gibasiewicz (Adam Mickiewicz University, Poznan, Poland), M Pajzderska (Adam Mickiewicz University, Poznan, Poland) Transient absorption spectroscopy with a time resolution of ~1 ns was applied to study the decay of the primary radical pair (PRP) P+Bphe– in Rhodobacter sphaeroides R-26 reaction centers with blocked electron transfer from Bphe– to QA. The block in electron transfer was realized in two ways: either by reducing or by removing QA. We have found very different kinetics for the PRP decay in these two cases. When QA was reduced to QA–, the decay was clearly biphasic with exponential lifetimes of t1 = 3 ns (63%) and t2 = 11 ns (29%). Fit with a single exponential gave a value of 5 ns. Reaction centers with QA removed were characterized by slower and monophasic decay with t = 12 ns. Addition of 10 mM o-phenantroline slowed down the PRP decay in reaction centers with QA– nearly twice, to t = 9 ns, and had almost no effect in QA-depleted reaction centers. O-phenantroline had also no effect on the PRP lifetime in chromatophores with QA reduced where t = 9 ns. We propose that the negative charge on QA accelerates the PRP decay by repulsive interaction with the negative charge on Bphe. This effect is partly removed by o-phenantroline-mediated protonation of the sites in the vicinity of QA.

PS2.67 The physiological relevance of electron transfer involving redox-active centres bound to the PsaA and PsaB subunits of Photosystem I. S Santabarbara (Queen Mary, University of London), P Bombelli (Queen Mary, University of London), S Purton (University College

156 14th Photosynthesis Congress - PS07 London), C Russell (Queen Mary, University of London), A Casazza (University of Milan), M Evans (Univesrity College London), P Heathcote (Queen Mary, University of London) Photosystem I (PS I) is a large macromolecular complex located in the thylakoid membranes of oxygenic phototrophs, and is an essential component of oxygenic photosynthesis. Recent literature is pointing towards the involvement of both the redox chains bound to the reaction centre subunits, PsaA and PsaB, in electron transfer in PS I. The effect of point mutations (histidine to methionine) of the axial ligand for the primary electron acceptor A0 on photosynthetic electron transfer in PS I is investigated in two mutants of the green alga C. reinhardtii (PsaA:M684H, PsaB:M664H). Both mutations affect the stability of the PS I reaction centre, the accumulation of which is approximately halved compared to the wild-type. PS II accumulation is not altered the mutants, compared to the wild type. In whole cells of the PsaA:M684H mutant the rate of linear electron transfer (ET) under saturating light conditions is decreased by only 5-10% compared to the wild-type, whereas in the PsaB:M664H mutant the rate is decreased by 90%. Under limiting light conditions these rates are decreased by 50% (PsaA:M684H) and 90% (PsaB:M664H) relative to WT. The maximal rate of PS I mediated ET is decreased by 30-35% in the PsaA:M684H mutant and by ~75% in the PsaB:M684H. These functional differences resulting from mutations affecting the PsaA-bound or the PsaB-bound cofactors are not fully explained by previous spectroscopic investigations. We interpret the results in terms of an asymmetric effect of the axial donor substitution to A0 on the maximal photochemical efficiency of PS I.

PS2.68 Redox potential of chlorophyll d M Kasahara (University of Tsukuba), S Ohashi (University of Tsukuba), S Fukuyo (University of Tsukuba), M Nakazato (Chlorophyll Research Institute), K Iwamoto (University of Tsukuba), Y Shiraiwa (University of Tsukuba), Y Kato (University of Tokyo), T Watanabe (University of Tokyo), M Kobayashi (University of Tsukuba) a plays a key role. In 1996, a Chl d-dominated cyanobacterium Acaryochloris marina was discovered. In the PS I RC of A. marina, Chl d' functions as the primary electron donor P740: a heterodimer of Chl d/d', like Chl a/a' for P700. The primary electron acceptor of PS II in A. marina has been defined as Phe a, however, whether Chl d acts as the primary electron donor in PS II is a matter of controversy. One of the reasons for this uncertainty is due to the absence of data about the redox potential of Chl d that is needed to be compared with that of Chl a. The oxidation potential of Chl d was found to be +0.88 V vs. SHE in acetonitrile, which was higher than that of Chl a (+0.81 V), and lower than that of Chl b (+0.94 V). The oxidation potential order, Chl b > Chl d > Chl a, can be explained by inductive effect of substituent groups on the conjugated p-electron system on the macrocycle. Corresponding pheophytins showed significantly high values around +1.2 V, which are rationalized in terms of an electron density decrease in the p-system by the replacement of magnesium with more electronegative hydrogen. Consequently, oxidation potential of Chl a was the lowest of all Chls. The results will help us to broaden our views on questions about photosystems in A. marina.

PS2.69 Oxidation potential of Chl a is the lowest of all Chls M Kobayashi (University of Tsukuba), S Ohashi (University of Tsukuba), S Fukuyo (University of Tsukuba), M Nakazato (Chlorophyll Research Institute), M Kasahara (University of Tsukuba), Y Kato (University of Tokyo), T Watanabe (University of Tokyo)

In 1996, a Chl d-dominated cyanobacterium Acaryochloris marina was discovered. In the PS I RC of A. marina, Chl d' functions as the primary electron donor P740: a heterodimer of Chl d/d', like Chl a/a' for P700, and Chl a as the primary electron acceptor. In PS II, however, whether Chl d acts as the primary electron donor in PS II is a matter of controversy, although the primary electron acceptor has been defined as not Phe d but Phe a. Quite recently, we first found that Chl d has higher oxidation potential than Chl a, and lower potential than Chl b. Phes a, b, and d showed much higher potentials than corresponding Chls. For water oxidation, very high oxidation power is believed to be needed, but oxidation power of Chl a is found to be the lowest of all Chls. To explain the enigma, we want to propose a unique model for O2 evolution. In our model, oxidation potential of Chl a(or d) is not high enough to oxidize water, but the stepwise positive shifts of oxidation potentials of the Mn-complex take place during the S-cycle to create the great high oxidation power to oxidize water. Lower oxidation states of the Mn-complex may accept holes from P680+, but higher oxidation state(s) cannot do this and should utilize photon energy to attain the final state to oxidize water. PS2.70 Electron and nuclear dynamics in many-electron atoms and molecules in bacterial V Shuvalov (Institute of Basic Biological Problems, RAS) L of each electron in many-electron atom is L = mvr = nh and similar to N. Bohr’s expression for one-electron atom. According to that the energy expression for many-electron atoms was found and used for the energy calculations in molecules, particularly with conjugated bonds, including chlorophyll like molecules and reaction centers (RCs). The nuclear wave packet motion was observed by fs-oscillations in stimulated emission from the primary electron donor P* at 900 nm and 940 nm which was accompanied by a coherent formation of the charge separated states P+BA-, P+HA- and P+HB- (where BA, HB, and HA are the primary and secondary electron acceptors, respectively) in native, pheophytin-modified and mutant RCs. Wave packet motion on the 130-150 cm-1 potential surface of P* was found to be due to the internal shift of the pyrrol ring I of PB with respect to PA molecule in the dimer P. This leads to the formation of an exciplex with an electron density shift from PA to PB accompanied by the stimulated emission spectrum shift from 900 nm to 940 nm and the electron density transfer to the primary electron acceptor BA with partial formation of the P+BA- state.

157 14th Photosynthesis Congress - PS07

PS3 - Structure and Function of Light Harvesting Complexes PS3.1 Photoprotective role of the IsiA chlorophyll-binding protein in cyanobacteria J Dekker (VU University Amsterdam), C van der Weij - de Wit (VU University Amsterdam), R Berera (VU University Amsterdam), S D'Haene (VU University Amsterdam), K Tsoukatos (VU University Amsterdam), E Romero (VU University Amsterdam), R van Grondelle (VU University Amsterdam), J Kennis (VU University Amsterdam), H Matthijs (University of Amsterdam) In many natural habitats, growth of cyanobacteria may be limited by a low concentration of iron. Cyanobacteria respond to this condition by expressing a number of iron-stress-inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43’. IsiA monomers assemble to ring-shaped polymers that encircle trimeric or monomeric photosystem I (PSI), or are present in supercomplexes without PSI, in particular upon prolonged iron starvation. Previously, we presented steady-state and time-resolved fluorescence measurements of isolated IsiA aggregates. We showed that these aggregates have a fluorescence quantum yield of about 2% compared to that of chlorophyll a in acetone, and that the dominating fluorescence lifetimes are more than an order of magnitude shorter than that of free chlorophyll a. Comparison of the temperature dependence of the fluorescence yields and spectra of the isolated aggregates and of the cells from which they were obtained suggests that these aggregates occur naturally in the iron-starved cells. We will present new spectroscopic data on isolated IsiA aggregates at room and cryogenic temperatures as well as on cells in various stages of iron deficiency, and conclude that the photoprotective function of IsiA occurs already in early stages of iron deficiency and that one or more carotenoids (zeaxanthin or β-carotene, but not echinenone) play important roles in the quenching process.

PS3.2 Purple Bacteria in the Light of Single-Molecule Spectroscopy J Köhler (University of Bayreuth) The primary reactions of purple bacterial photosynthesis take place within well defined light-harvesting complexes where the light energy is absorbed and transfered efficiently to the photochemical reaction centre initiating a charge separation that provides the free energy to drive the metabolic reactions. The photophysical properties of these complexes are determined not only by the intrinsic properties of the individual molecular building blocks but also by the intermolecular interactions between those units. These interactions depend strongly on the relative orientations and distances of the molecules and consequently the geometrical arrangement of the molecules has a crucial influence on the electronic excitations of the aggregate. Commonly, the great difficulty to determine the various parameters that play a role in the description of the electronic structure of light-harvesting complexes and the process of energy transfer is the fact that the optical absorption lines are inhomogeneously broadened as a result of heterogeneity in the ensemble of absorbing pigments. We circumvent this problem by employing single-molecule spectroscopic techniques which allows to uncover spectral details that would be completely masked by ensemble averaging in conventional optical experiments. The talk provides an overview over our work on this topic during the last years. R. J. Cogdell, A. Gall, J. Köhler Q. Rev. Biophysics 39, 3 (2006), 227

PS3.3

Structural aspects of the Assembly and Disassembly of the Phycobilisome N Adir (Technion), M Dines (Technion), L David (Technion), M Klartag (Technion), A McGregor (Technion), M Melamed-Frank (Technion), E Sendersky (Bar-Ilan University), R Schwarz (Bar-Ilan University) T. vulcanus and S. elongatus sp. PCC 7942. The monomer has a helix-hairpin-helix motif which dimerizes into an open four-helical bundle identical to the previously determined NblA structure from Anabena. Comparison between structures and homology based models indicate that while the NblA family is only weakly homologous, there are certain attributes of the protein that remain constant, and could be involved in its function. Random mutagenesis in the S. elongatus protein shows that critical residues affecting the disassembly process in vivo can be found at the polypeptide termini as well as at internal positions leading to a model for the NblA mode of action which is different than previously suggested. We will also describe structural information obtained on isolated components of the PBS that shed light on the process of PBS assembly and on the progress in determining the structure of the entire PBS complex by X-ray crystallographic methods.

PS3.4 Novel photo-protection mechanism in strongly coupled chlorophyll complexes: triplet excitons in chlorosomes and in artificial chlorophyll aggregates S Savikhin (Purdue University), H Kim (Purdue University), H Li (The Pennsylvania State University), J Maresca (The Pennsylvania State University), D Bryant (The Pennsylvania State University) This is the first report on the functional role of triplet excitons in photoprotection of strongly coupled artificial and native antenna systems. Bacteriochlorophyll (BChl) and chlorophyll (Chl) molecules are known to produce highly toxic singlet oxygen due to energy transfer from their excited triplet states to oxygen molecules. The monomeric (B)Chl molecules in a solution photo-degrade within minutes under sunlight. In (B)Chl pigment-protein complexes of photosynthesis, a carotenoid is typically positioned within a distance of 4 Å of individual (B)Chl or antenna arrays, allowing rapid triplet energy transfer from (B)Chl to the carotenoid. Our time resolved and steady state optical experiments reveal that strongly coupled BChl arrays of pigments are inherently protected due to the formation of triplet excitonic states. According to model simulations, the energy of the triplet exciton is substantially lower than that of the triplet state of an individual BChl, dropping below that of singlet oxygen, and blocking the triplet energy transfer to both carotenoid and to oxygen. This effect is observed experimentally in photosynthetic chlorosomes and in artificial BChl complexes. Triplet excited states in aggregates of BChl c are not quenched by oxygen and aggregated BChl appear to be >1000 times more stable than monomeric BChl under light illumination. This high intrinsic photostability makes artificial oligomers of BChls especially suitable candidates for the production of artificial antennae. A similar protection mechanism may function in other photosynthetic proteins that contain strongly interacting (B)Chl pigments.

PS3.5 Observing the ultrastructure of subunits c of chloroplast ATP synthase with atomic force microscopy and LB techniques

158 14th Photosynthesis Congress - PS07 J (Zhu) Compared to different traditional methods in ATP synthase purification came from many achieved workgroups, firstly introduced the newest simple pathway in high quality isolation of wheat leaf chloroplast, F1F0-ATPase complex, F1 and F0 moiety in details. Atomic force microscope (AFM) had been applied to study on ultrastructure and physical properties of chloroplast, F1F0-ATPase and its subunits. As a result, beautiful topographies and force figures have been saved from smart Shimadzu-SPM-9500J3, and reached the c subunits’ statistic number changed from 10 to 14(c10-c14) in the two dimensional crystal of F0 moiety which were constituted into lipid double layers. This result was different to the standard theoretic model (c12) from Walker J E and Boyer P, and we presumed that the ratio of F0 rotation cycle to H+ ion number was not the integer due to our repeating results and similar AFM data from Muller D J in plant cell at least, and given out possible revision for the theory. Then, we have gotten to the topography of purified F1 moiety followed Dimroth, from which sixfold structure were prominent to describe and there was one protuberance (cap) in middle of the sixfold structure, this result can be consistent with the results from Abrahams and Kagawa.

proteins surrounded by a lipid monolayer. To construct stable supramolecules mimicking chlorosomes by sol-gel processes, we prepare self-aggregates of a synthetic zinc chlorophyll derivative in the presence of a cationic surfactant cetyltrimethylammonium bromide (CTAB) and three kinds of organosilanes (tetraethoxysilane (TEOS), ethyltrichlorosilane (ETCS), and butyltrichlorosilane (BTCS)) in water. Zinc methyl 3-devinyl-3-hydroxymethyl- pyropheophorbide a (hereafter denoted as zinc chlorin 1) was synthesized from natural chlorophyll a in 6 steps. A methanol solution of zinc chlorin 1 was mixed with a methanol solution of CTAB, followed by dispersion into distilled water. Organosilane solution was treated with hydrochloric acid for partial hydrolysis, and was added to the aqueous solution of zinc chlorin 1 and CTAB. Their visible absorption and circular dichloism (CD) spectra were measured. Zinc chlorin 1 with CTAB and TEOS exhibited red-shifted Soret and Qy bands compared with monomeric 1, and large CD signals were observed in the Soret and Qy region. These indicate that zinc chlorin 1 can form chlorosomal self-aggregates in the presence of CTAB and TEOS. In contrast, addition of a methanol solution of ETCS and BTCS to the aqueous solution of self-aggregates of 1 in CTAB micelles caused the deaggregation. PS3.8 Ultrafast Time-resolved Absorption Spectroscopy Geometric Isomers of Open-Chain Carotenoids

of

PS3.6 Atomic Force Microscopy Reveals Multiple Patterns of Antenna Organization in Membranes of Purple Bacteria and Suggests Unanticipated Electron Transfer Mechanisms

H Frank (University of Connecticut), H Cong (University of Connecticut), G Gibson (University of Connecticut), R Birge (University of Connecticut), D Niedzwiedzki (University of Connecticut)

R Niederman (Rutgers University)

The strong visible absorption of carotenoids responsible for their yellow-orange coloration is associated with an S0 (11Ag-) → S2 (11Bu+)

James N Sturgis and Robert A Nie. Recent atomic force microscopy (AFM) studies have provided highly informative surface views of the intracytoplasmic membrane (ICM) of purple photosynthetic bacteria at submolecular resolution. The resulting AFM topographs reveal multiple, species-dependent patterns of organization for their light-harvesting (LH) complexes. These vary from the highly ordered linear arrays of dimeric LH1-reaction center (RC) core complexes in Rhodobacter sphaeroides, with the peripheral LH2 antenna either interspersed between them or arranged in large clusters, to less orderly arrangements in several other purple bacteria, where randomly organized, monomeric LH1-RC complexes coexist with large, paracrystalline LH2 domains. Neither the ATP synthase nor the cytochrome bc1 complex was observed in any of these images and they may localize at the poles of ICM vesicle out of view of the flat regions imaged by AFM. This does not support conventional electron transfer models which place the cytochrome bc1 complex in close association with RCs. Instead, the observed arrangements of LH2 and core complexes may specifically control quinol escape from the RC, with short-range diffusion within disordered regions of the membrane promoting passage of quinone, while the exclusion from large, ordered fields of LH2 antenna may augment the long-range flow of quinone species. Further details of these models will be discussed. PS3.7 Chlorosomal Self-Aggregation of Zinc Chlorophyll Derivatives in the Presence of Cationic Surfactant Cetyltrimethylammonium Bromide and Organosilanes in Aqueous Phase Y Saga (Kinki University), H Kida (Kinki University), Y Nishikawa (Kinki University), H Tamiaki (Ritsumeikan University) Green photosynthetic bacteria have unique extramembranous light-harvesting complexes called chlorosomes. In chlorosomes, bacteriochlorophyll(BChl) c, d, or e form self-aggregates without help of

transition. The lowest-lying singlet state, S1 (21Ag ), is a state into which absorption from the ground state is forbidden according to both symmetry (g → u) and pseudoparity (- → +) selection rules. A transition from the ground state, S0 (11Ag-), to another state, denoted S3 (11Ag+), becomes allowed when the symmetry (g → u) selection rule is relaxed upon trans → cis isomerization. The resulting absorption band appears in the UV region and is termed a “cis-peak”. In this work, we present the results of an ultrafast time-resolved spectroscopic study in the visible and NIR regions of HPLC-purified all-trans and central-cis geometric isomers of two open-chain carotenoids from photosynthetic bacteria, spheroidene (N = 10) and spirilloxanthin (N = 13). In addition, ultrafast time-resolved studies have been done on all-trans isomers of neurosporene (N=9), rhodopin glucoside (N=11) and rhodovibrin (N=12) in acetone and CS2 solutions at room temperature and in EPA (5:5:2 v/v/v ether: isopentane: ethanol) at 77 K to enhance the spectral resolution. Analysis of the data has revealed the energies and dynamics of the excited states and the spectral changes associated with their decay. The goal of this work is to understand the role geometric isomerization plays in controlling the photophysics and biological function of carotenoids. PS3.9 Origin and function of the long-wavelength chlorophylls of PSI in the cyanobacterium A. platensis. N Karapetyan (A.N. Bakh Institute of Biochemistry RAS, 119071 Moscow, Russia ), V A.N. Bakh Institute of Biochemistry RAS, 119071 Moscow, Russia (Shubin), M Department Plant Biochemistry, Ruhr-University-Bochum, 44780 Bochum, Germany (Roegner), E Max-Volmer Laboratory, Technical University Berlin, 10623 Berlin, Germany (Schlodder) The PSI core antenna of cyanobacteria is highly enriched with long-wavelength (or red) chlorophylls (LWCs); the relative content of

159 14th Photosynthesis Congress - PS07 LWC in the cyanobacterium A. platensis is about 10%. The red-most Chls in PSI trimers of A. platensis absorb at 740 nm and emit at 760 nm (77 K). The long-wavelength bands in the 77 K CD spectra of PSI trimers of A. platensis at 711 and 736 nm indicate the excitonic origin of red Chls. Distinct bands around 688, 697, 711, and 737 nm in the 77 K reduced LD spectrum of PSI trimers show that LWCs are highly oriented. The transition dipole moment of the red-most state is oriented parallel to the membrane plane as it is the case also for P700. Energy absorbed by red Chls migrates uphill with high efficiency to P700 causing its oxidation. To study the uphill energy transfer, we monitored the oxidation of P700 upon excitation with cw laser diodes emitting at 756 nm and 785 nm as a function of temperature. Oxidation of P700 in PSI trimers of A. platensis could be induced by 756 nm actinic light even at 77 K. Fluorescence of the red-most Chls is quenched under all conditions: by P700 via photochemistry, by P700 cation radical under oxidizing conditions, and by P700 in the triplet state. Since excitation energy is channeled towards the red-most antenna states, excess energy can always be dissipated efficiently in PSI giving evidence for the protective role of LWCs. Financial support of RFBR (grant 05-04-48526) is acknowledged.

physicochemical approaches. An external ligand can coordinate to the central metal atom of (B)Chls either from the chlorin macrocycle side where the C13^2-methoxycarbonyl moiety protrudes or from the other side. We found that among 151 (B)Chl a/b molecules found in 8 types of (B)Chl proteins, including PS1, PS2, LHC2, Chl-peridinin-protein, purple bacterial RC, LH2 and LH3, and FMO protein, 124 molecules (82%) are the former isomers. Computational examinations revealed that the diastereomeric preference in the side of the ligand-binding is inherent properties of (B)Chls. Crystal structures of the photosynthetic proteins suggests that many chlorophyll molecules in the photosynthetic proteins contact with neighboring chlorophyll molecules and/or carotenoide molecules. The conformation of the phytyl chain in the proteins can affect the orientation of the chlorin macrocycle. Previous mutagenesis studies suggested that the presence of a small cavity bearing the fifth ligand may determine whether Chl or Pheo binds in a site (Goldsmith et al., 1996). These findings together with our current examinations on pheophytinazation and Pheo-protein interaction may contribute to reveal the design strategies of (B)Chl)-proteins.

PS3.10 Solvation Effect of Bacteriochlorophyll Light-harvesting complex LH2

PS3.12 Regulation of antenna functions in chlorophyll assemblies

excitons

in

V Urboniene (Vilnius University), O Vrublevskaja (Institute of Physics), G Trinkunas (Institute of Physics), L Valkunas (Institute of Physics, Vilnius University), A Gall (DBJC/CEA and CNRS/URA), B Robert (DBJC/CEA and CNRS/URA) Possible effects of the protein surrounding on the spectral properties of the peripheral light-harvesting (LH2) complexes from photosynthetic bacterium Rhodobacter (Rba.) sphaeroides are considered. For this reason the comparative analysis of the temperature dependence of the absorption spectra is carried out for the LH2 containing membranes and for the LH2 complexes of Rba. sphaeroides 2.4.1 in 60% glycerol and for the LH2 complexes in buffer containing 0.1 % LDAO (w/v), and LH2 solvated in 70% and 80% glycerol/buffer solution. Temperature range has been chosen from 4K to room temperature. To describe the absorption spectra the exciton model has been applied. The spectral density functions for the excited bacteriochlorophylls in isolated (B800) as well as in aggregated (B850) states describing their coupling to the vibrational bath of the excitons has been found in parametric form. This has been accomplished from comparison of simulations and measurements of the temperature dependence of the absorption spectra. The absorption spectra of LH2 sphaeroides in 60% glycerol measured at 4K – 300K have been analysed in terms of linear combination of symmetric Gaussian sub-bands. All fitted absorption spectra of LH2 given by sixteen Gaussian sub-bands decomposition. With condition, that the ratio of band B850/B800 areas is independent of temperature and is close to 2. From the edge sub-bands of absorption spectra the value of nearest neighbour dipole-dipole coupling for B850 bacteriochlorophylls has been estimated to change with temperature. PS3.11 Pigment-protein interactions in photosynthetic proteins T Oba (Utsunomiya University), H Tamiaki (Ritsumeikan Unoversity), M Umetsu (Tohoku University) Evolutional supramolecular design of (bacterio)chlorophyll ((B)Chl)-proteins is still unclear. Folding processes of the pigment-protein complexes, regulations of spatial arrangements of pigments in antenna proteins, and biosynthesis of exotic pigments such as pheophytin-a (Pheo) and chlorophyll-a’ are still matters of dispute. For the basis to address these problems, we studies pigment-protein and pigment-pigment interactions in the proteins by in vitro and in silico

T Oba (Utsunomiya University), K Ogura (Utsunomiya University), M Hiraide (Utsunomiya University), S Ito (Utsunomiya University), K Hiratani (Utsunomiya University) Chlorophyll (Chl) molecules in some antenna apparatuses exist like aggregates with varieties of pigment-pigment distances and orientations for each neighbor, but the antenna functions look optimized. To elucidate evolutional strategy of the antenna structures and to develop useful artificial nano-systems, we have made model antenna systems and studied structure-function relationships in antenna chlorophyll-protein complexes to maximize photosynthetic efficiency. We synthesized chlorophyll (Chl) derivatives that have a series of alkyl chains at the 7-position or cationic substituents at the 17-position. The products were characterized by NMR, MALDI-TOF-MS, and visible absorption spectroscopy. An aqueous dispersion of the 7-alkyl compounds afforded aggregate nano-particles. The particle size, the position of Qy absorption, and the fluorescence intensity depended on the alkyl chain length. Mixing of lithocholic octadecanoate to the chlorophyllous pigment prior to dispersion afforded 670 nm absorbing nanoparticles, and the fluorescence intensity and the particles size were enhanced by increase of the mixed lithocholic ester. It is suggested that the lithocholic ester successfully mixed with the Chl derivative and formed the aggregates without phase separation inside each particle. The pigment concentration in each particle was diluted resulting in recovery of fluorescence intensity. It is noted that the fluorescence intensities of the cationic pigments were 60 times higher than the aggregate of methyl pyropheophorbide a. Mixing of lithocholic octadecanoate changed their particle sizes with keeping their fluorescence intensities. The results will be discussed in relation to structure-function relationships of photosynthetic antennas as well as potent applications of the nanoparticles as photoelectronic and clinical nanodevices. PS3.13 Exploring the structure of an antennae protein - High Resolution Crystal Structures of Phycocyanin A McGregor (Technion - Israel Institute of Technology), L David (Technion - Israel Institute of Technology) ), N Adir (Technion - Israel Institute of Technolo We present a high resolution structure of phycocyanin isolated from the thermophilic cyanobacterial species, T. vulcanus. Phycocyanin is an

160 14th Photosynthesis Congress - PS07 integral protein component of the phycobilisome, a huge antennae complex found in cyanobacteria and red alga. Elucidation of the mechanism by which the extremely efficient light capture and transfer process takes place, can benefit from exceptionally high resolution structural data. The structure presented here was obtained from a crystal, grown in ammonium sulfate and sucrose, which diffracted past 1.3 Å. The final structure refined to 1.43Å with an R factor of 21.43% (R free 23.01 %) and has been used for structural analysis including the deduction of stereochemistries around the cofactors. Current experiments aim to push the structural resolution higher to allow for more exact determination of hydrogen bond orientation and lengths, particulars not identifiable in the vast majority of protein structures. Improvement of the phycocyanin structure is a concerted process of refinement of crystallization and cryoprotection conditions and during this study a large number of high resolution structures of phycocyanin have been obtained from a wide range of crystallization conditions. Amazingly each of these structures, including those obtained both in salt and polymer precipitants crystallized in the same space group (R32) with a very low RMSD (<0.35). Comparison of these structures has allowed for identification of consistently observed water molecules with presumable structural significance and, additionally, structural features that are particularly susceptible to influence of the crystallization conditions. PS3.14 Functional Identification of GDP-Fucose Synthase Gene in Anabaena sp. PCC 7120 S Takaichi (Department of Biology, Nippon Medical School), M Mochimaru (Department of Natural Sciences, Komazawa University), H Masukawa (Department of Biological Sciences, Kanagawa University), T Maoka (Research Institute for Production Development) Carotenoids in cyanobacteria have diversity from species to species. Genera Anabaena and Nostoc have unique polar ketocarotenoids, ketomyxol glycosides. Anabaena 7120 contains ketomyxol 2'-fucoside (Takaichi et al., 2005, Plant Cell Physiol.). Ketolase, CrtW, is the only enzyme that functionally identified on the synthetic pathway from lycopene to ketomyxol 2'-fucoside (Mochimaru et al., 2005, FEBS Lett.). In this study, we investigated fucose synthase. Similarity search indicates that all4826 of Anabaena 7120 is a homologue of wcaG, the gene for GDP-fucose synthase from E.coli, with 35% identities. Its disrupted mutant contained polar carotenoid glycosides and also free myxol. Their glycoside moieties were identified to be rhamnose, isomer of fucose, by FD-MS and 1H-NMR, instead of usual fucose. This result suggests that in the mutant, fucose is not synthesized, and rhamnose is bound to myxol as a substrate by fucosyltransferase, so that all4826 is a functional gene for GDP-fucose synthase. We also identified ß-carotene hydroxylase, CrtR (Alr4009), in Anabaena 7120 catalyzed deoxymyxol to myxol. On the other hand, the disrupted mutant of sll1213 (a homologue of GDP-fucose synthase) of Synechocystis sp. PCC 6803 contains free myxol, but no myxol glycosides. It suggests that fucosyltransferase of Synechocystis 6803 has more precise substrate-specificity than that of Anabaena 7120, and can not use rhamnose as a substrate. The difference in substrate-specificity among enzymes from different species also observed in CrtR, and these might relate to the diversity of carotenoids in cyanobacteria (Takaichi and Mochimaru, 2007, CMLS, in press). This study was partly supported by NEDO. PS3.15 Molecular bases of antenna systems adaptation in a Chl dcontaining organism M Chen (School of Biological Scieences, University of Sydney), K Donohoe (University of Sydney), B Crossett (University of Sydney), M Schliep (University of Sydney), T Larkum (University of Sydney)

Acaryochloris marina is an ecologically important, biochemically interesting and evolutionarily fascinating marine cyanobacterium using the unique chlorophyll, Chl d. In addition to the Chl d-binding Pcb protein complexes as its major light harvesting system, which feeds energy to photosystems I and II, it possesses a primitive phycobiliprotein complex as an accessory peripheral antenna system. Our recent investigation reveals that the phycobilin pigment composition in A. marina is similar to that found in typical cyanobacteria, which contain allophycocyanin (APC), phycocyanin (PC) and phycoerythrin (PE). The ratios of these three major pigment proteins are shifted under various qualities of ambient light. With the aid of SDS-PAGE analysis we have identified a number of polypeptides and proposed a model structure in A. marina, phycobiliproteins and their associated linker proteins: the presence of different types of linker proteins, rod-linker, rod-core linker, core linker and core-membrane linker, suggested that there are phycobilisomes in A. marina. Acaryochloris marina is an enigmatic marine organism containing both an integral membrane Chl-based light-harvesting system and an accessory peripheral antenna system (PBS). Here we have explored the possible switching among all forms of antenna systems when the cells are cultured under different ambient light quality, such as white light (high and low intensities), green light, blue light and red light. The variation of optical properties of APC/PC/PE and level of Chl d were examined by spectroscopic methods in response to the cultural spectral light quality. The results of real time RT-PCR allowed us to elucidate the molecular mechanism of light acclimated capacity among Chl-binding light-harvesting complexes and phycobilin-antenna systems. The iron nutrient content in the medium also plays an important role in the processes of the shifting of control of the antenna system by ambient light quality. PS3.16 Chiral self-assembly of protobacteriochlorophyll-d derivatives

synthetic

zinc

H Tamiaki (Ritsumeikan University) Bacteriochlorophyll(BChl)-d molecules self-aggregate in a hydrophobic environment surrounded by a lipid monolayer including proteins to form the core part of major, peripheral and extramembranous light-harvesting antenna systems of green photosynthetic bacteria, chlorosomes. As chlorosomal models, self-aggregates of synthetic Zn-BChls-d in an aqueous solution of Triton X-100 (TX-100), a nonionic detergent have been reported [T. Miyatake, H. Tamiaki, J. Photochem. Photobiol. C: Photochem. Rev., 6, 89–107 (2005)]. Recently, Zn-protoBChls-d possessing a porphyrin π-system, the 17,18-dedihydro-form of Zn-BChls-d are available and their self-aggregates in an aqueous TX-100 micelle have similar supramolecular structures as in natural chlorosomes [H. Tamiaki, H. Kitamoto, T. Watanabe, R. Shibata, Photochem. Photobiol., 81, 170–176 (2005)]. Here we report synthesis of Zn-protoBChl-d derivatives possessing chiral hydrocarbon chains at the 17-propionate by modifying naturally occurring Chl-a and their self-aggregation in an aqueous TX-100 solution. Visible and circular dichroism spectral analyses indicate that the propionate group is important for the construction of their self-assembly as well as 31-OH, 13-C=O and central coordinatable metal on the Qy axis [H. Tamiaki, R. Shibata, T. Mizoguchi, Photochem. Photobiol., 83, 152–162 (2007)]. PS3.17 Energy transfer between antenna complexes: the “low energy” spectral forms of CP29. A Casazza (Istituto di Biofisica del CNR - Sezione di Milano, Dipartimento di Biologia, Università degli Studi di Milano, Italy), E Belgio (Sezione di Fisiologia Vegetale Fotosintesi, Dipartimento di

161 14th Photosynthesis Congress - PS07 Biologia, Università degli Studi di Milano, Italy), G Zucchelli (Istituto di Biofisica del CNR - Sezione di Milano, Dipartimento di Biologia, Università degli Studi di Milano, Italy), F Garlaschi (Sezione di Fisiologia Vegetale Fotosintesi, Dipartimento di Biologia, Università degli Studi di Milano, All PSII antenna complexes show similar absorption and fluorescence features. Ground state absorption and LD spectra decomposition analyses indicate the presence of similar spectral forms both in wavelength position and dipole orientation. Since excitation energy concentrates in the longer wavelength forms, it seems reasonable to suggest a critical function for these low energy states in excitation transfer among complexes. Ground state absorption spectral decomposition and non-linear spectroscopy in the frequency domain indicate that the longest chlorophyll spectral state peaks near 684 nm at physiological temperatures but is absent at cryogenic temperatures. Mutagenic analysis of specific chlorophyll sites has failed to demonstrated this long wavelength state. Little is known about the physical mechanism responsible for this phenomenon and it is still not clear if the low energy states are associated with specific Chla molecules or if they are vibrational bands associated with more than one chlorophyll. This is important for understanding a role in intercomplex energy transfer. We are investigating this using CP29 as the model complex by preparing site-directed mutants lacking one of the 8 Chls bound to the native complex and analysing the absorption spectra of the recombinant CP29s between 70 -300K. PS3.18 Sub-20 fs coherent spectroscopy of photosynthetic pigments

Y Ikeda (Graduate School of Life Science, University of Hyogo), Y Kashino (Graduate School of Life Science, University of Hyogo), H Koike (Graduate School of Life Science, University of Hyogo), K Satoh (Graduate School of Life Science, University of Hyogo) Diatoms play an important role as a primary producer, and ~20% of annual carbon fixation by photosynthesis in the ecological system is attributed to diatoms. Recently, whole genomes of two diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum, were published by Joint Genome Institute (http://www.jgi.doe.gov/ ). It has been reported that diatoms adapt to high light in a way different from that of cyanobacteria, green algae and higher plants. But antenna sizes of the two photosystems in diatoms is not known. Here, we estimated the antenna sizes and numbers of photosystem (PS) I and PS II complexes in thylakoids. Characterization of PS I complexes from a centric diatom, Chaetoceros gracilis was reported previously [1]. Here, we will show a simpler method to prepare PS I complexes from C. gracilis and T. pseudonana as well. Intact thylakoid membranes from the both centric diatom cells were obtained by freezing and thawing, and they were solubilized by n-dodecyl-b-D-maltoside (DDM). The solubilized supernatant was diluted to decrease the DDM concentration and then centrifuged. The resulting pellet was resuspended, and from the this suspensions PS I complexes were purified by anion exchange chromatography. Purified PS I complexes had more fucoxanthin-chlorophyll-binding protein (FCP) than a previous preparation [1], which was supported by SDS-PAGE and spectroscopic and pigment analyses. We succeeded in crystallizing such PS I complexes from C. gracilis. On the basis of these results, we will discuss the sizes and numbers of photosystems in two diatoms. [1] Proceedings of 13th International Congress on Photosynthesis, vol. 1, pp38-39, 2004

H Hashimoto (Osaka City University), M Sugisaki (Osaka City University), R Fujii (Osaka City University), R Cogdell (University of Glasgow)

PS3.20 Ultrafast relaxation dynamics of a keto-carotenoid, siphonaxanthin, probed by time-resolved fluorescence

Most previous laser spectroscopic studies on ultra-fast and highly efficient energy-transfer mechanisms have focused mainly on probing the population decay kinetics of the excited states. In this study, we applied sub-20 fs coherent spectroscopy to carotenoid and bacteriochlorophyll molecules free in solution. Coherence directly reflects the energy dissipation processes and by tracing the time evolution of the coherence the mechanism by which photosynthetic pigments dissipate excess energy to the surrounding molecules can be determined. For this purpose, we have performed the four-wave mixing spectroscopy of ß-carotene and bacteriochlorophyll a. ß-carotene measured under resonant excitation exhibits a clear coherent oscillation with a period of a few tens of femtoseconds. Assuming an adequate nonlinear optical response, simulation based on the Brownian oscillator model was successfully performed under the impulsive excitation limit resulting in the memory of the vibronic coherence generated in the S2 state being lost via relaxation processes that include the S1 state. The vibronic decoherence time of the system is estimated to be 1 ps, which is about five times longer than the population lifetime of the S2 state determined in previous studies. We have also performed coherent spectroscopy (3 pulse photo-echo peak shift) on bacteriochlorophyll a and find a long-lived (longer than carotenoid) vibronic coherence in this particular molecule. This is a good indication why highly efficient energy-transfer from carotenoid to bacteriochlorophyll occurs in the photosynthetic systems. Involvement of the two-photon process in the optical response of bacteriochlorophyll a is also discussed.

S Akimoto (Kobe University)

PS3.19 Purification and the antenna size of photosystem I complexes from a centric diatom, Chaetoceros gracilis

A keto-carotenoid, siphonaxanthin shows a characteristic in vivo absorption band around 535 nm in the pigment-protein complexes in a green alga Codium fragile. To reveal optical properties of the keto-carotenoid, we examined the relaxation dynamics of siphonaxanthin in solutions by femtosecond time-resolved fluorescence spectroscopy. Fluorescence kinetics after excitation to the S2 state was analyzed by multi-exponential functions with three components, independent of observed wavelengths: 35-fs and 200-fs components and a minor one longer than 10 ps. No rise component was resolved, which is in striking contrast to the fluorescence kinetics of lutein, a major carotenoid in green plants and consisting of the conjugated C=C bonds. The 35-fs component well corresponded to the mirror image of the absorption spectrum, therefore we assigned this component to the S2 fluorescence. The 200-fs component was much smaller than the 35-fs component in its amplitude, suggesting that its origin was different from the S2 state. Fluorescence anisotropy values of the 35-fs and 200-fs components were identical within an experimental error and obtained to be 0.39. An isotropy value of the S1 fluorescence was 0.32 after the S2 excitation, therefore the 200-fs component could not be assigned to the S1 fluorescence. In LHC II, siphonaxanthin exhibits a new excited state (Sx) between the S1 and S2 state, corresponding to the 535-nm absorption band (Chem. Phys. Lett. 390 (2004) 45). The 200-fs component might correspond to the Sx state of siphonaxanthin in solutions, although its intensity is smaller than that in the pigment-protein complexes. PS3.21 Theory of Optical Spectra - How Proteins Control Excitation Energy Transfer

162 14th Photosynthesis Congress - PS07 J Adolphs (Freie Universität Berlin), T Renger (Freie Universität Berlin), F Müh (Freie Universität Berlin), M Madjet (Freie Universität Berlin) A theory for structure based calculations of optical spectra of pigment-protein complexes has to describe the coupling between pigments and the coupling between each pigment and the protein. Three essential types of parameters of a theory of optical spectra [1] are: 1. The excitonic couplings between the pigments. 2. The local transition energies (site energies) of the pigments. 3. The spectral density that describes the dynamic modulation of pigment transition energies by the protein. We developed methods to obtain the above quantities and applied them to study optical properties of the FMO complex [2] of Prosthecochloris aestuarii. An electrostatic method was introduced [3] for calculating the excitonic couplings between the pigments, taking into account screening and local field effects by the dielectric protein environment. We used two independent methods to obtain site energies of the 7 bacteriochlorophyll a pigments: 1. A simple electrostatic method (improvement of [3]) that considers the charge density coupling between the ground and excited states of the pigments and the whole protein. 2. A fit of optical spectra [4] utilising a genetic algorithm [3]. From the calculation of exciton relaxation dynamics, evidence is obtained about which of the two possible orientations of the FMO complex with respect to the reaction center, inferred earlier [5,6] is realized. For efficient energy transfer from the FMO complex to the reaction center, bacteriochlorophyll 3 and 4 are the linker pigments [3]. Finally we comment on recent progress in calculating the spectral density of the pigment protein coupling from molecular dynamics and electrostatic calculations. [1] Renger, T. and R. A. Marcus (2002) J. Chem. Phys., 116, 9997. [2] Fenna, R. E. and B. W. Matthews (1975) Nature, 258, 573. [3] Adolphs, J. and T. Renger (2006) Biophys. J., 91, 2778. [4] Wendling, M. et al. (2002) Photosynth. Res., 71, 99. [5] Remigy, H. W. et al. (2002) Photosynth. Res., 71, 91. [6] Melkozernov, et al. (1998) Photosynth. Res., 56, 315. PS3.22 Why is the bacterial photosynthesis of Roseobacter denitrificans obligat aerob – a structural analysis of the RC-LH1 Core Complex S Schaefer (Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany), R Hite (Department of Cell Biology, Harvard Medical School, Boston MA, USA), T Walz (Department of Cell Biology, Harvard Medical School, Boston MA, USA), A Labahn (Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany)

core complex. 30,000 particles were selected from micrographs of negatively stained samples, aligned to each other and classified to produce class averages. The class averages showed different views of the core complex, which could thus be combined to calculate a three-dimensional reconstruction. The density map at 30 Å resolution shows that the LH1 complex surrounding the RC forms a closed circle. Although the resolution of the reconstruction is not yet sufficient to resolve possible gaps in the LH1 ring, a tight ring of LH1 around the RC may prevent the exchange of dihydroquinone and thus light-induced cyclic electron transfer. PS3.23 Spectral and biochemical analysis of interaction between CpcG2-phycobilisome and photosystem I in Synechocystis sp. PCC 6803 K Kondo (Univ. Tokyo), M Katayama (Univ. Nihon), M Ikeuchi (Univ. Tokyo) The phycobilisome is a supramolecular antenna complex required for photosynthesis in cyanobacteria and bilin-containing algae. Previously, we reported that the unicellular cyanobacterium Synechocystis sp. PCC 6803 possesses two types of phycobilisome which differ in their interconnecting ''rod-core linker'' proteins (CpcG1 and CpcG2). CpcG1-phycobilisome is equivalent to the conventional phycobilisome, whereas CpcG2-phycobilisome retains phycocyanin rods but is devoid of the central core. The current study describes the functional analysis of CpcG1-PBS and CpcG2-PBS. Specific energy transfer from phycobilisome to photosystems was estimated based on low temperature fluorescence and the results showed that CpcG2-phycobilisome transfers light energy preferentially to photosystem I compared to CpcG1-phycobilisome, although they are able to transfer to both photosystems. The preferential energy transfer was also supported by the increased photosystem I/photosystem II stoichiometry in the cpcG2 disruptant. The cpcG2 disruptant consistently showed retarded growth under weak PSII light, in which excitation of photosystem I is limited. Isolation of thylakoid membranes showed that CpcG2-phycobilisome is tightly associated with the membrane, while CpcG1-phycobilisome is partly released. Further, we solubilized and fractionated photosystems by glycerol density gradient centrifugation. The results suggest that at least a part of CpcG2 is directly associated with photosystem I trimer. Analysis of the contribution of CpcG1-phycobilisome and CpcG2-phycobilisome to the state transition is also currently being undertaken. We will discuss the specific interaction of CpcG2-phycobilisome and a possible mechanism of energy transfer to photosystem I. PS3.24 ECHINENONE IS NOT INVOLVED IN PHOTOPROTECTION IN THE ISI-A CHLOROPHYLL-BINDING PROTEIN OF CYANOBACTERIA

Roseobacter denitrificans belongs to the group of obligate aerobic photosynthetic bacteria. The typical bacterial photosynthesis is an anaerobic process, down-regulated in the presence of oxygen. Although Roseobacter denitrificans also produces bacteriochlorophyll a, it is incapable of synthesizing the protein components of the photosynthetic apparatus and performing photosynthetic energy transduction without oxygen. A key step in photosynthesis is the transfer of the light excitation energy from absorbed photons via the light harvesting complex 1 (LH1) to the bacterial reaction center (RC).

S D'Haene (Dept. of Biophysics, Faculty of Sciences, Vrije Universiteit, Amsterdam, The Netherlands), K Tsoukatos (Dept. of Biophysics, Faculty of Sciences, Vrije Universiteit, Amsterdam, The Netherlands), R Van Grondelle (Dept. of Biophysics, Faculty of Sciences, Vrije Universiteit, Amsterdam, The Netherlands), J Dekker (Dept. of Biophysics, Faculty of Sciences, Vrije Universiteit, Amsterdam, The Netherlands), H Matthijs (2Aquatic Microbiology, Institute of Biodiversity and Ecosystem Dynamics,

With the RC-LH1 complex from Roseobacter denitrificans, we have isolated for the first time the core complex from an obligate aerobic photosynthetic bacterium. The core complexes were solubilized with decyl-ß-D-maltoside and purified by anion exchange chromatography and sucrose gradient centrifugation steps. Single particle electron microscopy was used to perform a first structural characterization of the

The iron stress-inducible protein A IsiA is synthesized by cyanobacteria under stress conditions that affect photosynthesis like a lack of iron. In early stage of stress IsiA forms a ring of 18 subunits around photosystem I trimers. If the stress is prolonged, the relative amount of IsiA increases in the cells. Double rings around PSI monomers and IsiA rings aggregates of variable size without photosystem were found [1]. These

163 14th Photosynthesis Congress - PS07 IsiA aggregates were shown to be involved in photoprotection as they have a high efficiency to quench light energy [2]. In artificial dyads (zinc phthalocyanine molecules covalently linked to carotenoids) the one that has a carotenoid with the longest conjugation length was shown to be the strongest quencher [3]. In isolated IsiA aggregates from Synechocystis PCC 6803 echinenone is the carotenoid that has the longest conjugation length. We analyzed the carotenoid composition and the quenching properties in IsiA aggregates obtained from a Synechocystis PCC 6803 psaFJ-null mutant that contains mainly Isia after short term iron depletion and from a mutant crtO that is unable to synthesize echinenone. Pigment analysis indicates that echinenone is indeed absent in IsiA aggregates obtained from the crtO mutant and that it is not replaced by another carotenoid. The isolated IsiA aggregates from this mutant and the cells from which these aggregates were obtained show the same fluorescence quantum yields as in the corresponding materials from the psaFJ-null mutant. This suggests that echinenone is not involved in the quenching mechanism. PS3.25 Experimental evidence of polarization of the B798 antenna BChl a Qy transition dipoles of the green bacterium Chloroflexus aurantiacus in the antenna plane: femtosecond pump-probe studies Z Fetisova (M.V.Lomonosov Moscow State University), A Yakovlev (M.V.Lomonosov Moscow State University), A Taisova (M.V.Lomonosov Moscow State University), A Zobova (M.V.Lomonosov Moscow State University) We have recently shown by model calculations that polarization of the baseplate BChl a Qy transition dipoles (B798) of the green bacterium Chloroflexus aurantiacus in the antenna plane is biologically expedient, being optimal for excitation energy transfer B740?B798?B808-866. In this study, we show that this conclusion is confirmed with nonlinear spectroscopy. Room temperature isotropic and anisotropic pump-probe spectra were measured on the femtosecond through picosecond time scales for the chlorosome BChl a Qy band upon direct excitation of the band. The monomeric nature of B798 BChl a is manifested. The anisotropy in the B798 band decays from 0.4 to 0.1 and exhibits a fast component, reflecting the decay of coherences between the one-exciton B798 states that is followed by picosecond depolarization due to B798?B798 localized exciton migration. The data on spectra evolution evidences the polarization of the B798 BChl a Qy transition dipoles in the antenna plane. The B798/B808 and B800 antennae from green and purple bacteria exhibit some parallels in their spectra and exciton dynamics. We supposed that the Cf.aurantiacus building blocks of the B798 and B808 antennae are structurally similar and consist of ring-like aggregates (of ~20 nm in diameter) of N monomeric BChl a pigments (N=24) with the Mg-Mg separations of =25 Å between nearest B798/B808 pigments. This model enables to estimate the upper limit of the number of BChl c pigments per Cf.aurantiacus chlorosome which is equal to 25000. PS3.26 Compositional analysis of carotenoids in purple photosynthetic bacteria,Rhodopseudomonas sp. strain Rits, upon irradiated light intensity M Isaji (Ritsumeikan Univ.), T Mizoguchi (Ritsumeikan Univ.), J Hrada (Osaka Univ.), H Tamiaki (Ritsumeikan Univ.) Recently, we isolated the purple photosynthetic bacterium, Rhodopseudomonas (Rps.) sp. strain Rits, which is phylogenetiocally close to Rps. palustris. Both the species gave similar apparatus of the photosynthetic initial stage, which were dependent upon the cultured

irradiation light. Under normal- and high-light conditions, usual RC-LH1 and LH2 apparatus were produced as the core and peripheral components, respectively. Under low-light conditions, unique LH4 having a different absorption spectrum from LH2 was available as the peripheral antenna system. In this study, we analyzed the composition of carotenoids from the Rits strain that were grown by irradiation of different light intensity. Seven carotenoids (lycopene, rhodopin, 3,4-didehydororhodopin, anhydrorhodoviblin, rhodovibrin, OH-spirilloxanthin, spirilloxanthin) were detected from HPLC analyses. In the extract from the cell, dehydororhodopin was a major carotenoid under normal- and high-light conditions, while rhodopin was main under low light conditions. 3,4-Didehydororhodopin was found to be first accumulated in the Rits strain and the molecular structure was fully characterized by various spectroscopic measurements. We also analyzed the composition of carotenoids from the isolated photosynthetic apparatus including RC-LH1, LH2 and LH4. The RC-LH1 core contained mainly spirilloxanthin and anhydrorhodovibrin, which had 13 or 12 conjugated double bonds as well as two and one methoxy groups at the ends respectively. The isolated LH2 and LH4 contained mainly 3,4-didehydrorhodopin[(C=C)12] and rhodopin[(C=C)11] having only one terminal OH group. The biosynthetic pathway of spirilloxanthin is also discussed. PS3.27 The structure of the in-vitro reconstituted soluble light harvesting complex RFPCP. T Schulte (Ruhr-University Bochum) Dinoflagellates are eucaryotic algae which have a photosynthetic apparatus similiar to that of higher plants. Special for dinoflagellates is the use of the unique carotenoid peridinin in such amount that it is dominating the absorption properties of these algae. Peridinin is found mainly in the two antenna complexes LHC (light harvesting complex) and PCP (Peridinin-Chlorophyll a- Protein), a water soluble light harvesting complex only found in dinoflagellates. The structure of the main form of PCP (MFPCP) has been elucidated [1] a decade ago and helped a lot in understanding energy transfer in PCP on a molecular level. The energy transfer pathways within PCP are rather well understood and have been revealed by using fast spectroscopic methods (for a review on dynamics of carotenoid excited states see [2], review on PCP [3]). By comparing the main form of PCP with the high-salt form of PCP (HSPCP), which structure has recently been solved [4], one gets an idea of how the amino acid composition does influence the excited states of the pigments [5]. A betterway than comparing two forms of PCP to analyse the influence of the amino acids on the pigments is to mutate the ones of interest. For this purpose Miller and coworkers [6] developed a heterologous expression system of PCP (Refolded PCP, RFPCP). They showed by spectroscopic methods that RFPCP is correctly folded [6], but what is more important, they could reconstitute PCP with different chlorophylls to study its influence on energy transfer [6, 7]. In this work we show that the refolded PCP complex does not deviate from the native PCP complex. RFPCP was reconstituted, purified and crystallized. After optimizing crystal growth crystals diffracted to 1.5 °A. Phases were solved by molecular replacement, a model was built and refined up to a Rfree of 17.7%. Starting from this point our purpose is to confirm that the RFPCP proteins with different chlorophylls are not distinguishable from RFPCP with Chlorophyll a. Another target is to analyse the spectroscopic properties of RFPCP mutants and to confirm their structural consistence. References [1] E. Hofmann, P.M. Wrench, F.P. Sharples, R.G. Hiller, W. Welte, and K. Diederichs. Structural basis of light harvesting by carotenoids: Peridininchlorophyll- protein from amphidinium carterae. Science, 272:17881791, 1996. [2] Tomas Polivka and Villy Sundström. Ultrafast dynamics of

164 14th Photosynthesis Congress - PS07 carotenoid excited States-from solution to natural and artificial systems. Chem Rev, 104(4):2021–2071, Apr 2004. [3] Toma Polivka, Ivo van Stokkum, Donatas Zigmantas, Rienk van Grondelle, Villy Sundström, and Roger Hiller. Energy transfer in the major intrinsic light-harvesting complex from amphidinium carterae. Biochemistry, 45(28):8516–8526, Jul 2006. [4] T. Schulte, F.P. Sharples, R.G. Hiller, and E. Hofmann. Coordinates deposited in protein data bank under id 2c9e. unpublished. [5] Robielyn P Ilagan, Jeremy F Koscielecki, Roger G Hiller, Frank P Sharples, George N Gibson, Robert R Birge, and Harry A Frank. Femtosecond time-resolved absorption spectroscopy of main-form and high-salt peridininchlorophyll a-proteins at low temperatures. Biochemistry, 45(47):14052– 14063, Nov 2006. [6] David J Miller, Julian Catmull, Robert Puskeiler, Helen Tweedale, Frank P Sharples, and Roger G Hiller. Reconstitution of the peridinin-chlorophyll a protein (PCP): evidence for functional flexibility in chlorophyll binding. Photosynth Res, 86(1-2):229–240, Nov 2005. [7] Tomas Polivka, Torbjörn Pascher, Villy Sundström, and Roger G Hiller. Tuning energy transfer in the peridinin-chlorophyll complex by reconstitution with different chlorophylls. Photosynth Res, 86(1-2):217–227, Nov 2005. PS3.28 Structural and Functional Analysis of Light Harvesting Complexes from the Dinoflagellate Amphidinium carterae S Johanning (Ruhr-University Bochum) Photosynthetic active dinoflagellates use a two component light harvesting system to increase the energy flow to the photosystems. One is the membrane bound light harvesting complex LHC, which shows homology to higher plant CAB-proteins, but contains chl c instead of chl b [1]. The other complex is the unique water soluble peridinin-chlorophyll a-protein (PCP), located in the thylakoid lumen [2,3]. PCP exists in different isoforms of which the mainform with a pI of 7.5 (MFPCP) makes up more than 90%. Both kinds of complexes excessivly utilize the carotenoid peridinin for light harvesting. The structure of the MFPCP has been solved at a resolution of 2 Å. We have been able to improve the crystallization conditions and were able to refine the structure to a resolution of 1.35 Å. A new crystal form has also been found with the spacegroup P42212, solved at 2.9 Å. Both structures will be discussed. The LHC of Amphidinium carterea has been crystallized and initial data have been collected to 3.5 Å resolution. It has been postulated that PCP transfers energy directly to the membrane bound complex. In order to study this interaction a protocol for the reconstitution of LHC into liposomes has been developed. Initial characterization of the system will be presented. [1] Hiller, R.G., Wrench, P.M., Sharples, F.P. 1995. The Light-Harvesting Chlorophyll a-c-Binding Protein of Dinoflagellates: A Putative Polyprotein. FEBS Letters 363:175-178 [2] Sharples, F.P., Wrench, P.M., Ou, K., Hiller, R.G. 1996. Two distinct forms of the peridinin-chlorophyll a-protein from Amphidinium carterae. BBA 1276:117-123 [3] Hofmann, E., Wrench, P.M., Sharples, F.P., Hiller, R.G., Welte, W., Diederichs, K. 1996. Structural Basis of Light Harvesting by Carotenoids: Peridinin-Chlorophyll-Protein from Amphidinium carterae. Science 272:1788-1791 PS3.29 Unique optical properties of LHC II isolated from Codium fragile - its correlation to protein environment T Tsuchiya (Kyoto University), T Tomo (Kyoto University), S Akimoto (Kobe University), A Murakami (Kobe University), M Mimuro (Kyoto University) A marine green alga, Codium fragile, contains a specific

keto-carotenoid, siphonaxanthin (Siph), that shows a characteristic in vivo absorption band at 535 nm in LHC II. This band is ecologically advantageous under green light-rich underwater conditions but is not detected in solutions. In isolated LHC II complexes, an intensity of this absorption band of Siph was clearly enhanced, and the efficiency of energy transfer from Siph to Chl was observed to be very high. We confirmed that this band originated from a new excited state (Sx) between the S2 and S1 states based on fluorescence anisotropy decay of Codium chloroplasts [1] and isolated LHC II, and proposed that this Sx band derives from distortion of Siph in protein environment. To estimate the effect of amino acid residues around Siph, entire amino acid sequence was necessary. Because the amino acid sequences of LHC II of this alga have not been reported, we isolated a major lhcb gene by RT-PCR using degenerate oligonucleotide primers and determined the nucleotide sequence. Although the deduced amino acid sequence of Codium LHC II showed high homology to those of other LHC II, Codium-specific substitution was observed in several amino acids. We considered the local effect of the substitution to the electronic state of Siph. A possible model of the molecular interaction of Siph in Codium LHC II will be discussed. Reference 1. Akimoto, S. et al. Chem. Phys. Lett., 390, 45-49 (2004). PS3.30 Pigment-Pigment Interactions in Light-Harvesting Complexes Investigated by Nonlinear Polarization Spectroscopy in the Frequency Domain H Lokstein (Universität Potsdam), M Krikunova (Universität Hamburg), A Betke (Universität Potsdam), R Hiller (Macquarie University), D Leupold (Universität Potsdam) Nonlinear polarization spectroscopy in the frequency domain (NLPF) is a sensitive method to study pigment-pigment interactions in photosynthetic light-harvesting complexes (LHCs). NLPF has been employed previously to investigate strong excitonic interactions between chlorophylls. Here we show that NLPF is also suited to investigate carotenoid–chlorophyll interactions. Among the studied complexes were the peridinin-chlorophyll a-protein (PCP) and higher plant LHC II. Consequences for modes and paths of excitation energy transfer in the respective complexes are dicussed. PCP - a unique antenna complex in dinoflagellates employing the carotenoid peridinin as main light-harvesting pigment - is particularly well suited to study carotenoid-carotenoid and carotenoid-chlorophyll interactions: NLPF spectra indicate strong excitonic interactions between peridinin 11Bu+ states. Subbands of the peridinin absorption band were assigned to the two pigment clusters in PCP. Certain peridinin(s) corresponding to a ~487 nm-subband show specific interaction between their “optically dark” S1 (11Ag-) and/or intramolecular charge-transfer (ICT) state and chlorophyll a. Hence, this state is approximately isoenergetic to S1 of chlorophyll a. At high pump intensities an additional band at ≤ 660 nm appears in the NLPF spectra, which is assigned to a low-dipole moment S0 → S1/ICT transition of peridinin. Specific changes in chlorophyll-xanthophyll interactions upon altering the aggregational state of LHC II are indicated by the NLPF spectra. These changes might underly the chlorophyll-fluorescence quenching upon aggregation of LHC II as well as be the molecular basis of excess energy dissipation. PS3.31 The Monomeric Photosystem I-Complex of the Diatom Phaeodactylum tricornutum Binds Specific Fucoxanthin Chlorophyll Proteins (FCPs) as Light-Harvesting Complexes

165 14th Photosynthesis Congress - PS07 T Veith (University of Frankfurt), C Büchel (University of Frankfurt) A photosystem I (PSI)–fucoxanthin chlorophyll protein (FCP) complex with a chlorophyll a/P700 ratio of approximately 200:1 was isolated from the diatom Phaeodactylum tricornutum. Spectroscopic analysis proved that the bound FCP functions as a light-harvesting complex (LHC), actively transferring light energy from its accessory pigments chlorophyll c and fucoxanthin to the PSI core. Using an antibody against all FCP polypeptides of Cyclotella cryptica it could be shown that the polypeptides of the major FCP fraction differ from the FCPs found in the PSI fraction. Since these FCPs are tightly bound to PSI, active in energy transfer, and not found in the main FCP fraction, we suppose them to be PSI specific. Blue Native-PAGE and first electron microscopy studies of the PSI-FCP sample showed a monomeric complex comparable in size and shape to the PSI-LHCI complex of green algae. PS3.32 Self-organization of BChl d in Chlorosomes from 2D and 3D MAS NMR Correlation Spectroscopy S Ganapathy (Leiden University), M Reus (Max-Planck-Institut für Bioanorganische Chemie), A Gomez Maqueo Chew (Pennsylvania State University), D Bryant (Pennsylvania State University), A Holzwarth (Max-Planck-Institut für Bioanorganische Chemie), H de Groot (Leiden University) Magic angle spinning NMR spectroscopy has been used to investigate the self-organization of bacteriochlorophylls in chlorosomal light-harvesting antennae. Uniformly 13C enriched chlorosome preparations from two mutants of Chlorobium tepidum which produce BChl d and BChl c have been investigated. BChl d differs from BChl c only at the 20-methyl side chain. Using 2D and 3D solid state NMR MAS 13C-13C correlation spectroscopy in combination with the 1H-1H spin diffusion CP3/CHHC experiment we are able to obtain intermolecular correlations between BChl d molecules within a chlorosome, which suggests a more tight and flat arrangement of pigments in comparison to the chlorosome from the mutant producing BChl c and the wild type (1, 2). Since the solid state NMR data is quantitative at molecular levels, we are converging on a more accurate model of the 3D arrangement and the mechanism of self-assembly of bacteriochlorophylls in chlorosomes. References 1. van Rossum, B.-J., D. B. Steensgaard, F. M. Mulder, G.-J. Boender, K. Schaffner, A. R. Holzwarth, and H. J. M. de Groot. 2001. Biochemistry 40:1587-1595. 2. Balaban, T. S., A. R. Holzwarth, K. Schaffner, G. J. Boender, and H. J. M. de Groot. 1995. Biochemistry 34:15259-15266.

PS3.33 Structure-based Calculation of Pigment Transition Energies in Light-Harvesting Antennae F Müh (Free University of Berlin), M El-Amine Madjet (Free University of Berlin), J Adolphs (Free University of Berlin), T Renger (Free University of Berlin) The time scale and pathways of excitation energy transfer in antenna proteins are controlled by the protein scaffold, which holds the pigments at optimal geometry and tunes their excitation energies (site energies). The detailed understanding of the tuning of site energies by the protein has been an unsolved problem since the first high-resolution crystal structure of a light-harvesting antenna appeared more than 30 years ago.1 Recently, we developed a methodology to compute site energies that considers the whole protein in atomic detail and provides the missing

link between crystallography and spectroscopy. In our contribution, we summarize the results obtained by applying the method to the Fenna-Matthews-Olson protein of the green sulphur bacterium Prosthecochloris aestuarii,1–3 which revealed an unexpectedly dominating effect of the backbone dipole of two a-helices on the direction of excitation energy flow.4 The presentation is complemented by calculations concerning the LH2 complex of the purple bacterium Rhodopseudomonas acidophila.5,6 1. R.E. Fenna & B.W. Matthews, Nature 258, 573–577 (1975). 2. D.E. Tronrud, M.F. Schmid & B.W. Matthews, B. W, J. Mol. Biol. 188, 443–454 (1986). 3. M. Wendling et al., Photosynth. Res. 71, 99–123 (2002). 4. F. Müh et al., submitted. 5. M.Z. Papiz et al., J. Mol. Biol. 326, 1523–1538 (2003). 6. A.M. van Oijen et al., Science 285, 400–402 (1999). PS3.34 Characterization of the oligomeric antenna of the diatom P. tricornutum B Lepetit (University of Leipzig), D Volke (University of Leipzig)), M Szabó (Hungarian Academy of Sciences)), R Hoffmann (University of Leipzig)), G Garab (Hungarian Academy of Sciences)), C Wilhelm (University of Leipzig)), R Goss (University of Leipzig) The photosynthetic antenna system of diatoms contains fucoxanthin chlorophyll a/c binding proteins (FCPs), which exhibit high homology to the light harvesting proteins of vascular plants. Recent reports have indicated that the FCPs exist in an oligomeric state. To gain further information about FCP oligomerization we performed a detailed study of the antenna organization of the diatom P. tricornutum. Solubilization of thylakoids with different concentrations of the detergent n-dodecyl b-D-maltoside (DM) in combination with sucrose density centrifugation and gelfiltration yielded two FCP complexes with different oligomerization states. At low detergent concentrations we found an oligomeric complex which was termed FCPo, at higher DM concentrations an FCP complex with a lower oligomerization state was observed. Calculation of the molecular mass of these two complexes by gel filtration showed that the FCP complex is trimeric, whereas the FCPo consists of either hexamers or two tightly associated trimers. Monomeric antenna complexes which are typical for vascular plants could not be found. Spectroscopic characterization of both FCP complexes revealed that the FCPo most likely represents the native state of the peripheral antenna. The identity of the FCP proteins was confirmed by MS/MS, which further allowed the assignation of the FCPs to their respective genes. Our data also show that the peripheral antenna contains the main part of the xanthophyll cycle pigments diadinoxanthin and diatoxanthin, but that a small pool is additionally bound to the photosystem core fractions. This indicates that an additional xanthophyll binding protein exists which is tightly associated with the core complexes. PS3.35 Excitation energy transfer in the phycobiliprotein antenna of Acaryochloris marina studied by transient sub-ps absorption and fluorescence spectroscopy C Theiss (TU Berlin), F Schmitt (TU Berlin), S Andree (TU Berlin), C Cardenas-Chavez (TU Berlin), K Wache (TU Berlin), J Fuesers (TU Berlin), M Vitali (TU Berlin), M Wess (TU Berlin), S Kussin (TU Berlin), H Eichler (TU Berlin), H Eckert (TU Berlin) In addition to the unique pigment composition in PS I and PS II, the Chl d containing cyanobacterium A.marina also has a phycobiliprotein (PBP) antenna with a much simpler structure than in typical cyanobacteria. The phycobiliproteins are organized as rod shaped

166 14th Photosynthesis Congress - PS07 complexes consisting of three homohexamers containing phycocyanin (PC) and one heterohexamer with phycocyanin and allophycocyanin (PC/APC) (1). They do not aggregate in form of phycobiliproteins. Instead the excitation energy is funneled directly from the rod-antenna to PS II without involvement of an APC core as in typical cyanobacteria. This difference in the structure should also affect the excitation energy transefer. Therefore we studied the excited state dynamics of the PBP-antenna of A. marina by using transient sub-ps absorption spectroscopy and time-resolved fluorescence spectroscopy. The obtained results show, that the equilibration of the excitation energy over all PC and APC occurs with time constants of < 400 fs, 3 ps, and 14 ps which is followed by fast excitation energy transfer to Chl d with a 70 ps kinetics. The results will be discussed in comparison to the corresponding excitation energy transfer processes in the phycobilisomes of Chl a containing cyanobacteria.

and antenna function – helps outline the evolving functional role of antenna systems from the earliest green alga through the increasing complexity of green algae and higher plants. Discrete separation of pigment/protein complexes, using sucrose density gradient centrifugation, indicates that Lhcp antenna are associated with both photosystems. Most of these proteins appear peripherally associated, as indicated by the ease of dissociation under light cell disruption conditions. Localization and compositional analysis of minor photosystem proteins further clarifies the functional evolution of antenna proteins. Greater resolution of the relationship between antenna proteins throughout the green lineage will greatly expand our understanding of these essential adaptation tools.

(1) Q. Hu, J. Marquardt, I. Iwasaki, H. Miyashita, N. Kurano, E. Mörschel and S. Miyachi, Biohys. Acta, 1999, 1412, 250-261.

PS3.38 Excitation energy transfer in PS I of the cyanobacterium Gloeobacter violaceus PCC 7421 that lacks the long-wavelength form of chlorophyll

PS3.36 Thermal stability of LH2 and LH1 bacterial photosynthetic complexes in native membranes

M Mimuro (Kyoto University), M Higuchi (Kyoto University), H Inoue (Kyoto University), M Yokono (Hokkaido University), T Tsuchiya (Kyoto University), H Miyashita (Kyoto University), S Akimoto (Hokkaido University)

J SEGUIN (CEA), B ROBERT (CEA), M PATERNOSTRE (CEA) From the point of view of polypeptides association and pigments interaction within membrane, two of the best characterized membrane proteins are the core antenna protein LH1 from Rhodospirillum rubrum and peripherical protein LH2 from Rhodobacter sphaeroides purple bacteria. Light-harvesting 2 (LH2) ensures the capture of the solar photons and the efficient funnelling of the resulting excitation energy toward the photochemical reaction center via LH1 proteins. From the established structures and models, both LH2 and LH1 complexes are large oligomers of a basic structural unit composed of a heterodimer of two small integral membrane polypeptide (alpha and beta, ca. 50 amino acids) associated with bacteriochlorophyll and carotenoid molecules in symmetric circular aggregates. The electronic properties of these two complexes intimately depend on the association state of the pigments with polypeptides. LH1 and LH2, which are overexpressed in native environment, are good models to study membrane proteins stability in native membranes. We have investigated temperature effects on spectral properties of the two complexes in purified intracytoplasmic membranes and proposed models to explain different steps observed within temperature range. PS3.37 Understanding the evolution of eukaryotic light-harvesting through the analysis of the prasinophyte alga Ostreococcus tauri

Excitation energy transfer processes in photosystem (PS) I complexes isolated from the cyanobacterium Gloeobacter violaceus PCC 7421 were studied by steady-state and time-resolved fluorescence spectroscopy. The absence of the long-wavelength PS I components was demonstrated by low-temperature absorption spectroscopy, and no corresponding fluorescence components were detected at –196°C. Time-resolved fluorescence spectra and subsequent analysis of the fluorescence lifetimes of PS I complexes clearly showed that at 22°C, thermal equilibrium was established among antenna components within 10 ps after excitation. In contrast, at –196°C, energy flow to the trap was clearly resolved by global analysis on the basis of a single value decomposition method. It was difficult to discern reasons for the absence of the long-wavelength PS I components based on the primary structures of the major subunits of complexes in comparison with other cyanobacteria. The biological relevance of PS I fluorescence at physiological temperature is discussed based on the energy distribution among antenna components including P700, and on a comparative study with Synechocystis sp. PCC 6803. Acknowledgments: This study was supported by a Grant-in-Aid for Creative Scientific Research (No. 17GS0314) from the Japanese Society for the Promotion of Science to MM. PS3.39 Probing the carotenoid in its binding site in a reconstituted LH1 complex from the photosynthetic bacterium Rhodospirillum rubrum with electroabsorption spectroscopy

W Swingley (Hokkaido University), J Minagawa (Hokkaido University) M Nango (Nagoya Institute of Technology) Eukaryotic organisms have developed many unique light-harvesting antenna systems since the uptake of the cyanobacterial progenitor of the chloroplast. The availability of numerous algal genomes has greatly advanced the understanding of antenna evolution in modern plants and algae. One of the earliest divergent groups along the green lineage is the Prasinophyceae. These primitive alga contain a unique light-harvesting protein family, dubbed Lhcp, which is suspected to interact with both photosystems. Their LHCI library is similar to that in green plants and algae and, like green algae, prasinophytes contain only two minor [monomeric] LHCII antenna proteins, CP26 and CP29 (lacking CP24 found in higher plants); however, genes encoding major [trimeric] LHCII are absent. As a primitive member of the green lineage, the prasinophyte Ostreococcus tauri is a promising model organism for studying plant evolution. Our characterization of O. tauri light-adaptation mechanisms – the xanthophyll cycle, state transitions,

Energy transfer from LH1 to the reaction center (RC) complex is efficient because the distance and orientation of the co-factors involved (bacteriochlorophyll (BChl) and carotenoid) are precisely controlled at the nano-scale level. Recently, reconstitution of all-trans-spirilloxanthin into the LH1 complex from Rhodospirillum (Rs.) rubrum was reported. This study assumed that the reconstituted spirilloxanthin was bound into the same binding site as in the LH1 complex in vivo. The aim of the present study was to use electroabsorption spectroscopy (Stark spectroscopy) to investigate the structure and organization of all-trans-spirilloxanthin following reconstitution into the LH1 complex. This carotenoid was isolated from Rs. rubrum S1 and reconstituted into the LH1 complex from a carotenoidless mutant, Rs. rubrum G9+ in detergent micelles. The nonlinear optical parameters of carotenoid molecules, i.e. the change of polarizability (Da) and static

167 14th Photosynthesis Congress - PS07 dipole-moment (Dm) upon photoexcitation, in the reconstituted LH1 complex were determined using Stark spectroscopy. The nonlinear optical parameters of carotenoids in reconstituted LH1 complexes were somewhat different from those of the native LH1 complex, suggesting that the electrostatic environment around carotenoid molecules is different in reconstituted LH1 complexes compared with native complexes. This raises some questions about previous functional studies on carotenoids reconstituted into the LH1 complex from Rs. rubrum. It is apparent that Stark spectroscopy can be used as a much more stringent test of the quality of reconstitution than simple absorption, fluorescence, or CD spectroscopy. PS3.40 Diversity and localization of bacteriochlorophylls possessing different 17-propionate groups in purple bacterial antennae T Mizoguchi (Ritsumeikan University), J Harada (Ritsumeikan University), S Yoshida (Ritsumeikan University), M Isaji (Ritsumeikan University), H Tamiaki (Ritsumeikan University) a molecules possessing geranylgeranyl, dihydro- and tetrahydrogeranylgeranyl groups at the 17-propionate has been reported. However, the molecular structures of the latter two BChl-a intermediates have not yet been determined in terms of the positions of C=C double bonds in the 172-ester. The esterifying substituent is not directly conjugated with the p-system in BChl molecules and does not affect the electronic-absorption spectra of their monomeric states. As a result, the moiety has attracted less attention in comparison with other peripheral substituents, although it constitutes about 30% of the weight of a molecule. Recently, we isolated a purple photosynthetic bacterium, Rhodopseudomonas (Rps.) sp. strain Rits, which is phylogenetically close to Rps. palustris. This strain biosynthesized unusual peripheral antenna (LH4) at the expense of usual LH2 under low-light conditions, and accumulated BChl-a intermediates having different 17-propionate up to 40% of total BChls-a produced. In this study, first we isolated significant amounts of such pure BChls-a having different 17-propionate from Rps. sp. Rits and determined their structures by both mass spectrometry and 1H- and 13C-NMR spectroscopy [1]. Second, we analyzed the composition of the BChl-a intermediates in the isolated pigment-protein complexes (LH2, LH4 and RC-LH1) from Rps. sp. Rits, Rps. palustris DSM123 and Rps. palustris CGA009 by HPLC. The results indicated that the BChl-a intermediates were preferentially bound to the RC-LH1. [1] T. Mizoguchi, J. Harada and H. Tamiaki, FEBS Lett., 580 (2006) 6644-6648. PS3.41 Role of individual carotenoids bound to light-harvesting complexes of Photosystem II in triplets quenching: a Triplet-minus-singlet study M Mozzo (Department of Biophysical Chemistry, Groningen Bimolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.Istituto di Biofisica, CNR. c/o ITC via Sommarive 18. Povo (Trento) Italy), R Hienerwadel (Université d’Aix-Marseille II, LGBP- Faculté de Sciences de Luminy, Département de Biologie – Case 901-163 Avenue de Luminy, 13288 Marseille, France.), L Dall'Osto (Dipartimento Scientifico e Tecnologico, Università di Verona, St The antenna complexes of Photosystem II (Lhcb) coordinates four type of carotenoids: lutein, neoxanthin, violaxanthin and in stress conditions zeaxanthin. The main role of these chromophores is protecting the system from high-light damage via dissipation of the chlorophyll triplets and oxygen scavenging. We have investigated the photoprotective role

of the carotenoids bound to the different sites in individual Lhcb complexes via laser induced triplets-minus-singlet spectroscopy in aerobic and anaerobic conditions. Lhcb complexes either purified from plants with altered carotenoid composition or refolded in vitro have been analysed. The kinetics of triplet carotenoid decay of the Lhcb monomers, in the 420-580 nm range, are fitted with a single component having a lifetime between 1.53 and 1.96 μs in aerobic conditions. The triplet minus singlet spectra have maxima at 505 nm for Lhcb4, 507.5 nm for Lhcb5 and Lhcb1 and show increased broadening, indicating that the same binding site in different complexes have different influences of the carotenoids spectroscopic properties. Triplets minus singlet spectra of LHCII trimer demonstrate that the violaxanthin in V1 site does not participate to the triplets quenching and that the substitution of neoxanthin with violaxanthin in N1 site does not influence the T-S spectra. The effect of the removal of individual chlorophylls located in proximity of the carotenoids is also presented. PS3.42 Light-harvesting and Photoprotection in Diatoms: Identification and Role of L818-like Proteins and a Novel Member of the LHC Superfamily. B Green (University of British Columbia), S Zhu (University of British Columbia) Diatoms are unicellular photosynthetic eukaryotes responsible for a sizeable fraction of global CO2 drawdown, as well as being the basis for many marine food-webs. They have plastids surrounded by four membranes (a result of their secondary endosymbiotic ancestry), and use fucoxanthin Chl a/c proteins (FCPs) for light-harvesting. The diatom T. pseudonana has at least 30 members of the LHC superfamily, but PsbS is completely missing. However, it does have genes for four members of a clade that includes the green algal LI818 proteins. We are investigating the possibility that one or more of these LI818-like proteins (Lhcx1,2,4,5) takes the place of PsbS in contributing to NPQ and photoprotection. The expression patterns of the Lhcx genes under different light intensities were investigated by real-time RT-PCR and compared with several “standard” Lhcf genes encoding the major FCPs. Lhcx1 was up-regulated 4-5 times after 1 h high light (HL), then dropped 30% in the next five hours. Lhcx4 was also upregulated, although not as strongly, and Lhcx5 was unaffected. An antibody specific for the C-terminus of Lhcx1 (gift of E. Rhiel) identified it as a 23kDa protein, which remained elevated after 6 h HL.This suggests that the Lhcx1 protein, once expressed, remains stable for some time. We are also investigating a novel member of the LHC family, which is encoded by a single-copy gene and is found only in red algae, diatoms and haptophytes. (Supported by NSERC). PS3.43 Reconstitution of the Light Harvesting Antenna from Chloroflexus aurantiacus A Collins (Washington University), Y Xin (Washington University), R Blankenship (Washington University) A longstanding goal of our research is to fully understand the structure-function relationships of the light-harvesting architecture from green photosynthetic bacteria. To investigate this, we are purifying and characterizing the reaction center, membrane bound light harvesting antenna and chlorosome of the thermophilic filamentous anoxygenic bacterium Chloroflexus auranticus. We are reconstituting the individual components of the light harvesting system and investigating their properties using a number of types of spectroscopy. Recent experiments show that energy can be transferred from the peripheral chlorosome to the light-harvesting antenna in vitro. Another aim of this research is to fully characterize the baseplate complex from the chlorosome. The

168 14th Photosynthesis Congress - PS07 baseplate complex contains the CsmA protein, β-carotene, and bacteriochlorophyll a. It is an intermediary in the energy transfer pathway between the chlorosome and the membrane-bound antenna system. The current approach is to express the CsmA protein as a recombinant protein in E. coli and to reconstitute the complex by integrating purified pigments.

for bacteriochlorophyll organization within the chlorosome.

PS3.44 Electron Spin Density Distribution of the Carotenoid Triplet State in the Peridinin-Chlorophyll-Protein Antenna of Dinoflagellates Determined by Pulse ENDOR Spectroscopy

L Liu (Department of Biophysics, Leiden University, Niels Bohrweg 2, 2333CA Leiden, the Netherlands; State Key Lab of Microbial Technology, Shandong University, Jinan 250100, P. R. China), Y Zhang (State Key Lab of Microbial Technology, Shandong University, Jinan 250100, P. R. China), B Zhou (State Key Lab of Microbial Technology, Shandong University, Jinan 250100, P. R. China), T Aartsma (Department of Biophysics, Leiden University, Niels Bohrweg 2, 2333CA Leiden, the Netherlands)

W Lubitz (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany), J Niklas (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany), S Prakash (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany), T Schulte (Ruhr-University Bochu, Bochum, Germany), M van Gastel (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany), E Hofmann (Ruhr-University Bochum, Bochum, Germany) The triplet state of the carotenoid peridinin 3Pc in the refolded N terminal PCP antenna complex from Amphidinium carterae1 is investigated by orientation selected pulse ENDOR spectroscopy at 35 GHz. The peridinin triplet is created by triplet-triplet transfer from 3Chl a, which is generated by illumination at 630 nm. The 3Pc lifetimes fall in the range of 10 50 µs, which is close to the minimum duration of the pulse ENDOR experiment (~10 µs). Additionally, density functional theory (DFT) calculations of the magnetic resonance parameters of the carotenoid triplet state are presented, using the ORCA program package2. An assignment of the 1H hyperfine coupling constants (hfcs) to protons of the peridinin is achieved based on a comparison of the experimental and theoretical magnitudes, anisotropies and signs of the hfcs. This enables us to derive for the first time the delocalization of the triplet exciton (S = 1) over a carotenoid molecule, which is a sensitive probe for the electronic and geometric structure of this short-lived excited state. The application to triplet states of carotenoid molecules in other systems is discussed. Miller, D. J. et al. Photosyn. Res. 86, 229 (2005). 2 Ganyushin, D.; Neese, F. J. Chem. Phys. 125 024103, (2006). 1

PS3.45 Validation of chlorosome structure models by NMR and shift analyses S Ganapathy (Leiden University), I de Boer (Leiden University), M Reus (Max-Planck-Institut für Bioanorganische Chemie), A Gomez Maqueo Chew (Pennsylvania State University), D Bryant (Pennsylvania State University ), A Holzwarth (Max-Planck-Institut für Bioanorganische Chemie ), H de Groot (Leiden University) The NMR shifts of aggregated chlorophylls are very sensitive to the stacking. Here we present a procedure to validate stacking models for the self-organization of bacteriochlorophylls in chlorosomal light-harvesting antennae by Magic Angle Spinning NMR and ring current shift analyses. Uniformly 13C enriched chlorosome preparations of the mutant of Chlorobium tepidum which produces BChl d have been investigated. 2D and 3D solid state NMR MAS 13C-13C-1H and 13C-1H correlation spectroscopy yields narrow lines, much narrower than previously obtained for the wild type and allowing a complete proton resonance assignment. Comparison of the calculated and experimental proton aggregation shifts allows us to converge on the most likely model

PS3.46 Supramolecular architecture of hemiellipsoidal phycobilisome and thylakoid membrane in Porphyridium cruentum

Cyanobacteria and red algae use multimeric protein complexes, phycobilisomes (PBsomes), as their major light-harvesting complexes, which are directly attached to the stromal surfaces of thylakoid membranes. The shapes of PBsomes vary depending on their species, and sofar hemidiscoidal PBsomes has been the more extensively characterized category. In the present work, the morphology of hemiellipsoidal PBsome and the thylakoid membranes in unicellular red alga Porphyridium cruentum was studied by a combination of negative stain electron microscopy (EM) and atomic force microscopy (AFM). Investigation on native photosynthetic membranes under various illumination conditions shows both random and organized PBsome arrangements, reminiscent of the behavior of Photosystem II as reported in the literature. The presence of PBsome clusters in random distribution favored at higher light intensity and parallel PBsome arrangement at lower light intensity suggests light adaptation of the antenna organization for capturing maximum energy. Three-dimensional topographies achieved with AFM further reveal height variation of photosynthetic proteins and lead us to better understand the spatial configuration of thylakoid membranes. Additionally, the morphologically diverse types of PBsome-thylakoid membrane fragments provide the opportunity to resolve hemiellipsoidal PBsome conformation by EM, and an improved model is proposed. PS3.47 Carotenoids in the antenna complexes of Photosystem I R Croce (University of Groningen), M Mozzo (University of Groningen)), T Morosinotto (University of Aix-Marseille II)), A Romeo (University of Verona)), R Hienerwadel (University of Aix-Marseille II)), R Bassi (University of Verona) The spectroscopic characteristics of carotenoids associated to the antenna complexes of Photosystem I have been studied. Pigment composition, absorption spectra and laser induced triplet-minus-singlet (T-S) spectra were determined for native LHCI from wild type and lut2 mutant from Arabidopsis thaliana as well as for reconstituted individual Lhca WT and mutated complexes. All WT complexes bind lutein and violaxanthin, while b-carotene was found to be associated only to Lhca3. In the native complexes the main lutein absorption bands are located at 492 and 510 nm. It is shown that violaxanthin is able to occupy all lutein binding sites, but its absorption is blue shifted to 487 and 501 nm. A “red” lutein absorbing at 510 nm was found to be associated to Lhca3 and Lhca4 which also coordinate a second carotenoid, peaking around 490 nm. Both these xanthophylls are involved in triplet quenching and show two T-S maxima at 507 and 525 nm. The “blue” absorbing xanthophyll is located in site L1 and can receive triplets from chlorophylls (Chl) 1012, 1011 and possibly 1013. The red-shifted spectral component is assigned to a lutein molecule located in the L2 site. A 510 nm lutein was also observed in the trimers of LHCII but was absent in the monomers. In the case of Lhca the 510 nm band is present in both the monomeric and dimeric complexes. It is suggested that the

169 14th Photosynthesis Congress - PS07 large red shift observed for this xanthophyll is due to interaction with the neighbor Chl-1015. PS3.48 The influence of the second b-polypeptide upon B800 binding within the LH2 complex of Rba. sphaeroides. J Timney (University of Sheffield, UK) The purple non-sulphur bacterium Rhodobacter sphaeroides is used as a model organism for the study of light harvesting (LH) pigment-protein complexes. The structure of the LH2 complex has been shown by electron microscopy (Waltz et al., 1998) to be comprised of a circular array of nine heterodimers, which are pairs of transmembrane helices designated a and b. The structure observed is analogous to the 3D structure, solved using X-ray crystallography, of the LH2 complex from the related organism Rhodopseudomonas acidophila (McDermott et al., 1995). The pairs of a and b transmembrane polypeptides bind 9 B800 and 18 B850 bacteriocholorophyll molecules. Recent genome sequencing of Rhodobacter sphaeroides has uncovered a previously unknown LH2 gene pair (Zeng et al., 2003) designated puc2BA, which encodes a significant number of the b-polypeptides that are incorporated into the LH2 complex. This poster presents data demonstrating the optimal binding of B800 bacteriocholorophylls is highly dependent on the presence of the b-polypeptide encoded by the puc2BA operon. PS3.49 Kinetic Analysis of Energy and Electron Transfer Processes in PS I Particles from Arabidopsis thaliana C Slavov (Max-Planck-Institut für Bioanorganische Chemie, Mülheim a.d. Ruhr, Germany), M Ballottari (Dipartimento Scientifico e Tecnologico, Università di Verona, Italy), T Morosinotto (Dipartimento Scientifico e Tecnologico, Università di Verona, Italy), M Müller (Max-Planck-Institut für Bioanorganische Chemie, Mülheim a.d. Ruhr, Germany), R Bassi (Dipartimento Scientifico e Tecnologico, Università di Verona, Italy), A Holzwarth (Max-Planck-Institut für Bioanorganische Chemie, Mülheim a.d. Ruhr Photosystem (PS) I is a highly complex photosystem, fine-tuned to perform charge separation with a quantum yield close to one. A number of unique properties in PS I both control the energy trapping kinetics and complicate the study of the primary processes. Time-resolved fluorescence measurements were performed on higher plant PS I core and intact particles. Intact particles contain in addition the peripheral LHC I complexes where the “red Chls” are located. Their comparison allows a direct examination of the influence of these peripheral complexes and of “red Chls” on the trapping kinetics. The experimental data were analyzed in terms of kinetic modeling, resulting in effective rate constants and species associated spectra (1). The data confirm the previously proposed “charge recombination” model for the early electron-transfer steps (2;3) and, correspondingly, the charge separation in the reaction centre (RC) of PS I as the overall energy trapping bottleneck. Additionally, the fluorescence spectra of RC*, antenna* and the “red Chls”* compartments are resolved. The RC* spectra are nearly identical in both core and intact particles, which provides evidence for the validity of the proposed kinetic model. 1. Holzwarth, A. R. (1996) in Biophysical Techniques in Photosynthesis. Advances in Photosynthesis Research (Amesz, J. and Hoff, A. J., Eds.) pp 75, Kluwer Academic Publishers, Dordrecht. 2. Müller, M. G., Niklas, J., Lubitz, W., and Holzwarth, A. R. (2003) Biophys. J. 85, 3899. 3. Holzwarth, A. R., Müller, M. G., Niklas, J., and Lubitz, W. (2005) J. Phys. Chem. B 109, 5903.

PS3.50 Electron microscopy reveals a flexible multilamellar tubular arrangement of bacteriochlorophyll sheets in Chlorobium tepidum chlorosomes E Boekema (University of Groningen), G Oostergetel (University of Groningen), M Reus (MPI Muehlheim), A Holzwarth (MPI Muehlheim) The exact supramolecular organization of the bacteriochlorophyll (Bchl) layers within the chlorosomes has been a subject of long-standing discussion. It has been suggested that Bchl sheets are folded into single layered tubules with a defined diameter. Other researchers proposed an undulating lamellar for the arrangement of pigment aggregates of three different Chlorobium species. We have performed cryo-electron microscopy on intact chlorosomes of Chlorobium tepidum embedded in vitreous ice to directly reveal the arrangement of Bchl sheets. End-on views of chlorosomes give a direct clue to the packing of the sheets within the chlorosome. They show the presence of multi-lamellar tubules of variable size (20-30 nm) with some non-tubular locally undulating lamelllae in between. From the side-on views the tubules appear to have a length of the entire chlorosome. In conclusion, our data show that the Chlorobium chlorosomes do not have a highly regular supramolecular organization of either tubules or lamellae.

PS3.51 The structure of PufX from Rhodobacter sphaeroides and the effects of N-terminal truncation and mutagenesis in the transmembrane region of the protein I Ng (Sheffield University), E Ratcliffe (Sheffield University), R Tunnicliffe (Sheffield University), K Holden-Dye (Bristol University), M Jones (Bristol University), M Williamson (Sheffield University), N Hunter (Sheffield University) PufX is an integral component of the RC-LH1 core complex of Rhodobacter sphaeroides; it is involved in the organisation of this complex and facilitates quinol/quinone exchange between the RC and cytochrome bc1 complexes. The structure of PufX in organic solvent reveals two hydrophobic helices connected by a helical bend, and flanked by unstructured termini. The proposed location of the basic residues and tryptophans at the membrane interface orients the C-terminal helix along the membrane normal. The N-terminal helix is predicted to extend ~40 Å along the membrane interface (Tunicliffe et al., 2006). The nature of the structure would allow the N-terminus of PufX to facilitate the dimerisation of core complexes, as first suggested by Francia et al., (2002); rather than the membrane region. Data are presented that support the role of the PufX N-terminus in dimerisation of core complexes. Mutagenesis of a residue found in the hinge region of the protein has drastic effects on membrane morphology and photosynthetic growth. PS3.52 Binding Affinities of Chlorophylls in Hetero-Chlorophyllous Complexes of Peridinin-Chlorophyll-Protein as revealed by Ensemble and Single Molecule Spectroscopy H Scheer (Universität München), T Brotosudarmo (Ludwig-Maximilian-University), S Mackowski (Ludwig-Maximilian-University), S Wörmke (Ludwig-Maximilian-University), E Hofmann (Ruhr-University Bochum), R Hiller (Macquarie University), C Bräuchle (Ludwig-Maximilian-University) Peridinin-chlorophyll-proteins (PCP) containing differently absorbing chlorophyll derivatives are suitable models to study energy transfer

170 14th Photosynthesis Congress - PS07 among monomeric chlorophylls (Chls) by bulk and single-molecule spectroscopy (Wörmke et al., 2007). They are obtained by reconstituting the N-terminal domain of the protein (N-PCP) with chlorophyll mixtures. Upon dimerization of the “half-mers”, both homo- and heterochlorophyllous complexes are generated that correspond structurally to monomers of PCP that contain two different Chls in the two halves. Excitation into the peridinin bands populates independently excited states of both Chls, whose fate can be followed by fluorescence techniques (Brotosudarmo et al., 2006). We report reconstitution of N-PCP with binary mixtures of Chl a, Chl b, [3-acetyl]-Chl a and [Ni]-Chl a. The ratios of the pigments were varied in the reconstitution mixture, and relative binding constants determined from quantification of these pigments in the reconstituted PCPs. In most cases, the modified pigments have similar or even higher affinities that the native pigment, Chl a. Spectral shifts of the pigments in the complexes, as compared to monomeric solutions, are reported, as well as single molecule spectra. PS3.53 Mechanism of Redox Regulation of Energy Transfer of FMO Protein from Green Sulfur Bacteria J Wen (Department of Chemistry, Washington University in St. Louis), R Blankenship (Departments of Biology and Chemistry,Washington University in St. Louis) The Fenna-Matthews-Olson (FMO) antenna protein from green sulfur bacteria functions both as a light harvesting antenna and an energy mediator governing the energy transfer from chlorosome to the reaction center. Strong fluorescence quenching of the FMO protein was reported depending on either the redox conditions or thermal effects. A small regulating molecule was proposed to bind the protein tightly or covalently, sensitive to the local redox potential. Mass spectrometry and organic extraction are being used to try to understand the modifications of the protein that leads to redox-dependent regulation of energy transfer activity. PS3.54 Biochemical and spectroscopic properties of CP24. F Passarini (Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen), R Croce (Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen) CP24 is a minor antenna complex of Photosystem II present in higher plants but absent in green algae. It has been shown that CP24 is a crucial determinant of the structure and function of PSII; in fact the knock out mutation of this antenna protein leads to a major change in the supermolecular organisation of PSII and to a strong reduction of non-photochemical quenching, a mechanism that protects the system from high light damage. This antenna complex is poorly characterized owing to the impossibility of isolation of the native complex in sufficient amount and in stable form. In order to overcome this problem and study the role of CP24 in the energy transfer and the photoprotection we have reconstituted it in vitro upon overexpression in E. coli. We have also obtained a series of recombinant CP24 proteins mutated in single chlorophyll-binding residues. The in vitro mutational analysis has been performed with the aim of determine the functional and spectroscopic characteristics of each pigment coordinated to this complex. To extend the in vitro analysis we have overexpressed this subunit carrying an His-tag in the knock out mutant of Arabidopsis. This will allow the purification of the native protein in suitable amount for its characterization; this previously unavailable information will complete the in vitro data on its biochemical and spectroscopic properties.

PS3.55 Ultrafast relaxation dynamics of a keto-carotenoid, siphonaxanthin, probed by time-resolved fluorescence S Akimoto (Molecular Photoscience Research Center, Kobe University), M Yokono (Institute for Low temperature Science, Hokkaido University), M Higuchi (Graduate School of Human and Environmental Studies, Kyoto University), A Murakami (Kobe University Research Center for Inland Seas), S Takaichi (Biological Laboratory, Nippon Medical School), M Mimuro (Graduate School of Human and Environmental Studies, Kyoto University, Department of Technology and Ecology, Hall of Global Environmental Research, A keto-carotenoid, siphonaxanthin shows a characteristic in vivo absorption band around 535 nm in the pigment-protein complexes in a green alga Codium fragile. To reveal optical properties of the keto-carotenoid, we examined the relaxation dynamics of siphonaxanthin in solutions by femtosecond time-resolved fluorescence spectroscopy. Fluorescence kinetics after excitation to the S2 state was analyzed by multi-exponential functions with three components, independent of observed wavelengths: 35-fs and 200-fs components and a minor one longer than 10 ps. No rise component was resolved, which is in striking contrast to the fluorescence kinetics of lutein, a major carotenoid in green plants and consisting of the conjugated C=C bonds. The 35-fs component well corresponded to the mirror image of the absorption spectrum, therefore we assigned this component to the S2 fluorescence. The 200-fs component was much smaller than the 35-fs component in its amplitude, suggesting that its origin was different from the S2 state. Fluorescence anisotropy values of the 35-fs and 200-fs components were identical within an experimental error and obtained to be 0.39. An isotropy value of the S1 fluorescence was 0.32 after the S2 excitation, therefore the 200-fs component could not be assigned to the S1 fluorescence. In LHC II, siphonaxanthin exhibits a new excited state (Sx) between the S1 and S2 state, corresponding to the 535-nm absorption band (Chem. Phys. Lett. 390 (2004) 45). The 200-fs component might correspond to the Sx state of siphonaxanthin in solutions, although its intensity is smaller than that in the pigment-protein complexes. PS3.56 Changes in the lamellar organization of bacteriochlorophyll aggregates in chlorosomes upon hexanol treatment J Psencik (Charles University), J Arellano (Institute for Natural Resources and Agrobiology), M Torkkeli (University of Helsinki), P Laurinmaki (University of Helsinki), S Butcher (University of Helsinki), R Serimaa (University of Helsinki), R Tuma (University of Helsinki) Chlorosomes are main light harvesting complexes of green photosynthetic bacteria. In contrast to other antennae, the main pigments (bacteriochlorophyll c, d or e) in chlorosomes are not organized in pigment-protein complexes, but form aggregates. Recent results suggest that bacteriochlorophylls together with carotenoids and quinones self-assemble into lamellar nanostructures, rather than to tubular elements proposed formerly. Here we report changes in the arrangement of bacteriochlorophyll c in chlorosomes from Chlorobium tepidum induced by hexanol treatment. The results provide further support for the lamellar model. Hexanol was reported to reversibly convert the aggregated form of bacteriochlorophyll c (absorption maximum at ~740 nm) to a monomer-like form (absorption maximum at ~670 nm). We examined the effect of hexanol on chlorosome structure by X-ray diffraction and electron cryomicroscopy. At saturating hexanol concentrations, the lamellae persisted in the presence of hexanol although the short-range order and exciton interactions between chlorin rings were lost. The result suggested that the polar portion of hexanol solvated the chlorin rings while the hydrophobic tail partitioned into the lamellar structure. This

171 14th Photosynthesis Congress - PS07 partitioning further elongated the chlorosome along its long axis as observed in cryomicroscopy. Super-saturating hexanol concentrations produced round bacteriochlorophyll-hexanol droplets, which lost all lamellar order. After hexanol removal the lamellae reformed even in over-saturated samples although the shape and long-range order were not restored. PS3.57 Effect of quinones on formation bacteriochlorophyll c aggregates

and

properties

of

J Alster (Charles University), A Zupcanova (Biological Centre ASCR), F Vacha (Biological Centre ASCR), J Psencik (Charles University) Bacteriochlorophyll (BChl) c, d and e are the major pigments found in the main light harvesting complexes of green photosynthetic bacteria, the so-called chlorosomes. A unique property of chlorosomes is that the main BChl pigments are not present in pigment-protein complexes, rather they self-assemble into aggregates. Aggregates with very similar optical properties can be prepared also in vitro either in non-polar solvents or in aqueous buffers with addition of lipids and/or carotenoids. In this study we show that the aggregation of BChl c in aqueous buffers can be induced also by quinones, if they posses a hydrophobic tail. Such quinones are naturally present in chlorosomes in a stoichiometric ratio to main BChl approximately 1:10. Quinones in chlorosomes are involved in a redox-dependent excitation quenching mechanism, which may protect the cells against toxic substances produced as a consequence of charge separation under oxygenic conditions. BChl aggregates prepared with quinones also exhibit such an excitation quenching, as determined from fluorescence spectra. The amplitude of the quenching is proportional to concentration of quinones and is similar as for BChl aggregates prepared with quinones and lipids (lecithin). Although lecithin further improves the aggregation ability, it does not affect significantly the extent of quenching, which is thus determined mainly by interactions of BChl with quinones incorporated in the aggregate structure. PS3.58 Spectroscopic and structural studies of the light-harvesting complexes from photosynthetic purple sulfur bacteria A Gall (CEA), S Kereïche (University of Groningen), L Bourinet (CEA), W Keegstra (University of Groningen), A Arteni (University of Groningen), J Verbavatz (CEA), E Boekema (University of Groningen ), B Robert (CEA) In this work we have investigated the spectroscopic and structural properties of the light-harvesting complexes from closely related species of the genus Allochromatium. We will present our most recent findings on the pigment-protein interactions in the pigment-binding pockets of detergent-purified complexes and relate this to the overall quaternary structure. PS3.59 Single particle analysis of the dimeric RC-LH1-PufX complex from Rhodobacter sphaeroides P Qian (University of Sheffield), P Bullough (University of Sheffield), N Hunter (University of Sheffield) The reaction center-light harvesting 1-PufX (RC-LH1-PufX) complex from Rba. sphaeroides is a dimer, as shown by atomic force microscopy of native membranes (1), and by negative stain analysis of LH2-minus membranes (2). The purified complex has been crystallized and an 8.5 Å projection structure analysed by cryo-electron microscopy (3). The 3D shape of this

dimeric complex has been investigated using single particle EM analysis of negatively stained, purified RC-LH1-PufX complexes. The results show that the periplasmic faces of the two halves of the RC-LH1-PufX dimer are inclined towards each other forming a shallow ‘V’ shape. This information has important implications for structural parameters of the reconstituted tubular crystals and for native and mutant photosynthetic membranes. PS3.60 Electron microscopy of the photosynthestic membrane of Thermosynechoccus elongatus reveals new protein structures M Folea (University of Groningen), E Boekeme (University of Groningen), M Nowaczyk (University of Bochum) Phosynthetic membranes from Thermosynechoccus elongatus were solubilized with the detergent digitonin and the complete set of non-purified single particles was studied by electron microscopy to see if novel labile and/or transient structures could be found. Several new structures were observed. An novel U-shaped particle was found, which is very similar to NDH-1, the cyanobacterial counterpart of NADH-quinone oxidoreductase or Complex I, except for the fact that it has a loosely attached domain at the tip of the membrane domain, not observed after purification. This domain could be composed of CupA and/or other proteins. A large “rod-like” structure, composed of six disks, is a phycobilisome fragment, not observed and analysed previously. Another particle is a dimeric complex of the photosystem II dimer. The analysis shows that it is possible to find novel types of structures before further purification steps that otherwise might have been disrupted. PS3.61 Structural characterization of PSII membranes from an Arabidopsis knock-out mutant lacking Lhcb3 antenna S Kereiche (University of Groningen), J Dankjaer (Umea University), L Kovacs (University of Sheffield), S Jansson (University of Groningen), P Horton (University of Groningen), E Boekema (University of Groningen) The photosystem II light-harvesting antenna in higher plants contains a number of highly conserved gene products whose function is not precisely known. We have studied Arabidopsis thaliana plants depleted of one of these, Lhcb3. Knock-out Lhcb3 plants did not show a decrease in light-limited photosynthetic rate and growth, and the pigment and protein content of the thylakoid membranes were otherwise unchanged. We investigated ordered 2D arrays of grana membranes from the Lhcb3 mutant by electron microscopy. We found that the overall size of the photosystem II arrays is larger than seen before. The packing of the PSII particles is probably a particle equivalent to the C2S2M2 supercomplex particle, which could mean that the WT S-trimers (and/or M-trimers) with Lhcb3 have been replaced by similar (pseudo-) trimers without Lhcb3 in the mutant. We do not have a hint yet which antenna protein could replace Lhcb3. In the projection map the CP26 and the M-trimer are well resolved, and they are in the expected position, but the area of the S-trimer is slightly fuzzy and different. The S- and M-trimer differ in rotational position by 25°. In the new crystals it appears that this angle is about 10°. Because the position of the M-trimer has not significantly changed, it means that the (pseudo?) S trimer has been rotated 15°. This indicates that indeed this particle is not identical to the WT C2S2M2 particle. In conclusion, the mutant still can make supercomplexes, but with a slightly different structure. PS3.62 Zeaxanthin radical cation formation in minor light harvesting

172 14th Photosynthesis Congress - PS07 complexes of higher plant antenna R Bassi (University of Verona), T Avenson (University of California), T Ahn (Lawrence Berkeley National Laboratory; University of California), D Zigmantas (Lawrence Berkeley National Laboratory; University of California), L Zirong (Lawrence Berkeley National Laboratory; University of California), B Matteo (University of Verona), Z Li (Lawrence Berkeley National Laboratory; University of California), G Fleming (Lawrence Berkeley National Laboratory; University of California) Regulation of light capture is critical for survival of photosynthetic organisms. The predominant, short-term mechanism is termed energy-dependent quenching, or qE. qE is dependent upon de-epoxidized xanthophylls (i.e. zeaxanthin-Z), an antenna-associated protein (PsbS), and a transthylakoid pH gradient. However, their molecular interplay has been difficult to determine. We previously demonstrated transient zeaxanthin radical cation (Z∙+) formation in intact thylakoids (Holt et al. Science 307: 433-6), qE-dependent, using near infrared (NIR) transient absorption (TA) spectroscopy and proposed a molecular mechanism for qE involving a chlorophyll-zeaxanthin heterodimer ([Chl-Z]) undergoing charge separation and recombination, implying transient formation of a Z∙+ species . To localize the molecular site of this function, we systematically analyzed all light harvesting complexes of PSII antenna and demonstrated transient Z∙+ formation in all three minor complexes (CP24, CP26, and CP29) specifically binding Z. There was no evidence for Z∙+ formation in either Z-bound trimeric or monomeric LHCII complexes. We also present evidence that is inconsistent with PsbS itself mediating Z∙+ formation. In addition to evidence for Z∙+ formation in Z-bound CP26 complexes, a signal we propose is a lutein (Lut) radical cation (Lut∙+) was also found. Importantly, all of the LHCs exhibited Z-dependent quenching of chlorophyll excited states, including monomeric/trimeric LHCII, CP24, CP26, and CP29. Taken together, these data suggest that the minor complexes may serve as sites of qE in vivo, wherein all are capable of mediating ET quenching within [Chl-Z] heterodimers and in the case of CP26, ET quenching also could occur within a [Chl-Lut] heterodimer.

PS3.63 Modelling the IsiA-PSI supermolecular complex of cyanobacteria A Larkum (University of Sydney), Y Zhang (University of Sydney), B Churdh (University of Sydney), L Jermiin (University of Sydney) In some classical cyanobacteria poor nutrient conditions induced a reduction of the phycobilisome light harvesting system and an upregulation of an intrinsic chlorophyll antenna protein, IsiA, which can form an 18-mer ring around photosystem (PS) I trimers. The structure of this supercomplex has been obtained elsewhere at low resolution by electron diffraction from liquid crystals. The IsiA protein is closely related to CP43. We have used the recent high-resolution structure of PSII, which includes CP43, to obtain a better model of the molecular structure of IsiA. This model has been refined by further analysis of the sequence variations between CP43, PsaA and IsiA to recover a model in which there are 16 Chl, 2 more than predicted by the earlier models.

173 14th Photosynthesis Congress - PS07

PS4 - The Water Oxidising Enzyme PS4.1 Structure of the Photosynthetic Mn4Ca Cluster Using X-ray Spectroscopy J Yano (Lawrence Berkeley National Laboratory), J Kern (Technische Universitat, Berlin), M Latimer (Stanford Synchlotron Radiation Laboratory), K Sauer (University of California, Berkeley), Y Pushkar (Lawrence Berkeley National Laboratory), J Messinger (Max-Planck Institut fur Bioanorganische Chemie), A Zouni (Technische Universitat, Berlin), V Yachandra (Lawrence Berkeley National Laboratory) Single crystals of Photosystem II (PS II) isolated from thermophilic cyanobacteria have been studied by X-ray diffraction (XRD) with resolutions between 3.0 and 3.8 Å. These studies have localized electron density associated with the water-oxidizing Mn4Ca cluster within the large complex of PS II peptides, but the limited resolution is short of what is needed to place individual metal atoms precisely in the cluster. Examination of the orientation dependence of the EXAFS of single crystals of PS II can provide structural information about the Mn sites at a resolution higher than that is presently available from single-crystal X-ray diffraction. We have successfully collected single crystal XANES and EXAFS data from the native S1 state with the X-ray e-vector parallel to the a, b, and c axes of the crystal, under non-damaging conditions by monitoring the Mn K-edge for any X-ray induced Mn reduction. The EXAFS spectra show that the Fourier peaks are clearly dichroic, demonstrating an asymmetric Mn cluster. We have used the EXAFS dichroism to evaluate the Mn cluster geometry. Three Mn4Ca models which satisfy the trend of EXAFS dichroism were further fit into the ligand environment obtained from XRD, in order to discriminate between the several symmetry-related orientations which arise from the crystal symmetry. Furthermore, single crystals in the S1 state were illuminated either by continuous illumination or by laser flashes to create intermediate S-states (S2 and S3). Polarized XANES and EXAFS spectra from these crystals show unique orientational dependence.

PS4.2 DFT-QM/MM Structural Models of the Oxygen-Evolving Complex of Photosystem II V Batista (Yale University), E Sproviero (Yale University), J Gascon (Yale University), J McEvoy (Yale University), G Brudvig (Yale University) The mechanism of water splitting in photosystem II is described in terms of complete structural models of the oxygen evolving complex (OEC) in the S0-S4 states. The models include the influence of the surrounding protein environment according to state-of-the-art quantum mechanics/molecular mechanics (QM/MM) hybrid methods applied in conjunction with the X-ray crystal structure of PSII from the cyanobacterium Thermosynechococcus elongatus. The resulting structural models are validated through direct comparisons of simulated extendend-X-ray absorption fine structure (EXAFS) spectra with the experimental spectra of S-state intermediates. The models provide a detailed tentative description of the water splitting mechanism as determined by structural rearrangements in the oxomanganese cluster, rearrangements of water molecules around the metal cluster and changes in the oxidation states of metal centers. The formation of an additional mu-oxo bridge between Mn(3) and Mn(4) not present in the X-ray crystal structure of the S1 state is proposed for the S3, S4 and S0 states of the OEC. The models are consistent with the hypothesis that dioxygen is produced by the reaction of water with an oxyl radical in the S4 state

of the catalytic cycle.

PS4.3 Oxygen, water, proton and quinine channels in PSII J Murray (Imperial College London), J Barber (Imperial College London) The photosystem II (PSII) reaction centre must allow the entry and egress of substrates (water, plastoquinone), and products (protons, oxygen and plastoquinol), with the intermediate transfer of electrons within the enzyme. Using recent structural information on PSII a putative oxygen channel has been identified which is about 21Å in length, leading from the water splitting site to the lumen. This channel follows a path along the lumenal surface of CP43, passing across the interface of the large extrinsic loop which joins the fifth and sixth transmembrane helices of this chlorophyll binding protein. In so doing it seems to minimise interactions with the excited states of chlorophylls bound within the PSII complex, especially those that constitute the primary electron donor, P680. Two additional channels leading from the water splitting site, and also exiting at the lumen, were also identified. Their hydrophilic nature suggests that they probably facilitate the delivery of water to, and protons from, the water oxidising site. The acceptor side of PSII is associated with a cavity that is accessible to solvent on the stromal side and to the membrane. We use single particle electron microscopy to propose a possible role for this cavity.

PS4.4 Electronic and Geometric Structures of the Mn4OxCa Cluster in the S0 and S2 States of the Oxygen-Evolving Complex of Photosystem II Based on Pulse J Messinger (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany), L Kulik (Institute of Chemical Kinetics and Combustion, Novosibirsk, Russia), B Epel (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany), S Zein (Lehrstuhl für Theoretische Chemie, Institut für Physikalische und Theoretische Chemie, Universität Bonn, Bonn, Germany), J Yano (Melvin Calvin Laboratory, Physical Biosciences Division, Lawrence Berkeley National Laboratory) The heart of the oxygen-evolving complex (OEC) of photosystem II is a Mn4OxCa cluster that attains five different oxidation states (S0 to S4) during catalysis. Four very similar high resolution models of this cluster were recently obtained employing polarized EXAFS spectroscopy.1 Using DFT calculations we confirm that these models are chemically reasonable, and interpret on that basis our previously reported 55Mn-hyperfine coupling constants of the S and S states2 using 0 2 Y-shaped spin-coupling schemes with up to four non-zero exchange coupling constants. This analysis rules out the presence of one or more Mn(II) ions in S0, and thereby establishes that the oxidation states of the manganese ions in S0 and S2 are Mn4(III, III, III, IV) and Mn4(III, IV, IV, IV), respectively. By applying a ‘structure filter’ we (i) show that the above models are fully consistent with EPR and 55Mn-ENDOR data, (ii) assign the Mn oxidation states to individual Mn ions and (iii) propose that the known shortening3 of one 2.85 Å Mn-Mn distance in S0 to 2.75 Å in S1 corresponds to a deprotonation of a m-hydroxo bridge between MnA and MnB, i.e. between the outer Mn and its neighboring Mn, which belongs to the m3-oxo bridged moiety of the cluster. The results are

174 14th Photosynthesis Congress - PS07 summarized in a molecular model for the S0 → S1 and S1 → S2 transitions. Yano et al. (2006) Science 314, 821-825 Kulik et al. (2005) J. Am. Chem. Soc. 127, 2392-2393 3 Robblee et al. (2002) JACS 124, 7459-7471 1 2

PS4.5 pH dependence of the S0 Split EPR signal in photosystem II, induced by 5K illumination J Sjöholm (Uppsala University), K Havelius (Uppsala University), S Styring (Uppsala University) Upon illumination of PSII an electron is extracted from the CaMn4 cluster via tyrosine-161 on the D1 polypeptide, to form a neutral tyrosine radical YZ•. When illuminating at cryogenic temperatures, water oxidation is blocked but the primary charge separation can still occur. At 5K, illumination results in EPR signals originating from YZ• in magnetic interaction with the CaMn4 cluster, offering a way to probe for oxidation of YZ in active PSII. We have studied how pH affects the formation of the Split EPR signal from YZ in the S0 state. S0 was induced by 3 flashes. Then pH was adjusted between pH 4.0-9.0. The S0 split EPR signal was induced by illumination in the cavity at 5 K. Maximum signal intensity was observed around pH 6.3. On the acidic and alkaline sides the signal intensity decreased with apparent pKs of ~4.7 and ~7.9, respectively. The signal decayed in the dark on a similar time scale over the whole pH range. The EPR studies showed that cryogenic oxidation of Cyt b559, Car, and/or ChlZ can compensate for the loss of the split radical donor (YZ) at both high and low pH. As described earlier, the presence of methanol induces a change in the spectral shape of the Split S0 signal. Recent results from these methanol induced changes at different pH will be presented and discussed. PS4.6 Water binding to the Mn4OxCa cluster in Photosystem II studied by advanced pulse EPR spectroscopy J Su (Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34–36, D-45470 Mülheim an der Ruhr, Germany), W Lubitz (Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34–36, D-45470 Mülheim an der Ruhr, Germany), J Messinger (Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34–36, D-45470 Mülheim an der Ruhr, Germany) Mn4OXCa cluster acting together with the nearby D1-Tyr161. During the stepwise proton-coupled electron transfer, the Mn4OXCa cluster undergoes a cycle composed of five distinct intermediates termed Si states (i = 0-4). Molecular oxygen is released during the S3?[S4]?S0 transition. The ‘when’ and ‘where’ of substrate water binding belong to the important open questions about photosynthetic water splitting. Mass spectrometry data show that at least one water molecule is bound up to the S2 state, and that both substrate waters are bound in the S3 state. However, due to kinetic limitations, they are unable to determine if the fast exchanging substrate water molecule is also bound in the S0 to S2 states, and no direct information is obtained about the modes and sites of substrate binding in the OEC. In the paramagnetic S0 and S2 states binding of water to the Mn4OXCa cluster can be also studied by

advanced EPR spectroscopy via couplings of the unpaired spin on the cluster with directly bound or nearby isotopically labeled water. In the present study we extend previous EPR studies by employing advanced X- and Q-band pulse EPR spectroscopy, two-pulse ESEEM, two-dimensional three-pulse ESEEM, HYSCORE and Mims ENDOR and variously isotopically labeled water, H216O, D216O and H217O to investigate water binding to the S2-state.

PS4.7 A detailed structural model for the eukaryotic LHCII-PSII supercomplex J Neild (Imperial College London), J Barber (Imperial College London) Photosystem II (PSII) core complex X-ray structures have so far been derived from preparations isolated from prokaryotic cyanobacteria. These have provided significant information for understanding PSII functionality, including water splitting, but as yet no high-resolution structure has become available for eukaryotic PSII. Such knowledge will be required to reveal spatial excitonic linkages between the pigments of the intramembrane light-harvesting components (LHCII) and the chlorophylls of the reaction centre core. Furthermore, differences in the eukaryotic water splitting environment also need to be investigated. To this end we have utilised the published X-ray structures of a cyanobacterial PSII core (Ferreira et al., 2004), LHCII (Standfuss et al., 2005), PsbP (Ifuku et al., 2004) and PsbQ (Calderone et al., 2004) proteins to construct a model of eukaryotic LHCII-PSII supercomplex using 16 to 17 Å resolution 3D density maps from spinach and Chlamydomonas, as determined by electron cryo-microscopy and single particle analysis. We tentatively identify the positioning of chlorophylls, in order to consider energy transfer pathways between the different subunits, and discuss the positioning of the extrinsic protein subunits of the water-splitting site. Calderone V, et al., (2004) EMBO Rep. 4, 900-905 Ifuku K, et al., (2004) EMBO Rep. 5, 362-367 Ferreira K, et al., (2004) Science 303, 1831-1838 Standfuss J, et al., (2005) EMBO J. 24, 919-928 PS4.8 Structure of the Photosynthetic Mn4Ca Cluster Using X-ray Spectroscopy J Yano (Lawrence Berkeley National Laboratory), J Kern (Technische Universität), M Latimer (Stanford Synchlotron Radiation Laboratory), K Sauer (Lawrence Berkeley National Laboratory; University of California), Y Pushkar (Lawrence Berkeley National Laboratory; University of California), J Messinger (Max-Planck Institut fur Bioanorganische Chemie), A Zouni (Max-Planck Institut fur Bioanorganische Chemie), V Yachandra (Lawrence Berkeley National Laboratory) Single crystals of Photosystem II (PS II) isolated from thermophilic cyanobacteria have been studied by X-ray diffraction (XRD) with resolutions between 3.0 and 3.8 Å. These studies have localized electron density associated with the water-oxidizing Mn4Ca cluster within the large complex of PS II peptides, but the limited resolution is short of what is needed to place individual metal atoms precisely in the cluster. Examination of the orientation dependence of the EXAFS of single crystals of PS II can provide structural information about the Mn sites at a resolution higher than that is presently available from single-crystal X-ray diffraction. We have successfully collected single crystal XANES and EXAFS data from the native S1 state with the X-ray e-vector parallel to the a, b, and c axes of the crystal, under non-damaging conditions by monitoring the Mn K-edge for any X-ray induced Mn reduction.3 The EXAFS spectra

175 14th Photosynthesis Congress - PS07 show that the Fourier peaks are clearly dichroic, demonstrating an asymmetric Mn cluster. We have used the EXAFS dichroism to evaluate the Mn cluster geometry. Three Mn4Ca models which satisfy the trend of EXAFS dichroism were further fit into the ligand environment obtained from XRD, in order to discriminate between the several symmetry-related orientations which arise from the crystal symmetry. Furthermore, single crystals in the S1 state were illuminated either by continuous illumination or by laser flashes to create intermediate S-states (S2 and S3). Polarized XANES and EXAFS spectra from these crystals show unique orientational dependence. PS4.9 Electronic Structure and Oxidation State Changes in the Mn4Ca Cluster of Photosystem II V Yachandra (Lawrence Berkeley National Laboratory), J Yano (University of California), Y Pushkar (University of California), K Sauer (University of California), J Messinger (Max-Planck Institute), P Glatzel (European Synchrotron Radiation Laboratory), U Bergmann (Stanford Synchrotron Radiation Laboratory), V Yachandra (Lawrence Berkeley National Laboratory) A detailed electronic structure of the Mn4Ca cluster is required before two key questions for understanding the mechanism of photosynthetic water oxidation can be addressed. They are whether all four oxidizing equivalents necessary to oxidize water to O2 accumulate on the four Mn ions of the OEC, or do some ligand-centered oxidations take place before the formation and release of O2 during the S3 ® [S4] ® S0 transition, and what are the oxidation state assignments for the Mn during S-state advancement. X-ray absorption and emission spectroscopy of Mn, including the newly introduced resonant inelastic X-ray scattering spectroscopy have been used to address these questions. XAS and EPR of Mn model complexes and single crystals have also been used to understand the details of the electronic structure of the Mn4Ca cluster in PS II. The relevance of these data from Mn complexes to the Mn4Ca cluster in the OEC will be discussed. The present state of understanding of the electronic structure and oxidation state changes of the Mn4Ca cluster in all the S-states, derived from all these techniques will be presented. PS4.10 Role of phosphatidylglycerol in oxygen-evolving complex of photosystem II N Mizusawa (University of Tokyo), I Sakurai (University of Tokyo), H Kubota (University of Tokyo), H Wada (University of Tokyo) Our previous studies with pgsA mutant of Synechocystis sp. PCC6803 which is defective for the biosynthesis of phosphatidylglycerol revealed that PG is essential for electron transport from QA to QB on the acceptor side of photosystem II (PSII). In this study, we analyzed the properties of PSII with pgsA mutant both in vitro and in vivo in details to clarify the role of PG in the donor side of PSII, namely, oxygen-evolving complex (OEC). Analyses of purified PSII complexes indicated that PSII from PG-depleted pgsA mutant sustained only 50% of the oxygen-evolving activity compared to wild-type cells and dissociated the extrinsic proteins of PsbO, PsbV and PsbU, and manganese ions. The released PsbO re-bound to PSII when PG was added back to the PG-depleted mutant cells, even when de novo protein synthesis was inhibited. The extent of inactivation of oxygen evolution in PG-depleted mutant cells by heat treatment or dark incubation resembled those of ?psbO and ?psbV mutant cells. These results suggest that PG plays an important role in the binding of extrinsic proteins required for sustaining a functional Mn cluster.

PS4.11 Calcium requirement for S-state transitions M Miqyass (Biophysics Department, Huygens Laboratory, Leiden, Netherlands), H van Gorkom (Biophysics Department, Huygens Laboratory, Leiden, Netherlands) The functional role of the Ca2+ ion in the oxygen-evolving complex of photosystem II is still under debate. The S3 state is not formed in the absence of Ca2+. Re-interpretation of the literature on methods of Ca2+ depletion (Miqyass et al., 2007) led us to conclude that also the oxidation of S1 to S2 requires Ca2+. Here we confirm this interpretation. Ca2+ depletion of BBY membranes by the salt-wash procedure was studied by measuring flash-induced S-state transitions in UV absorbance. Using 1 M KCl rather than NaCl, we found that Ca2+ is replaced within 30 minutes in the dark, leaving PSII in an inactive S1 state. A saturating flash caused YZ oxidation only. Nevertheless, 30 minutes exposure to room light in the presence of 1 mM EGTA produced the inactive stable S2 state, in which also YZ oxidation was largely inhibited and flashes induced mainly P680+QA– recombination. Apparently, YZ• can still oxidize S1 to S2 but the flash yield is negligible, suggesting that the reaction takes much longer than the few seconds lifetime of YZ• in these conditions and is at least 5 orders of magnitude slower than in the presence of Ca2+. In spite of a good S2 yield after minutes of illumination, this indicates an essential functional role of Ca2+ in the S1 to S2 transition. Key words: Calcium, S-state, Oxygen evolution, Photosystem II.

PS4.12 Mono-manganese mechanism of dioxygen evolution catalyzed by unique Mn4Ca cluster in PSII M Kusunoki (Meiji University) The molecular mechanism of the water oxidation reaction in photosystem II (PSII) remains a great mystery in life science. This reaction, which takes place in the oxygen evolving complex (OEC) incorporating four manganese, one calcium and one chloride cofactors, is light-driven to cycle five intermediates, S0 through S4, to produce four protons, four electrons and lastly one dioxygen, as indispensable resources in biosphere. Despite of recent advancements of X-ray crystallography models, which established the existence of a catalytic Mn4Ca cluster ligated by seven protein amino acids, its functional structure has been a subject of intense debate. In this paper, we present a chemically complete model for the Mn4Ca cluster and its surrounding enzyme field and its predominant reaction intermediates, together with our theoretical interpretations of the S-state dependence of ^(18)O exchange rates of two substrate water molecules, by using the hybrid DFT/B3LYP geometry optimization method to confirm good agreements with the 3.0 Angstrom resolution PSII model. Remarkably, we could verified that two substrates are bound to asymmetric cis-positions on the terminal Mn ion being triply bridged (µ-oxo, µ-carboxylato, and µ-hydroxo) to the Mn3CaO3(OH) core, by developing a generalized theory of ^(18)O exchange kinetics in OEC to obtain an experimental evidence of the cross exchange pathway. Furthermore, it will be shown that the O-O bond formation can readily take place along the cross exchange pathway between neighboring oxo radical and hydroxo anion, which are both bound to the terminal Mn(IV) ion, but only the latter is H-bonding with the trimeric part.

176 14th Photosynthesis Congress - PS07 PS4.13 A Cluster of Carboxylic Groups in PsbO Protein is Involved in Proton Transfer from the Water Oxidizing Complex of Photosystem II

Bioinorganic Chemistry, Muelheim an der Ruhr, Germany)

T Shutova (Umeå University)

evidence suggesting that Cl- is a cofactor of photosynthetic water splitting, but its precise function and its putative binding site inside the OEC are unknown. While Ca2+ has been demonstrated to be part of the inorganic core, its functional role also remains to be fully established. Here we study the effects of Ca2+ and Cl- on substrate water binding, by replacing them with their functional surrogates Sr2+ and Br-. Substrate water exchange kinetics of the S3 state were measured in thylakoids isolated from T. elongatus grown in CaCl2, CaBr2, SrCl2 or SrBr2

The hypothesis presented here for proton transfer away from the water oxidation complex of Photosystem II (PSII) is supported by biochemical experiments on the isolated PsbO protein in solution, theoretical analyses of better understood proton transfer systems like bacteriorhodopsin and cytochrome oxidase, and the recently published 3D structure of PS II (Pdb entry 1S5L). We propose that a cluster of conserved glutamic and aspartic acid residues in the PsbO protein acts as a buffering network providing efficient acceptors of protons derived from substrate water molecules. The charge delocalization of the cluster ensures readiness to promptly accept the protons liberated from substrate water. Therefore protons generated at the catalytic centre of PSII need not be released into the thylakoid lumen as generally thought. This cluster is the beginning of a localized, fast proton transfer conduit on the lumenal side of the thylakoid membrane reaching all the way to the ATPase. Proton-dependent conformational changes of PsbO may play a role in the regulation of both supply of substrate water to the water oxidizing complex and the resultant proton transfer. PS4.14 Role of the psbA gene family of PSII from the thermophilic cyanobacterium Thermosynechococcus elongatus J Sander (Plant Biochemistry, Ruhr University Bochum), M Nowaczyk (Plant Biochemistry, Ruhr University Bochum), M Rögner (Plant Biochemistry, Ruhr University Bochum) The membrane protein complex photosystem II (PSII) catalyses the light-driven oxidation of water into molecular oxygen and protons. Electrons from the PSII donor site are transferred to the acceptor site via cofactors of which most are associated with the D1 core protein. In all sequenced cyanobacteria this D1 protein is encoded by a psbA gene family with species-dependant variation of the gene copy number. The gene family of the cyanobacterium Thermosynechococcus elongatus contains three copies (psbA1, psbA2 and psbA3) which all code for a distinct gene product. As shown for other organisms the exchange of these D1 proteins seems to be an adaptation of PSII to high light stress. Using quantitative real time PCR we can show an exchange of the psbA transcripts within 30 minutes under high light conditions (500µE). While transcription of psbA1 decreases from 90 % to 1.5 % the psbA3 transcription increases from 9 % to 98 %. Due to the high homology of the three D1 copies their discrimination on the protein level is extremely difficult and can only be achieved by an extremely high sensitive and high accurate mass spectrometry approach. This allows us to monitor and quantify the three genes and their products under conditions which might be the external trigger for the gene switch. In parallel, we can use isolated complexes gained from knock-out mutants which contain only one of the three gene copies. These enable for the first time a detailed functional characterisation of all three complexes on the molecular level.

The oxygen evolving complex (OEC) in photosystem II splits water to oxygen, protons and electrons. The inorganic core of the OEC is comprised of a Mn4OxCa cluster. There is ample of biochemical

containing media1 by time-resolved membrane-inlet mass spectrometry2. The measurements show a clear acceleration of the slow substrate water exchange rate after both Ca2+/Sr2+ exchange3 and after replacement of Clby Br-. Interestingly, the effects of Sr2+ and Br- are partially additive, so that the fastest exchange was found in the SrBr2 sample. These data support the idea that the binding sites of the slow substrate water, Ca2+ and Cl- are in close vicinity, but more indirect effects can not be excluded. 1 A. Boussac, F. Rappaport, P. Carrier, et al. J Biol Chem, 2004, 279 (22), 22809-22819 2 J. Messinger, M. Badger, T. Wydrzynski, P Natl Acad Sci USA, 1995, 92 (8), 3209-3213 3 G. Hendry, T. Wydrzynski, Biochemistry-US, 2003, 20, 6209-6217

PS4.16 Snapshots of Biological Proton-Coupled Electron Transfer: Tyrosine Intermediates in Photosystem II K Lakshmi (Rensselaer Polytechnic Institute), O Poluektov (Argonne National Lab), S Lee (Rensselaer Polytechnic Institute), S Chand (Rensselaer Polytechnic Institute), A Wagner (Argonne National Lab)

PS4.15 Effects of the Calcium/Strontium and Chloride/Bromide Substitution on Substrate Water Exchange Rates in Photosystem II

The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in Nature by using light energy to drive the catalytic oxidation of water. PCET reactions, which are exquisitely tuned by smart protein matrix effects, are central to this water-splitting chemistry. PSII contains two symmetrically placed tyrosine residues, YD and YZ. The functions of these tyrosines are quite distinct, a versatility provided by their distinct local environments in PSII. YZ is kinetically competent and is proposed to be directly involved in the PCET reactions of water oxidation. In contrast, the YD PCET redox poises the catalytic Mn4 cluster and may electrostatically tune the redox-active chlorophyll and ß-carotene in the secondary ET pathway of PSII. This study focuses on disentangling individual steps of the PCET events that lead to the formation of the tyrosyl radical, YD•, in PSII. It is our hypothesis that the electron transfer, proton movement and associated conformational changes of the YD-binding pocket are the individual steps of the PCET reaction. Using pulsed high-frequency EPR spectroscopy, we elucidate the sequence of events that lead to PCET at the YD site of PSII. These studies provide direct ‘snapshots’ of functional PCET intermediates and, for the first time, make it possible to detail the mechanism of PCET in biological energy transduction.

K Beckmann (Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany), N Ishida (Service de Bioénergétique, DBJC, URA CNRS 2096, CEA Saclay, Gif zur Yvette, France), A Boussac (Service de Bioénergétique, DBJC, URA CNRS 2096, CEA Saclay, Gif sur Yvette, France), J Messinger (Max Planck Institute for

PS4.17 Photoassembly of the Mn4-Ca cluster in mutants perturbed in the high affinity Mn-binding site of the H2O-oxidation complex of photosystem II

177 14th Photosynthesis Congress - PS07

R Burnap (Oklahoma State University), H Hwang (, Oklahoma State University), A McLain (, Oklahoma State University), R Debus (University of California Riverside, Riverside) The light-driven, oxidative assembly of Mn2+ ions into the H2O-oxidation complex of photosystem II is termed photoactivation. Three O2-evolving mutant strains of Synechocystis, D1-D170E, D1-D170H, and D1-D170V, were studied in terms of the kinetics of photoactivation under both continuous and flashing light. Photoactivation using single turnover flashes revealed D1-D170H and, especially D1-D170V, were prone to form substantial amounts (~40-50%) of inactive centers ascribed to photoligation of aberrant non-functional Mn based upon the reversibility of the inactivation and similarity to previous in vitro results. D1-D170E exhibited the lowest quantum yield of photoactivation with intermediates being highly unstable. It is concluded that occupancy of the high affinity site does not control the quantum yield of photoactivation, since D1-D170E has a lower quantum yield than D1-D170V, despite the fact that it has a higher affinity for Mn2+. The low quantum yield of photoactivation in D1-D170E appears to be due to inefficient binding and/or photooxidation of the second (or later) Mn2+ bound during the formation of the Mn4-Ca and this may be due to an alteration in the corresponding binding site evidenced by a large retardation in O2 release during the S3-S0 transition that is not seen in D1-D170H and D1-D170V.

PS4.18 Structural changes in the oxygen evolving complex of photosystem II upon S2 to S3 transition Y Pushkar (Lawrence Berkeley National Laboratory), J Yano (Lawrence Berkeley National Laboratory), A Boussac (CEA, iBiTec-S, SB2SM, URA CNRS 2096, 91191 Gif sur Yvette, Cedex, France), U Bergmann (Stanford Synchrotron Radiation Laboratory, Menlo Park, CA, USA), K Sauer (Lawrence Berkeley National Laboratory), V Yachandra (Lawrence Berkeley National Laboratory) The Mn4Ca cluster of the oxygen evolving complex (OEC) of photosystem II (PS II), cycles through five oxidation states (Si-states, i=0-4) coupling the one electron photochemistry of the reaction center with the 4 electron redox chemistry of water oxidation. While the structure of the OEC in S1-state is emerging from X-ray crystallography and spectroscopic studies [1-3], much less is known about the structural and oxidation state changes through the catalytic cycle, which are critical for understanding the mechanism of oxygen evolution. To study the changes in structure of the OEC along the catalytic pathway we used X-ray absorption spectroscopy and range-extended EXAFS in particular. The use of a high resolution crystal monochromator allows EXAFS collection beyond the Fe K-edge which improves distance resolution of Mn-backscatterer from 0.14Å to 0.09Å. For the S1 and S2 states in solution and S1 oriented membranes we show that there are three di-µ-oxo-bridged Mn-Mn distances of ~2.7Å and ~2.8Å in a 2:1 ratio [4], and that the orientation of the average of the two 2.7Å Mn-Mn vectors and the one 2.8Å Mn-Mn vector is ~60° relative to the membrane normal. Separation of the Mn-Mn ~3.2Å and Mn-Ca ~3.4Å vectors result in the finding that the average Mn-Ca ~3.4Å vector is directed toward the membrane normal, while the Mn-Mn ~3.2Å vector is not more than 30° out of the membrane plane [5]. Range-extended EXAFS data for the S3 state demonstrate a significant increase in Mn-Mn di-µ-oxo-bridged distances in the S2 to S3 transition; from ~2.7-2.8Å to ~2.8-2.9Å. Studies of oriented membranes in the S3 state suggest a change in the Mn-Mn di-µ-oxo-bridged vectors orientation relative to the membrane normal. Using PS II from Sr-medium grown Thermosynechococus elongatus, we detected considerable structural change in the Sr-Mn distances for S2 to

S3, S3 to S0 and S0 to S1 transitions. Since PSII cannot advance beyond the S2 state in preparations that lack Ca(Sr), these results show that Ca(Sr) is one of the critical requirements for the mechanism of the enzyme. The structural changes demonstrate that Ca(Sr) is not just a spectator atom involved in providing a framework but that it is actively involved in the mechanism of photosynthetic water oxidation. These results implicate the involvement of at least one critical bridging oxygen atom between the Mn-Mn and Mn-Ca(Sr) atoms in the complex. Theoretical analysis of the OEC indicate that the formation of a low-lying ligand oxygen radical precursor state may be required for forming the O-O bond [6]. To reach this state, a structural rearrangement is needed at the S2 to S3 transition. The structural rearrangements in the OEC that we detect favor such a model, involving oxidation centered predominantly on a bridging O atom in the S3 state. 1. Ferreira K. N.; Iverson T. M.; Maghlaoui K.; Barber J.; Iwata S. Science 2004 303; 1831-1838. 2. Loll B.; Kern, J.; Saenger, W.; Zouni A.; Biesiadka, J., Nature 2005 438; 1040-1044. 3. Yano J.; Kern J.; Sauer K.; Latimer M.J.; Pushkar Y.; Biesiadka J.; Loll B.; Saenger W.; Messinger J.; Zouni A.; Yachandra V. K. Science 2006 314; 821-825 4. Yano J.; Pushkar Y.; Glatzel P.; Lewis A.; Sauer K.; Messinger J.; Bergmann U.; Yachandra V. JACS (Communication), 2005 127(43); 14974-14975. 5. Pushkar Y.; Yano J.; Glatzel P.; Messinger J.; Lewis A.; Sauer K.; Bergmann U.; Yachandra V. K. JBC 2007 282, N 10; 7198-7208. 6. Siegbahn, P. E. M. Chem. Eur. J. 2006 12; 9217-9227. PS4.19 Insights into the Function of the Oxygen-Evolving Complex of Photosystem II from Manganese Model Chemistry G Brudvig (Yale University), C Cady (Yale University), R Tagore (Yale University), R Crabtree (Yale University) Manganese model complexes have played an important role in understanding the structure and function of the oxygen-evolving complex (OEC) of photosystem II. The complex [(terpy)(H2O)MnIII(O)2MnIV(H2O)(terpy)](NO3)3 (1, where terpy = 2,2':6',2''-terpyridine) catalyzes homogenous water oxidation and, thus, provides a structural and functional model of the OEC (Brudvig et al. (1999) Science 283, 1524). In an effort to determine the details of the water-oxidation reaction, we have examined complex 1, and related complexes, using stopped-flow UV-visible spectroscopy, electrospray mass spectrometry, and electrochemical methods. Here, we summarize recent studies of the redox and ligand-exchange properties of 1 and related manganese complexes and discuss the functional relevance of these studies to the rates of electron transfer between manganese ions, the rates of ligand exchange and the mechanism of proton-coupled electron-transfer reactions in the OEC. Supported by NIH grant GM32715. PS4.20 Substrate Water binding in the OEC: The Message is Clear from Flashes, Mutants and Strontium Effects. W Hillier (Australian National University), S Singh (Australian National University), A Boussac (CEA Saclay), R Debus (University of California at Riverside), I McConnell (Australian National University), T Wydrzynski (Australian National University) Photosynthetic water splitting is a reaction catalysed by Photosystem II and generates a profound footprint for life on earth by releasing ~9,000 ton/s of O2 as by-product. Recent structural insights from X-ray diffraction have constrained mechanistic proposals but have not

178 14th Photosynthesis Congress - PS07 definitively identified substrate water binding sites. In this poster we will outline our experiments using time resolved 18O exchange to follow the exchange behaviour of the two substrate water molecules. Though the use of point mutants in Synechocystis PC 6803 we have perturbed the Mn ions in the cluster at several locations in order to probe substrate water binding and have performed a thermodynamic analysis of water exchange in normal Ca and in vivo Sr reconstituted PSII centres. These results will provide some insights into where the catalytic site of water oxidation actually is located within the Mn4Ca cluster of the OEC in Photosystem II. PS4.21 Photosystem II – details of cofactor-protein interactions in the light of the 3.0 Å resolution crystal structure M Broser (Technische Universität Berlin), J Kern (Technische Universität Berlin), K Zimmermann (Charite-Universitätsmedizin), J Yano (Lawrence Berkeley National Laboratory), V Yachandra (Lawrence Berkeley National Laboratory), B Loll (Free University Berlin), J Biesiadka (Free University Berlin), W Saenger (Free University Berlin), A Zouni (Technische Universität Berlin) The large membrane intrinsic protein complex Photosytem II (PSII) catalyses light-driven charge separation accompanied by the oxidation of water during oxygenic photosynthesis. In this contribution we will discuss the recent X-ray crystallographic structural model at 3.0 Å resolution [Loll et al. (2005) Nature 438, 1040-1044] in relation to various spectroscopic and biochemical data. Special emphasis will be given on: a) the role of lipids as a new class of cofactors involved in PSII structure and function; b) the quinone binding pockets, the proposed diffusion pathway of quinones into the QB binding site and the possible binding of additional quinones within the complex; c) the catalytic center for light-induced water oxidation (the Mn4-Ca cluster). The arrangement of metal cations in the cluster, their coordination and protein surroundings are discussed with regard to spectroscopic and mutagenesis studies. Limitations of the presently available structural data are pointed out and the very recent results of X-ray spectroscopy (XANES and EXAFS) on PSII single crystals [Yano et al. (2006) Science 314, 821-825] are described in comparison with the X-ray crystallographic model of the water oxidizing complex.

are radiation damage and anisotropy in the crystal, that invoke deterioration of electron density maps. An outlook on possible strategies for further improvements of the structural data will be given. PS4.23 X-ray diffraction analysis to investigate chloride binding in the OEC K Maghlaoui (Imperial College London), J Kargul (Imperial College London), J Murray (Imperial College London), J Barber (Imperial College London) Photosystem II (PSII) uses light energy to oxidise water, the resulting electrons being used to reduce plastoquinone to plastoquinol. The thermodynamically demanding reaction takes place at a multinuclear metal site containing 4 manganese ions and a calcium ion. The reaction is inhibited by chloride depletion, but some other anions, notably bromide can restore activity in isolated particles. It has been proposed that chloride interacts directly with the metal ions of the water oxidation centre but the precise way in which chloride functions within the reaction cycle is unknown. The crystal structures of PSII available so far have not been of sufficient resolution to locate chloride ions unambiguously. Recent EXAFS results on particles purified from spinach suggest that chloride is not a primary ligand to the metal cluster (Haumann et al 2006). To investigate this directly by X-ray diffraction analysis we have used bromide substituted PSII, as bromine has an accessible X-ray absorption edge. We present results results from 3D crystals of cyanobacterial PSII, soaked with bromide, that suggest 2 possible sites for the binding of this halide within 7 Å of the oxygen evolving centre. The anomalous signal of the bromine K-edge allows for sensitive detection of atomic sites even at intermediate resolution. M Haumann et al, (2006). Bromide does not bind to the Mn4Ca complex in its S1 state in Cl(-)-depleted and Br(-)- reconstituted oxygen-evolving photosystem II: evidence from X-ray absorption spectroscopy at the Br K-edge. Biochemistry, 45(43):13101-13107. PS4.24 Insights Into The Photosynthetic Water Oxidation Mechanism: Determination of the Dissociation Constants for the Substrate Water Binding Sites from 18O Isotope Exchange Measuremen

PS4.22 X-ray crystallographic analysis of PSII from T. elongatus at 3.0 Å resolution

I McConnell (The Australian National University), L Andreasson (Göteborg University), T Wydrzynksi (The Australian National University), W Hillier (The Australian National University)

A Guskov (Free University Berlin), M Broser (Technical University Berlin), A Gabdulkhakov (Free University Berlin), J Kern (Technical University Berlin), A Zouni (Technical University Berlin), B Loll (Free University Berlin), J Biesiadka (Free University Berlin), W Saenger (Free University Berlin)

The exact nature of the substrate water binding sites and O-O bond formation during the oxidation of water in Photosystem II is unknown. We have employed 18O water isotope exchange measurements to address this problem. For samples in the S3 state the rate of 18O water exchange

Photosytem II (PSII) is a homodimeric protein-cofactor complex embedded in the thylakoid membrane that catalyses light-driven charge separation accompanied by the oxidation of water during oxygenic photosynthesis. In this contribution we will discuss the results of the X-ray crystallographic analysis of PSII from T. elongatus. The highest resolved published structure (at 3.0 Å, Loll et al. 2005) revealed the locations of and interactions between 19 protein subunits per monomer, and the positions of more than 75 cofactors, including the previously not described 14 lipids, which represent a new class of cofactors in PSII. This structural model allows for a detailed analysis of the role of cofactors in operation of this sophisticated complex – like electron and energy transfer, photoprotection and maintenance of spatial architecture. However, the quality of data suffers from different causes, the main ones

can be determined by measuring the amount of 18O incorporated into the O2 produced as a function of time between the rapid injection of enriched H218O and a turnover flash. At m/e=34 (16,18O2) two distinct kinetic phases (one fast and one slow) are observed while only one phase (slow) is found at m/e=36 data (18,18O2). The data can be fit to pseudo first order kinetics, with the 18,18O2 data fit to a single exponential function and the 16,18O2 data was fit to a bi-exponential function. The two distinct phases imply two separate and chemically distinct binding sites for substrate water. In a new approach each site is considered as involving an equilibrium with either a bound H216O or H218O. A model constructed from this approach infers specific koff and kon rate constants. These are used to derive the dissociation constants for the two substrate

179 14th Photosynthesis Congress - PS07 water binding sites. Surprisingly different values further support the concept that the two water binding sites are heterogeneous in character. PS4.25 Quantum requirement for oxygen evolution in photosystem II. New experimental data and theoretical solutions M Fragata (Univ. Québec, Trois-Rivières), V Viruvuru (Univ. Québec, Trois-Rivières) The determination of the quantum requirement for oxygen evolution (QRO2 ) in photosystem II (PSII) has given rise to controversial viewpoints since the publication by Warburg and Negelein of their 1923 paper. On the basis of investigations did mainly by Arnold, Emerson and Warburg from 1923 to 1969, Govindjee showed (Photosynth. Res. 1999, 59, 249-254) that the most probable QRO2 values are 8-12 quanta of light per oxygen molecule evolved. However, a review of old and recent literature shows that the QRO2`s frequently reported for various plant materials (e.g., algae cells, plant leaves) are between about 6-7 and 23. To clarify this question further, we performed a twofold inquiry. First, we studied the QRO2 in isolated PSII particles (PSIIp) and thylakoid membranes (Tm) from barley (Hordeum vulgare) in low light intensity conditions, i.e., 10-40 micromol of photons/(m2.s). The results show that QRO2 is approximately 12.5 in both isolated PSIIp and Tm. Secondly, using recent X-ray crystallography models of the PSII complex (e.g., 2AXT.pdb: Loll et al. Nature 2005, 438, 1040-1044), we demonstrated that the minimum theoretical QRO2 in the PSII reaction center is 8 photons per oxygen molecule evolved. Finally, we conclude from the afore discussion that the differences between the theoretical QRO2 = 8 calculated in this work and the experimental QRO2 = 12.5 that we observed in isolated PSII particles might result from the transient (or accessory) function of TyrD and the PD2 or ChlD2 molecules during energy and electron transfer in the PSII reaction center. (Work supported by NSERC Canada) PS4.26 A detailed structural model for the eukaryotic LHCII-PSII supercomplex

PS4.27 S-state dependence of turnover misses in the OEC probed by EPR spectroscopy of the individual S-states G Han (Molecular Biomimetics, Department of Photochemistry and Molecular Science, Ångström Laboratory, Uppsala University, Box 523, SE-751 20 Uppsala, Sweden), S Morvaridi (Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden), F Ho (Molecular Biomimetics, Department of Photochemistry and Molecular Science, Ångström Laboratory, Uppsala University, Box 523, SE-751 20 Uppsala, Sweden), F Mamedov (Molecular Biomimetics, Dep Photosynthetic water oxidation in Photosystem II (PSII) takes place at the CaMn4 cluster, during which the cluster cycles through five oxidation states, S0-S4, in the so-called S-cycle. Each S-state exhibits characteristic EPR signals. While perfectly synchronised S-state transitions in all PSII centres would lead to a continuous period-of-four oscillation of these signals, dampening occurs due to misses during individual flash-induced turnovers. We analysed this using the S1 and S3 split signals together with the S2 and S0 state multiline signals in order to quantify the proportion of centres in each S-state after the application of turnover flash(es). Each S-state formation could therefore be probed individually. Quantification of S-states using the split signals provides a novel probe for S-cycle efficiency. We found two types of misses, “real” and “apparent”. Real misses are caused by inherent equilibria in the intermediate steps during charge separation and are highly S-state dependent. These misses occur in the S2?S3 and S3?S0 transitions, involving ~25% of the centres at 0–2 ºC. By contrast, our results strongly indicate that there are no real misses in the S0?S1 and S1?S2 transitions at these temperatures. Apparent misses, on the other hand, reflect direct recombination reactions between a reduced species on the acceptor side and an oxidised species on the donor side. Such donor side species may include the CaMn4 cluster in the S2 or S3 states, YZ• and P680+. This miss fraction is temperature dependent and is high above 20ºC, leading to significant scrambling of S-states during flash-induced turnovers.

J Barber (Imperial College) Photosystem II (PSII) core complex X-ray structures have so far been derived from preparations isolated from prokaryotic cyanobacteria. These have provided significant information for understanding PSII functionality, including water splitting, but as yet no high-resolution structure has become available for eukaryotic PSII. Such knowledge will be required to reveal spatial excitonic linkages between the intramembrane light-harvesting components (LHCII) and the P680 reaction centre present. Furthermore, subtle differences in the eukaryotic water splitting environment also need to be investigated. To this end we have utilised the published X-ray structures of a cyanobacterial PSII core (Ferreira et al., 2004), LHCII (Standfuss et al., 2005), PsbP (Ifuku et al., 2004) and PsbQ (Calderone et al., 2004) proteins to construct a model of eukaryotic LHCII-PSII supercomplex using 16 to 17 Å resolution 3D density maps from spinach and Chlamydomonas, as determined by electron cryo-microscopy and single particle analysis. We tentatively identify the positioning of chlorophylls, in order to consider energy transfer pathways between the different subunits, and model the positioning of the extrinsic protein subunits of the water splitting site. The results will be compared with the latest developments in X-ray crystallographic data derived from cyanobacterial PSII. V. Calderone et al. EMBO Rep. 4 (2003) 900-905. K.N. Ferreira et al. Science 303 (2004) 1831-1838. K. Ifuko, et al. EMBO Rep. 5 (2004) J. Standfuss et al. EMBO J. 24 (2005) 919-928.

PS4.28 Eight steps towards dioxygen formation - Time-resolved X-ray experiments and a basic reaction cycle of photosynthetic water oxidation H Dau (Freie Univ. Berlin), M Haumann (Freie Univ. Berlin), A Grundmeier (Freie Univ. Berlin), P Loja (Freie Univ. Berlin), B Suess (Freie Univ. Berlin), J Buchta (Freie Univ. Berlin), I Zaharieva (Freie Univ. Berlin) The classical S-state cycle was proposed by Bessel Kok in 1970. It soon became a central paradigm in photosynthesis research and today is found in numerous student textbooks. The four semi-stable S-states of the Kok cycle (S0 to S3) have been well characterized whereas the postulated transient formation of the S4-state in the S3->S0 transition remained enigmatic. In time-resolved X-ray absorption experiments, an intermediate formed prior to dioxygen formation has been detected and assigned to an S4-state formed by deprotonation of the Mn4Ca complex of Photosystem II [Haumann et al., 2005, Science 310, 1019]. In time-resolved studies on the deuterium influence, we now obtained first evidence for a further reaction intermediate transiently formed in the S2->S3 transition. The existence of this additional reaction intermediate supports a basic reaction cycle of alternate electron-proton removal from the Mn4Ca complex [Dau and Haumann, 2006, Science 312, 1471]. This

180 14th Photosynthesis Congress - PS07 novel reaction cycle of dioxygen formation and possible mechanistic implications are discussed.

Possible role of bicarbonate in the photosynthetic water oxidation V Klimov (Institute of Basic Biological Problems, Russian Academy of Sciences)

PS4.29 Photosystem II function and bicarbonate Y Feyziyev (Institute of Botany, 40 Patamdar Shosse, Baku AZ-1073, Azerbaijan) Photosystem II (PSII) is a multisubunit protein complex, localized in thylakoid membrane of the oxygenic organisms and catalyzes light-induced electron transfer from water-splitting reactions to plastoquinol with concomitant release of molecular oxygen to the atmosphere. The potential required for water splitting accumulates in its Ca(Mn)4 cluster through the S-state cycle driven by light-induced charge transfer through the Ca(Mn)4-YZ-P680-QAFe network. It is known that bicarbonate (BC, HCO3-) is needed for PSII function. Its presence accelerates the electron flow in PSII. Although large amounts of the experiments have been reported, the function of bicarbonate in PSII is still unclear. Either the acceptor (on the QAFe level) side or donor (on the Ca(Mn)4 level) side of PSII were suggested earlier as a target for BC action. The object of present work is a place and function of bicarbonate in PSII. Comparative studies performed between native O2-evolving PSII and PSII lacking Ca(Mn)4 in presence of exogenous donors show that BC stimulate FV, slowing electron flow in PSII in its acceptor side. In addition, presence of BC does not stimulate the photoreduction DCPIP via PSII. A comparison of above results with results obtained from DCMU-poisoned PSII suggests that the acceptor side of PSII is a BC-functional site, while the effects, ascribed previously, to the PSII donor side function of BC arises through the acceptor side action of BC. A possible new mechanism for bicarbonate function in PSII is proposed. PS4.30 A view of the damped flash-induced period-four oscillations of oxygen evolution: The interredox S-states J Jablonsky (Palacký University, Faculty of Science, Department of Experimental Physics, Laboratory of Biophysics) The damped flash-induced period-four oscillations of the oxygen evolution, catalyzed by photosystem II (PSII), was empirically described in the Kok model by introducing two parameters, the so-called misses and double hits. 30 years later after that, positive proof of the mechanisms hidden within these damping parameters is still lacking. In this respect, a reliable simulation tool would be useful to gain insight into these parameters. Therefore we analyzed these oscillations with the aid of a kinetic model of the PSII based on the experimentally accessible rates of the forward and backward reactions involved in the successive light induced turnover of the PSII. We shown that in the kinetic models heretofore-unconsidered four transient steps of the so-called interredox S-states, each time-associated with SiYZ• formation (i = 0, 1, 2, 3; YZ• is neutral radical), enable for the first time the description of oxygen oscillations without consideration of the misses and the double hits. The assumption of the interredox S-states is, together with S2QA- charge recombination, prerequisite for simulation of the flash-induced period-four oscillations of the chlorophyll fluorescence. Moreover, new kinetic model of PSII is suitable for testing of many others experimental data such oxygen evolution in dependency on the flash frequency, herbicide activity, high illumination or some site-directed mutagenesis of PSII. [Supported by grant MSM 6198959215] PS4.31

QA and QB, in formate-inhibited PS II; the non-heme Fe between QA and QB plays an essential role in BC binding. Strong evidence for BC requirement within the water-oxidizing complex (WOC) (both O2-evolving and assembling from apo-WOC and Mn(II)), of PSII is presented. The following explanations for the involvement of BC in the events within the WOC are considered: 1) BC facilitates re-assembly of the WOC from apo-WOC and Mn(II); 2) BC is a constituent of the WOC essential for its function and stability; it may serve as a direct ligand to the inorganic core of the WOC modifying the redox properties of Mn; 3) BC is an easily accessible base (with an appropriate pK) involved in the removal of protons released during water oxidation. Comparative studies of electrochemical and functional properties of complexes of Mn(II) and Mn(III) with BC and other carboxylates (formate and acetate) show that the unique capability of BC to initiate the assembly of the tetramanganese cluster of the WOC from Mn(II) and apo-WOC-PSII can be attributed to formation of electroneutral, easily oxidizable Mn/BC-complexes that serve as building blocks for the WOC. PS4.32 Access channels and methanol binding site to the CaMn4 cluster in Photosystem II based on solvent accessibility simulations, with implications for substrate water access F Ho (Molecular Biomimetics, Department of Photochemistry and Molecular Science, The Ångström Laboratory, Uppsala University, Box 523, SE-752 10 Uppsala, Sweden), S Styring (Molecular Biomimetics, Department of Photochemistry and Molecular Science, The Ångström Laboratory, Uppsala University, Box 523, SE-752 10 Uppsala, Sweden) Biochemistry 45:7617-7627) we showed that the EPR split signals for the S1-, S3-, and S0-states of the CaMn4 cluster in Photosystem II (PSII) were affected by methanol in a concentration dependent manner, and proposed that methanol binds to one and the same Mn ion in all S-states. We have tested this hypothesis by simulating methanol solvent access surfaces based on the latest 3.0 Å resolution crystal structure. Two channels of the correct dimensions were found to make contact with the CaMn4 cluster, at the ions 3Mn and 4Mn. The other two Mn ions showed no methanol accessibility. We propose here that 3Mn is the ion to which methanol binds, based on these simulation results and spectroscopic data in the literature. In addition, a continuously open channel from the luminal side of PSII was found to lead all the way to the Ca ion of the CaMn4 cluster, but did not make contact with any Mn ions. However, this channel was found to be separated only by a small gap from one of the other channels that lead to Mn ions. Interestingly, the gap was formed by the mechanistically very significant residues. Given the high degree of similarity between the methanol access surfaces and surfaces analogously calculated for water access, we discuss the possible routes of substrate water access to the CaMn4 cluster, including the possibility of a gating mechanism across the above gap. PS4.33 Isolation of the diatom PSII retaining high oxygen-evolving activity from a marine diatom, Chaetoceros gracilis R Nagao (Department of Biology, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3, Shinjuku-ku, Tokyo, 162-8601 Japan), T Suzuki (Biomolecular Characterization Team, Discovery Research Institute, RIKEN, Hirosawa 2-1, Wako, Saitama, 351-0198 Japan JST

181 14th Photosynthesis Congress - PS07 CREST), N Domae (Biomolecular Characterization Team, Discovery Research Institute, RIKEN, Hirosawa 2-1, Wako, Saitama, 351-0198 Japan JST CREST), A Okumura (Department of Integrated Science of Physics and Biology, College of Humanities and Science, Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan) Diatoms are one of the most important constituents of phytoplankton in aquatic ecosystems and the global carbon cycle. In spite of their significance, little is known about Photosystem II (PSII) in diatoms or other chromophytic chlorophyll a/c containing algae. Detailed studies of diatom PSII have been blocked at the level of breaking open the silica-based rigid cell wall without damaging intracellular structure. In this study, thylakoid membranes retaining oxygen-evolving activity were prepared from a marine diatom, Chaetoceros gracilis, after disruption of the cells by freeze-thaw. We succeeded in partial purification of diatom PSII (PSII particles) by differential centrifugation of the thylakoid membranes treated with Triton X-100. The diatom PSII particles showed the oxygen-evolving activity of about 850 µmol O2 (mg Chl)-1h-1. The PSII particles contained fucoxanthin chlorophyll a/c-binding proteins in addition to main intrinsic proteins of CP47, CP43, D2, D1 and a large subunit of cyt b559, indicating that the PSII particles are similar to BBY-type PSII membrane particles from spinach. Five extrinsic proteins were released by alkaline Tris-treatment of the diatom PSII particles. These extrinsic proteins were identified to be PsbO, PsbQ’, PsbV and PsbU and a new hypothetical protein by their immunoblotting analysis and N-terminal amino acid sequences. This indicates that the diatom PSII contains the red algal-type extrinsic proteins, corresponding with the fact that chromophytic chlorophyll a/c containing algae such as diatoms and brown algae resulted from red algal secondary endosymbiosis. A remarkable feature of the diatom PSII is to contain a new extrinsic protein. PS4.34 Identification of functional domain of PsbU in red algal PSII by site-directed mutagenesis M Sano (Department of Biology, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3, Shinjuku-ku, Tokyo, 162-8601 Japan), A Okumura (Department of Integrated Sciences in Physics and Biology, College of Humanities and Science, Nihon University, 3-25-40 Sakurajousui, Setagayaku, Tokyo 156-8850, Japan), T Suzuki (Biomolecular Characterization Team, Discovery Research Institute, RIKEN, Hirosawa 2-1, Wako, Saitama, 351-0198 Japan JST CREST), M Iwai (Department of Applied Biological Science Red algal Photosystem II (PSII) purified from Cyanidium caldarium contains PsbO, PsbQ’, PsbV and PsbU as the extrinsic proteins. Among the four extrinsic proteins, PsbU functions to minimize the chloride and calcium requirement of oxygen-evolving activity. In the previous paper (1), we showed that the C-terminal peptides of PsbU constituted an important domain for its function. In this study, we first constructed PsbU lacking one or two residues from C-terminus (K93), and reconstituted their PsbU mutants with red algal PSII reconstituted with PsbO, PsbQ’ and PsbV in order to identify the amino acid in the C-terminal peptides for retaining the function. From recovery of oxygen-evolving activity in the absence of NaCl and CaCl2 of these reconstituted PSII, we concluded that Tyrosine-92 (Y92) of second residue from C-terminus in PsbU was important for the function. Second, we constructed a series of site-directed mutants of PsbU in order to determine the function of Y92. The substitution of Y92 with glycine, threonine, asparatic acid, lysine or arginine totally abolished the function of PsbU. In contrast, the substitution of Y92 with phenylalanine retained the function of PsbU. These indicate that aromatic structure of Y92 in PsbU is important for the maintenance of functional structure of PsbU. Now, we are furthermore constructing various mutants of PsbU. On the basis of these results, we will discuss the functional structure of PsbU in relation to the interaction of intrinsic proteins of PSII.

(1) Okumura et al. (2001) Plant Cell Physiol. 42: 1331-1337. PS4.35 Cloning and sequence analyses of the five extrinsic proteins in diatom PSII A Okumura (Department of Integrated Science of Physics and Biology, College of Humanities and Science, Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan), K Nakazato (Department of Integrated Science of Physics and Biology, College of Humanities and Science, Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan), S Yamagoe (Department of Bioactive Molecules, National Institute of Infectious Diseases, 23-1 Toyama, Shinjyuku-ku, Tokyo 162-8640, Japan) Recently, Enami et al. succeeded in the preparation of Photosystem II (PSII) particles having high oxygen-evolving activity from a marine diatom Chaetoceros gracilis, and found that the diatom PSII particles contained five extrinsic proteins of PsbO, PsbQ', PsbV, PsbU and a new hypothetical protein. In this study, we cloned and sequenced the genes encoding these five extrinsic proteins. N-terminal amino acid sequences of the five extrinsic proteins released by alkaline Tris-treatment of the diatom PSII particles were determined. Oligonucleotides synthesized on the basis of their N-terminal amino acid sequences were used as primers for degenerate RT-PCR. The full-length of cDNAs that encode PsbO, PsbQ', PsbU, and the new hypothetical protein were obtained by RNA ligase mediated-RACE. On the other hand, DNA fragment containing psbV was isolated using inverse PCR. The results show that the former genes are encoded in the nuclear genome, while psbV is encoded in the plastid genome. The deduced proteins consist of 307 (PsbO), 211 (PsbQ'), 151 (PsbU), 179 (the hypothetical protein) and 163 (PsbV) amino acids, including 59, 56, 58, 55, and 26 amino acid-targeting peptides at the N-terminal region, and have predicted molecular masses of the mature proteins of 26.7 kDa, 17.1 kDa, 10.1 kDa, 13.3 kDa, and 15.5 kDa, respectively. Their targeting peptide analysis showed that all of the extrinsic proteins have typical features for thylakoid transfer peptides. This suggests that the new hypothetical protein is one of the luminal extrinsic proteins functioning in oxygen evolution. PS4.36 Effects of P680 ligand substitution in the oxygen-evolving Photosystem II of Thermosynechococcus elongatus M Sugiura (Osaka Prefecture University) The chlorophyll dimer P680 traps the light energy and its redox potential (E’0> +1.2 V) fixes the driving force available for water oxidation. In this study, to know the effect of a ligand of D1 side of P680 (PD1) to its redox potential in PSII, we analyzed PSII core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus in which a normal His ligand of PD1 was substituted for either Ala or Gln by site-directed mutagenesis. Both mutant cells grew photo-autotrophically. However, the amounts of phycobilliproteins in the mutants were smaller than in the WT*. The energy transfer from phycocyanin to PSII via allophycocyanin in both mutants was not efficient as that in WT*. Purified PSII core complexes were fully active in oxygen-evolution as high as 4000 µmoles O2/mg Chl/h, and kinetics of oxygen release of both mutants were the same as that of WT*. FTIR spectra shows molecular stractural changes of ligand of PD1. Flash-induced absorption changes and temperature shifts in themoluminescence spectra suggested possible changes in the redox potential of P680+/ P680. However, the changes were not so large as the results in the mutants of Synechocystis. These results suggested that 1) the ligand amino acid of PD1 does not effect to the redox potential of P680+/ P680 or 2) PD1 and PD2 do not correspond to a primary electron

182 14th Photosynthesis Congress - PS07 donor P680.

PS4.37 Cytochrome b-559 is important for modulating electron transfer on the acceptor side of Photosystem II and for photoprotection during assembly of the Mn4Ca complex M Hamilton (Imperial College London), Z Deak (Hungarian Academy of Sciences), E Franco (Imperial College London), I Vass (Hungarian Academy of Sciences), P Nixon (Imperial College London) Cytochrome b-559 (Cyt b-559) is an essential part of photosystem II (PSII), the light driven water-plastoquinone oxidoreductase found within the thylakoid membrane of oxygenic photosynthetic organisms. Cyt b-559 is a heterodimer composed of a and ß subunits of 9 kDa and 6 kDa respectively. The function of Cyt b-559 is still unknown although most hypotheses have centred on a role in photoprotection. To investigate the physiological role of Cyt b-559 we have constructed a PsbE-H23C mutant of C. reinhardtii in which the His-ligand to the haem provided by the the a subunit has been replaced by Cys. The H23C mutant assembles PSII at 15-20% of wild type (WT) levels, is able to evolve oxygen at around 20% of WT rates but is unable to co-ordinate haem in the membrane. More detailed analyses of PSII electron transfer indicate that reduction of QB is impaired in the mutant. The mutant is also more sensitive to photoinhibition than WT cells especially under conditions when the Mn cluster is being photoactivated. Overall our results indicate that Cyt b-559 plays a role in PSII electron transfer, PSII photoprotection and PSII repair. PS4.38 FTIR Study on the Proton Release Pattern during Water Oxidation in Photosystem II Core Complexes from Thermosynechococcus elongatus H Suzuki (Institute of Materials Science, University of Tsukuba), M Sugiura (Department of Plant Bioscience, Osaka Prefecture University), T Noguchi (Institute of Materials Science, University of Tsukuba) Photosynthetic water oxidation takes place in the water oxidizing center (WOC) of photosystem II (PSII) through a light-driven cycle of five intermediates called S states (S0-S4). During this S-state cycle, two water molecules are oxidized to split into four protons and one oxygen molecule. Although oxygen is known to evolve during the S4?S0 transition, the definite conclusion has yet to be reached about the proton release pattern for individual S-state transitions. In this study, we have estimated the proton release pattern during water oxidation by means of flash-induced Fourier transform infrared (FTIR) difference spectroscopy. We have used the PSII core complexes of Thermosynechoccocus elongatus from its TyrD-less mutant, in which D2-160Tyr was replaced with Phe, to accurately estimate proton release from WOC. The core complexes were suspended in a Mes buffer (pH 6.0) at a high concentration (200 mM). Twelve consecutive flashes were applied to the sample and FTIR difference spectra upon individual flashes were recorded. The obtained spectra included the signals of the Mes buffer that trapped protons from WOC as well as protein signals. The protein bands were eliminated from the spectra by subtracting the corresponding spectra recorded in a buffer of deuterated-Mes (D13-Mes). The flash-number dependence of the intensities of the buffer bands showed a typical period-four oscillation. Simulation of the oscillation indicated that the proton release pattern was approximately 1.0 : 0.3 : 1.0 : 1.7 for the S0?S1?S2?S3?S0 transitions, being consistent with the generally accepted 1:0:1:2 pattern.

PS4.39 Roles of lipids in photosystem II: Possible differences between cyanobacteria and higher plants J Shen (Graduate School of Natural Science and Technology, Okayama University), J Leng (Graduate School of Natural Science and Technology, Okayama University), I Sakurai (Graduate School of Arts and Sciences, The University of Tokyo) Lipids are one of the important constituents of membrane protein complexes, and 14 lipid molecules have been identified in the crystal structure of photosystem II (PSII) from a thermophilic cyanobacterium. In order to study the roles of lipids in PSII, we treated PSII dimer complexes from cyanobacteria and higher plants with phospholipase A2 (PLA2) and lipase, and compared their effects on PSII activity and organization. PLA2- and lipase-treatments of PSII from a thermophilic cyanobacterium Thermosynechococcus vulcanus decreased the oxygen-evolving activity by 40% and 16%, respectively. The same treatments of spinach PSII, however, decreased the activities by more than 80% in both cases. Analysis of lipid contents indicated that PLA2-treatment decreased PG to around 1 molecule per PSII, and lipase-treatment decreased MGDG to less than 1 molecule per PSII, in spinach PSII, whereas the numbers of PG and MGDG molecules remained after PLA2- and lipase-treatments are 3 and 5, respectively, in T. vulcanus PSII. These results indicate that the association of lipids in spinach PSII is significantly weaker than that in the thermophilic cyanobacterium, which leads to the significantly larger decrease of activity after both treatments in spinach PSII. The differences in lipid binding may account for, at least in part, the significantly larger instability of the spinach PSII compared with the thermophilic cyanobacterial PSII as usually observed. PS4.40 Purification and crystallization of photosystem II complex from a red alga Cyanidium caldarium. H Adachi (Graduate School of Natural Science ,Okayama University), I Enami (Department of Biology, Tokyo University of Science), T Henmi (Graduate School of Science,Osaka City University), N Kamiya (Graduate School of Science,Osaka City University), J Shen (Graduate School of Natural Science ,Okayama University) Photosystem II (PSII) consists of around 20 subunits, most of them are highly conserved from prokaryotic cyanobacteria to eukaryotic organisms. There are, however, some apparent differences in the extrinsic proteins involved in oxygen evolution among different organisms. For example, prokaryotic cyanobacterial PSII contains PsbO, PsbU, PsbV as the major extrinsic proteins, whereas PSII of red algae, one of the eukaryotic algae most closely related to cyanobacteria, contains, in addition to these three proteins, a 20 kDa protein as the fourth extrinsic protein. Furthermore, higher plant PSII has PsbO, PsbP, PsbQ as its extrinsic proteins. So far, the crystal structure of PSII from cyanobacteria has been reported, but no reports have been published on the structure of any eukaryotic PSII. In order to elucidate the structure of PSII from eukaryotic organisms and their differences with cyanobacterial PSII, we initiated the crystallization trials with PSII from an acidophilic, thermophilic red alga Cyanidium caldarium which grows at pH 2-3 and 42-44°C. In order to do so, we improved the previously published procedure for purification of PSII from the red alga, which yielded a highly purified PSII dimer preparation with high oxygen-evolving activity. We obtained three-dimensional crystals of the dimeric PS II in several conditions, and will report the characteristics of the crystals obtained. PS4.41 Relationship between Main Reaction and Side-Pathway Reaction at Cryogenic Temperature in Intact Photosystem II

183 14th Photosynthesis Congress - PS07

C Zhang (Institute of Chemistry, Chinese Academy of Sciences), H Bao (Institute of Chemistry, Chinese Academy of Sciences), Y Ren (Institute of Chemistry, Chinese Academy of Sciences), J Zhao (Institute of Chemistry, Chinese Academy of Sciences) TyrZ oxidation by P680+∙ formed after primary charge separation is the main electron transfer reaction on donor side of intact PSII, while P680+∙ can also drive the oxidation of Car/ChlZ/Cytb559. The latter is the so called side-pathway reaction. It was suggested that the main reaction and side-pathway reaction competed with each other in PSII. However, detailed characterization on the relationship between these two types of reactions remains unclear because the rate and quantum yield of side-pathway reaction are much less than that of the main reaction at physiological conditions. Here we study the possible relationship between these two types of reactions by monitoring the yield and kinetics of TyrZ and Car/ChlZ/Cytb559 oxidation, respectively, in intact PSII at cryogenic temperature with the EPR method developed recently (ref. Zhang, C.; Styring, S. Biochemistry 2003, 42, 8066-8076; Zhang, C.; Boussac, A.; Rutherford, A. W. Biochemistry, 2004, 43, 13787-13795). We found that these two types of reactions did not compete with each other; instead, they may occur separately in different fractions of PSII reaction centers. One may argue that it may arise from the possible cooling-induced heterogeneity in the sample. However, we have evidence that the selection of main or side-pathway reaction in difference reaction centers are directly related to the activities of the intact PSII samples at room temperature. PS4.42 Crystal Structure Analysis of a Mutant Photosystem II Complex Lacking PsbI from Thermosynechococcus vulcanus. K Kawakami (Graduate School of Natural Science and Technology/Fuculty of Science; Okayama University), Y Kawabata (Graduate School of Natural Science and Technology/Fuculty of Science; Okayama University), T Henmi (Graduate School of Natural Science, Osaka City University), M Iwai (Fuculty of Science and Technology, Tokyo University of Science), T Suemasu (Graduate School of Natural Science and Technology/Fuculty of Science; Okayama University), M Ikeuchi (Graduate School of Arts and Science, The University of Tokyo) PsbI is one of the low molecular mass subunits of PSII with an approximate molecular mass of 4 kDa and single trans-membrane helix, and is tightly associated with PSII reaction center, since it is present in the isolated reaction center complex. PsbI is located in the vicinity of D1 and CP43 in the current crystal structure. Deletion mutants lacking PsbI have been constructed with cyanobacteria, green alga C. reinharadtii, and higher plant tobacco. All these mutants were capable of photo-autotrophic growth, with the mutants of C. reinharadtii and tobacco showing larger effects on the PSII activity and stability, and higher sensitivity toward photoinhibition, than the cyanobacterial mutant. In order to further study the role of PsbI from a structural point of view, we purified PSII dimer complexes from a PsbI deletion mutant of Thermosynechococcus vulcanus. PSII dimer purified from the PsbI-deletion mutant showed slightly lower activity than that of wild-type. We crystallized the PSII dimer from the PsbI-deletion mutant by hanging drop vapor diffusion method, and collected X-ray diffraction data to a 3.45 Å resolution with a synchrotron radiation source. Using previously determined phase information for wild-type PSII, we solved the structure of the mutant PSII. The resulted difference Fourier map between the wild type and mutant PSII indicated that the electron density corresponding to PsbI disappeared in the mutant, confirming the position

and deletion of this subunit in the mutant. We will also report the characteristics of PSII from the PsbI-deletion mutant. PS4.43 Functional Characterization of PsbO, the PSII Manganese Stabilizing Protein C Yocum (University of Michigan), H Popelkova (University of Michigan), A Commet (University of Michigan) Efficient oxygen evolution by eukaryotic PSII requires the extrinsic subunits PsbO, PsbP and PsbQ. While additions of calcium and chloride can reconstitute this activity after extraction of the latter two subunits, removal of PsbO has detrimental effects on both activity and on stability of the Mn cluster. It has also been shown that in plants and algae, the presence of this protein is required for stable assembly of PSII. A number of studies to characterize PsbO structure and function in plants have shown that two copies of this subunit are present, in contrast to the situation in cyanobacteria, where a single copy is found. We have characterized the function of spinach PsbO using a series of recombinant proteins that were overexpressed in E. coli. Sequential mutations (introduction of Met residues) were employed to engineer a series of N-terminal truncations. These proteins rebind to PSII with altered stoichiometries (2, 1, or <<1 copy/reaction center; Popelkova, et al., Biochemistry 41, 10038 (2002)). Results of a series of reconstitution experiments show that a single copy of PsbO bound to PSII is sufficient to restore stability to the Mn cluster, as evidenced by retention of activity after long periods of incubation in darkness. Maximum restoration of activity and efficient retention of chloride under steady state conditions, on the other hand, requires both copies of PsbO. PS4.44 Time-resolved delayed chlorophyll fluorescence to study the donor-side of phtotsystem II J Buchta (Freie Universität Berlin), T Shutova (Umea University), I Zaharieva (Freie Universität Berlin), G Samuelsson (Umea University), H Dau (Freie Universität Berlin) Inter alia this method can provide insights in the functionally important inner-protein proton movements, which are hardly detectable by conventional spectroscopic approaches. The delayed emission of chlorophyll fluorescence was measured on both wild type and mutant of green algae PSII membrane particles in the time domain from 10 µs to 60 ms after each flash of a train of nanosecond laser pulses. The influence of the PSII-associated carbonic anhydrase (CA) on the donor side reactions was studied for a Chlamydomonas reinhardtii wild type and CA-free mutant. PS4.45 The photosynthetic Mn complex in its reaction cycle: An attempt to obtain pure FTIR difference spectra for the four transitions between semi-stable S-states B Süss (Freie Universität Berlin), H Dau (Freie Universität Berlin) Mn4Ca complex bound to the proteins of Photosystem II (PSII). The water-oxidation cycle of the Mn complex may involve nine distinct states of the Mn complex [Dau and Haumann, 2006, Science 312, 1471], but only four of these are stable for several seconds (S2, S3), several hours (S0), or fully dark-stable (S1). The four transitions between these semi-stable S-states have been studied by a variety of spectroscopic techniques. Due to the pioneering work of several groups, today also FTIR spectroscopy plays a prominent role¾frequently in fruitful conjunction with mutagenesis studies (see, e. g., studies of Debus and

184 14th Photosynthesis Congress - PS07 coworkers). After application of ns or µs flashes of saturating light and subsequent FTIR measurements, difference spectra can be calculated which are mostly interpreted as S-state difference spectra. However, potential complications come from (i) acceptor side contributions to the FTIR spectra, (ii) PSII with a partially defect donor side, and (iii) significant mixing of S-state populations caused by so-called miss events. We have measured FTIR spectra on PSII membrane particles in the intervals between Laser flashes of an extended flash sequence. Taking into account also complementary results obtained by time-resolved prompt and delayed fluorescence measurements, we attempt a correction (or deconvolution) for (i) to (iii). These investigations aim at a rationale to get the pure S-state difference spectra in FTIR investigations on the reaction cycle of PSII water oxidation. First results are presented; problems and perspectives are discussed.

Thermosynechococcus elongatus at 10 °C. Along with the numerous bands arising from Tyr and protein backbones in the 1800-1100 cm-1 region, differential signals were observed at 3635/3617 and 3595/3584 cm-1 in the weakly H-bonded OH stretching region. These signals downshifted by 12-13 cm-1 upon H218O substitution and disappeared in a deuterated film, and hence they were definitely assigned to the water OH vibrations. The presence of these water bands in the YD•/YD spectrum indicate that at least two water molecules are structurally coupled to YD and their H-bond interactions are perturbed upon YD oxidation. These water molecules are probably involved in the H-bond network around YD; thus, they might play a significant role in the proton release reaction of YD.

PS4.46 On the structure of the manganese complex of photosystem II: extended-range EXAFS data and specific atomic-resolution models for four S-states

PS4.48 Structural and mechanistic studies of the oxygen-evolving complex and cytochrome b559 in photosystem II

A Grundmeier (Freie Universität Berlin), P Loja (Freie Universität Berlin), M Haumann (Freie Universität Berlin), H Dau (Freie Universität Berlin) In plants and cyanobacteria, water is oxidized at the Mn4Ca complex bound to the proteins of photosystem II (PSII). This manganese complex was studied by X-ray absorption spectroscopy on PSII membrane particles using an extended range for collection of EXAFS data (up to 16.6 Å-1). The extended-range EXAFS suggests the presence of two Mn-Mn distances close to 2.7 Å (per Mn4Ca complex); the existence of a third Mn-Mn distance below 2.9 Å is at least uncertain. Interestingly, a distance of 3.7 Å is clearly resolved in the extended-range data and tentatively assigned to a Mn-Mn distance. Taking into account the above EXAFS results (inter alia), we present a model for the structure of the PSII manganese complex, which differs from previous atomic-resolution models. Emphasizing the hypothetical character, we propose for all semi-stable S-states (i) a structure of the Mn4Ca(µ-O)n core, (ii) a model of the amino-acid environment, and (iii) assignments of distinct Mn oxidation states to all the individual Mn ions. This specific working model may permit discussion, verification, and invalidation of its various features by comparison with experimental and theoretical findings.

H Chu (Institute of Plant and Microbial Biology, Academia Sinica, Taiwain) NH3 is a structural analog of substrate H2O and an inhibitor to the water oxidation reaction in Photosystem II (PSII). We have applied light-induced FTIR difference spectroscopy to study NH3-induced structural changes of the OEC in PSII. We found that NH3-induced spectral changes of the S2QA-/S1QA spectrum of PSII were dependent on the temperature of FTIR measurement. Particularly, NH3-induced spectral changes in the S2QA-/S1QA spectrum of PSII were diminished when the temperature of FTIR measurement was raised from 250K to 277K. In addition, we have performed low-frequency FTIR measurement on NH3-induced structural changes of the OEC in PSII. The implication of our results on the structural mechanism of photosynthetic water oxidation will be discussed. Furthermore, we have constructed a series of site-directed mutants on the heme ligands (His-22 of a and ß subunits) of cyt b559 by using cyanobacteria Synechocystis 6803 G. We identified two cyt b559 mutants that were able to grow photoautotrophically and assembled the heme of cyt b559 and the stable PSII. We have performed structural and functional analysis on these cyt b559 mutants and their PSII particles. The redox role(s) of cyt b559 in the photoprotection mechanism of PSII will be discussed.

PS4.47 Structural coupling of water molecules with YD in photosystem II as revealed by FTIR spectroscopy R Takahashi (Institute of Materials Science, University of Tsukuba, Japan), M Sugiura (Department of Plant Biosciences, Osaka Prefecture University, Japan ), T Noguchi (Institute of Materials Science, University of Tsukuba, Japan) The redox-active tyrosine YD (D2-Tyr160) in photosystem II (PSII) serves as an accessory electron donor to P680. It undergoes proton-coupled electron transfer; when it is oxidized, a proton is released and a neutral radical YD• is formed. The recent X-ray structure of the PSII core complex revealed a H-bond network around the OH group of YD. This H-bond network must play an important role in the proton-transfer reaction upon YD oxidation. In this study, we have detected water molecules coupled to YD probably through a H-bond network by means of light-induced Fourier transform infrared (FTIR) spectroscopy. YD/Y D• FTIR difference spectra was obtained by applying five flashes to a moderately hydrated (deuterated) film of PSII core complexes from

PS4.49 Spectroscopic studies of the Mn4Ca cluster in mutants and biosynthetically modified Photosystem II from Thermosynechococcus elongatus A Boussac (CEA-CNRS), M Sugiura (Osaka Prefecture University), N Ishida (CEA), T Lai (CEA), S Un (CEA), F Rappaport (CNRS), A Rutherford (CEA-CNRS) The structure of PSII isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus has now been solved to 3.5 Å and then to 3.0 Å resolution [1, 2]. However, the structure of Mn4Ca cluster is still under debate since its redox state is undefined in the crystallographic structures due to the reductive effect of the x-rays used [3]. More fundamentally, these crystallographic structures by themselves, remain ambiguous concerning the various important protein-cofactor and cofactor-cofactor interactions that influence the electronic properties of the cofactors and enzyme function. By combining the detailed physical structure obtained from crystallography with the electronic structural information from spectroscopy, a more precise functional picture of structure can be obtained. In this respect the PSII isolated from Thermosynechococcus elongatus provides an ideal framework. The

185 14th Photosynthesis Congress - PS07 protein itself is robust [4], the genomic sequence of this organism has been determined and molecular biological technologies are well under development [5]. We will present spectroscopic studies on site-directed mutants both in the D1 and D2 proteins and biosynthetically modified [e.g. 6, 7] and biochemically disrupted photosystem II [e.g. 8]. [1] Ferreira et al. Science 2004, 303, 1831-1838. [2] Loll et al. Nature 2005, 438, 1040-1044. [3] Yano et al. Proc. Natl. Acad Sci. USA 2005, 102, 12047-12052. [4] Sugiuraet al. Plant Cell Physiol. 1999, 40, 1919-1931. [5] Sugiura et al. Biochemistry 2004, 43, 13549-13563. [6] Boussac et al. J. Biol. Chem. 2004, 279, 22809–22819. [7] Suzuki et al. Biochemistry, 2006, 45, 13454-13464. [8] Un et al. Biochemistry, 2007, 46, 3138-3150. PS4.50 Expression of the Manganese Stabilising Protein from a Primitive Cyanobacterium A Williamson (ANU) The Manganese Stabilising Protein (MSP) is an extrinsic subunit of Photosystem II which is essential for optimal levels of oxygen production during photosynthesis under physiological conditions. In addition to its function in stabilising the catalytic manganese cluster, the MSP has been proposed to be part of a channel conducting substrate water to the oxidation site, and allowing the exit of protons. In the crystal structure of PSII from Thermosynechococcus elongatus the D158, D222, D233, D224, H228 and E229 residues of the MSP have been suggested to be involved in the putative channel. The MSP is present in all oxygenic phototrophs including Gloeobacter violaceus, which is the most primitive of contemporary cyanobacteria, having diverged very early from the common ancestor. However, G. violaceus is the only known species which does not conserve the D222 and D224 residues, making it a natural variant to test the function of the putative channel through reconstitution (2) and 18O-exchange measurements (3). Because G. violaceus is difficult to culture and is extremely slow growing, it has not yet been isolated biochemically. Instead we report here on our protocols for the expression and purification of rMSP in E. coli inclusion bodies. Structural and unfolding data will be presented. Funded by the Australian Research Council (DP0450421) and in part by an APA (AW) 1. Murray & Barber (2007) J. Struct. Biol 2. Williamson et al (2007) Photosynth Res 3. Hillier & Wydrzynski (2004) Phys Chem Chem Phys PS4.51 Crystallization of Photosystem II under controlled degrees of supersaturation K Nakazato (Department of Integrated Science of Physics and Biology, College of Humanities & Science, Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo 156-8550,Japan), A Okumura (Department of Integrated Science of Physics and Biology, College of Humanities & Science, Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo 156-8550,Japan) The degree of supersaturation is an important factor for the preparation of high quality protein crystals, because it determines the driving force for nucleation and growth processes. It is generally considered that crystals grown in a metastable region are preferred over those grown at higher supersaturation. However, there has been no system for the definite control of supersaturation levels on protein crystallization. Supersaturation is itself a function of an array of experimental variables such as precipitant concentration, protein concentration, temperature, or other physical and chemical factors. From a viewpoint that

oxygen-evolving photosystem II crystals have been prepared by reducing the solubility of PSII through the use of various PEGs, it is desirable to develop a system to control the levels of supersaturation as a function of PEG concentration. For this purpose, we made a unique thin micro dialysis cell which permits a direct optical measurement of PSII concentration therein. The PEG concentration in the dialysis cell was changed forcibly to a desired concentration with the advantage of dialysis. With this dialysis cell and several devices including a spectrophotometer, micro-pumps and a personal computer, we constructed a system to control the degrees of supersaturation based on the solubility curve of PSII as a function of PEG. This system was applied to the crystallization of oxygen-evolving PSII from Thermosynechococcus vulcanus. The results demonstrated that the dynamical control of supersaturation degrees with this system is useful for crystallizing PSII from its solution and growing micro PSII crystals under a metastable region. PS4.52 What can give us the simple method of the ligand field theory in studing the complicated oxygen evolving complex A Rychkova (Moscow State University) At the present time there are many experimental data that give us information about the structure of the oxygen evolving complex (OEC). Subsequently, these data are used for theoretical calculations and constructing the structure models of the OEC. Recently scientists from Yale University published the paper [1], where they presented two possible models of the OEC based on the quantum mechanics/molecular mechanics (QM/MM) hybrid methods applied in conjunction with the recent X-ray crystal structure of PSII [2]. The authors predict high-valent configurations of the S1 state of the OEC of PSII, with oxidation numbers Mn4(IV, IV, III, III) and Mn4(IV, III, III, IV) for model structures A and B, respectively. However, the authors mention that predicting the correct stability of low-lying spin states in multinuclear oxomanganese complexes might be beyond the capabilities of the implemented DFT/B3LYP method. In our research we use the ligand field theory to construct the model of the OEC of PSII. We apply the eighteen- and twelve-electron rules for the stable shells of coordination complexes with central manganeses and calcium relatively. Previously we already published the results of this approach in [3, 4]. There we presented the new model of the OEC based on the two X-ray crystal structures of PSII [2, 5] and the scheme of redox states at the transitions between discrete S-states of OEC at excitations by shot pulse of light. In this paper we would like to present the results of application of our approach to the model A and model B, proposed recently in [1]. We used the semi-empirical ZINDO and DFT methods to calculate the energies of complexes. It was shown that our model of the OEC with low-lying spin states is more stable than model A and model B with high-lying spin states. This means that the ligand field theory can be used as a convenient instrument for constructing complexes with transition metals. References: [1] E. Sproviero, J. Gascon, J. McEvoy, G. Brudvig, V. Batista, J. Chem. Theory Comput., 2006, 2, 1119-1134. [2] K. Ferreira, T. Ivenson, K. Maghlaoui, J. Barber, S. Iwata, Science, 2004, 303, 1831-1838. [3] A.S. Denisenko, A.K. Kukushkin, Biophysics, Vol. 50, No. 5, 2005, pp. 728–735 [4] A. Rychkova, A. Kukushkin, International Meeting “Photosynthesis in the Post-Genomic Era: Structure and Functions of Photosystems” in honor of Professor James Barber, Pushchino, 20-26 August 2006, pp. 278 [5] B. Loll, J. Kern, W. Saenger, A. Zouni, J. Biesiadka, Nature, 2005, 438, 1040-1044.

186 14th Photosynthesis Congress - PS07 PS4.53 Oxidative photosynthetic water splitting: energetics, kinetics and mechanism G Renger (Max Volmer Laboratory, Technical University Berlin) Thermodynamic and kinetic constraints of oxidative water splitting into molecular oxygen and four protons require a balanced fine tuning of the reaction coordi-nates. Hydrogen bond network(s) and protein flexibility are essential parameters. Four topics will be discussed: A. Multiphasic kinetics of P680+• reduction by YZ. The characteristic feature reflects a sequence of three different types of rate limitations: (i) non-adiabatic electron transfer ("fast" ns kinetics), (ii) local "dielectric" relaxation ("slow" ns kinetics), and (iii) "large scale" proton shift (µs kinetics). B. Coupling between electron (ET) and proton transfer (PT) reactions. The mode of coupling probably depends on redox state Si of the water oxidizing complex (WOC) where S0 and S1 oxidation comprises separate pathways of ET and PT, while S2 and S3 undergo proton-coupled electron transfer (PCET) [1,2]. C. O-O bond formation. This key reaction is postulated to occur within a multistate equilibrium at the redox level of S3, comprising both redox isomerism and proton tautomerism, where state S3(P) attains the electronic configuration and nuclear geometry of a hydrogen bonded peroxide and acts as the entatic state for the generation of complexed molecular oxygen through S3(P) oxidation by [2,3]. D. Protein flexibility and electron transport activity. References [1] Renger G (2001) Biochim. Biophys. Acta 1503: 210-228 [2] Vrettos JS, Limburg J, Brudvig GW (2001) Biochim. Biophys. Acta 1503: 229-245 [3] Renger G (2007) Photosynth. Res. (in press) PS4.54 IR-induced Changes in Flash-induced Fluorescence of Photosystem II

Variable

Chl

PS4.55 The S2YZ• intermediate of the OEC in the functional Photosystem II at temperatures close to the onset of the S2 to S3 transition. An EPR investig N Ioannidis (Institute of Materials Science, NCSR "Demokritos", Aghia Paraskevi, Athens 15310, GREECE), V Petrouleas (Institute of Materials Science, NCSR "Demokritos", Aghia Paraskevi, Athens 15310, GREECE) Photosystem II preparations (BBY) poised in the S2...QA state produce no detectable intermediate by visible-light illumination at liquid helium temperatures. However, upon flash illumination at 77-190 K, they produce a transient state which can either advance to S3 by warming up to -10 oC, or give rise to a 116 G wide metalloradical EPR signal by rapid cooling to 10 K. Formation of the metalloradical signal is accompanied by reversible changes in the Mn multiline signal. The intermediate has been assigned to YZ • magnetically interacting with the Mn cluster in the S2 state, S2YZ•. A temperature dependence study of its spectrum was done using slow and rapid scanning EPR spectroscopy. At liquid helium temperatures the metalloradical intermediate is dominated by the magnetic interaction with the Mn cluster. The magnetic splitting decreases above 30-35 K, due to rapid enhancement of the relaxation rate of Mn, and averages out completely above 100 K, revealing the unperturbed spectrum of YZ•. This is the first time that S2YZ• is trapped in the functional system at high temperatures. Its spectrum shows distinct differences from that of the stable YD• radical. The spectrum of Yz• was simulated by varying the rotational conformation of the phenoxyl ring and the spin density r on carbon C1. The latter can be related to the strength of the hydrogen bond between tyrosine and its base partner (presumably D1 H190). Our results indicate that the hydrogen bond of YZ• is stronger than that of YD•.

a

A Thapper (Uppsala University), F Mamedov (Uppsala University), S Styring (Uppsala University) The flash-induced variable Chl a fluorescence of PSII, FV, and the decay kinetics of FV relaxation, give valuable information about the activity of the PSII acceptor side. We have used PSII enriched BBY membranes from spinach to study changes in FV rise and decay after giving a number of flashes of defined wavelengths between 650-800 nm using a MOPO laser. The maximally inducible FV in the samples, as obtained in the presence of 10 mM dithionite, was not reached giving one laser flash of a wavelength in the range 650-740 nm. Instead FV increases with a train of flashes and reaches a maximum after about 10 flashes. The decay kinetics of FV changes as more laser flashes are given. When FV is maximally induced the relative amplitude of the two fastest phases that are normally associated with forward electron transfer from QA- to QB has decreased compared to after just one flash. Furthermore, the second of these two phases, reflecting QB-binding, has slowed down. These two observations indicate that the plastoquinone pool is limited and the re-oxidation is not fast enough under these conditions. More interestingly, the increase in FV and the altered decay kinetics can also be induced using flashes in the range 750-800 nm, but more flashes have to be given to the sample to reach the same effect. The involvement of NIR light, which is not absorbed by the chlorophylls in PSII, in the build of high FV is interesting and its implications will be discussed.

PS4.56 Temperature dependence of the S1YZ•, S0YZ•(+MeOH) and S2YZ (+MeOH) intermediate states of Photosystem

•

G Zahariou (Institute of Materials Science, NCSR "Demokritos", Aghia Paraskevi, Athens 15310, GREECE), V Petrouleas (Institute of Materials Science, NCSR "Demokritos", Aghia Paraskevi, Athens 15310, GREECE) Visible-light illumination of dark-adapted (S1-state) oxygen-evolving (PSII) samples at liquid helium temperatures produces a transient “split” EPR signal, assigned to the S1Yz• intermediate. The signal decays in the min time range, due to charge recombination with QA--. Addition of methanol eliminates the observation of this signal. However, illumination at liquid helium temperatures of methanol treated samples, poised in the S0…QA and S2…QA states results in the production of 230 G and 160 G wide EPR signals, assigned to S2Yz• and S0Yz•, respectively. We studied the temperature dependence of the above metalloradical EPR signals in the temperature range 11 – 200 K, using slow and rapid scans. As the temperature increases, the lifetime of the transients decreases, due to the progressive increase of the recombination rate. In one case, S1Yz• intermediate, the decay rate increases rapidly above about 100 K, presumably due to advancement to the S2 state. Besides the lifetime changes, above 25-30 K, the spectra narrow progressively and above 100 K collapse to a signal somewhat broader than “signal II”. The narrowing of the SnYz• EPR signals is

187 14th Photosynthesis Congress - PS07 consisted with an increase of the Mn spin-lattice relaxation rate, with increasing temperature. The spectra do not reach saturation at microwave powers up to 100 mW over the entire temperature range, and the signal intensity multiplied by temperature remains approximately constant. We conclude that the broad EPR signals observed at low temperatures and the narrow signals at elevated temperatures are manifestations of the same intermediate SnYz• (n = 0, 1, 2).

reactions will be discussed. (B) The critical S3 state of the OEC (an integer-spin state) gives perpendicular and parallel mode signals at X band. Furthermore it has the unusual property to undergo light-induced (near IR) spin transitions at l. helium temperatures. We have recently extended the S3 studies to Q-band. As a first step to providing EPR-based information on S3 we will present simulations of the various EPR spectra yielding the ground spin state of the Mn cluster.

PS4.57 The mechanism(s) behind the formation of the “Split S3 EPR signal” in Photosystem II induced by visible or near infrared light.

PS4.59 ESEEM and ESE-ENDOR investigation of the light inducible Split S1 EPR signal from Photosystem II

K Havelius (Uppsala University), J Su (Max Plank Institute for Bioinorganic Chemistry), F Ho (Uppsala University), F Mamedov (Uppsala University), S Styring (Uppsala University) 1

and S0 the signal induction involves charge separation oxidizing YZ.

The signals decay by recombination with QA- formed in the same photon event. A split EPR signal can be induced in the S3 state samples both with visible light (430-690nm) and NIR light (750-900 nm). The question is how the radical is formed. The NIR induced signal is unlikely to involve charge separation and is most likely induced by excitation of the CaMn4 cluster, which would oxidize the radical. The NIR signal is stable, but can be forced to decay quantitatively if QA- is present before the induction. The Split S3 signal is also induced over the entire visible spectrum. In this case, about 25% of the signal decays rapidly with formed QA-. Can the “NIR-mechanism” explain the visible induced signal or must charge separation be considered? We designed a series of experiment to find out if the same mechanism is responsible for the formation of the Split S3 signal induced by NIR or 560 nm flashes. Induction with NIR or visible illumination and decay in the dark at 5 K of the Split S3 signal was followed in the absence and presence of preformed QA-. The results provide new insights about the origin of the Split S3 signal and different mechanistic implications will be discussed in the light of the experimental results. PS4.58 Recent EPR studies of the OEC of Photosystem II. (A) Trapping tyrosyl Z• in action. (B) The critical S3 integer-spin state of the Mn cluster V Petrouleas (NCSR Demokritos), N Ioannidis (NCSR Demokritos), G Zahariou (NCSR Demokritos), J Sarrou (NCSR Demokritos), G Sioros (NCSR Demokritos), Y Sanakis (NCSR Demokritos) (A) Tyr Z is closely associated with the WOC. Significant interest has been shown on its properties, but the EPR characterization of tyr Z• has been restricted, until recently, to inhibited (unable to evolve O2) preparations. An important advance in recent years is the trapping at liquid helium temperatures, of metalloradical intermediates of the S-state transitions, assigned to tyr Z• magnetically interacting with the Mn cluster. We have studied tyr Z as a function of temperature in various S states and at temperatures approaching the onset of the respective S-state transitions. The magnetic interaction with Mn averages out completely above about 100 K, revealing for the first time the unperturbed spectrum of Tyr Z•. These experiments provide information about the conformation of tyr Z in the functional system. The electronic parameters of Tyr Z• as derived by spectral simulations are compared to tyr D• and related to the strength of their respective hydrogen bondings. Implications about the coupling of the tyr Z and Mn (de)protonation

F Mamedov (Uppsala University), K Havelius (Uppsala University), S Styring (Uppsala University) Upon oxidation, the secondary donor in Photosystem II, TyrZ interacts magnetically with the CaMn4-cluster of the water oxidizing complex. This results in splitting of the TyrZ∙ EPR radical spectrum making TyrZ∙ observable when reduction by the CaMn4-cluster is slowed down or closed. Such split EPR signals can be induced from the S0, S1, S3- states, and, at certain conditions, from the S2-state when Photosystem II is illuminated at liquid He temperatures. Due to the interaction nature of the signals, important information about the structure and function of both the CaMn4-cluster and TyrZ∙ in the different S-states could be obtained. We have performed 2 pulse and 3 pulse ESEEM (electron spin echo envelope modulation) and proton ESE-ENDOR (electron spin echo – electron nuclear double resonance) measurements on the Split S1 signal induced by illumination at 5 K in the Photosystem II membranes, predominantly poised in the S1-state. The results will be discussed in terms of the structural configuration of the S1 state of the water oxidizing complex. PS4.60 The S-state dependence of the location and affinity of the protons bound to the manganese cluster in photosystem II. H Mino (Nagoya university), H Yamada (Nagoya university), S Itoh (Nagoya university), A Kawamori (Agape-Kabutoyama Institute of Medicine) Protons in the vicinity of the oxygen-evolving manganese cluster in photosystem II were studied by proton matrix ENDOR. Six pairs of proton ENDOR signals were detected in both the S0 and S2 states of the Mn-cluster. Two pairs of signals that show hyperfine constants of 2.3/2.2 and 4.0 MHz, respectively, disappeared after D2O incubation in both states. The signals with 2.3/2.2 MHz hyperfine constants in S0 and S2 state multiline disappeared after 3 hours of D2O incubation in the S0 and S1 states, respectively. The signal with 4.0 MHz hyperfine constants in S0 state multiline disappeared after 3 hours of D2O incubation in the S0 state, while the similar signal in S2 state multiline disappeared only after 24 hours of D2O incubation in the S1 state. The different proton exchange rates seem to be ascribable to the change in affinities of water molecules to the variation in oxidation state of the Mn cluster during the water oxidation cycle. The short distances estimated by hyperfine constants suggest the protons belong to the water molecules ligated to the manganese cluster. Based on the crystal structural studies, the locations of the protons were estimated by the ENDOR spectra in the oriented PS II membranes. We propose a model for the binding of water to the manganese cluster based on these results.

188 14th Photosynthesis Congress - PS07

PS5 - Electron Transport; operation, organisation and regulation PS5.1 Structure of the Cytochrome b6f Complex: Quinone Analogue Inhibitors as Ligands of Heme

introduced to modulate the relation between heme ci and its ligands and to altered chlorophyll position and/or stability. We will discuss the consequences of these changes in C. reinhardtii mutants both in vivo and in vitro.

W Cramer (Purdue University), E Yamashita (Osaka University), H Zhang (Purdue University), D Baniulis (Purdue University) A 3.00 Å native structure of the cytochrome b6f complex from the thermophilic cyanobacterium, M. laminosus, was obtained from crystals grown with divalent cadmium (PDB: 2E74). One Cd2+ binding site bridges His143 of cytochrome f and the acidic residue, Glu75, of cyt b6; (ii) a second site has three identified ligands, Asp58 (subunit IV), Glu3 (PetG subunit) and Glu4 (PetM subunit). Binding sites of quinone analogue inhibitors map the transfer pathway of the lipophilic quinone across the complex. Two sites were found for the chromone ring of the tridecyl-stigmatellin (TDS) quinone analogue inhibitor, one near the p-side [2Fe-2S] cluster (PDB: 2E76). A second TDS site faces the quinone exchange cavity as an axial ligand of heme cn. A similar binding site proximal to heme cn was found for the n-side quinone analogue inhibitor, NQNO (PDB: 2E75). Binding of these inhibitors required addition before lipid used to facilitate crystallization. Binding of NQNO and TDS as axial ligands to heme cn implies that cn utilizes plastoquinone as a natural ligand, thus defining an n-side electron transfer complex consisting of hemes bn, cn, and PQ in the reduction pathway of PQ in the cavity. The NQNO binding site explains effects associated with its inhibitory action: a negative shift in heme cn Em, increased amplitude of light-induced reduction of heme bn, and g value shifts in the EPR spectrum attributed to interaction between hemes cn and bn. The b6f structure allows prediction of pathways for H+ uptake and a potential site of ferredoxin binding.

PS5.2 Inside the cytochrome b6 f complex: where is Nessie? F Zito (Institut de Biologie Physico-Chimique, UMR 7099, CNRS/Université Paris-7), A de Lacroix de Lavalette (Institut de Biologie Physico-Chimique, UMR 7099, CNRS/Université Paris-7), L Barucq (Institut de Biologie Physico-Chimique, UMR 7099, CNRS/Université Paris-7), F Rappaport (Institut de Biologie Physico-Chimique, UMR 7141, CNRS/Université Paris-6), D Picot (Institut de Biologie Physico-Chimique, 1UMR 7099, CNRS/Université Paris-7), D Picot (Institut de Biologie Physico-Chimique, UMR 7099, Oxidoreductases of the cytochrome bc1/b6 f family transfer electrons from a liposoluble quinol to a water-soluble acceptor protein and contribute to the formation of a transmembrane electrochemical potential. In addition, cytochrome b6 f complex (Cyt b6 f) is thought to play specific roles in oxygenic photosynthesis, namely its involvement in cyclic electron transfer around photosystem I, in the regulation of the so-called 'state transitions', and in the pumping of additional protons. The crystal structure of Cyt b6 f has revealed the presence in the Qi site of a hitherto unsuspected, atypical c-type heme, heme ci. Unusually, the protein does not offer any axial ligand to the iron of this heme which becames accessible to exogenous ligands. In addition Cyt b6 f contains other cofactors, including one molecule of chlorophyll a (Chla) whose role represents one of the most mysterious features of the b6 f complex. In this work we will describe a mutagenesis approach aimed at characterising heme c and its interaction with the Qi site environment, and the role of chlorophyll a. More specifically, mutations were

PS5.3 Photosystem I does not contribute to photosynthetic flux control M Schottler (Max-Planck-Institut für Molekulare Pflanzenphysiologie), C Flügel (Max-Planck-Institut für Molekulare Pflanzenphysiologie), W Thiele (Max-Planck-Institut für Molekulare Pflanzenphysiologie), K Piepenburg (Max-Planck-Institut für Molekulare Pflanzenphysiologie), R Bock (Max-Planck-Institut für Molekulare Pflanzenphysiologie) In recent years, the contribution of all components of the photosynthetic electron transport chain except for photosystem (PS) I to photosynthetic flux control has been analysed. Flux control is predominantly exerted at the level of the cytochrome-bf-complex and of plastocyanin, whose contents vary strongly in response to growth conditions and the developmental state in higher plants. Different to these components, the contents of PSI are rather constant (2.0 to 2.5 mmoles mol chlorophyll-1), already suggesting a minor contribution of PSI to flux control. Using tobacco-, sunflower- and Arabidopsis-mutants suffering from either reduced stability or impaired accumulation of PSI, we here demonstrate that in saturating light, a reduction of PSI down to 50% of wild-type contents does not impair linear electron flux capacity. Rather, a mild reduction of PSI results in increased flux, due to complementary increases of the rate-limiting components of the electron transport chain. However, already a relatively mild reduction of PSI results in an over-reduction of the electron transport chain at limiting light intensities, due to an imbalance of excitation distribution between the photosystems. We conclude that PSI accumulates to much higher contents than are required to sustain linear electron flux at saturating light because of a rather inflexible PSI antenna system (Lhca1-4). Despite a severe reduction of PSI contents, the antenna cross section cannot be sufficiently increased per PSI unit to compensate for the reduced overall PSI accumulation. State transitions are not sufficient to balance excitation between both photosystems at low light intensities.

PS5.4 Oxygen as an alternative electron sink, a photo-protective mechanism in leaves? S Driever (University of Essex) In many situations the rate of light absorption exceeds the rate of CO2 assimilation and the possibility of direct reduction of oxygen by PSI arises. Photo-reduction of oxygen produces the potentially harmful reactive oxygen species (ROS) superoxide (O2-) and hydrogen peroxide (H2O2). Although such ROS production can be damaging to cells, provided the ROS can be effectively scavenged, the photo-reduction of O2 is a potentially important alternative electron sink. This may act as an important protection mechanism against photo-inhibition and damage to the photosynthetic apparatus. In this study we examined the generation of ROS and O2 photo-reduction in leaves under a range of physiological conditions using a range of techniques. In both C3 and C4 plants, light dependent formation and in vivo localization of O2- and H2O2 has been assessed by imaging of leaves fed with probes specific for O2- and H2O2.

189 14th Photosynthesis Congress - PS07 Oxygen exchange and oxygen evolution were determined by mass spectrometric measurements; CO2 gas exchange and chlorophyll fluorescence were also measured to assess the photosynthetic performances of the leaves. Increasing light intensity produced an elevated production of both O2- and H2O2 primarily observed around the vascular tissues of both C3 and C4 leaves. This study has demonstrated that increased electron transport occurs with increasing light intensity in the absence of similar increases in CO2 assimilation. The data support the contention that photo-reduction of oxygen functions as a photo-protective mechanism in both C3 and C4 leaves. PS5.5 Reconstitution of cyclic electron transport in Synechocystis sp. PCC 6803 by a cell-free system M Watanabe (University of Hyogo), Y Kashino (University of Hyogo), K Satoh (University of Hyogo), H Koike (University of Hyogo) Cyanobacteria possess a hight activity of cyclic electron transport. The several pathways have been proposed: electron (i) from NAD(P)H to the plastoquinone (PQ) pool via multi-subunit NAD(P)H dehydrogenase (NDH), NDH-1 or a single polypeptide NDH, NDH-2; (ii) from ferredxin (Fd) to the PQ pool mediated by a Fd-quinone reductase (FQR); or (iii) direct transfer from Fd to the cytochrome b6/f complex to which Fd-NADP+ oxidreductase (FNR) is bound stoichiometrically. Many components are nominated as candidates for the cyclic electron transport, but they are not confirmed yet. For the first step to identify the possible component(s) of the pathway in cyanobacteria, we tried to reconstruct the cyclic electron transport in the cell-free system. The Synechocystis sp. PCC 6803 cells were disintegrated by an improved method and thylakoid membranes and soluble fractions were separated from the homogenate. P700+ was rereduced rapidly by electrons from photosystem II. It was also rereduced with a significant rate in the presence of DCMU, suggesting that cyclic electron transport activity was also preserved. On addition of the soluble fraction, the rereduction rate of P700+ was accelerated much more. This indicates that cyclic electron transport activity was reconstituted by addition of the soluble fraction to the thylakoid membranes. Further addition of NAD(P)H yielded faster rereduction of P700+. This suggests that the fraction contains some component(s) which mediate(s) electrons from NAD(P)H to P700+ possibly via PQ pool.

was lower, higher activity of PGR5-dependent PSI cyclic electron transport was detected. This result indicated a competition between NADP+ photoreduction and PGR5-dependent PSI cyclic electron transport. From these results, it is suggested that the rate of PGR5-dependent PSI cyclic electron transport is high enough to contribute to balancing the production ratio of ATP and NADPH during steady-state photosynthesis as well as under stress conditions, consistently with the phenotype of the pgr5 mutant. The activity of this pathway is regulated by the redox state of NADPH pool. PS5.7 Regulation of superoxide dismutase expression by copper availability C Cohu (Colorado State University), M Pilon (Colorado State University) The most abundant copper proteins in green tissues are plastocyanin (PC) in thylakoids and copper/zinc superoxide dismutase (Cu/ZnSOD) of which the major isoforms are found in the cytosol and in the chloroplast stroma. An iron superoxide dismutase (FeSOD) can also be found in the stroma. The expression of superoxide dismutases (SODs) has been studied mainly in the context of abiotic stress. However, the availability of metal cofactors may also determine SOD expression patterns. Indeed, in Arabidopsis thaliana, Cu/ZnSOD enzymes were only expressed when copper was sufficient. This observation was made for plants grown on sucrose-containing tissue culture media and regulation of SOD expression by copper was not tested for other species. To investigate the effect of copper on SOD expression, we used a hydroponic set-up in which plants grew without any evident stress symptoms. We observed that A. thaliana, Brassica juncea, Lycopersicum lycopersicum, Zea mays and Oryza sativa, downregulated Cu/ZnSOD in response to copper limitation. Under this condition, FeSOD expression was upregulated to replace Cu/ZnSOD in the stroma in all plants except Z. mays, in which FeSOD was not detectable. Copper limitation did not affect PC accumulation in any of the plants except Z. mays. Comparisons of leaf copper contents and SOD expression suggest that Cu/ZnSOD and FeSOD expression levels are good indicators of impending copper deficiency. Plants that downregulate Cu/ZnSOD and upregulate FeSOD under copper limitation can maintain superoxide scavenging and save copper for use in PC, which is essential for photosynthesis.

PS5.6 Characterization of regulatory factors of PGR5-dependent PSI cyclic electron transport

PS5.8 Heterogeneity of xanthophyll-independent non-photochemical quenching during photosynthetic induction in shade-grown leaves of avocado (Persea americana L.)

Y Okegawa (Graduate School of Agriculture, Kyushu University), Y Kagawa (Graduate School of Agriculture, Kyushu University), Y Kobayashi (Graduate School of Agriculture, Kyushu University ), T Shikanai (Graduate School of Agriculture, Kyushu University )

K Takayama (Ehime University), B Osmond (The Australian National University), K Omasa (The University of Tokyo)

Photosynthetic electron transport consists of two main routes: linear and photosystem I (PSI) cyclic electron transport. Although PSI cyclic electron transport has been proved to be essential for photosynthesis and photoprotection, our knowledge of this route is very limited. In higher plants, the PGR5-dependent pathway is the main route taken by electron in PSI cyclic electron transport. Although a small thylakoid protein, PGR5 (PROTON GRADIENT REGULATION 5), is essential for this pathway, the function of the PGR5 protein is still unclear and there are debates on the rate of electron transport in vivo and its regulation. We evaluated the activity of Fd-dependent PQ reduction in ruptured chloroplasts by using an Arabidopsis mutant, pgr5. We detected high activity of Fd-dependent PQ reduction even in the presence of linear electron transport. In the wild type, when the concentration of NADP+

Shade-grown avocado plants rich in lutein epoxide, were used to image chlorophyll fluorescence quenching, photosynthetic rate and stomatal conductance during photosynthetic induction for 20 min after transition from dark to light similar to growth irradiance were performed under 100, 400 and 700 ppm CO2. The duration and extent of transients in NPQ and FPSII, photosynthetic rate and stomatal conductance were sensitive to CO2 concentration. Chlorophyll fluorescence imaging revealed that NPQ was initially heterogeneously distributed across the avocado leaf, with marked differences between cells in tissues defined by minor veins and those adjacent to primary and secondary veins. In 400 ppm CO2 this phenomenon was accentuated and prolonged in areas of the leaf in which stomatal conductance was occluded by a layer of Vaseline. The extent of local heterogeneity in NPQ was often of the same scale as the average value, and similar to that observed in spot

190 14th Photosynthesis Congress - PS07 measurements. On the other hand, FPSII was relatively uniform and did not show marked association with the vascular elements, independent of the CO2 concentrations. Analysis of the pigment composition of avocado leaves transfer to similar light treatments showed that the dynamic changes in NPQ observed in the initial stages of photosynthetic induction was independent of detectable changes in the epoxidation status of both xanthophyll pools (V+A+Z and Lx+L). These results imply that the dynamic, heterogeneously-distributed, stomatally dependent and CO2 responsive NPQ measured and imaged here mainly involves quenching processes that occur in reaction centres prior to stabilization of heat dissipation in the antennae. PS5.9 Identification of photosynthesis-related genes in rice using FOX hunting system M Higuchi (Plant Functional Genomics Research Team, RIKEN PSC), K Matsui (Plant Functional Genomics Research Team, RIKEN PSC), T Ichikawa (Plant Functional Genomics Research Team, RIKEN PSC), Y Kondou (Plant Functional Genomics Research Team, RIKEN PSC), Y Hasegawa (Plant Functional Genomics Research Team, RIKEN PSC), M Kawashima (Plant Functional Genomics Research Team, RIKEN PSC), S Muto (NEC Soft, Ltd), H Hirochika (National Institute of Agrobiological Sciences), M Matsui (Plant Functional Genomics Research Team, RIKEN PSC) Photosynthesis is one of the most important determinants of crop productivity. Although many studies have been conducted to improve yield, very little progress has been made. In this study, we used rice FOX lines that rice full-length cDNAs were over-expressed in Arabidopsis to identify novel photosynthesis-related genes of rice. We have used imaging of chlorophyll fluorescence to screen candidates. Time course changes of chlorophyll fluorescence intensity were compared with WT. So far, we screened about 7,000 FOX lines and isolated 28 candidates with altered chlorophyll fluorescence kinetics. Isolated candidates could be classified into several categories depending on chlorophyll fluorescence parameters. Moreover, chlorophyll fluorescence was also measured after high-light treatment (photon flux density: 1,000 μmol photons m-2 s-1, duration: 1 hour) to isolate high-light tolerant mutants. We isolated 2 lines that showed tolerance to high light from about 3,000 lines. Various full-length rice cDNAs were transformed in candidate lines, such as cDNAs encoding enzymes that have a chloroplast transit peptide, transcriptional-related proteins and unknown proteins. To confirm over-expression of these cDNAs cause the observed phenotype, we generated transformants overexpressing each cDNA, resulting that 8 lines showed the same phenotype as original lines. We analyzed gene expression profile to predict gene function using DNA microarray. We will report the results of microarray analysis in this meeting. This research is supported by the Special coordination funds for Promoting Science and Technology entitled Rapid identification of useful traits using rice full-length cDNAs. PS5.10 Thioredoxin potential target proteins in Green sulfur bacterium Chlorobaculum tepidum N Matsuda (Dept. Biol. Sci., Kanagawa Univ.), T Hisabori (Chem. Res. Lab., Tokyo Tech.), K Inoue (Dept. Biol. Sci., Kanagawa Univ.) Thioredoxin (Trx) is a small ubiquitous protein that involves in the dithiol-disulfide exchange reaction using two reactive cysteines on the molecule. Reduced Trx easily react with oxidized target protein and reduce disulfide bond on it. By using the immobilized mutant Trx on the resin, in which one cysteine was substituted with serine, the target proteins was efficiently captured. In higher plants, green alga and cyanobacteria, many Trx target proteins

were already identified using this method. In contrast, redox regulation mediated by Trx is not studied very much in photosynthetic bacteria so far. Green sulfur bacterium Chlorobaculum tepidum (formerly designated as Chlorobium tepidum) requires strict anaerobic condition for growth. This bacterium uses various inorganic sulfur compounds and H2S ----as electron donors. The complete genome of C. tepidum was determined in 2002 by the Institute for Genomic Research, USA. Then typical Trx homologues were found (Trx1 and Trx2) in the genome. To elucidate Trx system in C. tepidum, we first expressed the wild type and cysteine mutants of Trx1 and Trx2 in E. coli. Both wild type Trxs showed thiol-disulfide oxidoreductase activity. By using immobilized cysteine mutants, we could obtain Trx target candidate proteins from C. tepidum. The captured proteins were then separated by 2-D SDS/PAGE, and the protein spots on the gel were digested with trypsin, and identified by using matrix assisted laser desorption/ionization time of flight mass spectrometry and the MASCOT peptide mass fingerprint search engine (Matrix Science). We report the possible Trx system in C. tepidum cell. PS5.11 Redox regulation of chloroplast gene expression in wheat plants M Ismayilov (Institute of Botany)

PS5.12 Oscillating yield of flash-induced chlorophyll fluorescence decay in intact cells of Thermosynechococcus elongatus Z Deák (Institute of Plant Biology, Biological Research Center, Szeged, Hungary ), I Vass (Institute of Plant Biology, Biological Research Center, Szeged, Hungary ) Flash-induced chlorophyll fluorescence increase and subsequent decay provides a useful tool in PSII electron transport studies in a wide range of experimental objects ranging from isolated PSII core complexes to cyanobacterial or algal cells and intact leaves. Typically increased fluorescence yield reached due to excitation by a saturating light pulse decreases monotonically in time from its maximal level to Fo. Decay curves can be explained by assuming the well-known two electron gate model of PSII acceptor side, and can be resolved to three decay components. Intact cells of the thermophilic cyanobacterium Thermosynechococcus elongatus do not show the monotonically decreasing yield of flash-induced fluorescence decay. Instead the relaxation curves possess a transient drop with minimum around 100 ms after the flash, followed by an overshoot after few seconds resulting in dampened oscillation of the fluorescence yield. This very interesting phenomenon was investigated utilizing different acceptors and/or inhibitors of electron transport, temperature dependence and light/dark adaptation conditions. Possible mechanisms behind the oscillating yield of flash-induced fluorescence decay in intact cells of Thermosynechococcus elongatus will be discussed. PS5.13 Modulation of photosynthetic capacity and thylakoid protein composition in Arabidopsis thaliana FNR knock-out mutants M Lintala (University of Turku), P Mulo (University of Turku) Ferredoxin-NADP+-oxidoreductase (FNR) is a hydrophilic, FAD-containing enzyme that catalyses the final step of linear photosynthetic electron transfer reducing NADP+ to NADPH. In addition to linear electron transfer FNR is assumed to participate in cyclic electron flow around Photosystem I (PSI). In Arabidopsis thaliana

191 14th Photosynthesis Congress - PS07 two distinct nuclear genes encode leaf-targeted isoforms of FNR, AtLFNR1 and AtLFNR2. We are currently studying the specific functions of these isoforms using mutant plants lacking either AtLFNR1 or AtLFNR2. Since both mutants are viable it seems that both FNR isoforms are functional, although reduced growth of the mutant plants implies that both isoforms are needed to guarantee optimal photosynthetic capacity. Additionally, slow re-reduction rate of P700 in the mutant plants indicates that both isoforms are involved in cyclic electron transfer as well. In wild type chloroplast FNR isoforms are localised in two compartments: in the stroma as soluble protein and as thylakoid membrane-bound form. In the thylakoid membrane FNR appears in several protein complexes detected after Blue Native gel electrophoresis. Absence of AtLFNR1 protein leads to dissociation of FNR from thylakoid membrane whereas AtLFNR1 can be found both as thylakoid-bound as well as soluble form when AtLFNR2 has been knocked out. This implies that AtLFNR1 is needed for thylakoid membrane association of AtLFNR2 protein, most probably by forming FNR1-FNR2 heterodimer, which is supported by structural model. PS5.14 Enhanced sensitivity to photoinhibition of PSI in the Costata-type mutant of pea is due to PSI-acceptor side limitation A Ivanov (Department of Biology and The Biotron, University of Western Ontario, 1151 Richmond Street, N., London, Ontario, Canada N6A 5B7), M Krol (Department of Biology and The Biotron, University of Western Ontario, 1151 Richmond Street, N., London, Ontario, Canada N6A 5B7), R Morgan-Kiss ( Department of Microbiology, Miami University, Pearson Hall, Oxford, OH 4505 , USA), Y Zeinalov (Institute of Biophysics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, 1113 Sofia, Bulgaria), Comparative analysis of in vivo chlorophyll fluorescence imaging revealed that PSII photochemical efficiency (Fv/Fm) of leaves of the Costata 2/133 pea mutant did not differ from that of WT. However, the yield of PSII electron transport (Fe) and the photochemical quenching coefficient (qP) were decreased to 73% and 78%, respectively relative to WT. This was associated with 64% lower steady state light inducible P700 (P700+) signal, a higher intersystem electron pool size (e-/P700) and higher rate of P700+ re-reduction, which indicate an increased capacity for PSI cyclic electron transfer in the 2/133 mutant than WT. Exposure of WT and the Costata 2/133 mutant to high light stress resulted in a slightly higher photoinhibition of PSII in the mutant. Surprisingly, under the same photoinhibitory conditions PSI photochemistry (P700+) was reduced by 82% in the 2/133 mutant compared to only 28% in the WT. The lower level of oxidizable P700 (P700+) in Costata 2/133 mutant is consistent with a lower amount of PSI related chlorophyll protein complexes (CP1) and lower abundance of PsaA/PsaB heterodimer and PsaD polypeptides as revealed by non-denaturating SDS-PAGE and immunoblot measurements. In addition, this mutant exhibited a limited capacity for state transitions and an up-regulation in the stromal reduction of the plastoquinone pool suggesting electron flux of a cyclic or chlororespiratory nature. The role of PsaD protein in the function, composition and/or stability of the PSI complex as well as the overall organization of the photosynthetic apparatus and its susceptibility to photoinhibition is discussed. PS5.15 CRR1 is specifically involved in accumulation of the chloroplast NDH complex despite its homology to DHPR in Arabidopsis H Shimizu (Kyushu University)

Chloroplast NAD(P)H dehydrogenase (NDH) is a homolog of the bacterial NADH dehydrogenase NDH-1 and is involved in the cyclic electron transport around photosystem I. Although 14 subunits have been identified in the chloroplast NDH complex, its entire subunit composition, including the subunits involved in electron donor binding, has not been elucidated yet. Arabidopsis crr1 (chlororespiratory reduction 1) mutants were isolated by chlorophyll fluorescence imaging on the basis of their lack of NDH activity. CRR1 was homologous to dihydrodipicolinate reductase (DHPR), which functions in a lysine biosynthesis pathway. However, the DHP-binding motif was not conserved in CRR1, and the crr1 defect specifically caused the destabilization of the chloroplast NDH complex, implying that CRR1 is not involved in lysine biosynthesis in Arabidopsis. CRR1 contained a chloroplast targeting signal and an NAD(P)H-binding motif. In addition, CRR1 was expressed only in photosynthetic tissue as other nuclear-encoded genes for NDH subunits. These characteristics indicated that CRR1 was a candidate for the electron donor-binding subunit of the NDH complex. However, CRR1 was detected in the stroma but not in the thylakoid membranes, where the NDH complex is localized. Furthermore, CRR1 was stable even in absence of the NDH complex. These results suggest that CRR1 is involved in biogenesis or stabilization of the NDH complex, possibly via the reduction of an unknown substrate. PS5.16 Energy dissipation and photoprotection in deletion mutants of the minor Lhcb proteins CP24 and CP26. S de Bianchi (University of Verona - Italy), L Dall'Osto (University of Verona - Italy), R Bassi (University of Verona - Italy) Lhcb proteins act in light harvesting and energy dissipation of Photosystem II. In particular, minor complexes CP29, CP26 and CP24 have been suggested to be of particular importance for NPQ and photoprotection. However, their specific role in these processes is still not clear. The analysis of koCP26Arabidopsis thaliana plants only slightly affects non-photochemical quenching while plants koCP24 plants shows a decrease capacity of non-photochemical quenching and limited growth. We analyzed growth and NPQ capacity of the double mutants koCP26xkoCP24-, which recovers near-WT phenotype. We conclude that CP24 protein is not directly involved in the NPQ process. The mechanism for the decrease of NPQ and growth rate in koCP24 plants is discussed in detail. PS5.17 Preferential decay of the CF1 epsilon subunit induces thylakoid uncoupling in wild watermelon under drought stress K Kohzuma (NAIST), K Akashi (NAIST), Y Munekage (NAIST), T Hisabori (TIT, ERATO), A Yokota (NAIST) Photosynthetic energy conversion in plants involves the formation of proton gradient across thylakoid membranes, but the mechanisms for balancing the membrane potential has been poorly elucidated. We found that drought stress induces selective decomposition of the epsilon subunit in the CFoCF1 ATP synthase. Thylakoid membranes from the stressed leaves showed reduced efficiencies in proton gradient formation and energy coupling, but addition of the recombinant epsilon subunit significantly suppressed their leaky property. We conclude that the selective decomposition of the epsilon subunit induces partial uncoupling of the thylakoid membranes under drought, and hence contributes to the excess energy dissipation in chloroplasts.

192 14th Photosynthesis Congress - PS07 PS5.18 Effects of the PsbP knockdown on the photosynthetic electron transfer in Nicotiana tabacum. K Ido (Graduate School of Biostudies, Kyoto University), K Ifuku (Graduate School of Biostudies, Kyoto University), S Ishihara (Graduate School of Biostudies, Kyoto University), Y Yamamoto (Graduate School of Biostudies, Kyoto University), C Miyake (Research Institute of Innovative Technology for the Earth), F Sato (Graduate School of Biostudies, Kyoto University) PsbP is an extrinsic protein associated with the luminal side of photosystem II in higher plants and some green algae. In vitro experiments suggest that PsbP facilitates retention of Ca2+ and Cl- within PSII, which are essential cofactors for the water splitting reaction. In our laboratory, tobacco plant in which PsbP was knocked-down by RNAi was established to analyze the physiological function of PsbP in vivo [Ifuku K. et al., Plant Physiology 139, 1175-1184, 2005]. In the PsbP-knockdown tobacco (ΔPsbP plants), Fv/Fm value, an indicator of maximal PSII activity, was severely decreased, whereas most of PSII subunits were normally accumulated. On the other hand, the level of photosystem I was markedly decreased, while that of subunits of NDH and Cyt b6/f complex were increased. These observations were not observed in Chlamydomonas mutant lacking PsbP. Therefore, knockdown of PsbP caused particular effects on the property of the photosynthetic electron transfer machinery in higher plants. Photosynthetic electron flow in ΔPsbP leaves was further analyzed by means of chlorophyll fluorescence and thermoluminescence. In ΔPsbP leaves, the redox state of PSI reaction center (P700) was rather oxidized even when significant accumulation of the reduced primary acceptor QAwas observed. This indicated that the back-electron flow around PSII would be dominated in ΔPsbP leaves. Thermoluminescence study supported the stabilization of the redox pair of S2/QA- in ΔPsbP leaves. Effects of PsbP knockdown on the protein-supercomplex formation in thylakoid membranes are being analyzed. PS5.19 The plastoquinone pool involving in the oxygen reduction in the photosynthetic electron transport chain M Mubarakshina (Institute of Basic Biological Problems RAS) The oxygen reduction in the photosynthetic electron transport chain increased with an increase of the light intensity. The share of the plastoquinone pool participation in the oxygen reduction in the electron transport chain reached 50-70% at high light intensities. The hydrogen peroxide production in thylakoids was studied in the presence of cytochrome C preventing superoxides dismutation outside thylakoids. The considerable amount of the hydrogen peroxide is generated inside the thylakoid membranes. The oxidation of reduced plastoquinone pool after switching off the light consisted of two phases: the first quick and the second slower. The share of the first quick phase was higher after plastoquinone pool reduction at high light intensity. The oxidation of the plastoquinone pool didn’t observe under anaerobic conditions. The reduction level of the plastoquinone pool was the same at both light intensities. It was concluded that the first quick phase depended on the oxidation of the plastoquinone pool by superoxides generated in the light in the thylakoid membrane, while the second phase was autocatalytic oxidation of the plastoquinone pool through the oxygen reduction in the plastoquinone pool. The data are explained as the result of ?2 reduction in the plastoquinone pool as well as in the Photosystem I followed by reduction of the superoxides generated in both processes, by plastohydroquinone, to H2O2.

PS5.20 The oxidation of the plastoquinone pool in the thylakoids under areobic conditions M Mubarakshina (Institute of Basic Biological Problems Russian Academy of Sciences) The oxidation of the plastoquinone pool, reduced during illumination of the thylakoids, consisted of two phases after switching off the light. The proportion of the phases contributions depended on the light intensity: the contribution of the first quick phase in the pool oxidation was higher after illumination with stronger light. The initial phase could not be explained by the electron flow toward FeS-Rieske centre, cyt f and plastocyanin since the light-induced redox changes of cyt f were not observed at both light intensities. The reduction of the plastoquinone pool was the same in both strong and weak lights, and therefore the level of the plastoquinone pool reduction was not responsible for the difference in the contributions of the quick phase in various lights. The oxidation of the plastoquinone pool was not under anaerobic conditions. A putative plastoquinone oxidase was not involved in the process determining the biphasic kinetics of the plastoquinone pool oxidation. The rate of oxygen reduction in the light increased with an increase in the light intensity. The both the dependence of the plastoquinone pool oxidation on the oxygen presence and the higher size of the first quick phase after illumination with stronger light lead to the conclusion that this phase represents the appearance of PQ from PQ•- produced in the course of PQH2 oxidation by the superoxides accumulated in the light within the thylakoid membrane. The second slow phase is the autocatalytic oxidation of the plastoquinone pool through the oxygen reduction in the pool. PS5.21 Ultrafast Optical Studies of the Cytochrome b6f Complex in Solution and Crystalline States S Savikhin (Purdue University), N Dashdorj (Purdue University), E Yamashita (Purdue University), J Schaibley (Purdue University), W Cramer (Purdue University) The cytochrome b6f complex of oxygenic photosynthesis contains a single chlorophyll a molecule whose function is presently unknown. The singlet excited state of the Chl a molecule is quenched by the surrounding protein matrix, and thus the Chl a molecule in the b6f complex may serve as an exceptionally sensitive probe of the protein structure. For the first time, singlet excited state dynamics were measured in well-diffracting crystals using femtosecond time-resolved optical pump-probe methodology. Lifetimes of the Chl a molecule in crystals of the cytochrome b6f complex having different space groups were 3-6 times longer than those determined in detergent solution of the b6f. The observed differences in excited state dynamics may arise from small (~ 1 Å) changes in the local protein structure caused by crystal packing. The Chl a excited state lifetimes measured in the dissolved cytochrome b6f complexes from several different species are essentially the same, in spite of differences in the local amino acid sequences around the Chl a. This supports an earlier hypothesis that the short excited state lifetime of Chl a is critical for the function of the b6f complex. PS5.22 Plastocyanin rather than cytochrome f is responsible for their specific electrostatic interactions in the cyanobacterium C Albarran (IBVF, Universidad de Sevilla & CSIC, Sevilla, Spain), J Navarro (IBVF, Universidad de Sevilla & CSIC, Sevilla, Spain), M De

193 14th Photosynthesis Congress - PS07 la Rosa (IBVF, Universidad de Sevilla & CSIC, Sevilla, Spain), M Hervas (IBVF, Universidad de Sevilla & CSIC, Sevilla, Spain) b6f complex to photosystem I. The plastocyanin-cytochrome f complex from Nostoc exhibits relevant structural and functional differences when compared with the homologous complexes from other cyanobacteria and plants. The Nostoc complex shows an apparent electron transfer rate constant value much larger than in other organisms, with electrostatic and hydrophobic interactions being differently involved in complex formation. A wide set of site-directed mutants of Nostoc plastocyanin and cytochrome f – in which some of the residues located at the interface area of the complex were changed – has been analyzed by laser flash photolysis to determine the role of such residues in the protein-protein interaction. The mutations on plastocyanin have a similar effect on the interaction with both cytochrome f and photosystem I, thus indicating that the copper protein uses the same surface areas to interact with its two physiological partners. On the other hand, cytochrome f mutants with some negative charges replaced by neutral residues show an apparent electron transfer rate constant with wild-type plastocyanin similar to or slightly higher than the wild-type species, whereas the mutants with negative charges replaced by positive residues show a significantly lower reactivity. Taking together, these results indicate that the effects of neutralizing residues at the electrostatically charged patch of cytochrome f are smaller than those previously observed for mutants of plastocyanin, thus suggesting that it is the copper protein which determines the specificity of the electrostatic interaction with the heme protein. PS5.23 Dark inactivation of ferredoxin-NADP reductase and cyclic electron flow under far-red light in sunflower leaves E Talts (Tartu University, Institute of Molecular and Cell Biology), V Oja (Tartu University, Institute of Molecular and Cell Biology), A Laisk (Tartu University, Institute of Molecular and Cell Biology) The oxidation kinetics under far-red light (FRL) of photosystem I (PSI) high potential donors P700, plastocyanin (PC) and cytochrome f (Cyt f) were investigated in sunflower leaves with the help of a new high-sensitivity photometer at 810 nm. The slopes of the 810 nm signal trace were measured immediately before and after FRL was turned on or off. Provided that the intensity and leaf absorptance of FRL were quantified, such measurements were sufficient to quantify the pools of P700, PC and Cyt f, as well as cyclic electron flow (CET) and the quantum efficiency of PSI electron transport. The 810 nm signal derivatives (slopes) were calculated from a mathematical model based on redox equilibrium between P700, PC and Cyt f and the parameters of the model were varied until the model fitted to the measurements. In sunflower the typical pool sizes were 1.0 – 1.5 mmol m-2 of P700, 3 PC/P700 and 1 Cyt f/P700. The best-fit apparent equilibrium constants (averaged over the inhomogeneous leaf cross-section) were 15 between P700 and PC and 3 between PC and Cyt f. CET activated as soon as electrons accumulated on the PSI acceptor side, provided that Cyt f was sufficiently oxidized. After longer dark exposures CET was remarkably fast because linear e- transport was temporarily hindered by the dark inactivation of ferredoxin-NADP reductase. It is suggested that the dark inactivation of FNR may reflect the complexing of the enzyme with Cyt b6f.

PS5.24 Dual-wavelength analysis of photosystem I electron transport V Oja (Tartu University, Institute of Molecular and Cell Biology), H Rämma (Tartu University, Institute of Molecular and Cell Biology), A Anijalg (Tartu University, Institute of Physics), A Laisk (Tartu

University, Institute of Molecular and Cell Biology) We describe a new modulated dual wavelength spectrophotometer with noise level <10-5 for the measurement of leaf transmittance changes at 810 and 950 nm. At 810 nm the full redox signal is about 1000, at 950 nm about 150 A/D converter bits with the noise of 1 bit. At 810 nm the PC+/P700+ signal ratio is about 0.3/0.7 and Fd- may contribute up to 15%, at 950 nm the PC+/P700+ ratio is about 0.7/0.3 and Fd- is not detected. The measuring beams of the spectrophotometer are incorporated into a specially designed fibber-optic light guide along with Chl fluorescence measurements and three light sources for actinic leaf illumination, forming a computer-controlled optical and gas exchange system for simultaneous measurements of leaf transpiration, CO2 uptake, O2 evolution, Chl fluorescence and transmittance at two wavelengths. The sensitivity is sufficient to replace the 950 nm for 520 nm, indicating electrochromic shift. The system was used to analyze the reduction/oxidation kinetics in sunflower leaves under far-red light (FRL). During the slow (20 s) reduction by stromal reductants the 810 vs. 950 nm signal dependence could be best fit with a homogeneous redox-equilibrium model assuming an equilibrium constant KP of 50 between P700 and PC, however, then the ratio of extinction coefficients of P700+ and PC+ at 810 nm of 3 was required, instead of 6.4 reported in literature. Assuming the extinction ratio of 6.4, the best-fit KP was 15, but the fit was worse. During the following oxidation under FRL the 810 vs. 950 nm trajectory declined from the homogeneous redox equilibrium, indicating inhomogeneity of the PC/P700 ratio, probably the fractions of PSI in stroma and grana thylakoids in the leaf. During fast photosynthesis under saturating light and post-illumination re-reduction, the 810 vs. 950 nm signals remained on the same homogeneous equilibrium trajectory as during the slow re-reduction from FRL. No indications about the limitation of steady-state leaf photosynthesis by PC diffusion could be detected when Cyt b6f regulation was established, but the diffusional limitation was evident during the initial period of dark-light transition, when Cyt b6f was not yet downregulated. PS5.25 Inclusion of an oxygen electron-transport chain

pool

into

photosynthetic

B Ivanov (Institute of Basic Biological Problems, Russian Academy of Sciences), S Khorobrykh (Institute of Basic Biological Problems, Russian Academy of Sciences), M Mubarakshina (Institute of Basic Biological Problems, Russian Academy of Sciences), K Marina (Institute of Basic Biological Problems, Russian Academy of Sciences) The experimental data on the light-dependent transfer of electrons to oxygen as well as a hydrogen peroxide formation in the thylakoid membranes are presented. It is shown that only 30-50 % of an electron flow to oxygen goes through ferredoxin in the absence and in the presence of NADP+. With oxygen as the sole electron acceptor a PQ-pool contribution in the total oxygen reduction in the photosynthetic electron-transport chain at high light intensities achieved 50 % at ?? 5.0, and was higher than 60 % at ?? 6.5 and ?? 7.8. The hydrogen peroxide formation occurs mainly inside the thylakoids. The analysis of the data implies that a superoxide formed within the membrane as a result of O2 reduction by components of the acceptor side of Photosystem 1, is reduced to the hydrogen peroxide by plastoquinol molecules of the plastoquinone pool (co-operative oxygen reduction). The hypothesis is put forward, that the photosynthetic electron-transport chain includes an «oxygen pool». PS5.26

194 14th Photosynthesis Congress - PS07 Relation between ADP inhibition and e inhibition of rotation of the chloloplast-type F1-ATPase T Hisabori (CRL, Tokyo Tech.), T Murakami (CRL, Tokyo Tech.), H Konno (CRL, Tokyo Tech.) The chloroplast type F1 ATP synthase synthesizes ATP coupled with the photosynthetic electron flow in the chloroplasts and works as the key enzyme of energy conversion. This enzyme is known to be highly regulated by the endogenous inhibitor e, tightly bound ADP, the membrane potential, and the redox state of the g subunit. In order to understand e inhibition at the molecular level, we adopted the a3b3g subcomplex in thermophilic cyanobacteria, which was expressed in E. coli. We found that e inhibition of this ATPase was ATP-independent, and was different to that observed for bacterial F1-ATPase. Since the chloroplast type F1-ATPase contains the additional region on the g subunit, the function of this region was also examined though the g subunit of cyanobacteria lacks the regulatory two cysteines. By the analysis of single molecule rotation, we succeeded in assigning the pausing angular position of the g subunit in e inhibition. The observed position was found to be identical to that observed for ATP hydrolysis, product release and ADP inhibition, but distinctly different from the waiting position for the binding of the next substrate ATP (Konno H. et al., EMBO J. (2006) 25, 4596-4604). Based on these observations, we discuss about the significance of the regulation of ATP synthase and the variety of the regulation mechanisms as well. PS5.27 Insights into the function of PsbR protein in Arabidopsis thaliana Y Allahverdiyeva (Department of Biology, Plant Physiology and Molecular Biology, University of Turku, FIN-20014 Turku, Finland), F Mamedov (Department of the Photochemistry and Molecular Science, Ångström Laboratory, Box 532, Uppsala University, Uppsala 751 20, Sweden), M Suorsa (Department of Biology, Plant Physiology and Molecular Biology, University of Turku, FIN-20014 Turku, Finland), S Styring (Department of the Photochemistry and Molecular Science, Ångström Laboratory, Box 532, Uppsala University, Uppsala 751 20, Sweden) The functional state of the Photosystem II complex in Arabidopsis psbR T-DNA insertion mutant was studied. The DPsbR thylakoids showed about 34% less oxygen evolution than WT, which correlates with the amounts of PSII estimated from YDox radical EPR signal. The increased time constant of the slow phase of flash fluorescence (FF)-relaxation and upshift in the peak position of the main thermoluminescence (TL)-bands, both in the presence and in the absence of DCMU, confirmed that the S2QA- and S2QB- charge recombinations were stabilized in DPsbR thylakoids. Furthermore, the higher amount of dark oxidized Cyt-b559 and the increased proportion of fluorescence, which did not decay during the 100s time span of the measurement thus indicating higher amount of YD+QA- recombination, pointed to the donor side modifications in DPsbR. EPR measurements revealed that S1-to-S2-transition and S2-state multiline signal were not affected by mutation. The fast phase of the FF-relaxation in the absence of DCMU was significantly slowed down with concomitant decrease in the relative amplitude of this phase, indicating a modification in QA to QB electron transfer in DPsbR thylakoids. It is concluded that the lack of the PsbR protein modifies both the donor and the acceptor side of the PSII complex. PS5.28 Do alternative electron donors protect heat-inactivated photosystem II from photoinhibition?

S Toth (Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, PO Box 521, H-6701 Szeged, Hungary), L Kovacs (Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, PO Box 521, H-6701 Szeged, Hungary), J Puthur (Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, PO Box 521, H-6701 Szeged, Hungary), G Garab (Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, PO Box 521, H-6701 Szeged, Hungary) Oxygen-evolution can be fully inhibited with a heat pulse (submerging leaves in water bath of 48-50 °C for 40-60 s) without inducing visible symptoms and secondary effects, such as dessication. Under these conditions, a limited linear electron transport is observed which is due to the presence of alternative electron donors that donate electrons to photosystem II with a t1/2 of ~ 30 ms (Tóth et al. 2007, Biochim. Biophys. Acta 1767: 295-305). The pool of electrons is large (half-depletion occurs after ~500 charge separations) and regenerates after a few minutes of darkness. It has been suggested that ascorbate is the alternative electron donor and its possible role is to prevent the accumulation of highly oxidizing charges in photosystem II. Keeping heat-treated plants in the light for 30 min at 100 µmol m-2 s-1 PPFD does not cause significant damage compared to plants kept in the dark for the same duration after the heat pulse. It takes about 4 hours in the light until all linear electron transport activity disappears (Tóth et al. 2005, J Plant Physiol 162: 181-194). These data suggest that ascorbate has a protective role against photoinhibition. This hypothesis is being further tested by measuring the dependence of the residual photosystem II acitivity in leaves after heat pulse and by using ascorbate-deficient mutants. PS5.29 Development of 3D confocal laser scanning microscope for applying saturation pulse method of chlorophyll a fluorescence K Omasa (The University of Tokyo), A Konishi (The University of Tokyo) To analyze three-dimensionally chlorophyll a fluorescence of the leaf tissue using the saturation pulse method, a newly real-time confocal laser scanning microscope was developed. In the microscope, light intensities of the saturation light pulse and the actinic light were controlled at 1500 to 6000 μmol m-2 s-1 and 50 to 250 μmol m-2 s-1, respectively. The high sensitive CCD camera was used for capturing the clear focal planes even under the weak light. A high-speed z-scanning device was installed to gain many focal planes enough to reconstruct clear 3D images of chlorophyll fluorescence parameters within an irradiance of the saturation light pulse about 2 second using an objective of ×40 magnification. Using the system, chlorophyll fluorescence parameters, which responded to light intensity of the actinic light, of guard cell chloroplasts, epidermic cell chloroplasts and mesophyll chloroplasts of a fern were compared each other in a 3D image. It was demonstrated that FPSII values of the guard cell chloroplasts was lower than not only those of the just adjacent mesophyll chloroplasts but also those of mesophyll chloroplasts which stayed away from the guard cells. Additionally, FPSII values of epidermic cell chloroplasts were lower than those of subjacent mesophyll chloroplasts, whereas NPQ values of the former were lower than those of the latter. Consequently, it was implied that functions of chlorophyll fluorescence electron transport were different between the guard cell chloroplasts, the epidermic cell chloroplasts and mesophyll cell chloroplasts. PS5.30 Three-dimensional chlorophyll fluorescence imaging for

195 14th Photosynthesis Congress - PS07 detecting effects of a herbicide in leaves. A Eguchi (The University of Tokyo), A Konishi (The University of Tokyo), F Hosoi (The University of Tokyo), K Omasa (The University of Tokyo) The spatiotemporal movements and effects of a herbicide in leaves of a potted eggplant were examined by mapping chlorophyll a fluorescence image on a 3D polygon image derived from lidar (light detection and ranging) data. In the experiment, the herbicide containing DCMU (3-(3,4-Dichlorophenyl)-1,1-dimethylurea) was dissolved in water and it was watered to the pot. Images of chlorophyll a fluorescence intensity were captured every 15 minutes during 5 hours in lighting conditions. The DCMU in the herbicide obstructed a photosynthetic electron transport, consequently the chlorophyll a fluorescence quenching disappeared. In the concrete, the increases in chlorophyll a fluorescence intensity firstly appeared along the veins of the leaves, and gradually expanded to the mesophylls. It was implied that the herbicide was transferred by water flow from the roots to the leaves through the stems, and arrived at the veins and mesophylls of the leaves. The increase rates in fluorescence intensity at the upper young and lower aging leaves were slower than those at the middle mature leaves. It would appear that the transpiration in the mature leaves adequately functioned but the young and aging leaves did not efficiently transpiration because of immaturity and senescence of stomata. PS5.31 Spatiotemporal changes in PRI and NPQ under different light intensity gradients on leaf surfaces A Konishi (The University of Tokyo), M Munehiro (The University of Tokyo), K Omasa (The University of Tokyo) PRI (photochemical reflectance index) reflects changes of xanthophyll pigments, therefore it was regarded as a useful index which assesses a degree of heat dissipations of plant leaves. NPQ (non-photochemical quenching of chlorophyll fluorescence) also is an indicator of the degree of heat dissipations. We have therefore compared spatiotemporal changes in PRI and NPQ under different light intensity gradients on attached leaf surfaces. As compared with temporal changes of PRI and NPQ at one measurement point, a linear relationship was observed between PRI and NPQ at strong actinic light intensity (180 μmol m-2 s-1), whereas such relationship was not observed at weak actinic light intensity (800 μmol m-2 s-1), because the change of PRI was very small in comparison with that of NPQ. When a large gradation of light intensity was generated on the leaf by illumination from an oblique direction, gradations of both PRI and NPQ corresponded to those of the light intensity were observed. However, when the leaf was illuminated from above just after the illumination from an oblique direction was lighted off, the gradation of NPQ remained, meanwhile that of PRI disappeared. These results suggest that PRI was affected by physical factors, such as leaf inclination and surface structure, relating to leaf reflectance as well as physiological factors. Therefore, we should pay more careful attention to explanation of spatiotemporal changes of PRI. PS5.32 Genetic manipulation of PSI biogenesis in higher plants D Bednarczyk (Max-Planck-Institut für Molekulare Pflanzenphysiologie), R Bock (Max-Planck-Institut für Molekulare Pflanzenphysiologie), M Schöttler (Max-Planck-Institut für Molekulare Pflanzenphysiologie) In higher plants, the contents of PSII, the cytochrome-bf complex and plastocyanin vary strongly under different growth conditions and developmental states of leaves. In case of plastocyanin and the

cytochrome-bf complex, content variations by a factor of up to ten have been observed, resulting in comparable changes in linear electron flux capacity, as the cytochrome-bf complex is the predominant site of photosynthetic flux control in higher plants. On the other hand, PSI contents of higher plants are relatively constant, varying between 2 to 2.5 mmoles (mol chl.)-1 independent of the environmental and metabolic state of the plant. Always, a surplus number of PSI units is present, relative to the rate-limiting cytochrome-bf complex. Due to its faster turn-over halftime per electron, the higher PSI content results in a several fold potential overcapacity of PSI, relative to the limiting step of linear electron flux. It is not understood why such a surplus number of PSI is required. To address this problem, we have generated transplastomic tobacco plants with decreased accumulation of PSI with wild-type subunit composition. For this purpose, we have altered the translation initiation side (Shine-Dalgarno-sequence and start codon) of the psaA mRNA, as the PsaAB reaction center core dimer is supposed to be rate-limiting for the overall accumulation of PSI. We have also depressed essential (Ycf3) and non-essential chaperones of PSI assembly. Using these approaches, we have generated and physiologically characterized tobacco transformants with a wide array of PSI contents, varying from almost wild-type like to knock-out like phenotypes. PS5.33 Bifurcated electron donations from quinol oxidoreductase and soluble CycA to cytochrome cz of the photosynthetic reaction center complex in the green sulfur bacterium Chlorobium tepidum C Azai (Department of Biological Sciences, Graduate School of Science, Osaka University), Y Tsukatani (Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology), S Itoh (Division of Material Science, Graduate School of Science, Nagoya University), H Oh-oka (Department of Biological Sciences, Graduate School of Science, Osaka University) Chlorobium (Chl.) tepidum, the photo-oxidized primary electron donor P840+ is rapidly rereduced by cytochrome (cyt) cz, one of subunits of the reaction center (RC) complex. Previous studies have revealed that two different electron donors function to rereduce the oxidized cyt cz. The membrane-bound quinol oxidoreductase has been shown to donate electrons directly to cyt cz in the purified membranes, while soluble low-molecular-weight cyt c, CycA, has been done in the in vitro reconstitution experiment using the purified RC complex. In this study, we investigated the characteristics of the electron transfer reactions from quinol oxidoreductase and CycA to cyt cz when they co-exist together as in vivo. The reconstitution system was set up by adding CycA into membranes prepared from the ?cycA mutant lacking CycA. The oxidized cyt cz, immediately formed after flash excitation, was rereduced by both CycA and quinol oxidoreductase independently. The reaction mode from the former to cyt cz appeared to obey a second-order reaction. Unlike the electron transfer reaction from cyt bc1 complex to cyt c2 in purple bacteria, the Chl. tepidum quinol oxidoreductase never operates as an electron donor to CycA. To elucidate its physiological meanings, we examined how the photosynthetic electron transfer pathways were linked to the sulfur oxidation ones. Analysis of mutants related to sulfur oxidations in addition to a ?cycA mutant one indicated that quinol oxidoreductase and CycA seemed to serve as electron carriers in the different sulfur oxidation pathways. PS5.34 Resolving Protein-Cofactor Interactions in the Cytochrome bc1 Complex F MacMillan (UEA), C Lange (Max-Planck Institute for Biophysics), C

196 14th Photosynthesis Congress - PS07 Hunte (Max-Planck Institute for Biophysics)

E Gross (The Ohio State University)

Ubiquinol:cytochrome c oxidoreductases are found in photosynthesis where the bc1 complex is associated with bacterial reaction centres and its homologue in higher plants, the b6f complex, is located between photosystems I and II. The recent 3D structures of these bc1-type complexes now allows the possibility to investigate the relationship between their structure and their catalytic function, an important issue since the mechanism of electron-coupled proton transfer in such proteins is still poorly understood. Quinones molecules are bound at specific binding sites within the bc1 complex. Their biological function is determined by their ability to be reduced and/or oxidised in two successive one-electron steps. In this study, we focus on the redox-active ubiquinones from the cytochrome bc1 complex from S. cerevisiae where the location and/or exact binding of such molecules is still unclear. Here we apply EPR techniques to characterise these quinone-binding sites. High-field EPR is used to resolve the g-tensor,while pulsed Electron Nuclear DOuble Resonance (ENDOR) is used to map out the spin density distribution. Selective isotopes (13C, 15N, 2H, 17O) and site directed mutants are used to characterise binding within the protein by both ENDOR and 2-dimensional proton-HYperfine Sub-level CORrElation (HYSCORE) and DONUT spectroscopy. Further ESEEM spectroscopy together with an inversion recovery filter (REFINE) is applied to resolve whether observed 14N modulations arise from interactions to semiquinones or to other underlying paramagnetic centres. These results are compared with DFT calculations and are discussed with regard to the location and catalytic function of these quinones within the enzyme.

Brownian Dynamics (BD) computer simulations were used to study the interaction between Chlamydomonas cytochrome f (cyt f) and plastocyanin (PC). The question addressed was whether an electrostatic encounter complex is formed before an electron-transfer-active dock. To do this, the kinetics of complex formation was studied. In BD simulations, the motion of proteins is determined by both electrostatic forces and random Brownian motions. No hydrophobic interactions are included. Simulations were carried out at pH7 and 10mM ionic strength either in the presence or absence of an electrostatic field determined by the charges on both proteins. The simulations are started with the Cu on PC ~90 Å from the Fe on cyt f where the electrostatic field is negligible. Once PC is caught in the electrostatic field, it is rapidly drawn into an electrostatic well forming electrostatic encounter complexes with a Cu-Fe distance between 15 and 40Å. PC stays in this well between 50 and 200 ms. Occasionally, once in the electrostatic well, the PC approaches to withing 15-18 Å and are close to forming electron-transfer-active docks. The structures of both types of complexes will be discussed. In contrast, in the absence of the electrostatic field, PC, no encounter complexes are formed.

PS5.35 The oxidation state of the manganese cluster modulates the kinetics of chlorophyll fluorescence induction R Carpentier (Université du Québec à Trois-Rivières), S Boisvert (Université du Québec à Trois-Rivières), A Gauthier (Université du Québec à Trois-Rivières), D Joly (Université du Québec à Trois-Rivières) The oxygen evolving complex (OEC) provides electrons to the primary electron donor of photosystem II (PSII), P680. Previous works demonstrated that changes in the initial population of the S-states induced by flashes affected PSII fluorescence. A popular way to study PSII is fluorescence induction (FI) because it is fast, reliable and non invasive. There are three apparent steps in the FI curve of thylakoid membranes denoted O-J, J-I and I-P, which are generally related to peak accumulation of the different reduced species in PSII. A recent study has confirmed that O-J is related to the accumulation and equilibrium of QAwhile I-P is related to plastoquinone pool reduction. However, while the origin of J-I remains unclear, it was observed that the amplitude of O-J (AO-J) and J-I (AJ-I) are closely related. In the present work, we analyzed the effects of the initial S-state population on FI of thylakoid membranes pre-illuminated with a sequence of flashes. The results indicate that the intensity of AJ-I correlates with the S2+S3 population and that AO-J pattern is exactly the opposite. Both amplitudes follow a period four oscillation, related to the different S-states. From these observations, we suggest that both O-J and J-I are related to QA reduction but they represent two different bottlenecks, strongly regulated by the S-state, prior to the complete closure of the reaction center. PS5.36 Complex Formation Between Chalmydomonas Cytochrome f and Plastocyanin Studied Using Brownian Dynamics Computer Simulations

PS5.37 Structure and Function of Cytochrome c6A R Nimmo (University of Cambridge), D Bendall (University of Cambridge), C Howe (University of Cambridge), B Schlarb-Ridley (University of Cambridge) Cytochrome c6A is a recently identified chloroplast protein homologous to the well-characterized photosynthetic electron carrier cytochrome c6. The latter, like plastocyanin, transfers electrons from the cytochrome bf complex to Photosystem I. It is found in cyanobacteria and algae but not higher plants. Until recently, the accepted view was that higher plants had dispensed with cytochrome c6 entirely. However, cytochrome c6A has now been identified in green algae and land plants. The major differences between the two proteins are the presence in cytochrome c6A of a 12-residue loop containing a disulphide bond and a significant reduction in midpoint potential in cytochrome c6A (~200 mV) compared to cytochrome c6. Knock-out experiments performed in higher plants have shown that cytochrome c6A is unable to function as a substitute for plastocyanin. In addition, the midpoint potential of cytochrome c6A is such that it is unable to accept electrons from the cytochrome bf complex and it is unable to reduce Photosystem I in vitro. We have determined the crystal structure of cytochrome c6A and have proposed a model for its function that accounts for the observed differences between cytochromes c6 and c6A. We suggest that cytochrome c6A acts to catalyse the formation of disulphide bonds within the chloroplast lumen by acting as a "funnel" for electron transfer. PS5.38 PetP, a new regulatory subunit of cyanobacterial cytochrome b6f complexes? M Gendrullis (Ruhr-Universität Bochum), D Gomolla (Ruhr-Universität Bochum)), G Bernát (Ruhr-Universität Bochum)), M Rögner (Ruhr-Universität Bochum) Using a new, rather gentle purification procedure for the cytochrome b6f complex from the cyanobacterium Thermosynechocococcus elongatus (T. elongatus) we could co-isolate a 7.2 kDa protein. This protein had been reported first by us for the mesophilic cyanobacterium Synechocystis PCC 6803 (S. 6803) as product of the open reading frame

197 14th Photosynthesis Congress - PS07 ssr2998. ESI-MS analysis showed that in T. elongatus this protein is encoded by open reading frame tlr0524 - with rather high homology to ssr2998 of S. 6803 and to hypothetical proteins of other cyanobacteria. There are several indications that this 7.2 kDa protein is both structurally and functionally associated with the cyt b6f complex from S. 6803 and T. elongatus: a) Similar to already published data on S. 6803 disruption of tlr0524 leads to seriously reduced growth rates and apparently affect the function of the cyt b6f complex in T. elongatus. b) Also, a slower reduction of the cyt b6f complex by the PQ pool is observed with implications on state transitions and slight change in the PS1-PS2 ratio. c) The isolated cyt b6f complex of T. elongatus also contains the 7.2 kDa protein analog of S. 6803 as observed by mass spectrometry analysis. In contrast to S. 6803, we succeeded in heterologous overexpression of this protein which now enables functional (reconstitution to isolated cyt b6f complex) and structural studies. In summary, we suggest that the 7.2 kDa protein is a new - possibly regulatory - subunit of the cyt b6f complex and we propose to name it "PetP". PS5.39 Experimental resolution and theoretical complexity determine the amount of information extractable from the chlorophyll fluorescence transient OJIP M Tsimilli-Michael (University of Geneva), R J Strasser (University of Geneva) What we know for any aspect in experimental sciences is the models we make for it. Models of any theoretical complexity level can be formulated, but they are meaningful only if the experimental signals provide the according resolution. We here present the contribution of our laboratory in utilising chlorophyll fluorescence transients to formulate photosynthetic models. The starting complexity levels were those of the open/closed reaction centres (RCs) model, supported by the experimental determination of the fluorescence rise extremes (F0, FM), and the separate/grouped units model, assessed from the fluorescence rise shape in DCMU-treated samples. Using fluorimeters with improved time resolution (10µs) and actinic-light intensity, the polyphasic shape (OJIP) of the fluorescence rise was revealed in detail. This permitted us to formulate models of higher complexity, composed of more functional building blocks, hence assessing/describing higher heterogeneity levels: Detailed analysis of the O-J phase allowed calculation of electron transport yields, recognition/estimation of silent RCs-“heat-sinks” and determination, also in DCMU-absence, of the grouping probability pG. We could therefore formulate and check a 3-types-model (open/closed/silent RCs) with energetically connected/grouped units (any pG value), applicable for different physiological states. The later recognised K-band (0.3ms) was related with RCs dissociated from the oxygen-evolving-complex and the I-step (30ms) to PSI-RCs inactive in supplying electrons into the CO2-fixation process. The model complexity was enriched with more functional building blocks from studies during State 1-State 2-transition. Simultaneous P700 and plastocyanin absorption measurements and delayed fluorescence signals permit us now to check conclusions made from the OJIP multilevel utilisation.

Geneva) All the time we are surrounded by a varying electromagnetic field. This field of background noise can be picked-up by a coil and stored in a computer for further analysis. The aim of the current work is to investigate whether the presence of substances, placed inside the detecting (pick-up) coil, modifies the background electromagnetic field and whether the modifications can be registered with physical methods and analysed mathematically. Our interest was focused on water solutions of compounds that affect the photosynthetic electron transfer reactions. We designed an experimental setup that includes a sensitive coil and a signal amplifier in order to register the electromagnetic field with and without water solutions and/or living plant material. The electric signal was recorded by a 24-bit sound card of a laptop computer and was analysed by the WaveLab 4.0 computer software. By a Fast Fourier Transformation the signal was translated into spectra (intensity vs frequency) in the frequency range from 20 Hz to 20 kHz. The resulted electromagnetic spectra in the presence of the electron acceptors 1,4-benzoquinone, potassium ferricyanide and methylviologen, and the inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone were obtained. For all the chemicals used, the differential spectra (spectrum of water solution minus spectrum of water) showed the existence of different maxima in the frequency range from 20 to 500 Hz. These maxima appear to be specific for each of the tested compounds. This finding opens the way to study photosynthetic activities with an analytical set-up that does not depend on a detecting light beam. PS5.41 Modulated sink-source interactions preserve the PSII electron transport from senescence-induced inactivation in model system with expanded life span induced by decapitation of bean plants R Strasser (University of Geneva), Y Ivan (Bulgarian Academy of Sciences), V Goltsev (University of Sofia), P Chernev (University of Sofia), I Zaharieva (University of Sofia), D Stefanov (Bulgarian Academy of Sciences)

PS5.40 How to derive electromagnetic frequency spectra of substances placed in a coil that senses the electromagnetic field of background noise: Application for leaves and molecules useful in photosynthesis

One of the approaches to create different physiological states in plants of the same ontogenetically age is decapitation, i.e. removal of the top stem bud without or in combination with consecutive removal of trifoliate leaves. Decapitation procedure can be used as model system for investigation of defoliation, which is often induced by various factors as insects, herbivores, hail, etc. Decapitation was performed immediately after appearance of the primary leaves or after appearance of the 1st, 2nd, 3rd composite leaf. We studied also a variant with primary leaves and stem with apical bud, but without composite leaves. Analyses of prompt chlorophyll fluorescence kinetics OKJIP and millisecond-delayed fluorescence of primary leaves were undertaken to investigate the alterations in PSII electron transport during the decapitation-induced senescence of the non-detached leaves. Analysis of the OKJIP transient by the JIP-test showed an increase in several biophysical parameters of photosystem II, namely the density of reaction centers on cross-section basis, the yields for trapping and electron transport and the performance index, in decapitated plants; the effect was mostly expressed when all leaves except the primary were detached. The effect was weaker when decapitation was done on higher leaf stages. The variant with preserved apical bud showed values similar to those of decapitated plants with primary leaves only. From all cases studied, the observed changes in the induction curves and thermograms of delayed fluorescence, similarly to the results from the analysis of OKJIP, indicated an acceleration of the electron transport beyond PSII in the decapitated plants.

V Goltsev (University of Sofia), M Tsimilli-Michael (University of Geneva), P Chernev (University of Sofia), I Zaharieva (University of Sofia), M Kousmanova (University of Sofia), R Strasser (University of

PS5.42 Thiosulfate-oxidizing multi-component system in the green

198 14th Photosynthesis Congress - PS07 sulfur bacterium Chlorobaculum tepidum T Ogawa (Dept. Biol. Sci., Grad. Sch. Sci., Univ. Tokyo), D Seo (Dept. Chem., Fac. Sci., Kanazawa Univ.), H Sakurai (Dept. Biol., Kanagawa Univ.), I Kazuhito (4Dept. Biol., Kanagawa Univ.) Green sulfur bacteria grow phototrophically using sulfur compounds such as sulfide, sulfur, or thiosulfate as electron donors. The components of the thiosulfate oxidoreductase system, and the functions of each component are controversial. The thiosulfate-dependent mammalian cytochrome (cyt) c reducing activity of the cell extract from Chlorobaculum tepidum was resolved into four fractions (Fraction I, II, III and IV, in the order of elution from a DEAE-Toyopearl column) by ammonium sulfate fractionation, anion-exchange chromatography and cation-exchange chromatography. Fraction I is a heterodimer of SoxY and SoxZ, Fraction II a monomeric SoxB, and Fraction III a heterotrimer of SoxA SoxX and a new component (referred to as γ). For reduction of mammalian cyt c by thiosulfate, all of Fraction I-III is indispensable. The optical spectrum of dithionite-reduced SoxAX-γ showed characteristic of cyt c with an α peak at 551 nm. SoxYZ and SoxB were colorless. Fraction IV is a yellow monomeric SoxF2, and its absorption spectrum suggests that SoxF2 contains flavin. We have also purified a soluble cyt c-554 (about 10 kDa). Addition of SoxF2 and cyt c-554 to Fraction I-III enhanced the thiosulfate oxidizing activity. The functions of each component will be discussed. We propose that thiosulfate is oxidized by the above described thiosulfate oxidoreductase system and that the electrons are then transferred to cyt c-554, which in turn reduces the photooxidized special pair P840 via the reaction center bound cyt c-551 (different from SoxAX-γ). PS5.43 Investigation of the function of a nuoE homologue, sll1220 in Synechocystis sp. PCC6803 by means of mutagenesis H Koike (University of Hyogo), R Islam (University of Hyogo) A multi-subunit complex, NAD(P)H dehydrogenase (NDH1), functions in a cyclic electron transport around photosystem I in cyanobacteria. The mitochondrial or bacterial complex is divided into three subcomplexes; soluble, peripheral and membrane integral part. Genes corresponding to peripheral and membrane integral parts are identified in chloroplast or cyanobacterial genomes. However, three subunits constituting soluble part have not been identified yet. In Synechocystis sp. PCC6803, sll1220 homologous to one of the components of soluble subcomplex, nuoE, is annotated. Thus, the gene product could act as an electron input device to NDH1 complex in cyanobacteria. In order to investigate the possible function, sll1220 was inactivated and phenotype of the mutant was analyzed. The cells grew at the same rate as wild type under various conditions such as low (30 mE×m-2×s-1) or high (300 mE×m-2×s-1) light intensities, low CO2 (0.038%) or high salt (0.5 M NaCl) conditions. The room temperature absorption and 77K fluorescence emission spectra were also not different from those of the wild type. Cyclic electron transport activity was measured by monitoring re-reduction of P700+ in the presence of DCMU. P700+ was rapidly re-reduced after cessation of actinic illumination indicating that the mutant cells still possess cyclic electron transport activity. The rates of re-reduction were severely inhibited by diphenyleneiodonium, an inhibitor of flavoprotein. The cells were more sensitive to the inhibitor than the wild type. On the other hand, phenylmeruric acetate was less inhibitory to the cells. These results suggest that the cyclic electron transport pathway is modified in the mutant. PS5.44 NADPH feeds back to regulate electron transport – a novel mechanism for the regulation of photosynthesis under environmental stress.

S Hald (University of Manchester), P Gallois (University of Manchester), G Johnson (University of Manchester) When plants experience an imbalance between the absorption of light energy and the use of that energy to drive metabolism, they are liable to suffer from oxidative stress. Such imbalances arise due to environmental conditions, (e.g. heat, chilling or drought) and can result in the production of reactive oxygen species (ROS). Various mechanisms have been characterised which help to limit ROS production, including non-photochemical quenching and the accumulation of antioxidants. Here we demonstrate a novel protective process – feedback redox regulation via the NADP+/NADPH pool – and demonstrate that this is vital in avoiding oxidative stress. Photosynthetic electron transport was studied in two transgenic tobacco (Nicotiana tabacum) lines – one having reduced levels of ferredoxin NADP+-reductase (FNR), the enzyme responsible for reducing NADP+, and the other reduced levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the principle consumer of NADPH. Both had a similar degree of inhibition of carbon fixation and impaired electron transport. However, whilst FNR antisense plants were obviously stressed, with extensive bleaching of leaves, GAPDH antisense plants showed no visible signs of stress, beyond having a slowed growth rate. Examination of electron transport in these plants showed that this difference is due to feedback regulation from the NADP+ acceptor pool, preventing over-reduction of the electron transport chain and so controlling ROS production. PS5.45 Kinetic Simulations of the Roller Coaster Landscape of the C-Subunit of the Bacterial Reaction Center T Becker (University Bayreuth), R Ullmann (University Bayreuth), G Ullmann (University Bayreuth) Electron transfer in various systems were shown to include uphill steps in the underlying energy landscape. Kinetic properties of such roller coaster landscapes were measured for several mutants of the tetraheme subunit of the bacterial reaction center [1]. Accompanying simulations based on the experimentally derived energetics allowed to relate the kinetic data to changes in the observed energy landscape. The question remains whether these roller coaster landscapes are common in electron transfer systems since they do offer advantageous properties or whether it is a feature hardly to avoid due to small distances between cofactors required for efficient electron transfer. Continuum electrostatic calculations can be used to study the influence of the protein on the redox properties of the cofactors [2]. The same theoretical framework can be used to simulate the kinetics of electron transfer between cofactors [3], thus allowing to directly relate theoretical calculations to the transfer properties of the system. Here, we used this method to investigate the influence of the protein on the kinetic properties of electron transfer between the C-subunit and the special pair of the bacterial reaction center. The electron flux through the C-subunit is discussed with hinsight to influences of the protein on the cofactors such as solvent exposure, fixed charges of the protein and coupling to its titration behavior. [1] Alric et. al.; J. Am. Chem. Soc. 2006, 128, 4136-4145 [2] Gunner, Honig; Proc. Natl. Acad. Sci. 1991, 88, 9151-9155 [3] Becker,Ullmann, Ullmann; J. Phys. Chem. B 2007, 111, 2957-2968 PS5.46 The Mehler reaction in Chlamydomonas reinhardtii : efficiency during photosynthetic induction and steady-state photosynthesis in wild-type and in a mitochondrial mutant shifted to energy distribution state 2.

199 14th Photosynthesis Congress - PS07 F Franck (University of Liège), P Houyoux (University of Liège) The effects of short (30 s) exposure to anaerobiosis on the light-induced 820 nm absorption transients and on the kinetics of electron transport rate (ETR), measured through chlorophyll fluorescence, were investigated. In wild-type cells, anaerobiosis had large effects on the redox state of PSI donor side (P700 and plastocyanin) and caused a strong decrease in ETR during photosynthetic induction, which lasted for about 1 min. As indicated by their insensitivity to inhibitors, these effects were not dependent on mitochondrial electron transport or on chlororespiration. We therefore took the results as evidence that oxygen is the main electron acceptor at PSI during photosynthetic induction, and we used the anaerobiosis-induced decrease of ETR as a measure of the electron flux through the Mehler reaction. By this method, light-saturation curves of the O2-dependent electron flow were established in different conditions. This study shows that O2-dependent electron flow is triggered within a few seconds and amounts to up to 80 % of total electron flow during photosynthetic induction in algal cells grown on minimal medium, while it represents less than 15 % at steady-state. It was significantly weaker in the presence of acetate and in a mitochondrial mutant devoided of complexes I and III. In both cases a transition towards the energy distribution state 2 was observed. Since cyclic electron transport is promoted in state 2, we suggest that cyclic electron transport efficiently competes with the Mehler reaction. PS5.47 -G and temperature dependencies of the electron transfer rates between P700+ and A1- or FeS- in photosystem I containing different quinones H Murakami (Nagoya University), Y Shibata (Nagoya University), H Mino (Nagoya University), S Itoh (Nagoya University) The rate of the electron transfer (ET) between P700+ and A1- or FeS- (Fx, FA and FB) have been studied in the plant photosystem I (PS I) reaction center complex, which contains different artificial quinones, at 77 - 277 K by transient absorption spectroscopy and ESR. We reconstituted 7 different artificial quinones after the extraction of the intrinsic A1 phylloquinone by the ether extraction method. The redox potential of artrtificial quinones at the A1-site varies between -1.0 and -0.4 V in contrast to the A1 phyllquinone (about -0.8 V). We investigated the rates of charge recombination (CR) from A1- or FeS- to P700+ in these RCs, as well as of the ET between A1 and FeS. We obtained the following results: (1) The CR rates from A1- to P700+ showed almost no -?G and temperature dependencies. Their rates had t1/2 of 200~300 µs. (2) The CR rates from FeS- to P700+ slowed down with decreasing temperature (t1/2 = 0.03 - 1 s at 277 - 77 K). The CR rate depended on the redox potential of each A1 that apparently is not involved in this reaction. (3) The spin polarized signals of P700+A1radical pair states were somewhat different depending on quinone species at the A1-site and temeperature. From these results, we will discuss the electron transfer mechanism between A1-, FeS- and P700+.

PS5.48 Ultrafast Electron Transfer in Photosynthesis: Reduced Pheophytin and Quinone Interaction Mediated by Conical Intersections G Olaso-González (Instituto de Ciencia Molecular (ICMol)-Universitat de València), M Merchán (Instituto de Ciencia Molecular (ICMol)-Universitat de València), L Serrano-Andrés (Instituto de Ciencia Molecular (ICMol)-Universitat de València)

The mechanism of electron transfer (ET) from reduced pheophytin (Pheo-) to the primary stable photosynthetic acceptor, a quinone (Q) molecule, is addressed by using high-level ab initio computations and realistic molecular models. The results reveal that the ET process involving the (Pheo- + Q) and (Pheo + Q-) oxidation states can be essentially seen as an ultrafast radiationless transition between the two hypersurfaces taking place via conical intersections (CIs). According to the present findings, an efficient ultrafast ET implies that the Pheo and Q move toward each other in a given preferential parallel orientation, reaching the most effective arrangement for ET at intermolecular distances (R) around 5-3 Å, where the lowest CIs are predicted. Favored donor/acceptor interactions are related to orientations with some overlap between the lowest occupied molecular orbitals (LUMO) of the two systems, and they lead to state-crossings at an earlier stage of the movement (larger R). Furthermore, when the topology of the interacting moieties does not make possible the LUMOs overlap, the corresponding diabatic potential energy curves do not intersect. Thus, it is anticipated that large scale motions, which are difficult to monitor experimentally, are actually occurring in the photosynthetic reaction centers of bacteria, algae, and higher plants, to fulfill the observed ultrafast ET processes. PS5.49 Photoinactivation of Ascorbate Peroxidase in Isolated Tobacco Chloroplasts: Galdieria partita APX Maintains the Electron Flux through the Water-Water Cycle in Transplastomic Tobacco Plants H Kanamoto (Research Institute of Innovative Technology for the Earth), Y Shinzaki (Research Institute of Innovative Technology for the Earth), M Nishioka (Research Institute of Innovative Technology for the Earth), S Horiguchi (Research Institute of Innovative Technology for the Earth), K Tomizawa (Research Institute of Innovative Technology for the Earth), C Miyake (Research Institute of Innovative Technology for the Earth) We evaluated the H2O2-scavenging activity of the water-water cycle (WWC) in illuminated intact chloroplasts isolated from tobacco leaves. Illumination under conditions that limited photosynthesis [red light, 250 μmol photons m-2 s-1 in the absence of HCO3 -] caused chloroplasts to take up O2 and accumulate H2O2. Concomitant with the O2 uptake, both ascorbate peroxidase (APX) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) lost their activities. However, superoxide dismutase (SOD), monodehydroascorbate radical reductase (MDAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) activities remained unaffected. The extent to which the photosynthetic linear electron flow decreased was small compared with the decline in APX activity. Therefore, the loss of APX activity lowered the electron flux through the WWC, as evidenced by a decrease in relative electron flux through PSII [Φ(PSII)xPFD]. To verify these interpretations, we created a transplastomic tobacco line in which an H2O2-insensitive APX from the red alga, Galdieria partita, was overproduced in the chloroplasts. In intact transplastomic chloroplasts which were illuminated under conditions that limited photosynthesis, neither O2 uptake nor H2O2 accumulation occurred. Furthermore, the electron flux through the WWC and the activity of GAPDH were maintained. The present work is the first report of APX inactivation by endogenous H2O2 in intact chloroplasts. PS5.50 Alteration of O-J-I-P chlorophyll induction kinetics by chromium effect on water splitting system R Popovic (University of Quebec in Montreal, Department of Chemistry, TOXEN), D Dewez (University of Quebec in Montreal, Department of Chemistry, TOXEN), F Perreault (University of Quebec in Montreal,

200 14th Photosynthesis Congress - PS07 Department of Chemistry, TOXEN), P Juneau (University of Quebec in Montreal, Department of Biology, TOXEN) The rapid chlorophyll fluorescence transients induced by saturating flash was investigated when alga Chlamydomonas reinhardtii was exposed to chromium inhibitory effect on water splitting system. The “Pulse-Amplitude-Modulated” fluorometer and the “Plant Efficiency Analyser” system were simultaneously used to analyse under different light condition the yield of variable fluorescence and the rapid fluorescence transients. In this study we showed that variation of transition steps appearing over time during the “Kautsky effect” reflected water splitting system activity as electron donor to photosystem II. We found for chromium effect to change the yield of variable fluorescence and the appearance time of the different transients. We used mathematical approach to identify amplitude and half time of the different fluorescence transients. The change of apparent activation energy (EA) for each fluorescence transients was related to chromium effect on water splitting system and such change was also related to different state of the “Kautsky effect”. The gradual inhibition of water splitting system by chromium effect permitted to interpret the relationship between differences in the fluorescence induction traces and water splitting system activity. PS5.51 Relationship between the in vivo bacteriochlorophyll fluorescence and the state of the photosynthetic apparatus in purple bacteria. D Bina (BC ASCR), R Litvin (BC ASCR), F Vacha (BC ASCR) We have studied the light-induced electron transport and changes in the membrane potential in intact, living cells of the purple bacterium Rhodobacter sphaeroides by means of kinetic difference absorption spectroscopy. In addition, parallel measurements of kinetics of fluorescence yield were performed to obtain information on the energy transformation in the photosynthetic apparatus. Experiments were performed on photoheterotrophically grown cells of Rhodobacter sphaeroides, strain Y, using a laboratory-built multichannel kinetic spectrophotometer-fluorimeter equipped with a pulsed measuring light (Xe-flash lamp, pulse length 1.5 μs) and photodiode array detectors. Kinetics of redox changes of the primary donor (around 600 nm and 870 nm), cytochromes (420 nm), the electrochromic bandshift of carotenoids (around 500 nm) and bacteriochlorophyll (800 nm and 850 nm) were measured. In addition, a light-induced increase of the light scattering was observed. This depended on functioning photosynthetic electron transport. Sensitivity of this feature to gramicidin indicated its connection to the membrane permeability for ions. The fluorescence yield was shown to be modulated by the membrane potential in a complex way: both enhancement and quenching of fluorescence was observed depending on the state of the reaction centre. A model for of this effect was developed. Our results indicate regulatory effect of the photoinduced electric field in bacterial photosynthesis in vivo. PS5.52 An axial ligand for heme ci Phenylalanine to tyrosine substitution in SUIV of the cytochrome b6f complex from Chlamydomonas reinhardtii W Nitschke (BIP/CNRS), F Baymann (BIP/CNRS), L Barucq (IBPC/CNRS), Z Francesca (IBPC/CNRS), A de Lacroix de Lavalette (IBPC/CNRS), D Picot (IBPC/CNRS) Heme ci of the b6f complex, as revealed by the crystallographic structure [1,2] has no axial protein ligand. One side of the heme plane is exposed to the quinone reduction site Qi. It is partially covered by a

phenylalanine residue F40 of subunit IV. This particular arrangement sterically hinders perpendicular access of a potential ligand to the heme plane. EPR spectroscopy showed that heme ci has a complex high spin signature. Addition of NQNO, an inhibitor of the Qi site, strongly simplifies the spectrum. The ensemble of the data suggested that the inhibitor becomes a (5th) axial ligand to the heme iron [3]. More recently, we constructed a mutant where the phenylalanine residue was substituted by tyrosine. Here we present the caracterization of the resulting b6f complex by EPR spectroscopy. The obtained results indicate that the tyrosine residue ligates heme ci. Addition of NQNO alters the spectral parameters and results in a spectrum close to that of the wild-type enzyme in the presence of NQNO, indicating that the tyrosine can be replaced by NQNO as 5th ligand to heme ci. These results are supported by the X-ray structure of the mutant in the presence of the inhibitor. [1] D. Stroebel, Y. Choquet, J.L. Popot, D. Picot, Nature 426 (2003) 413. [2] G. Kurisu, H. Zhang, J.L. Smith, W.A. Cramer, Science 302 (2003) 1009. [3] F. Baymann, F. Giusti, D. Picot, W. Nitschke, Proc Natl Acad Sci U S A 104 (2007) 519. PS5.53 Characterization of ferredoxin-NADP-oxidoreductase stably associated on the thylakoids from the green alga Chlamydomonas reinhardtii A Okamuro (Okayama University), H Tkahashi (Okayama University), M Iwai (Hokkaido University), J Minagawa (Hokkaido University), Y Tkahashi (Okayama University) Ferredoxin-NADP-oxidoreductase (FNR) catalyzes NADPH production in the presence of ferredoxin using reducing power from photosystem I (PSI) in the light. A part of FNR in chloroplasts is stably associated on isolated thylakoids and is fractionated with cytochrome b6f complex in higher plants (1). In order to localize FNR in the thylakoids of C. reinhardtii, we estimated FNR stably associated on the thylakoids using wild type and photosynthetic mutants. The thylakoids purified from wild type retained 40% of total FNR and showed a light-induced NADP photoreduction activity in the presence of ferredoxin. In fact the bound FNR was copurified with PSI-LHCI supercomplex on sucrose density gradient. Accordingly the PSI deficient mutant (delta-psaA/B) accumulated FNR at wild type level but the isolated thylakoids lacked FNR. These results indicate that the association of FNR with the thylakoids is stabilized by the presence of PSI complex. However the isolated thylakoids from a cytochrome b6f deficient mutant also lacked the bound FNR although the mutant cells accumulated normal amount of FNR. Because the cytochrome b6f deficient mutant cells are generally locked in state 1 and wild type cells obtained under our standard condition are usually in state 2, the stable association of FNR on the thylakoids might depend on state transitions (2). We will report a more detailed characterization of the FNR stably bound on the thylakoids. 1. Zhang, H. et al. J. Biol. Chem. 276 (2001) 38159-38165 2. Takahashi, H. et al. Proc. Natl. Acad. Sci. USA 103 (2006) 477-482 PS5.54 Insights into the function of haem ci in the Qi site of the b6f complex, from the binding of NQNO F Rappaport (Institut de Biologie Physico-Chimique, CNRS-UPMC, Paris), L Barucq (Institut de Biologie Physico-Chimique, CNRS-UPMC, Paris), F Baymann (BIP, CNRS, Marseille), F Giusti (Institut de Biologie Physico-Chimique, CNRS-UPMC, Paris), C Léger (BIP, CNRS, Marseille), D Picot (Institut de Biologie Physico-Chimique,

201 14th Photosynthesis Congress - PS07 CNRS-UPMC, Paris) The 3D structure of the cytochrome b6f has revealed the presence of an additional haem, with respect to its mitochondrial homolog. This heme is part of the quinone binding pocket Qi. It is strongly atypical, being covalently bound by a single thio-ether bond to the thiol side chain of Cys35 of cytochrome b6, and having no axial protein ligand. The spectroscopic and redox properties of this additional haem have recently been characterized, opening the way to study its functional implication in the electron transfer sequences catalyzed by cytochrome b6f. In particular, addition of NQNO, a semiquinone analog known to inhibit the reduction of quinone at the Qi site, strongly alters both the redox and spectroscopic properties of haem ci. At least two midpoint potentials are found, one similar to that obtained in the absence of inhibitor, and the other one being strongly down-shifted. This suggests a strong interaction between NQNO and haem ci. Crystallographic and EPR data also indicate an interaction between NQNO and the iron of haem ci. In the light of recent data, the nature of this interaction will be discussed in terms of: i) different affinity constants of the semiquinone analog for the reduced and oxidized form of the haem, ii) a redox-coupling between the different partners in the site. The mechanistic implications for the reduction of quinones by the cytochrome b6f complex will be discussed.

202 14th Photosynthesis Congress - PS07

PS6 - Assembly and Repair of Pigment/Protein Complexes PS6.1 Genetic dissection Arabidopsis thaliana

of

photosystem

II

assembly

in

L Zhang (Chinese Academy of Sciences), L Peng (Chinese Academy of Sciences), J Ma (Chinese Academy of Sciences), W Chi (Chinese Academy of Sciences), J Guo (Chinese Academy of Sciences) Photosystem II is a pigment-protein complex that drives the electron transfer from water to plastoquinone with concomitant oxygen evolution. In higher plants photosystem II consists of more than 20 subunits including both membrane integral and extrinsically associated proteins. Because of the structural complexity of PSII, the assembly process is likely to consist of multiple steps. We are beginning to understand the different steps of PSII assembly during thylakoid membrane biogenesis. However, very little is known about accessory factors (chaperones or assembly factors) involved in these complex processes. To identify genes and factors required for efficient assembly of photosystem II, various Arabidopsis mutants with low PSII accumulation (named lpa mutants) that affecting PSII assembly at different stages were identified. In these mutants the levels and processing patterns of the RNAs encoding the PSII subunits are unaltered in the mutant. The newly synthesized PSII proteins were assembled into functional protein complexes, but the assembly was less efficient in these mutants. The genes identified were intrinsic thylakoid membrane proteins, which are involved in the different steps of PSII assembly, such as D1 precursor processing, incorporation of D1 or CP43 into photosystem II. The possible functions of these factors were investigated through yeast two-hybrid assays and immunoprecipitation analyses. Thus, it appears that a number of nuclear-encoded proteins are likely to participate in the assembly of PSII and PSII assembly at each step is assisted by one or more assembly factors.

PS6.2 Advances in understanding mechanisms underlying Photosystem II assembly and repair in cyanobacteria J Komenda (Institute of Microbiology Trebon, Czech Republic and Institute of Physical Biology, University of South Bohemia, Nove Hrady, Czech Republic), M Dobakova (Institute of Microbiology Trebon, Czech Republic and Institute of Physical Biology, University of South Bohemia, Nove Hrady, Czech Republic), M Tichy (Institute of Microbiology Trebon, Czech Republic and Institute of Physical Biology, University of South Bohemia, Nove Hrady, Czech Republic) The Photosystem II complex (PSII) represents the key component of the oxygenic photosynthetic apparatus which is responsible for water oxidation. It is a multi-subunit protein complex consisting of large subunits D2, D1, CP47 and CP43, and a number of small polypeptides. During PSII assembly the reaction center heterodimer D1-D2 binds initially CP47 and later CP43 while the small subunits are incorporated at various stages of the process. Some of them are initially bound to a single large binding partner before they assemble with other proteins into more complex assembly intermediates. In contrast, small proteins belonging to a family of scp‘s („small cap-like proteins“) or hlip‘s („high light induced proteins“) are attached to assembled PSII when the cells are exposed to stress conditions. In the genome of Synechocystis there are four genes coding for these small proteins and their possible importance for resistance of PSII to stress will be discussed. The most important protective mechanism counterbalancing stress-induced damage to PSII is the repair based on the selective replacement of the D1 protein. In the cyanobacterium Synechocystis PCC 6803 a homologue of the bacterial protease FtsH encoded by the slr0228 gene plays a crucial

role in the selective replacement of D1. Characterization of a Synechocystis mutant lacking 20 amino-acid residues at the exposed N-terminus of D1 showed that it assembles functional PSII complexes but is unable to effectively degrade D1. The results suggested the new concept for PSII repair relying on FtsH-mediated processive degradation of damaged D1 starting from the N-terminus.

PS6.3 Metamorphosis of PS2 as basis for biogenesis and stress adaptation - a molecular approach M Roegner (Ruhr-University Bochum) Photosynthesis and especially Photosystem 2 (PS2) has to respond to extreme environmental conditions by dynamic adaptations. Focus of our work is on membrane protein networks and the life cycle of individual subunits under normal and stress conditions -including biogenesis, degradation and the network of transiently involved factors. As shown for PS2, the combination of sophisticated isolation procedures and time-dependent mass spectrometry with other methods yields first-hand information on assembly, repair and degradation of membrane proteins : 1) On the donor side, a transient Psb27-PS2 subcomplex was shown to be involved in the repair-cycle of PS2 (Nowaczyk et al., 2006): The lipoprotein Psb27 replaces the three extrinsic proteins PsbO, PsbU, PsbV and might also shield PS2 during the repair process, as supported by new EPR- and fluorescence spectroscopy data (Mamedov et al., 2007). 2) On the acceptor side, iron deficiency was shown to induce an IdiA-PS2 complex (Lax et al., 2007) and light stress may induce a potential Peroxiredoxin-PS2 complex (unpublished). 3) Internally, PS2 responds to light stress by switching between 3 copies of the PsbA gene. We can monitor their turnover relative to all major PS2 subunits both on the RNA- and the protein level, which enables us to identify the trigger conditions for the "gene switch". In parallel, we use isolated PS2 complexes from knock-out mutants which contain only one of the three PsbA copies. These enable for the first time a detailed functional characterisation of all three complexes on the molecular level.

PS6.4 Small Cab-like Proteins (SCPs) are involved in pigment turnover in the cyanobacterium Synechocystis sp. PCC 6803 W Vermaas (Arizona State University), D Vavilin (Arizona State University), D Yao (Arizona State University) Cyanobacteria contain a family of small Cab-like proteins (SCPs) that are stress-induced and that have sequence similarity to the transmembrane regions of plant LHCII; in Synechocystis sp. PCC 6803, at least one of them is associated with photosystem (PS) II. PS I-less Synechocystis strains lacking one or more SCPs were labeled with 13C-glucose/bicarbonate or 15N ammonium/nitrate to monitor synthesis and degradation rates of PS II-associated pigments. Without SCPs, the rate of chlorophyll degradation (consisting of dephytylation leading to the formation of free chlorophyllide and phytol and subsequent chlorophyllide degradation) was increased up to 5-fold. However, even in the absence of SCPs the majority of the chlorophyllide formed upon chlorophyll dephytylation still was recycled and used for the biosynthesis of new chlorophyll. In the PS I-less strain, 15N incorporation into pheophytin was delayed compared to the labeling of chlorophyll, suggesting that upon repair of damaged PS II some pheophytin molecules are formed from chlorophyll associated with damaged complexes. In contrast, in PS I-less/SCP-less strains the

203 14th Photosynthesis Congress - PS07 kinetics of pheophytin labeling with 15N exactly matched the labeling kinetics of chlorophyll. Carotenoid levels are low in the PS I-less/SCP-less strain; consistent with this, synthesis of β-carotene (the main PS II carotenoid) was slower than in the PS I-less control. However, β-carotene conversion to zeaxanthin and echinenone occurred faster, suggesting increased exposure of β-carotene to hydroxylase/ketolase. These data support the emerging concept that SCPs are involved in temporary pigment binding during PS II repair and may be important for stabilizing PS II complexes.

observed in WT under the same conditions. Western blot analysis revealed that the mutated enzyme was strongly damaged by oxidation. Interestingly, similar oxidative damage of FeCH was observed also in mutants lacking small stress-induced SCP proteins also containing CAB domain that are PSII associated. From this circumstantial evidence and from the fact that similar oxidative damage was observed specifically in proteins associated with PSII we speculate that FeCH may interact with SCP proteins and/or with PSII via its CAB domain, and that putative Chl-binding residues are important for this interaction and for protection of FeCH against photooxidation.

PS6.5 Construction of an in vivo assay system for activity of mutagenized spinach CtpA and its application for characterization of highly conserved residues in the CtpA family.

PS6.7 Arabidopsis PsbOs differ in their GTPase activity

N Inagaki (Natl. Inst. Agrobiol. Sci.), N Yuzurihara (Natl. Inst. Agrobiol. Sci), M Takano (Natl. Inst. Agrobiol. Sci.), R Burnap (Oklahoma State Univ.)

B Lundin (Division of cellbiology in Linköpings University), S Thuswaldner (Division of cellbiology in Linköpings University), C Spetea (Division of cellbiology in Linköpings University)

Carboxyl-terminal processing protease (CtpA) catalyzes processing of precursor D1 protein (pD1), a pivotal subunit of photosystem II. The proteolytic process is requisite for assembling photosynthetic oxygen evolving machinery. CtpA exhibits a narrow substrate specificity, a common property of intracellular processing proteases. As a first step to approach the molecular basis of the high specificity, we constructed a facile in vivo assay system for activity of mutagenized CtpA, in which we transformed spinach CtpA gene with a signal sequence for directing into lumen, into a Synechocystis heterotrophic mutant deficient in its native ctpA gene. The transformants with WT spinach CtpA regain photoautotrophic growth competence, suggesting the introduced spinach CtpA cleaves the pD1 at the canonical position. To evaluate the system, we introduced the mutant CtpA library in which four residues that are highly conserved among members of the CtpA family, K466, I469, V485 and Y488, were substituted randomly, into the system. Transformants were screened for autotrophy and CtpA genes isolated from surviving cells were sequenced. These analyses revealed V485 and Y488 are not critical for CtpA action, because these positions accepted various substitutions without deactivation of CtpA. In contrast, no genes with substitutions at K466 and few genes with cognate substitutions at I469 were recovered, indicating that these residues are critical for CtpA activity. The importance of K466 is reasonable because the residue has been suggested as the catalytic center. We are performing further characterization regarding the aliphatic residue, I469, to determine how this chemically inert residue contributes to CtpA function.

Crucial for the optimal function of the oxygen-evolving complex is the PsbO subunit of the photosystem II (PSII) complex. Recently we have reported the ability of PsbO in spinach to bind and hydrolyze GTP [1]. GTP stimulates the dissociation of PsbO from PSII following illumination and induces the degradation of the D1 protein. We have predicted four plant-specific binding motifs for GTP, which are not conserved in the sequences of the cyanobacterial or green algae PsbO proteins. We have proposed a location of the GTP-binding site inside the ß-barrel exposed to the lumenal side. Arabidopsis thaliana has two PsbO isoforms encoded by two different genes: psbO1 and psbO2. Here we have measured and compared the GTPase activities in PSII membranes isolated from Arabidopsis knockouts mutants containing T-DNA insertions in one or the other of the psbO genes. The specific GTPase activity of PsbO2 is three fold higher than that of PsbO1. Furthermore, PsbO2 is more efficient released than PsbO1 from PSII following light treatment. We conclude that PsbO2 is a much more efficient GTPase than PsbO1.

PS6.6 Putative chlorophyll-binding amino acid residues in cyanobacterial ferrochelatase are important for the enzyme protection against light-induced oxidative damage

[1] Lundin B, Thuswaldner S, Shutova T, Eshaghi S, Samuelsson G, Barber J, Andersson B, Spetea C (2006) Subsequent events to GTP binding by the plant PsbO protein: structural changes, GTP hydrolysis and dissociation from the photosystem II complex. Biochim Biophys Acta Bioenerg. doi:10.1016/j.bbabio.2006.10.009 (2006). PS6.8 Functional Analysis of PsbZ in Photosystem II from the Thermophilic Cyanobacterium Thermosynechococcus elongatus BP-1

R Sobotka (Institute of Microbiology), M Tichy (Institute of Microbiology), J Komenda (Institute of Microbiology)

M Iwai (Fuculty of Science and Technology, Tokyo University of Science), T Suzuki (Biomolecular Characterization, RIKEN), N Dohmae (Biomolecular Characterization, RIKEN), Y Inoue (Fuculty of Science and Technology, Tokyo University of Science)

Ferrochelatase is a ubiquitous enzyme that catalyses the insertion of ferrous iron into porphyrin ring to form heme. In plant, algae and cyanobacteria, where heme and chlorophyll (Chl) are produced via a common pathway, FeCH is extended by a membrane domain with a Chl-binding motif (CAB domain). We have overexpressed this domain as a small His-tagged protein in the cyanobacterium Synechocystis 6803. The purified protein was green, demonstrating that FeCH is a Chl-binding protein. In order to investigate the significance of putative Chl ligands in the CAB domain for FeCH function we have replaced them by residues that are unlikely to serve as Chl ligands. Although these mutations did not influence in vitro FeCH activity in cells grown at normal light (30 μE), the enzyme activity disappeared after exposure to strong light (2 h, 2000 μE). Only a slight decrease in FeCH activity was

PsbZ is a membrane protein of ~6.5 kDa found in Photosystem II (PSII) complexes from cyanobacteria to higher plants. The two helices of PsbZ are located on the perimeter of the dimer near CP43 and PsbK in the cyanobacterial PSII structure. In tobacco and Chlamydomonas reinhardtii, psbZ knockout leads to reduce amounts of the minor antenna protein CP26. However, CP26 is not found in the cyanobacterial thylakoids. The function of PsbZ in cyanobacteria is still not clear. In this work, we deleted psbZ gene from the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1 to analyze the function of PsbZ. No difference was observed in the growth under the normal light or low light condition, and in the oxygen-evolving activities between the wild type and the mutant. However, the oxygen-evolving activity in the mutant PSII complexes purified by Ni-affinity chromatography was

204 14th Photosynthesis Congress - PS07 lower than those in the purified wild-type PSII complexes. Electrophoretic profile of the mutant PSII complexes revealed that most of PsbK band was disappeared but no significant release of the extrinsic proteins was observed. N-terminal amino acid sequences of PsbK and PsbM/Ycf12 bands showed that most of Ycf12 as well as PsbK was lost in the mutant PSII complexes. These indicate that PsbZ is important for the stable binding of PsbK and Ycf12 with PSII core intrinsic proteins, and suggest that Ycf12 is unidentified X1 (Loll et al. 2005) observed between PsbZ and PsbK in crystal structure of the PSII core complex of Thermosynechococcus elongatus. PS6.9 Genetic and biochemical characterization of early carotenoid biosynthesis mutants of Chlamydomonas reinhardtii

than in wild type after dark-induced senescence. Immunoblotting analysis revealed that degradation of photosystem core and reaction centre proteins were faster than those in wild type. Interestingly, we found that hmc1 accumulated a substantial amount of pheophorbide a after dark-induced senescense. These results suggest that a common factor is involved in the metabolism of 7-hydroxymethyl chlorophyll a and pheophorbide a after dark-induced senescence. PS6.11 Psb27 is required for photoinactivation and recovery of photosystem II in Synechocystis sp. PCC 6803 cells lacking PsbV F Bentley (University of Otago), J Eaton-Rye (University of Otago)

P Tran (University of CA, Berkeley, USA), R Dent (University of CA, Berkeley), S McCarthy (University of CA, Berkeley), M Kobayashi (University of CA, Berkeley), S Poddar (University of CA, Berkeley), K Niyogi (University of CA, Berkeley) Photosynthetic organisms, such as plants and green algae, synthesize carotenoids for harvesting light energy, photoprotection, and maintaining the structure and function of photosynthetic membranes. Carotenoid-deficient mutants were isolated in the green alga Chlamydomonas reinhardtii and found to be genetically linked to predicted early carotenoid pathway genes: phytoene desaturase (PDS), zeta-carotene desaturase (ZDS), and carotene isomerase (CRTISO), respectively. Together PDS, ZDS, and CRTISO catalyze the desaturation and isomerization of phytoene to all-trans-lycopene. Unexpectedly, pds1, zds, and crtiso null mutants still synthesize low levels of downstream colored carotenoids, suggesting that there is another pathway for the conversion of phytoene to all-trans-lycopene. These phenotypes are consistent with the incomplete inhibition of carotenoid biosynthesis in wild-type cells by norflurazon, a specific inhibitor of PDS. Cycloheximide treatment of zds mutants in the dark and light also supports the existence of a second desaturase activity and a role for photoisomerization in the early steps of carotenoid biosynthesis. To isolate mutants affecting the alternative desaturation pathway, pds1 mutants were mutagenized and screened for the presence of pigments using HPLC. The screen produced a mutant, pds1 pds2, that accumulates only phytoene, the intermediate accumulated in pds1 single mutants, and no other carotenoids. Genetic characterization is in progress to determine the molecular basis of this mutation. PS6.10 Analysis of an Arabidopsis mutant that accumulates 7-hydroxymethyl chlorophyll a T Nagane (Institute of Low Temperature Science,Hokkaido University), R Tanaka (Institute of Low Temperature Science,Hokkaido University,CREST,JST), A Tanaka (Institute of Low Temperature Science,Hokkaido University,CREST,JST) In higher plants, chlorophyll a is converted to chlorophyll b through 7-hydroxymethyl chlorophyll a, and chlorophyll b returns to chlorophyll a again through 7-hydroxymethyl chlorophyll a. This conversion system is called the chlorophyll cycle. Aiming at identifying the enzymes and the regulatory factors that are involved in the chlorophyll cycle, we screened EMS-mutagenized Arabidopsis thaliana, and we isolated a mutant that accumulated 7-hydroxymethyl chlorophyll a. We named this mutant hmc1 (7-hydroxymethyl chlorophyll a accumulation). This mutant showed slow growth, and accumulated 7-hydroxymethyl chlorophyll a more than 10 times in its early developmental stage compared with those in wild type. Light and electron microscopy revealed that most of the chloroplasts in hmc1 were broken and the remaining chloroplasts accumulated a greater amount of plastoglobules

The 9-kDa Psb27 lipoprotein co-purifies with photosystem II (PSII) but is absent form X-ray structures of PSII from cyanobacteria. Evidence from several groups suggests that Psb27 is associated with inactive monomeric PSII: and PsbO, PsbU and PsbV are unable to bind when Psb27 is present. We have examined the effect of removing Psb27 in Synechocystis sp. PCC 6803 strains lacking PsbO and PsbV and found it did not affect photoautotrophic growth, assembly of PSII or oxygen evolution in cells grown under light at 30 µE m-2 s-1. Exposure of ∆PsbO and ∆PsbV cells to high light at 2 mE m-2 s-1 resulted in >80% reduction of oxygen evolution in <15 min, whereas the wild type readily acclimated to these conditions. Both photoinactivated strains recovered to their initial rates of oxygen evolution when returned to light of 30 µE m-2 s-1. Removal of Psb27 in the ∆PsbO strain had no effect on either photoinactivation or recovery. In contrast, after 15 min at 2 mE m-2 s-1, the ∆PsbV:∆Psb27 strain evolved oxygen at ~70% of the initial rate, and after 45 min exposure the rate had still only been reduced by ~55%. Additionally, when these cells were returned to light of 30 µE m-2 s-1, recovery was prevented and oxygen evolution did not exceed 50% of the initial rate even after 2 h. Our results suggest removal of Psb27 in the ∆PsbV strain decreases the rate and extent of photoinactivation; moreover, recovery is also blocked in this mutant. PS6.12 Mutations in CP47 and PsbL that target putative hydrogen bonds with sulphoquinovosyl-diacylglycerol at the monomer-monomer interface of photosystem II H Luo (University of Otago), J Eaton-Rye (University of Otago) The X-ray crystal structure of photosystem II (PSII) from Thermosynechococcus elongatus at 3.0 Å [Loll et al. (2005) Nature 438:1040-1044] identified three lipid molecules at the monomer-monomer interface of the PSII dimer. In particular, a sulphoquinovosyl-diacylglycerol (SQDG) is hydrogen bonded through sulfate to Arg-18 of CP47 and Arg-14 of PsbL from the adjacent monomer. To examine if this stromal side interaction is important in PSII function in Synechocystis sp. PCC 6803, we have created three mutants carrying substitutions at Arg-18 of CP47. The R18A and R18K strains had a similar phenotype to wild type; however, the R18E mutant exhibited retarded photoautotrophic growth and impaired oxygen evolution together with a 68% reduction in the number of assembled PSII centers. In parallel, we have constructed a psbL mutagenesis system allowing us to introduce mutations throughout PsbL. We are introducing substitutions that target the putative hydrogen bond with SQDG and at other conserved residues within PsbL. Characterization of these new PsbL mutants will also be presented. PS6.13 The PsbU and Psb27 proteins are required for assembly of

205 14th Photosynthesis Congress - PS07 photosystem II in a Synechocystis sp. PCC 6803 strain lacking PsbM

A Yamashita (Gruduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan)

J Eaton-Rye (University of Otago), F Bentley (University of Otago)

The reaction center binding protein D1 of photosystem II (PSII) is a target of various stresses such as high temperature and strong visible light. The D1 protein damaged is degraded by specific protease, most probably by FtsH proteases, and then removed from the PSII complexes. Although the details of proteolysis of the damaged D1 protein are becoming clear, the fate of the damaged D1 protein seems to be more complex than was previously thought. Not only cleavage but also aggregation of the D1 protein takes place. In this study, we investigated the effect of moderate heat stress (40 for 30 min) or light stress (1,000μEm-2s-1 for 30 min) on the D1 protein of spinach thylakoids and PSII-enriched membranes, as well as on the thylakoids of the cyanobacterium Synechocystis sp. PCC6803, with Western blot analysis with specific antibodies, under the aerobic and anaerobic conditions. Under the anaerobic conditions, heat-induced degradation (or cleavage) and aggregation of the D1 protein were suppressed significantly. We also detected formation of hydroxyl radicals in the spinach PSII membrane by the heat stress, which was monitored by EPR spin trapping spectroscopy. These data suggest that reactive oxygen species are involved in the heat-induced damage to the D1 protein.

The PsbM and PsbT proteins of photosystem II (PSII) are single transmembrane helices found at the PSII monomer-monomer interface. This position suggests a role in dimer stabilization. Removal of either of these proteins from Synechocystis sp. PCC 6803 does not prevent dimer formation; however, the ∆PsbM:∆PsbT mutant is composed exclusively of PSII monomers although it remains photoautotrophic. The ∆PsbM and ∆PsbT strains are readily photoinactivated and we have observed photodamaged cells lacking PsbT require Psb27 for recovery while cells lacking PsbM require Psb27 for PSII assembly. We therefore investigated the functional cooperation of PsbM and PsbT with the extrinsic proteins of the oxygen-evolving complex by creating the ∆PsbM:∆PsbO, ∆PsbM:∆PsbU and ∆PsbM:∆PsbV mutants and a similar series lacking PsbT. Removal of PsbO or PsbV from strains lacking PsbM or PsbT slowed photoautotrophic growth and reduced assembly of PSII and oxygen evolution. Similarly, removal of PsbU from a strain lacking PsbT increased the photoautotrophic doubling time by 6 h and lowered oxygen evolution by 40%. In contrast, the ∆PsbM:∆PsbU mutant was unable to assemble functional PSII centers. Hence in the absence of PsbM, the removal of PsbU unexpectedly has a more dramatic impact than the removal of PsbO. We have therefore observed cells lacking PsbM fail to assemble PSII in the absence of either PsbU or Psb27 and this suggests an important role for PsbU in PSII assembly. PS6.14 Quality control of Photosystem II: recovery of Photosystem II from heat-damage depends on an FtsH protease (slr0228) in Synechocystis sp. PCC6803 D Takenaka (Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan) Daichi Takenaka, Hideki Hiramoto, Takashi Kamata, Noriko Morita and Yasusi Yamamoto Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan Members of FtsH family of proteases are found widely in nature and are shown to play an important role in the proteolysis of misfolded and damaged proteins. Recent studies with the cyanobacterium Synechocystis sp. PCC 6803 indicated that an FtsH protease (slr0228) is involved in degradation of photo-damaged D1 protein. We analyzed the effect of moderate heat stress (40 for 30min) on the wild type cells and ΔFtsH (Δslr0228) cells, and showed that the FtsH protease is responsible for the degradation of heat-damaged D1 protein. We further studied the relationship between the content of FtsH in the cell and recovery of the light- or heat-damaged D1 from the damage. PSII activity was assayed with wild type cells and ΔFtsH cells using an oxygen electrode and a PAM. We found that the rate of the recovery increased when the amount of FtsH increased by pre-treatment of the wild type cells with low temperature (4 , 12h) , while ΔFtsH cells did not show any recovery after light and heat stress. We also monitored the changes in the D1 and FtsH levels during light or heat-treatment and recovery process with wild type cells and ΔFtsH cells by Western blotting. Our results suggest that the FtsH protease (slr0228) is essential for degradation of the D1 protein during heat stress, as well as light stress. PS6.15 Quality control of Photosystem II: degradation and aggregation of the D1 protein induced by moderate heat stress depend on oxygen

PS6.16 Effects of amino-terminal deletions of Ycf4 on photosystem I complex assembly M Funakawa (Okayama University), T Onishi (Okayama University), Y Takahashi (Okayama University) Photosystem I (PSI) is a large chlorophyll-protein complex composed of about fourteen subunits and more than one hundred cofactors. In the green alga Chlamydomonas reinhardtii, chloroplast-encoded proteins, Ycf3 and Ycf4, are essential for PSI synthesis (1). We are interested in understanding the function of these factors in the assembly process of PSI complex. We focused on a small hydrophilic region of N-terminus of Ycf4 and have generated several chloroplast mutants with deletion of 2-9 amino acid residues. Deletion of the amino acid residues gradually reduced the steady-state level of Ycf4, suggesting that the N-terminal region is important for the Ycf4 stability. However deletion of four amino acid residues (ND4) neither significantly affect photosynthetic growth nor decrease the accumulation of PSI complex. In contrast five or six amino acids deletion apparently decreased photosynthetic growth and slightly reduced the accumulation of PSI complex. More amino acid deletions significantly reduced the accumulation of PSI complex and the mutant cells did not grow photosynthetically. Separation of PSI complexes from the thylakoid extracts from ND7 as well as ycf4 and/or ycf3 deficient mutants revealed the presence of a small amount of unusual PSI complexes that are smaller than that of mature PSI complex in wild type cells. According to the apparent size, these PSI complexes could be deteriorated or assembly intermediate complexes. We will discuss a possible involvement of Ycf4 and Ycf3 in an initial assembly stage of PSI complex. 1. Boudreau, E. et al. EMBO J. 16 (1997) 6095-6104 PS6.17 Identification and characterization of a semi-stable assembly intermediate of photosystem I complex Y Takahashi (Okayama University), S Ozawa (Okayama University), T Onishi (Okayama University) Photosystem I (PSI) complex in higher plants and green algae consists of 14-15 subunits and a number of cofactors, and tightly associates light harvesting complex I (LHCI) to form PSI-LHCI supercomplex. It is believed that PSI-LHCI supercomplex would be assembled step-by-step.

206 14th Photosynthesis Congress - PS07 Several factors have been reported to be involved in assembly or stability and regulation of the complex (1). However, little is known the molecular process how the assembly of this complicated complex proceeds. Here we report a new assembly intermediate of PSI complex in the green alga Chlamydomonas reinhardtii. Solubilization of the thylakoids and subsequent fractionation of the extracts on sucrose density gradient yields four chlorophyll-protein complexes (A1 - A4) from the top to the bottom of the gradient (2-4). While mature PSI-LHCI supercomplex was found in A-3, a smaller amount of PSI complex was also separated in A-2. This PSI complex in A2 is smaller than the mature PSI-LHCI supercomplex in A3 and lacks some peripheral PSI subunits. Pulse-chase labeling experiments of total cellular proteins revealed that PSI complex in A2 is a semi-stable assembly intermediate and becomes a mature PSI complex in A3 after integration of some peripheral subunits as the final assembly step. We will discuss assembly steps of PSI complex. 1. Hippler, M. et al. Protist 153 (2002) 197-220 2. Sugimoto, I. and Takahashi, Y. J.Biol. Chem. (2003) 278 45004-10 3. Takahashi et al. Biochemistry 43 (2004) 7816-23 4. Takahashi, H. et al. Proc. Natl. Acad. Sci. USA103 (2006) 477-482 PS6.18 Specific binding of D1 protein degradation products to the psbAI promotor in Synechococcus PCC 7942 * C Stelljes (University of Bremen), F Koenig (University of Bremen) Synechococcus strain PCC 7942 the reaction center protein D1 is encoded by a psbA gene family (Golden et al. 1986). Transcription of the three psbA genes is differentially regulated by light. Under high light conditions, the transcription of psbAI is repressed and that of psbAII and psbAIII is induced (Kulkarni et al. 1992, Golden 1995). Regulatory sequences of the 5´upstream region of the psbAI gene, coding for the D1:1 isoform of the reaction center protein, have been demonstrated long time ago (Nair et al. 2001). Protein binding to this region was proven. However, the respective protein could not be identified by the authors so far (Nair et al. 2001). The present investigation shows that the D1:1 protein and its naturally occurring degradation products, the latter in vivo mediated by the protease FtsH (Nixon et al. 2005), bind to the 5´upstream region of the psbAI gene. Some of them bind to the promotor. Additionally, C-terminal fragments of the D1:1 protein bind to a sequence with similarity to isiB, a gene which encodes a flavodoxin-like protein. The experimental approach was the following: The D1:1 protein and its potentially occurring degradation products were overexpressed in Escherichia coli. Protein–DNA interaction was shown by means of electrophoretic mobility shift assays. * Journal of Bacteriology 189, 1722-1726, March 2007 PS6.19 Chromophore attachment to biliproteins H Scheer (Ludwig Maximilians Universität, München, Germany), K Zhao (Huazhong University of Scinece and Technology, Wuhan, PR China) The linear tetrapyrrole chromophores of phycobiliproteins are covalently bound to the protein. The mode and sequence of attachment of the various chromophores to the different apoproteins and binding sites is only poorly understood. The covalent linkage has also hampered reconstitution and directed modification of these systems. Recently, several reconstitution systems have been established, progress in this field is reviewed. Autocatalytic chromophore attachment is established only for the core-membrane linker, ApcE, and phytochromes. Three types of lyases are currently known. The heterodimeric E/F-type serves only a single

binding site, a-84 in phycocyanins and phycoerythrocyanins; it can have a secondary isomerase function. T-type lyases serve the ß-155 binding site in phycocyanins and phycoerythrocyanins. The more universal S-type lyases attach chromophores to all binding sites of allophycocyanins, to ß-84 of phycocyanins and phycoerythrocyanins, and to a-84 and ß-84 of C-phycoerythrin. We report properties of the lyases from Anabaena PCC7120, the reaction mechanisms, and optimum reconstitution conditions. We emphasize two problems: One is the spontaneous chromophore addition that can occur with all apoproteins, but is of low fidelity regarding the correct chromophore structure. It is minimized in E. coli, and can be overcome by a multiple-plamid system expressing up to 8 genes for chromophore synthesis, lyase(s) and apoproteins(s). The second is the sequence of attachment with apoproteins carrying multiple binding sites. With CpcB, chromophore binding at cysteine-84 inhibits subsequent binding at cysteine ß-155, care has therefore to be taken to maintain the proper binding sequence, viz. ß-155 followed by ß-84. PS6.20 Thylakoid Lumen Immunophilin TLP40 is Crucial for PSII Assembly and a Prerequisite for Plant Survival under Short Day Conditions A Khrouchtchova (University of Copenhagen), S Sirpiö (University of Turku)), Y Allahverdiyeva (University of Turku)), M Hansson (Linköping University)), A Vener (Linköping University)), H Vibe Scheller (University of Copenhagen)), P Jensen (University of Copenhagen)), A Haldrup (University of Copenhagen)), E Aro (University of Turku) TLP40 is a complex peptidyl-prolyl isomerase localized to the thylakoid lumen. The protein was previously shown to associate with the inner side of stroma thylakoids - the site of insertion of the newly synthesised photosynthetic proteins. Arabidopsis thaliana plants devoid of TLP40 are unable to survive under 8 hour photoperiod, but increasing the duration of illumination allows the plants to develop. Analysis of the mutant thylakoid preparations using Western blot showed an approximately 50% decrease in the amounts of PSI and PSII proteins. 2D gels revealed a decrease in the amounts of PSII supercomplexes and an increase in the amounts of CP43-less PSII monomer in the TLP40-deficient plants. Flash induced fluorescence relaxation, monitored in the absence or presence of DCMU, indicated a pronounced effect of the mutation on both the donor and the acceptor side of PSII, suggesting a general destabilisation of the PSII complex. Oxygen generation capacity of the PSII complexes in the TLP40-deficient plants was reduced and analysis of the publicly available DNA-array experiments indicated a clear co-expression of tlp40 transcript with the genes encoding the components of the oxygen-evolving complex. Altogether, the results obtained point clearly towards the importance of TLP40 in the formation of functional PSII complexes and, directly or indirectly, also of PSI through assembly/stabilisation steps. However, the reason the mutant plants are dependant on the photoperiod might not only be the insufficient capture of light energy due to PSII malfunction, but also additional processes are likely to be involved that require further examination. PS6.21 A versatile heterologous system in E. coli for phycobiliprotein chromophorylation H Scheer (Universität München), K Zhao (Huazhong University of Science Technology), J Tu (Huazhong University of Science and Technology), H Liu (Universität München), P Su (Universität München), S Böhm (Universität München), M Zhou (Huazhong University of Science and Technology) After cloning of apophycobiliprotein genes, phycobilin biosynthesis

207 14th Photosynthesis Congress - PS07 genes and lyase genes from several cyanobacteria, various phycobiliproteins could be biosynthesized in the heterologous E. coli system using dual plasmids containing the respective genes. BL21(DE3) cells could be transformed to express up to eight proteins in the desired combination. This heterologous system in E. coli is more advantageous than reconstitution in vitro in the following two respects: 1) On reconstitution in vitro, where the respective genes were first overproduced in E. coli, and then reconstituted in vitro, both some lyases and some apoproteins were difficult to dissolve and refold, so reconstitution yields in vitro were low and poorly reproducible. An example is the phycoerythrin reconstitution: apo-phycoerythrin is insoluble when overexpressed in E. coli, and phycoerythrin biosynthesis is therefore very difficult to study. Using the heterologous system in E. coli, both a-phycoerythrin and b-phycoerythrin could be correctly chromophorylated with PEB at the C84 site (consensus sequence). 2) In vitro reconstitutions are complicated by the fact that most apo-phycobiliprotein bind phycobilins spontaneously, but with low fidelity. This results in unwanted products and mixtures that are difficult to analyze and handle, and hinder enzymatic studies. By using the heterologous system in E. coli, the spontaneous phycobilin binding is generally inhibited. By using the E. coli system, we succeeded in biosynthesizing phycoerythrocyanin, phycocyanin and allophycocyanin in E. coli. PS6.22 Phycocyanobilin-CpeS lyase complex, an intermediate for phycobiliprotein chromophorylation H Scheer (Universität Münch), K Zhao (Huazhong University of Science and Technology), J Tu (Huazhong University of Science and Technology), J Zhang (Huazhong University of Science and Technology), M Kupka (Universität München), M Zhou (Huazhong University of Science and Technology), S Böhm (Universität München) CpeS, encoded by alr0617 in the cyanobacterium Anabaena PCC 7120, is a bilin:Cys84-phycobiliprotein lyase, that catalyzes phycocyanobilin (PCB) and phycoerythrobilin attachment to Cys84 (or consensus site) of phycoerythrin, phycoerythrocyanin, phycocyanin and allophycocyanin. Site-directed mutants of CpeS were constructed to determine the functional amino acid residues. CpeS(C51T), CpeS(H22V), CpeS(R149L) and CpeS(W14I) had much lower enzymatic activities for PCB attachment to CpcA and PecA, showing that C51, H22, R149 and W14 are essential for CpeS activity. CpeS can bind PCB non-covalently as well as covalently, as assayed by Ni2+ chelating affinity chromatography and Zn2+-induced fluorescence on denaturing gels. Binding and spectral chase experiments of the enzymatic reaction showed that PCB is first bound covalently CpeS, and subsequently transferred to the respective apophycoproteins. The fast PCB binding to CpeS was analyzed by stop-flow techniques. An enzymatic mechanism is proposed of CpeS. PS6.23 The occurrence of native monomer protochlorophyllide forms and their physiological roles

Recently, we described the occurrence of protochlorophyllide forms in naturally grown organs where the outer tissue layers behave as optical filters thus they partially or fully shade the inner regions. Such arrangement was found in case of cabbage, buds of numerous trees and in sunflower achene (where the exocarpium often remains attached to the cotyledon after germination). In these tissues, besides the POR dimers and oligomers, monomer protochlorophyllide forms were found often in much higher amounts than in most leaves of dark-germinated seedlings. The abundance of monomers was found also in pea epicotyls. These monomers fall into two categories. One group is a POR unit in which protochlorophyllide can directly transform into a 676 nm emitting chlorophyllide form under 632.8 nm laser light and was artificially aggregated into photoactive dimers and oligomers. The second group is non-POR bound protochlorophyllide which cannot directly transform into chlorophyllide but sensibilize photoxidation processes. The production of ROS and lipid peroxidation was detected in these tissues on illumination; cooling of samples amplified the photooxidation. A model is designed to explain these reactions. PS6.24 Probing the structure of a Ycf4-containing protein complex involved in green algal Photosystem I assembly M Cullen (Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, UK), S Ozawa (Department of Biology, Faculty of Science, Okayama University, Okayama, Japan), Y Takahashi (Department of Biology, Faculty of Science, Okayama University, Okayama, Japan), J Nield (Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, UK) Synthesis of the Light Harvesting Complex I (LHCI)-Photosystem I (PSI) supercomplex, a large macromolecular complex of at least 23 polypeptides (>700 kDa), is currently believed to proceed by a process of sequential integration. The molecular mechanisms in play remain shrouded in mystery, however, as the various protein factors involved are only partially characterized and undergo rapid integration. This has so far inhibited the successful detection of assembly intermediates (Hippler et al., 2002; Rochaix et al., 2004). Here we have focused on the chloroplast-encoded Ycf4 protein, known to be essential for PSI synthesis/assembly (Boudreau et al., 1997). We have identified, isolated and purified a large Ycf4-containing complex (>1 MDa) from Chlamydomonas, which also contains small quantities of Psa gene products. This does indeed suggest that such a complex is part of an assembly apparatus, or scaffold, which aids initial PSI assembly. We subjected this preparation to structural analysis for the first time by imaging, in negative stain, the individual protein complexes present using transmission electron microscopy (Ruprecht & Nield, 2001). A dataset of ~11,000 single particles was image-processed in order to identify averaged 2D views pertaining to these large Ycf4-containing complexes, a series of which will be shown and discussed. Boudreau E et al., (1997) EMBO J. 16, 6095-6104. Hippler M et al., (2002) Protist 153, 197-220. Rochaix JD, et al., (2004) Biochem Soc Trans. 32, 567-570. Ruprecht J & Nield J (2001) Prog Biophys Mol Biol. 75, 121-164.

B Böddi (Eötvös University), K Solymosi (Eötvös University), A Kósa (Eötvös University), A Szenzenstein (Eötvös University), B Vitányi (Eötvös University), É Hideg (Biological Research Centre)

PS6.25 Structural analysis of the FtsH2 homologue of Synechocystis sp. PCC6803

The protochlorophyllide – chlorophyllide phototransformation is usually studied in dark-germinated seedlings of higher plants in which this step is light-regulated. The NADPH:protochlorophyllide oxidoreductase (POR) is accumulated in etioplast inner membranes, mainly in dimer and oligomer forms with fluorescence emission maxima at 644 and 655-657 nm, respectively. These complexes are flash-photoactive, i.e. they transform into chlorophyllide within a few ms of illumination. The monomer POR unit is traditionally considered as non-photoactive.

J Nield (Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, UK), M Barker (Division of Biology, Faculty of Natural Sciences, Imperial College London, UK), M Boehm (Division of Biology, Faculty of Natural Sciences, Imperial College London, UK), P Nixon (Division of Biology, Faculty of Natural Sciences, Imperial College London, UK) FtsH Zn2+-metalloproteases are known to be key players in the

208 14th Photosynthesis Congress - PS07 II

photosystem II (PSII) repair cycle which operates to maintain PSII efficacy in the light. Sequencing of the Synechocystis sp. PCC6803 genome has revealed the presence of four FtsH homologues. One of these, FtsH2 or FtsH (slr0228), has been implicated in PSI complex assembly (Mann et al., 2000) and turnover of the PSII D1 protein (Silva et al., 2003). To clarify the structure of FtsH2 we isolated a GST-tagged derivative by affinity chromatography. Biochemical analysis indicated that FtsH2 formed a hetero-oligomeric complex with FtsH (slr1604). These complexes were visualised by negative stain and room temperature electron microscopy. High-quality micrographs were recorded and single particle analysis then identified over 3,000 particles in two dimensions. These were the largest observed and of a relatively uniform shape and size. Reference-free alignment, multi-variate statistical analysis and iterative refinement followed with no symmetry enforced upon the structure. The overall structure was revealed at a resolution of ~25 Å as a pseudo-hexameric particle comprised of three dimeric domains. Furthermore, it was possible to attribute density to the GST-tag employed and hence distinguish between the two types of FtsH subunit in the complex. This work shows for the first time that intact FtsH complexes are indeed hexameric as often assumed in the literature. Mann NH, et al., (2000) FEBS Lett. 479, 72-77 Silva P, et al., (2003) Plant Cell 15, 2152-2164

Functional photosynthetic complexes only have a short life-span as a means of quality control. Turn-over of pigment-binding proteins and changes in the composition of light-harvesting complexes are major tools for light adaptation. Recently we have identified the LHC II degrading protease in A. thaliana as a Zn2+-dependent metalloprotease belonging to the FtsH family. T-DNA KO A. thaliana mutants, lacking ftsH6, were unable to degrade either Lhcb3 during dark-induced senescence or Lhcb1 during high light acclimation (1). The A. thaliana ftsH6 gene has a clear orthologue in the genome of Populus trichocarpa. It is likely that FtsH6 is a general LHC II protease and that FtsH6-dependent LHC II proteolysis is a feature of all higher plants. Studies on AtFtsH6 and related members of the FtsH family will be presented. 1) Zelisko, A., Garcia-Lorenzo, M., Jackowsky, G. Jansson, S., Funk, C. (2005) AtFtsH6 is involved in the degradation of the light-harvesting complex II during high light acclimation and senescence. Proc. Natl. Acad. Sci. USA 102, 13699-13704.

PS6.26 Characterization of an Arabidopsis chl27-t mutant on chlorophyll synthesis and chloroplast development

PS6.28 TLP18.3, a novel thylakoid lumen protein regulating photosystem II repair cycle

W Bang (Division of Applied Life Sciences (BK21) and Environmental Biotechnology National Core Research Center (EB-NCRC), Graduate School of Gyeongsang National University, Jinju 660-701, Korea.), I Jeong (Division of Applied Life Sciences (BK21) and Environmental Biotechnology National Core Research Center (EB-NCRC), Graduate School of Gyeongsang National University, Jinju 660-701, Korea.), D Kim (Division of Applied Life Sciences (BK21) and Environmental Biotechnology National Core Research Ce

S Sirpiö (University of Turku), Y Allahverdiyeva (University of Turku), M Suorsa (University of Turku), V Paakkarinen (University of Turku), J Vainonen (University of Turku), N Battchikova (University of Turku), E Aro (University of Turku)

Chloroplast development depends on chlorophyll biosynthetic pathway. Mutants defective in chlorophyll synthesis are a well-defined class of chloroplast mutants. Here, we report the characterization of an Arabidopsis mutant, chl27-t, defective in both chlorophyll synthesis and chloroplast development. The mutant allele contained a T-DNA insertion within promoter of CHL27, encoding a membrane-bound component in the aerobic Mg-protoporphyrin IX monomethyl ester cyclase for the synthesis of protochlorophyllide at the chlorophyll biosynthetic pathway, and thus, this insertion dramatically reduced CHL27 mRNA level. The homozygous chl27-t lines showed a dwarf phenotype with pale green appearance. It was represented that total chlorophyll content decreased and chlorophyll a/b ratio increased in chl27-t lines, compared with those of wild-type. The increase of chlorophyll a/b ratio seems to be due to the decreased level of AtCAO (chlorophyll a oxygenase) mRNA in chl27-t mutants. Transmission electron microscopy of leaf sections of chl27-t mutants exhibited abnormal chloroplastic morphology. By using fluorescence quenching analyses, chl27-t mutants represented much lower photosynthetic activity than that of wild-type. Complementation of the mutant with the CHL27 cDNA resulted in restoration of the green phenotype. These results indicate that the reduced level of CHL27 mRNA leads to defective in chlorophyll biosynthesis and to abnormal development of chloroplast. Further, to get more information on the chloroplast targeting of CHL27, we performed protoplast transient assays. Our results demonstrated that the N-terminal 39-amino acid region was necessary and sufficient for chloroplast targeting. [This work was supported by BK21 program and EB-NCRC at Gyeongsang National University] PS6.27 Degradation of the Light Harvesting Complex of Photosystem

C Funk (Umeå University), A Pruzinska (Umeå University), M Garcia-Lorenzo (Umeå University)

A proteome analysis of Arabidopsis thaliana thylakoid-associated polysome-nascent chain complexes was performed to find novel proteins involved in the biogenesis, maintenance and turnover of thylakoid protein complexes, particularly the photosystem II (PSII) complex exhibiting a high turnover rate. Four unknown proteins were identified, of which TLP18.3 (thylakoid lumen protein of 18.3 kDa) was selected for further analysis. The Arabidopsis mutants (SALK_109618 and GABI-Kat 459D12) lacking the TLP18.3 protein showed higher susceptibility of PSII to photoinhibition. The increased susceptibility of DTLP18.3 plants to high light is likely to originate from an inefficient reassembly of PSII monomers into dimers in the grana stacks. This, in turn, is at least partially due to an impaired capability of the DTLP18.3 plants to replace the damaged D1 protein with a new copy in the stroma-exposed thylakoids. Indeed, the TLP18.3 protein is evenly distributed between the grana and stroma thylakoids. Notably, the lack of the TLP18.3 protein does not lead to a severe collapse of the PSII complexes, suggesting a redundance of proteins assisting these particular repair steps to assure functional PSII. The TLP18.3 plants showed no clear visual phenotype under standard growth conditions but when challenged by fluctuating light during growth, the retarded growth of DTLP18.3 plants was evident.

PS6.29 A Role of THYLAKOID FORMATION 1 in the Assembly of Trimeric LHCII in Arabidopsis thaliana Z Lingang (National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, 200032 Shanghai, People's Republic of China) Lingang Zhang Jirong Huang National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science,

209 14th Photosynthesis Congress - PS07 Chinese Academy of Sciences, 200032 Shanghai, People's Republic of China Descended from ancestral cyanobacteria by endosymbiosis, chloroplast development is under the control of orchestrating gene expression of chloroplast and nucleus. Thylakoid Formation 1 (THF1) is nuclear-encoded protein mainly localized in the chloroplast. Disruption of the THF1 gene results in a phenotype in variegated leave, in which chloroplast development is impaired in yellow sectors while normal in green sectors. The development of these impaired plastids appear to be blocked at various steps in chloroplast biogenesis because the plastids lack organized internal membrane structures or contain rudimentary lamellae. Noninvasive fluorometric analysis showed that the proportion of uncoupled proximal antennae in the Photosystem II (PSII) rose faster in the thf1 mutant than in wild-type (WT) plants with the increase in light intensity. To date, it is unclear whether THF1 mutation causes a change in protein complexes in the thylakoid membrane. We used Blue Native PAGE (BN-PAGE) to investigate structural alterations of thylakoid proteins in nature conditions. Our results showed that trimeric light-harvesting complex II (LHCII) was significantly reduced in thylakoid preparations from the thf1 mutant compared with those from WT plants on the equal basis of chlorophyll, whereas the monomeric LHCII accumulated more in the mutant than in WT. However, Western blot analysis revealed that the levels of Lhcb1, Lhcb2 and Lhcb3 in the thylakoid membrane was the same as those in WT. Two-dimensional electrophoresis analysis combined with immunoblotting showed that THF1 was detected in the monomeric LHCII. Based upon these results, it is likely that THF1 is involved in the trimerization of LHCII directly or indirectly. PS6.30 Does photoinactivation of Photosystem II occur in low light conditions? R Oguchi (The University of Tokyo), I Terashima (The University of Tokyo), W Chow (Australian National University) -2 s-1) for two days or in high white light (950 mmol m-2 s-1). The results showed that there was a difference in the extent of photoinactivation per photon exposure between low- and high-light conditions, and that photoinactivation did occur in low light. They suggest that both mechanisms occur in the photoinactivation process. Further elucidation of the mechanism of photoinactivation of PSII will be achieved by the use of red, green or blue lights in the photoinhibition treatment. aPlant Sciences, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan bResearch School of Biological Sciences, Australian National University, Canberra, ACT 0200, Australia

PS6.31 The photorespiratory pathway is important for the repair of photodamaged photosystem II under CO2 limiting conditions S Takahashi (RSBS), H Bauwe (University of Rostock), M Badger (RSBS) The photorespiratory pathway plays an important role in preventing photoinhibition of photosystem II (PSII). To understand its role, we investigated the mechanism of photoinhibition upon interruption of the photorespiratory pathway using four different Arabidopsis mutants that lack the activity of Fd-glutamate synthase (Fd-GOGAT), plastidic glutamate/malate translocator (DiT2), serine hydroxymethyl transferase (SHMT) or glycerate kinase (GLYK). After transfer from high CO2 to air, the extent of photoinhibition was enhanced in all photorespiratory mutants but not in wild type. However, in the presence of

chloramphenicol, that inhibits the de novo synthesis of proteins in chloroplasts, there was no differences in the rate of damage between mutants and wild-type. After photoinhibition treatment by strong light, photodamaged PSII was repaired in wild type in air, but the repair was significantly suppressed in all photorespiratory pathway mutants. We found that interruption of the photorespiratory pathway inhibits the synthesis of D1 protein at the step of translation. These results suggest that the operation of the photorespiratory pathway minimizes inhibition of the de novo synthesis of D1 protein, which is important for the repair of photodamaged PSII, under conditions where the oxygenase reaction of Rubisco occurs. PS6.32 The PsbI protein stabilizes the newly synthesized D1 protein and assists its stable incorporation into Photosystem II in the cyanobacterium Synechocystis PCC M Dobakova (Institute of Microbiology Trebon and Institute of Physical Biology Nove Hrady, University of South Bohemia, Czech Republic), J Komenda (Institute of Microbiology Trebon and Institute of Physical Biology Nove Hrady, University of South Bohemia, Czech Republic), M Tichy (Institute of Microbiology Trebon and Institute of Physical Biology Nove Hrady, University of South Bohemia, Czech Republic) Function of the PsbI protein during the assembly of Photosystem II (PSII) complex has been studied in the cyanobacterium Synechocystis sp. PCC 6803. Analysis of the PSII complexes in the wild-type strain showed that the PsbI protein is present in dimeric and monomeric core, core lacking CP43 and in reaction center complexes consisting of D1, D2 and cytochrome b-559. PsbI is not a limiting factor in the formation of the reaction center complexes although its absence leads to destabilization of CP43 binding within the core complex. In the mutant strain that is unable to assemble larger PSII complexes due to the absence of D2 the PsbI protein is found in a small complex with the D1 protein or with its incompletely processed forms pD1 and iD1. All three D1 forms can be co-immunoprecipitated with PsbI and mutual complex D1-PsbI can be isolated using nickel chelating chromatography from the strain expressing His-tagged PsbI. Despite the close structural relationship between both proteins PsbI turns-over much slower than D1. On the other hand, the turnover of D1 is accelerated in the absence of PsbI. The results show that the PsbI is an important stabilizing factor of the newly synthesized D1 protein and supports its correct incorporation into PSII. PS6.33 Association of the small Cab-like proteins with Photosystem II M Hernandez (Umeå university), W Vermaas (Arizona State University), C Funk (Umeå University) ) The small Cab-like proteins (SCPs) resemble the first or third helix of the light-harvesting complex of higher plants. They are involved in the stabilization of chlorophyll in the cyanobacterium Synechocystis sp. 6803 under stress conditions; however, their role in this process is not clear. The co-isolation of ScpD and ScpB with Photosystem II components of high-light stressed cells may be an indication that the function of these proteins is related to the repair cycle of this system (1). Recently, the ORF slr1544 was found to co-transcribe with ScpD, suggesting a collaborative role between these proteins. To test this hypothesis, slr1544 was modified to include a Strep-tag in the N-terminal region of the protein and used to transform different Synechocystis sp. 6803 mutants. Experimental results and their implications will be discussed. 1) Yao, D., Kieselbach, T., Komenda, J., Promnares, K., Hernandez-Prieto, M., Tichy, M., Vermaas, W., Funk, C. (2007)

210 14th Photosynthesis Congress - PS07 Localization of the small CAB-like proteins in the photosynthetic complexes, J. Biol. Chem. 282, 267-276. PS6.34 Small chlorophyll binding proteins overexpressed cyanobacterium, Synechocystis Sp. PCC 6803

in

G Kufryk (Umeå University), W Vermaas (Arizona State University), C Funk (Umeå University) Chlorophyll a/b binding (CAB) proteins are ubiquitous among cyanobacteria and higher plants where they participate in light harvesting, non-photochemical quenching, photoprotection and regulation of chlorophyll biosynthesis. Proteins of this group have between one to four transmembrane helices which are highly conserved. CAB proteins have a chlorophyll-binding motif (Glu-X-X-His/Asn-X-Arg), and some of them were experimentally shown to bind chlorophyll, as well as carotenoids and lipids. Small CAB proteins (SCPs) with a molecular weight about 8 kDa were found in cyanobacteria and their homologues – in higher plants. These proteins are not abundant in normal growth conditions whereas their expression increases dramatically under the stress conditions, including high light, low temperature, and nutrient deficiency. They have been implied in chlorophyll binding and regulation of chlorophyll biosynthesis. Low expression level of the genes that code for small CAB proteins creates a serious obstacle for studying localization and function of these proteins in the cell. To overcome it, these proteins have to be over-expressed. In order to over-express SCP B and SCP E from Synechocystis sp. PCC 6803, we created a construct where coding part of the corresponding genes was put under a psbA3 promoter with antibiotic resistance cassette downstream of the gene and psbA3 flanking regions. PsbA3 is one of the three genes coding for the D1 protein of photosystem II in Synechocystis, and it is dispensable under most growth conditions. To facilitate extraction and localization of the over-expressed SCPs, a strep tag II was introduced into the N-terminus of these proteins. This construct was successfully used to transform a wild type strain of Synechocystis sp. PCC 6803 and obtain completely segregated strains where psbA3 gene was replaced with gene coding for a strep-tagged SCP B or SCP E. Localization of these SCPs in the cell and their interaction with other proteins in vivo was investigated.

211 14th Photosynthesis Congress - PS07

PS7 - Membrane Dynamics and Organisation PS7.1 Architecture, membranes.

connectivity

and

dynamics

of

thylakoid

V Brumfeld (Weizmann Institute), D Charuvi (Weizmann Institute), S Chuartzman (Weizmann Institute), V Kiss (Weizmann Institute), E Shimoni (Weizmann Institute), R Nevo (Weizmann Institute), O Rav-Hon (Weizmann Institute), Z Reich (Weizmann Institute), A Kaplan (The Hebrew University of Jerusalem), I Ohad (The Hebrew University of Jerusalem), R Schwarz (Bar-Ilan University) Using dual-axis electron microscope tomography (EMT) we determined the three-dimensional organization of the thylakoid membranes in two cyanobacterial and two higher-plant species. Atomic force- and scanning electron microscopy studies complemented the EMT analyses by providing detailed information on the surface topography of isolated intact thylakoid membranes. The structures revealed by the different techniques will be described and their implications on the function, formation, and chromatic adaptation of the thylakoid membranes will be discussed. The question of how trafficking of molecules and inclusions proceeds in cells or chloroplasts filled by extensive thylakoid membrane systems will also be addressed. References 1. Nevo, R., et al. Thylakoid membrane perforations and connectivity enable intracellular traffic in cyanobacteria. EMBO J. 26,1467-73, 2007. 2. Shimoni, E., et al. Three-dimensional organization of chloroplast thylakoid membranes revealed by electron tomography. Plant Cell 17, 2580-2586, 2005. 3. Kaftan, D., et al. From chloroplasts to photosystems: in situ scanning force microscopy on intact thylakoid membranes. EMBO J. 21, 6146-6153, 2002.

PS7.2 Protein diffusion and macromolecular crowding in grana. H Kirchhoff (Institüt fur Botanik) Protein diffusion and macromolecular crowding in grana thylakoids Helmut Kirchhoff, Silvia Haferkamp, John Allen, Conrad Mullineaux Abstract For the survival in an ever-changing and oxidizing environment vascular plants must control the functionality of their light harvesting photosynthetic machinery. In the course of evolution dynamic molecular adaptation mechanisms were invented which are based on a flexible response of the protein composition / arrangement and distribution in the thylakoid membrane system of chloroplasts. These mechanisms require lateral protein traffic e.g. between stacked and unstacked thylakoid regions over several 100 nm. In this respect a key question is how efficient protein migration depends on the complex and dynamic supramolecular protein architecture in thylakoids. Thylakoid membranes are crowded by photosynthetic proteins (estimated protein area 70 to 80%) which can act as very efficient diffusion obstacles. Interestingly macromolecular crowding seems to be a common feature of all bioenergetic membranes. Experimental tools are required to study diffusion processes in thylakoid membranes and correlate them with the structural protein organization to understand adaptation mechanisms on the molecular level. We introduced Fluorescence Recovery After Photobleaching (FRAP) for isolated grana thylakoids from spinach to analyze diffusion characteristics of photosystem II (PSII) and light harvesting complex II (LHCII). This technique was established for cyanobacteria and reveal very interesting information on phycobilisom mobility. To analyze the influence of molecular crowding on PSII and

LHCII diffusion in grana membranes of higher plants FRAP measurements were carried out with isolated grana membranes with different lipid/protein ratios. This ratio was "titrated" by fusion of BBY membranes with different amounts of liposomes. Interestingly the data indicated the coexistence of very mobile and very immobile protein pools in grana thylakoids which are strongly dependent on the lipid/protein ratio. Implications on regulatory adjustments, the biogenesis and turnover of the complex thylakoid architecture will be discussed.

PS7.3 A Line-Scanning Multiphoton Fluorescence Spectromicroscope Applied to the Study of the Thylakoid Membrane in Cyanobacteria and Chloroplasts S Kumazaki (Kyoto University), M Hasegawa (Kyoto University), M Ghoneim (Kyoto University), M Terazima (Kyoto University), H Oh-oka (Osaka University), T Shiina (Kyoto Prefectural University) We have constructed a line-scanning semiconfocal multiphoton fluorescence microscope with a capability of simultaneous broadband spectral acquisition from a whole linear region. The spatial resolution of the system was 0.3, 0.4 and 0.6 micrometer along the x, y and z axes. It was applied to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120 and chloroplasts in plant, Zea mays. In the case of Anabaena, it was found that the fluorescence intensity ratio between chlorophyll molecules mainly of photosystem II and phycobilin molecules of phycobilisome (chlorophyll/ phycobilin), in the thylakoid membranes, became lower as one probed deeper inside the cells. This was attributable not to position dependence of re-absorption or scattering effects, but to an intrinsic change in the local physiological state of the thylakoid membrane, with the help of a transmission spectral measurement of the subcellular domains. In the case of Z. mays, we have determined spectral regions by which photosystem I and II including their associated antenna complexes are almost exclusively probed, and the spectroimages were found to exhibit clear differences in spatial fluorescence distribution between photosystem I and II. The observed phenomena in both Anabaena and Z. mays are not yet well explained, but they demonstrate unique potentials of the spectromicroscope to reveal intrachloroplast or subcellular thylakoid membrane in live cells in terms of its constitution and physiological state. The observed representative spectroimages as well as the technical details of the home-made spectromicroscope will be described.

PS7.4 Mutation of a single residue, glutamate ß10, alters protein-lipid interactions of light harvesting complex II P Braun (LM-University, Munich), L Kwa (LM-University), G Wanner (LM-University), D Wegmann (Biochemie Zentrum Heidelberg) It is well established that LH2 complex assembly is required for proper biogenesis of the vesicularised intracytoplasmatic membranes of purple bacterium R. sphaeroides. The underlying interactions are, as yet, still not understood. Here we examined the specificity of LH2-lipid interactions and its effect on membrane morphology. Our data demonstrate that non-bilayer lipid, phosphatidylethanolamin, is clearly enriched at the LH2-lipid interface. Remarkably, replacement of a single residue, ß-glutamate 10 with alanine, results in a reduction of the phosphatidylethanolamin content (~15%) in the annular lipid phase of LH2 without altering the lipid composition of the bulk phase. Alignment

212 14th Photosynthesis Congress - PS07 studies indicate that ß-glutamte 10 which is located at the N-terminal edge of the ß-subunit's transmembrane stretch, may be part of a lipid binding motif, preferentially interacting with phosphatidylethanolamin enriched membranes. The morphology of the LH2 housing membranes is, however, unaffected by the mutation ßE10A at the LH2 lipid interface. In contrast, simultaneous mutagenesis of glutamate 10 and replacement of the closely adjacent carotenoid sphaeroidenone with neurosporene results in significant changes of the size and shape of the vesicularised membrane invaginations. In particular, the rounded membrane invaginations are significantly enlarged (duplication of the radius) and additionally elongated membrane structures are observed. Taken together, these findings suggest that the N-terminal site of the LH2 complex, comprising glutamate 10 of the ß-subunit and the carotenoid's polar head group synergistically contribute to the morphogenesis of the vesicularised intracytoplasmatic membranes by specific interactions with the surrounding lipids. A possible model for the membrane shaping effect of the LH2-lipid interaction site will be discussed. PS7.5 The three-dimensional chloroplast structure of Ostreococcus tauri M Hohmannn-Marriott (National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health), L Dorward (National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health), M Aronova (National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health), G Zang (National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health) Ostreococcus tauri is a eukaryotic alga that possesses features that make it a very attractive model system for organelle biogenesis and function. A complete organelle system that includes nucleus, mitochondrion, Golgi apparatus and chloroplast is contained within a 1-2 µm cell. We have explored the possibility of imaging an entire cell by electron tomography. Employing this technique we demonstrate that the complete membrane systems of the chloroplast can be visualized. PS7.6 Structural organization of individual chloroplasts investigated with harmonic generation microscope V Barzda (University of Toronto), R Cisek (University of Toronto), A Tuer (University of Toronto), N Prent (University of Toronto), A Major (University of Toronto) The efficiency of light-harvesting is influenced by the macro-organization of pigment-protein complexes in thylakoid membranes of chloroplasts. It has been shown that LHCII, the major light-harvesting chlorophyll a/b pigment-protein complex associated with the photosystem II, is largely responsible for the chiral macroorganization of the grana. Furthermore, light-induced changes in the macrochirality have been correlated with the changes in non-photochemical quenching of chlorophyll fluorescence. In this work, the orientation dependent structural organization of the grana has been studied with the second and third harmonic generation, and multiphoton excitation fluorescence laser scanning microscope. The generation of harmonics and two-photon excitation fluorescence of carotenoids was provided by the 300fs, 14.7MHz repetition rate Yb:KGW laser operating at 1040nm. The second harmonic was generated in the grana and showed the orientation dependency with respect to the linear polarization of the laser. The second harmonic was sensitive to the chiral organization of the stacked thylakoid membranes. The third harmonic was also efficiently generated in chloroplasts from a multilamellar structure of the grana. Simultaneous second and third harmonic generation imaging gives complementary structural information about the macroorganization

of the grana. Combination of harmonic generation with the multiphoton excitation fluorescence enables to perform simultaneous measurements of the structural changes in the grana and variations of the fluorescence quenching in individual chloroplasts. PS7.7 Significance of molecular crowding for the functional protein organisation in grana membranes H Kirchoff (Institut für Botanik), S Haferkamp (Institut für Botanik), C Mullineaux (Queen Mary University), H van Amerongen (Wageningen University), W Haase (MPI of Biophysics) Grana membranes are crowded by photosystem (PS) II and its antenna system (LHCII) (~ 75% area occupation). Although it is expected that this extreme packing of proteins causes serious problems for membrane functions its significance for the supramolecular arrangement of PSII and LHCII as well as its effect on the light-harvesting properties and the stability of the PSII supercomplex is poorly understood. We studied the influence of molecular crowding on the antenna organization of PSII by "diluting" the high protein density in grana thylakoids. Therefore BBY membranes were fused with unilamellar liposomes consisting of native lipids. Successful incorporation of extra lipids and the molecular organisation of protein complexes were analysed by density gradient centrifugation, lipid analysis, electron microscopy and fluorescence lifetime imaging microscopy. Time-resolved and steady state chlorophyll a fluorescence spectroscopy at RT and 77K indicate a detachment of LHCII in "diluted" membranes. The detachment correlate almost linear with the lipid/chlorophyll ratio, indicating weak interaction forces between the antenna proteins. In contrast threshold behaviour on the lipid/chlorophyll ratio is apparent for the quantum yield of PSII photochemistry, the reduction in connectivity of PSIIa centres and the PSIIa to b conversion. We postulate a hierarchy in protein interactions which becomes manifest in a differential dependency on protein density. Molecular crowding in grana membranes in combination with interplay between weak and strong interactions could reflect a compromise between the flexibility of the protein arrangement and a high efficiency of light utilization. PS7.8 Impaired Isoprenoid Biosynthesis: a Competitive Disadvantage under Light Stress in Synechocystis PCC 6803 E Gantt (University of Maryland), K Poliquin (University of Maryland), F Cunningham Jr (University of Maryland), I MacDonald (University of Maryland), R Gantt (University of Maryland), E Gantt (University of Maryland) C-methyl-D-erythritol 4-phosphate pathway. The C5 building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are required for biosynthesis of carotenoids, in prenylation of quinones, and in phytylation of chlorophylls. The role of Sll1556, previously designated as an IPP isomerase (Barkley et al. 2004, J. Bact. 186:8156), was investigated in Synechocystis PCC 6803 with a deletion mutant ?sll1556. Genes homologous to sll1556 are present in many, but not in all cyanobacteria. The ?sll1556 mutant was not impaired in growth under low light phototrophic conditions (20 µmol photons m-2 s-1). However, at 200 µmol photons m-2 s-1 the ?sll1556 cells were impaired in growth and were out-competed by wild-type (WT) cells in mixed cultures. Consistent with these results, Sll1556 protein expression in WT was greater in cells under high light than under non-stress conditions. Furthermore, ?sll1556 cells grown under high light had altered cell structure and a greater reduction in chlorophyll and carotenoid content relative to WT. In vitro stimulation of isoprenoid biosynthesis by pentose phosphate cycle (PPC) compounds, as evident in WT

213 14th Photosynthesis Congress - PS07 supernatant, was lacking in ?sll1556 (Poliquin et al. 2004, J. Bact. 186:4685). In WT, such stimulation resulted in a progression of isoprenoid synthesis from C5 to C10 to C20, although PPC compounds did not directly serve as substrates. The collective results suggest that the Sll1556 protein, while dispensable, is advantageous to Synechocystis PCC 6803 upon increased metabolic demand for isoprenoids.

PS7.9 Arrangement of chlorophyll – protein complexes determines chloroplast structure I Rumak (Warsaw University) Gieczewska Katarzyna, 2Koziol-Lipinska Joanna, 3Kierdaszuk Borys, Mostowska Agnieszka, 1Garstka Maciej 1 Department of Metabolic Regulation, Warsaw University, Miecznikowa 1, 02 096 Warsaw, PL 2 Department of Plant Anatomy and Cytology, Warsaw University, Miecznikowa 1, 02 096 Warsaw, PL 3 Department of Biophysics, Warsaw University, Zwirki I Wigury 93, 02 089 Warsaw, PL 1,2 2

It is believed that there is close relation between chloroplast structure and function. To support this statement we studied chloroplast structure and function in two plant species with different susceptibility for chilling – bean (Phaseolus vulgaris) – CS (chilling sensitive) plant and pea (Pisum sativum) – CT (chilling tolerant) plant. The ratio between fluorescence intensities of PSI and PSII (F736/F681) at 120K was 1.5 – times higher in thylakoid membranes isolated from bean as compared with pea. Besides that the fluorescence difference spectrum indicated that there was relatively less PSII complexes, antenna complexes associated with PSII and PSI complexes in bean than in pea. The appearance of the emission band at around 705 nm suggested however that the aggregated form of LHCII was more frequent in bean thylakoid membranes than in pea ones. The composition of CP (chlorophyll – protein) complexes shown by mild - denaturing electrophoresis was substantially different. Structure of chloroplasts was probably influenced not only by LHCII trimers content but also protein composition of particular CP complexes. Moreover ultrastructural measurements of grana size revealed significant differences between those species. The fact that bigger appressed domains (grana) occurred in pea than in bean was confirmed by 3D structure of chloroplasts obtained using confocal laser scanning fluorescence microscopy. PS7.10 3D chloroplast structure I Rumak (Department of Metabolic Regulation and Department of Plant Anatomy and Cytology, Warsaw University, Miecznikowa 1, 02 096 Warsaw, PL), K Gieczewska (Department of Metabolic Regulation and Department of Plant Anatomy and Cytology, Warsaw University, Miecznikowa 1, 02 096 Warsaw, PL), B Kierdaszuk (Department of Biophysics, Warsaw University, Zwirki I Wigury 93, 02 089 Warsaw, PL), W Gruszecki (Department of Biophysics, Maria Curie-Sklodowska University, Marii Curie-Sklodowskiej 1, 20-031 Reconstruction of stacking in vitro of thylakoid membranes isolated from two plant species with different susceptibility for chilling – bean (Phaseolus vulgaris) – CS (chilling sensitive) plant and pea (Pisum sativum) – CT (chilling tolerant) plant was investigated as a function of Mg2+ concentration in the range from 0 to 6 mM MgCl2. Arrangement of CP (chlorophyll – protein) complexes were analyzed using low temperature (120K) fluorescence emission spectra and Fourier transform infrared spectroscopy (FTIR). 3D structures of intact chloroplasts were

constructed by computer analyses of chloroplast images obtained using confocal laser scanning fluorescence microscopy (CLSM). Experiments revealed that bigger appressed domains (grana) occurred in pea compared with bean. Moreover a reorganization of the chloroplast structure caused by the increase of MgCl2 concentration was different in pea and bean plants. PS7.11 Membrane dynamics during the CT and CS plants chilling revealed by FTIR spectroscopy K Gieczewska (Department of Plant Anatomy and Cytology, and Department of Metabolic Regulation, Warsaw University, Miecznikowa 1, 02 096 Warsaw, PL), A Kuta (Department of Metabolic Regulation, Warsaw University, Miecznikowa 1, 02 096 Warsaw, PL), W Gruszecki (Department of Biophysics, Maria Curie- Sklodowska University, Maria Curie-Sklodowska sq. 1, 20 031 Lublin, PL), I Rumak (Department of Plant Anatomy and Cytology, and Department of Metabolic Regulation, Warsaw University) The thylakoid membranes, which comprise delicate assemblies of lipids and proteins, change their properties depending – among other agents – on temperature. An attempt was made to study the dynamics of thylakoid membranes in two plants that differ in response to chilling stress, namely Phaseolus vulgaris, the chilling sensitive plant (CS) and Pisum sativum, the chilling tolerant one (CT). FTIR spectra of thylakoid membranes were analysed in the Amid I and the symmetric CH2 stretching vibration regions providing data on membrane proteins’ secondary structures and lipid acyl chain conformations, respectively. A difference in Amid I region between the CS and CT plants during dark-chilling and subsequent reactivation was observed. The level of ß-sheet compound of Amid I spectra indicates a degree of membrane proteins aggregation. Having increased after chilling, it soared even higher after reactivation in Phaseolus vulgaris. On the contrary, in Pisum sativum it decreased, and after reactivation only slight changes were observed. PS7.12 Below ambient levels of UV-A,B induces chloroplast structural change and alters starch metabolism W Fagerberg (University of New Hampshire) Above ambient levels of UV-B radiation (290-320nm) under high PAR conditions elicits responses in chloroplasts similar to their being exposed to low PAR levels (W. Fagerberg and J. Bornman 1997. Physiol Plant 101: 833-844.). Here we provide evidence that even below ambient levels of UV-B (1/28th ambient; Durham, NH, USA 1200hr March) are capable of inducing an increase in thylakoid surface area relative to the chloroplast volume typical of a low PAR response (shade response) in sunflowers. This response occurred even though leaves were concurrently exposed to PAR levels that normally induce a "sun"or high PAR response in the absence of UV-B. Sub-ambient levels of UV-B were also associated with a decrease in chloroplast and starch volume. Exposure to levels of UV-A 1/10 of ambient enhanced the high PAR response of the chloroplast characterized by an increase in the amounts of stored starch, increase in chloroplast volume density ratio (Vv), and a decrease in thylakoid surface density ratios (Sv) relative to the high light controls. These effects were opposite to those seen in UV-B exposed tissue. Sub-ambient levels of UV-B evoked a response similar to that elicited by low PAR irradiance while sub-ambient UV-A elicited responses similar to those typical of high PAR irradiance. The fact that below ambient levels of UV altered a normal chloroplast structural response to PAR provides evidence that UV may be an important

214 14th Photosynthesis Congress - PS07 environmental signal for plants. PS7.13 The serine-type chloroplast protease is involved in Lhcb6 polypeptide degradation in Arabidopsis leaves in response to wounding R Lucinski (Adam Mickiewicz University, Department of Plant Physiology Poznan, Poland), G Jackowski (Adam Mickiewicz University,Department of Plant Physiology, Poznan, Poland) Chloroplast proteases are considered to be an important component of cellular protein quality system responsible for degradation of inherently stable chloroplast proteins damaged either as a result of exposition of plants to stressing environmental conditions or spontaneously. Photosystem II (PS II) polypeptides have been shown to be among targets of chloroplast proteases under stressing conditions but there is no experimental data concerning their potential engagement in degradation of Lhch6, the apoprotein of CP24, one of PS II minor peripheral energetic antennae. In the present work it was demonstrated that Lhcb6 displayed an ample disappearance in detached Arabidopsis leaves exposed to a number of short-time stresses, i.e. salt, dessication, cold and wounding ones. In order to address the question of possible involvment of proteolytic events in Lhcb6 disappearance an experimental system was designed composed of thylakoids isolated from leaves stressed for 3h and then incubated in vitro in darkness. When the thylakoids isolated from the cold-treated and wounded leaves were incubated in vitro for a period of 6h a remarkable reduction of Lhcb6 abundance was observed. Aprotinin, a potent inhibitor of serine-type endoproteases significanty arrested in vitro Lhcb6 disappearance in thylakoids isolated from wounded leaves and it is inferred that wounding-dependent disappearance of Lhcb6 is a proteolytical phenomenon and that a thylakoid-bound, serine-type endoprotease is responsible for this degradation. PS7.14 FtsH5 as the novel main light harvesting complex of photosystem II (LHC II) degrading protease G Jackowski (Adam Mickiewicz University, Department of Plant Physiology, Poznan, Poland), R Lucinski (Adam Mickiewicz University, Department of Plant Physiology, Poznan, Poland) Arabidopsis mutant VAR1 in which AtFtsH5 has been inactivated. Namely, changes in the level of Lhcb1-3 LHC II apoproteins have been traced in VAR1 mutant vs. wild type plants in response to six environmental stresses applied to detached Arabidopsis leaves. It was established that during the application of desiccation and wounding (3 and 6h) as well cold shock and excessive irradiance (6 and 24h) significant and reproducible disappearance of Lhcb1 and Lhcb2 apoproteins took place in the leaves of wild type plants whereas both these apoproteins resisted the disappearance in the leaves of VAR1 mutants. Therefore, clearly, AtFtsH5 is involved in Lhcb1 and 2 degradation under the four stresses. In addition, the level of Lhcb3 in the VAR1 mutant leaves which underwent high salt concentration and cold (6h) as well as oxidation (6 and 24h) stresses did not decline although it was decreasing significantly and reproducibly in the stressed leaves of wild type plants. Therefore we conclude that AtFtsH5 is responsible for the degradation of Lhcb1-3 apoproteins under various environmental stresses with 6h-long cold treatment being the one inducing the degradation of all three apoproteins. PS7.15 Arabidopsis ANTR1 is a thylakoid phosphate transporter -functional characterization in E. coli

L Ruiz-Pavón (Division of Cell Biology,Linköping University, SE-581 85 Linköping, Sweden), A Mirsha (Department of Physics and Measurement Technology, Linköping University, SE-581 85 Linköping, Sweden), F Lundh (Department of Chemistry and Biomedical Sciences, Kalmar University, SE-39182 Kalmar, Sweden), S Thuswaldner (Division of Cell Biology,Linköping University, SE-581 85 Linköping, Sweden), B Persson (Department of Chemistry and Biomedical Sciences, Kalmar University, SE-39182 Kalmar, Sweden Arabidopsis thaliana . An active nucleotide metabolism in the thylakoid lumen implies the existence of additional, yet unidentified transporters, such as those exporting phosphate back to the soluble stroma. In A. thaliana, there are six genes encoding anion transporters (ANTR1 to 6), sharing homology with the type I of mammalian Na+-dependent inorganic phosphate (Pi) cotransporters (NaPi-I). In this study, the putative ANTR1 was immunolocalized to the chloroplast thylakoid membrane. By heterologous expression and uptake of radioactive Pi into Escherichia coli cells, the function of ANTR1 as a NaPi cotransporter was demonstrated. The expression of ANTR1 conferred increased growth rates to the transformed cells, and stimulated Pi uptake in a Na+-dependent and ionophore-sensitive manner, as compared to the control cells. A role of ANTR1 in exporting Pi produced during nucleotide metabolism in the chloroplast thylakoid lumen is proposed. PS7.16 Protein shape and crowding drive domain formation and curvature in photosynthetic membranes R Frese (Leiden University), J Pàmies (Amolf Amsterdam)), J Olsen (Sheffield University)), N Hunter (Sheffield University)), D Frenkel (Amolf Amsterdam)), T Aartsma (Leiden University)), R van Grondelle (Vrije Universiteit Amsterdam) Packed proteins in varying crystalline order are observed in all energy generating membranes, and it is often assumed that attractive forces are responsible for the appearance of such domains. However, it is well known in colloidal science that entropy alone is sufficient to produce ordered states of non-interacting hard bodies, as well as bulk or microphase separation through what is known as depletion-induced attraction or macromolecular crowding. Besides, colloidal bodies may also experience elastic interactions resulting from curvature mismatch. We have investigated these colloidal effects in photosynthetic membranes of Rhodobacter sphaeroides, whose organization can be precisely determined by means of atomic force microscopy and polarized light spectroscopy. Results on sphaeroides membranes with varying content (RCLH1 +/- PufX +/- LH2) are modeled using Monte Carlo simulations and demonstrate that the size and shape asymmetry of the protein complexes, for example dimerisation, are sufficient to induce lateral segregation with varying order. We also show that domain formation is coupled to the local curvature of the membranes and can account for the budding of sphaeroides membranes. Our results suggest that depletion interactions between the proteins in densely packed membranes results in the partitioning into fluid and ordered domains which are of key importance in photosynthesis, where both close packing and diffusion are a functional necessity. PS7.17 Fluorescence lifetime imaging microscopy on individual chloroplasts in intact leaves K Broess (Wageningen University), J Borst (Wageningen University), H van Amerongen (Wageningen University) Fluorescence lifetime imaging microscopy (FLIM) provides the possibility to perform picosecond fluorescence measurements at a microscopic level. We started to explore the possibilities to perform

215 14th Photosynthesis Congress - PS07 FLIM measurements on individual chloroplasts within a leaf, with the aim to monitor the response of the chloroplast to changes of the environmental conditions. This was done on a fluorescence lifetime imaging microscope with 2-photon excitation (840 nm, 100 fs pulses). We performed FLIM on Arabidopsis thaliana leaves where either ferricyanide or DCMU was added by vacuum infiltration to keep the reaction centers of photosystem II open or closed, respectively. The results were compared with the results of macroscopic time-correlated single photon counting measurements on chloroplasts. The kinetics show good agreement, demonstrating that FLIM can indeed be used to study individual chloroplasts in leaves. As a next step we performed singlet-singlet annihilation measurements on individual chloroplasts. The fluorescence kinetics appears to depend strongly on the laser intensity. The results can be related to domain sizes of connected pigments and thus provide information on the organization within the membrane in planta. This will provide an extra tool to study the response of chloroplasts to changes in their direct environment. PS7.18 Heterogeneity of the lipid phase of chloroplast thylakoid membranes S Krumova (Wageningen University, Laboratory of Biophysics, P.O Box 8128, 6700 ET Wageningen, The Netherlands ), C Dijkema (Wageningen NMR Center Wageningen P.O. 8128, 6700 ET Wageningen, The Netherlands), R Koehorst (Wageningen University, Laboratory of Biophysics, P.O Box 8128, 6700 ET Wageningen, The Netherlands), T Páli (Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences P.O. Box 521, Szeged H-6701, Hungary), H van Amerongen (MicroSpectroscopy Centre Wagening In chloroplast thylakoid membranes the non-bilayer forming lipids account for about half of the lipid content. It is generally believed that non-bilayer lipids in biological membranes do not form non-lamellar phases; however, they might be transiently formed and/or associated with the membrane. It has been suggested that such non–bilayer phases have physiological relevance for the operation of violaxanthin de-epoxidase, might regulate the lipid content of the membranes and might provide additional structural flexibility to the thylakoid membrane. In order to get better insight in the lipid phase behaviour of thylakoid membranes, we applied 31P-NMR measurements and fluorescence investigations using merocyanine 540. Our 31P-NMR data show that at physiological temperatures, the contribution of the lamellar phase is very weak and the spectrum is dominated by an isotropic peak, which suggests that the motion of the lipids is not restricted to the bilayer phase. The spectra also contained a signal which might be attributed to the inverted hexagonal phase of lipids; however, its assignment remains ambiguous because of possible contributions from phosphorylated proteins in the same spectral region. Steady-state and time-resolved measurements using merocyanine 540 also reveal strong heterogeneity of the lipid phases in the thylakoid membrane. A comparison with purified lipid systems shows that these phases exhibit spectral and lifetime features which closely resemble those in bilayer, and inverted hexagonal and gel phases, respectively. PS7.19 Difference in SQDG metabolism between green algae and cyanobacteria under the sulfur-starved condition N Sato (Tokyo University of Pharmacy and Life Sciences), R Kamimura (Tokyo University of Pharmacy and Life Sciences), K Sugimoto (Tokyo University of Pharmacy and Life Sciences), M Tsuzuki (Tokyo University of Pharmacy and Life Sciences) The sole sulfolipid sulfoquinovosyl diacylglycerol (SQDG) is included predominantly in thylakoid membranes, shown to contribute to the

functioning of photosystem (PS) II probably through associating with the PSII complex for its normal conformation [1]. Besides its crucial role as the membrane lipid, SQDG has recently been found subjective to degradation for providing the major S-source for synthesis of proteins in a green alga Chlamydomonas reinhardtii upon transfer of the cells to the sulfur (S)-starved condition, thereby playing the other role as the sulfur (S)-storage lipid. Here, we investigated whether or not this regulation operates also in the other green alga Chlorella kessleri or two strains of cyanobacteria postulated to represent the ancestor of chloroplasts in plants. The cells of respective species preincubated in the presence of [35S]sulfate for universal labeling of cellular S-compounds were transferred to the growth condition starved of S for chasing of the radioactivity of SQDG. C. kessleri showed a decrease in the radioactivity of SQDG, in line with that in the SQDG content, whereas little decrease was evident in the two strains of the cyanobacteria as to either the radioactivity or abundance of SQDG. These results indicated the induction of SQDG degradation in C. kessleri similar to that in C. reinhardtii, but definitely not in either strain of cyanobacteria. The induction mechanism of SQDG degradation thus seems to occur after evolutionary appearance of green algae at latest, but not in cyanobacteria before the primary endosymbiosis. [1] Sato, N. (2004) J. Plant Research 117, 495-505. PS7.20 Three dimensional architecture of the granum-stroma thylakoid membrane system revealed by electron tomography G Garab (Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary), L Mustárdy (Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged), K Buttle (Resource for Visualization of Biological Complexity, Wadsworth Center, Albany, N.Y., USA) We investigated the three-dimensional architecture of isolated granal thylakoid membranes by using high voltage electron tomography on 250 nm thick sections of isolated intact thylakoid preparations fixed and stained with conventional electron microscopy techniques. High resolution reconstructed tomographic images were obtained which clearly resolved the membranes and the lumenal spaces and thus also the connections between the stacked and non-stacked membranes. Based on the series of tomographic sections and the computer assisted 3D models, and also using the electron tomography data of chloroplasts within cryo-immobilized, freeze substituted lettuce leaves (Shimoni et al. 2005, Plant Cell 17: 2580), we propose a refined model of the granum-stroma assembly. The model takes into account the following structural factors and membrane properties: (i) the size differences between the stroma exposed sides of the two photosystems, i.e., the fact that the stromal sides of photosystem II (PSII) and its main light harvesting complexes (LHCII) are flat, while PSI and the ATP synthase protrude significantly into the stroma liquid; (ii) the ability of LHCII and PSII to self-assemble into large domains, i.e., the ability of LHCII and PSII, and PSI and the ATP synthase for lateral segregation; (iii) the stacking of membranes possessing flat membrane surfaces, which stabilizes the lateral segregation of the (super)complexes; (iv) the fusion and overlap of membranes during the growth of membranes. These factors might bring about the self-assembly of a quasi-helical organization of the granum-stroma contiguous thylakoid membrane system that encloses a single interior aqueous phase. PS7.21 Probing the organization of photosystem II in photosynthetic membranes by atomic force microscopy H Kirchhoff (Institute of Botany) Efficient photosynthetic energy transduction and its regulation depends

216 14th Photosynthesis Congress - PS07 on a precise supramolecular arrangement of the plant photosystem II complex in grana membranes of chloroplasts. The topography of isolated photosystem II supercomplexes and the supramolecular organization of this complex in grana membrane preparations are visualized by high resolution atomic force microscopy in air with tapping mode and an active feedback control to minimize tip-sample interactions. Systematic comparison between topographic characteristics of the protrusions in the atomic force microscopic images and well established high resolution and freeze fracture electron microscopic data shows that the photosystem II organization can be properly imaged by AFM in air. Taking the protruding water splitting apparatus as a topographic marker for PSII, its distribution and orientation in isolated grana membranes was analyzed. For the latter a new mathematical procedure was established which reveals a preference for a parallel alignment of PSII that resembles the organization in highly ordered semicrystalline arrays. Furthermore, when analyzing the height of grana membrane stacks we conclude that lumenal protrusions of adjacent photosystem II complexes in opposing membranes are displaced relative to each other. The functional consequences for lateral migration processes are discussed. PS7.22 The Conditional Role of Membrane Sulfolipid as Sulfur-Storage Compound in Chlamydomonas reinhardtii

a

K Sugimoto (School of Life Sciences, Tokyo University of Pharmacy and Life Sciences), N SATO (School of Life Sciences, Tokyo University of Pharmacy and Life Sciences), M TSUZUKI (School of Life Sciences, Tokyo University of Pharmacy and Life Sciences) The sulfolipid, sulfoquinovosyl diacylglycerol (SQDG), which is present in thylakoid membranes, possesses a sulfonate residue in its head group, sulfoquinovose. In this study, we will report the fate of sulfur (S) atom of SQDG in a green alga, C. reinhardtii, after transfer of the cells to the S-starved condition. SQDG was found to account for 11% of total cellular S through measurement of the radioactivity of SQDG relative to that of the cells that had been grown in the presence of [35S] sulfate for universally labeling of S-compounds. The radioactivity was relatively stable during further incubation of the cells under the 32S-replete condition. Exposure of the cells to the S-starvation, however, decreased the radioactivity of SQDG to 15% of the initial level within 6h, indicating degradation of SQDG under the S-starved condition. Interestingly, the loss of 35S in SQDG by its degradation could explain a large part of an increase in the radioactivity of protein fraction of the cells, which suggested redistribution of S from SQDG to a major part of de novo synthesized proteins. Accordingly, little increase in the radioactivity of the protein fraction was observed with an SQDG-deficient mutant of C. reinhardtii, hf-2, after transfer of the cells from 35S-replete to S-starved conditions. Moreover, accumulation of proteins was repressed in hf-2 relative to the wild type under the S-starved condition. These results indicated that the membrane lipid, SQDG, is degraded as a storage molecule of S for protein synthesis under the S-starved condition in C. reinhardtii. PS7.23 Thylakoid Membrane Proteomics and Spectroscopic Studies of Vegetative and Isolated Heterocyst cells from Nostoc punctiforme T Cardona (Uppsala University), Å Agervald (Uppsala University), P Zhang (University of Turku), N Battchikova (University of Turku), E Aro (University of Turku), S Styring (Uppsala University), P Lindblad (Uppsala University), A Magnuson (Uppsala University) The thylakoid membranes of nitrogen-fixing multicellular cyanobacteria have not been investigated in detail; especially the changes that occur during cell differentiation and the possible functional differences that arise as a consequence of nitrogen fixation. Protocols for the purification

of thylakoid membranes, from vegetative cells and isolated heterocyst from Nostoc punctiforme sp. PCC 73102 have been developed to investigate their biochemical and biophysical properties. Light microscopy proved that the heterocyst were intact and deprived from vegetative cells; confocal microscopy was implemented to further estimate the purity of the heterocyst preparations using the stronger phycobilisome emission of vegetative cells as a marker when excited at 488 nm. It confirmed the quality of the heterocyst samples and revealed variations in fluorescence due to the difference in phycobilisome content. The purified membranes from vegetative cells showed high oxygen evolution and the heterocyst thylakoids electron transfer through PSI. 77 K fluorescence emission spectroscopy detected the presence of PSII antenna in heterocyst thylakoids; however, the remaining phycobilisomes appear to be connected only to PSI. A proteomic study using 2D BN/SDS PAGE followed by MALDI-TOF has been initiated to identify the protein composition of both types of membranes. A full set of electron transfer chain protein complexes together with other integral and membrane associated proteins were found as well as relevant differences. This study have confirmed previous observations in other types of heterocystous cyanobacteria, added knowledge about the physiology of heterocyst, and opened the door for cell-type specific research using state-of-the-art technologies which were not accessible before. PS7.24 Galactolipid biosynthesis is essential for proper chloroplast biogenesis and embryogenesis K Kobayashi (University of Tokyo), M Kondo (National Institute for Basic Biology), H Fukuda (Tokyo Institute of Technology), M Nishimura (National Institue for Basic Biology), H Ohta (Tokyo Institute of Technology) Photosynthetic reactions of plants rely on a well-developed membrane system of thylakoids inside chloroplasts. In chloroplasts, monogalactosyldiacylglycerol (MGDG) is the most abundant lipid of thylakoid membranes. The final step in MGDG biosynthesis occurs in the plastid envelope and is catalyzed by MGDG synthase. In Arabidopsis, three functional MGDG synthases have been identified, namely MGD1, MGD2 and MGD3, which differ substantially in many aspects such as substrate specificity, subcellular localization and gene expression profiles. Among them, MGD1 is regarded as a major isozyme involved in chloroplast biogenesis. In this study, we newly isolated a MGD1 knockout mutant mgd1-2, which shows severe defect in embryogenesis and seedling development. mgd1-2 seedlings have a dwarf and albino phenotype and show neither chlorophyll accumulation nor photosynthetic activity. In the mutant, MGDG content was reduced by 98% compared with wild type, indicating that MGD1 function underlies the bulk of MGDG biosynthesis and that MGD2/MGD3 cannot substitute for MGD1 function. Transmission electron microscopic analysis shows that the mutant leaves contain only abnormal plastids with no or severely underdeveloped internal membrane structures, demonstrating that the galactolipid deficiency caused severe impairment of thylakoid membrane development. In addition, invagination of inner envelope membranes was observed in the mutant plastids. Together with an old observation that young undifferentiated plastids displayed inner envelope invagination, this result suggests that inner envelope invagination initiates the formation of thylakoid membranes from undifferentiated proplastids. In conclusion, MGDG biosynthesis catalyzed by MGD1 is a key step for chloroplast biogenesis and is indispensable for plant development.

217 14th Photosynthesis Congress - PS07

PS8 - CO2 Diffusion, Gas Exchange and the Role of Stomata PS8.1 Carbon-water balance in the future: are stomata up to the task? T Buckley (The University of New South Wales) Stomata tend to track photosynthesis so that the ratio of intercellular to ambient CO2 concentrations (ci/ca) is conserved, despite broad variation in photosynthetic capacity in relation to light environment. However, stomata also tend to preserve the balance between hydraulic supply and demand, preventing leaf water potential from dropping low enough to cause runaway xylem embolism. Therefore, the actual value of ci/ca is constrained by carbon allocation, which determines the balance between hydraulic supply and the drivers of photosynthetic CO2 demand (nitrogen and light). It is unclear how a doubling of atmospheric CO2 will affect ci/ca and other key features that emerge from the adaptive regulation of carbon allocation. I will present a range of predictions based on the assumption that resource coordination will be regulated in the future to maximise growth (using models that permit resource substitution and exclude ad hoc assumptions about functional balance). I will then discuss what these predictions would require, in terms of the reduced processes involved in the leaf-level regulation of gas exchange, in order to be borne out in the future.

PS8.2 The internal resistance to CO2 movement in C3 plants

J Flexas (Universitat de les Illes Balears), A Díaz-Espejo (Instituto de Recursos Naturales y Agrobiología)), J Galmés (Universitat de les Illes Balears)), R Kaldenhoff (Darmstadt University of Technology)), H Medrano (Universitat de les Illes Balears)), M Ribas-Carbó (Universitat de les Illes Balears) The effects of short-term (minutes) variations of CO2 concentration on mesophyll conductance to CO2 (gm) were evaluated in six different C3 species by simultaneous measurements of gas exchange, chlorophyll fluorescence, on-line carbon isotope discrimination and a novel curve-fitting method. Depending on the species, gm varied from 5 to 9-fold, along the range of sub-stomatal CO2 concentrations typically used in photosynthesis CO2-response curves (AN-Ci curves), i.e., 50 to 1500 mmol CO2 mol-1 air. Although the pattern was species-dependent, gm strongly declined at high Ci, where photosynthesis was not limited by CO2, but by regeneration of ribulose-1,5-bisphosphate or triose phosphate utilization. Moreover, these changes on gm were found to be totally independent of the procedure used to induce variations of Ci. The response of gm to Ci resembled that of stomatal conductance (gs), but kinetic experiments suggested that the response of gm was actually faster than that of gs. Transgenic tobacco plants differing in the amounts of aquaporin NtAQP1 showed different slopes of the gm-Ci response, suggesting a possible role for aquaporins in mediating CO2-responsiveness of gm. The importance of these findings is discussed in terms of their effects on parameterization of AN-Ci curves.

C Warren (University of Sydney) There is a large resistance to CO2 movement between the sub-stomatal cavities and sites of carboxylation. This resistance, generally termed internal resistance (or internal conductance), has been measured in a large number of species and in all cases it has been shown to pose a large limitation to photosynthesis. Internal conductance was originally thought to be largely constitutive and a function of leaf anatomy, but we now know it can change rapidly in response to temperature, soil water availability, and [CO2]. Most previous literature reviews have implicitly assumed an infinite internal conductance and/or that internal conductance scales with the biochemical capacity for photosynthesis. Examination of published data for 54 species and a detailed examination for three well-characterised species - Eucalyptus globulus, Pseudotsuga menziesii and Phaseolus vulgaris - show these assumptions to be incorrect. The reduction in concentration of CO2 between the sub-stomatal cavity (Ci) and the site of carbon fixation (Cc) varies greatly among species and limitations to photosynthesis due to internal resistance are not constant among species. Importantly, there is a general trend for plants with small internal conductance to have a larger draw-down from Ci to Cc, further confounding efforts to scale photosynthesis and other attributes with internal conductance. Variation in the relationship of photosynthesis with internal conductance contributes to variation in rates of photosynthesis per unit N (PNUE) and water (WUE). A priority for future research should be elucidation of relationships between internal and stomatal conductances and how these vary in response to environmental conditions (e.g. soil water, leaf-to-air vapour pressure deficit, temperature) and among species.

PS8.3 Rapid variations of mesophyll conductance in response to changes in CO2 concentration around leaves

PS8.4 CO2 transfer in the mesophyll: a carbonic anhydrase perpective B Genty (CEA Cadarache/CNRS/Université Aix-Marseille), I Reiter (CEA Cadarache/CNRS/Université Aix-Marseille), N Fabre (CEA Cadarache/CNRS/Université Aix-Marseille), S Driever (CEA Cadarache/CNRS/Université Aix-Marseille), D Rumeau (CEA Cadarache/CNRS/Université Aix-Marseille) Using 18O labelled CO2 and mass spectrometry, estimations of the carbonic anhydrase (CA) activity and the CO2 transfer conductance between the atmosphere and the site of CA have been obtained in various C3 plants. Remarkably, the CO2 concentration estimated at the site of catalysed hydration in folio was always closer to the CO2 concentration estimated at the evaporative site than to the CO2 concentration predicted in the chloroplast contrary to the current view assuming catalysis by chloroplastic activity only. In order to dissect the basis of this observation, we analysed expression and function of genes encoding putative CA in Arabidopsis thaliana. 6 β-CA genes were found to be expressed in leaf tissues. We provided evidence that the each of the corresponding β-CAs are targeted to specific subcellular compartments (chloroplast, cytosol, plasma membrane and mitochondria) (Fabre et al., 2007). By using null mutants depleted in various CA isoforms, the role of CA activity with respect to their specific cellular compartments has been addressed. We showed that CO2 hydration in the mesophyll is not only catalysed by chloroplastic CA but also by extrachloroplastic CAs. In particular, the main cytosolic isoform represented up to a quarter of the whole leaf CA activity. For several null mutants, contrasting growth phenotypes which depended on CO2 concentration during growth have been obtained. From CA-based estimations of mesophyll conductance

218 14th Photosynthesis Congress - PS07 together with estimations based on Rubisco in these mutants, the relationship between CAs (activity and cellular localisation) mesophyll CO2 transfer, CO2 uptake and growth will be discussed.

PS8.5 Mesophyll conductance to CO2 in Arabidopsis thaliana M Ribas-Carbo (Universitat Illes Balears), J Flexas (Universitat Illes Balears), M Ortuño (CEBAS-CSIC Murcia), A Díaz-Espejo (CSIC Sevilla), I Florez-Sarasa (Universitat Illes Balears), H Medrano (Universitat Illes Balears) The close rosette growth form, short petioles and small leaves of Arabidopsis thaliana make measurements with commercial gas exchange cuvettes difficult. This difficulty can be overcome by growing Arabidopsis thaliana plants in “ice-cone like” soil pots. This design has permitted simultaneous gas exchange and chlorophyll fluorescence measurements from which the first estimates of mesophyll conductance to CO2 (gm) in Arabidopsis were obtained and used to determine photosynthetic limitations during plant ageing from about 30 to 45 days. Estimations of gm showed maximum values of 0.2 mol CO2 m-2 s-1 bar-1, lower than expected for a thin-leaved annual species. The parameterization of the response of AN to chloroplast CO2 concentrations (Cc) yielded estimations of the maximum velocity of carboxylation (Vc,max_Cc) which were also lower than those reported for other annual species. As Arabidopsis thaliana plants aged from 30 to 45 days, there was a 40% decline of AN that was entirely due to increased diffusional limitations to CO2 transfer, with gm being the largest. In conclusion, the results suggest that in Arabidopsis thaliana AN is limited by a low gm and a low capacity for carboxylation. Decreased gm is the main factor involved in early age-induced photosynthesis decline. PS8.6 Seasonal evolution of biochemical and diffusional limitations to photosynthesis in olive A Diaz-Espejo (IRNASE (CSIC)), J Fernández (IRNASE (CSIC)), E Nicolás (CEBAS (CSIC)) This study was undertaken to test the hypothesis that diffusional limitation of photosynthesis, rather than light, determine the distribution of photosynthetic capacity in olive leaves under drought conditions. The crowns of four olive trees growing in an orchard were divided in two sectors: one sector absorbed most of the radiation early in the morning (MS) while the other absorbed most in the afternoon (AS). When the peak of radiation absorption was higher in MS, vapor pressure deficit of the air (VPD) was not high enough to provoke stomatal closure. In contrast, peak radiation absorption in AS coincided with the daily peak in VPD. In addition to this, two soil water treatments were evaluated: irrigated trees (I) and non-irrigated trees (nI). Seasonal diffusional limitation of photosynthesis was mainly increased in nI trees, especially due to stomatal limitation, although mesophyll conductance (gm) was found to decrease in summer in both treatments and sectors. A positive relationship between leaf nitrogen content, photosynthetic capacity of leaves and the daily integrated quantum flux density was found in spring, but not in summer. The relationship between photosynthetic capacity and gm was curvilinear. Leaf temperature affected also to gm with an optimum temperature at 29 ºC. AS showed larger biochemical limitation than MS in August in both treatments. All this suggested that both diffusional limitation and the effect of leaf temperature could be involved in the seasonal reduction of photosynthetic capacity of olive leaves.

PS8.7 Arabidopsis mutants showing open stomata in the dark J Costa (CEA-Cadarache /CNRS / Aix-Marseille II University), F Monnet (CEA-Cadarache /CNRS / Aix-Marseille II University), I Reiter (CEA-Cadarache /CNRS / Aix-Marseille II University), N Leonhardt (CEA-Cadarache /CNRS / Aix-Marseille II University), B Genty (CEA-Cadarache /CNRS / Aix-Marseille II University) A screen based on thermal imaging was designed to identify Arabidopsis thaliana mutants with open stomata in the dark. Forty dark unresponsive stomata (dus) mutants were isolated among a total of 72,000 EMS-mutageneised Col-0 M2 seeds. The dus mutants were grouped into two categories: one including 8 mutants with growth characteristics closely related to the wild type (WT) and another including mutants with retarded growth and/or deformed or chlorotic leaves. Within the first category, we have found a dominant mutant, the dus16, characterized by the coolest rosette temperature in the dark. Remaining mutants were recessive. The dominant mutant exhibited the highest stomatal conductance and aperture in darkness, about 9.5 and 2.5 times of the WT respectively, while the recessive ones showed an intermediate stomatal phenotype. The physiological characterization of the mutants will be presented, in particular the stomatal response to light, CO2 or ABA. The genetic characterization has revealed that the dus16 is allelic to the open stomata mutant ost2 previously isolated in the Ler background (Merlot et al. 2002). J.M. Costa acknowledges the support of LEM-ITQB-UNL and FCT (ref.POCI2010/SFRH/BPD/ 14498/2003), Portugal. PS8.8 A highly embryogenic line of the legume model Medicago truncatula, M9-10a: performance under water deficit. C Nunes (Faculdade de Ciências, Universidade de Lisboa), S Araújo (Instituto de Tecnologia Química e Biológica), J Marques da Silva (Faculdade de Ciências, Universidade de Lisboa), P Salema Fevereiro (Instituto de Tecnologia Química e Biológica), A Bernardes da Silva (Faculdade de Ciências, Universidade de Lisboa) Drought resistance was analysed in a line of Medicago truncatula, M9-10a, isolated at ITQB Oeiras, Portugal. This highly embryogenic line is being efficiently used for transformation of this model legume, including genetic transformations to improve osmotic stress resistance. In order to permit the future comparison of M9-10a and transgenic lines performance under water deficit conditions, photosynthesis and water relation parameters were analysed in M9-10a adult plants. Assays were performed in 8 week-old plants grown under controlled conditions with adequate water supply or under progressive water deficit imposed by suppression of soil irrigation. A decrease of SWC values from 75% to 15% induced a non-linear decline of leaf RWC values from 75% to 40%. However, only below 30% of SWC did the leaves RWC began to reduce. Gas exchange analyses showed reduced net CO2 assimilation under water deficit. Analyses of A/Ci curves show reduced stomatal conductance, Rubisco carboxilation efficiency and RuBP regeneration indicating that both stomatal and non-stomatal limitations occur under these conditions. In vitro assays are being performed in order to clarify Rubisco efficiency limitations. The A/I curves show that water deficit decreased the apparent quantum yield and the maximum net photosynthetic rate but increased the compensation irradiance. These results are in agreement with chlorophyll a fluorescence parameters. Pressure-volume curve analyses suggest the existence of osmotic adjustment, both osmotic potential at full turgor and at turgor loss point decreased under water deficit. PS8.9

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14th Photosynthesis Congress - PS07 Relationship between mesophyll CO2 gas diffusion conductance and leaf plasma-membrane-type aquaporin contents in tobacco plants grown under drought conditions S Miyazawa (RITE), T Kawasaki (RITE), Y Shinzaki (RITE), M Maeshima (Nagoya Univ.), C Miyake (RITE) CO2 concentration in the chloroplasts (Cc) is often lower than half the ambient CO2 concentration. Such reduction of Cc is attributed to CO2 gas diffusion conductance from stomatal cavities to the chloroplasts (gm). Some recent studies have indicated that plasma-membrane-type aquaporin contents have positive relationships with gm using transgenic plants overexpressing aquaporins. The result suggests that leaf aquaporin content regulates gm. Drought treatment decreases gm, which has been reported for some plant species. However, it is uncertain that leaf aquaporin contents explain the changes in gm. In this study, tobacco (Nicotiana tabacum cv. Xanthi) plants were intermittently irrigated (drought-stressed) while some plants were constantly irrigated (control). gm was estimated from concurrent measurements of leaf CO2 gas exchange rates with chlorophyll fluorescence intensity. Plasma-membrane-type aquaporin was immunochemically quantified with the antibody of radish PIP2-type aquaporin. gm in drought-stressed plants was about one-third that in control. On the other hand, relative aquaporin content on a leaf area basis was about 1.5 times lager in drought-stressed plants than that in control. gm had a negative correlation with the relative leaf aquaporin contents. When leaves were fed with 0.5 mM HgCl2, an inhibitor of aquaporins, from the petioles, gm in control plants was reduced while that in drought-stressed plants was not significantly affected. These results imply that a decrease in a specific activity of aquaporins leads to the reductions of gm in response to drought.

PS8.10 Do homobaric and heterobaric leaves differ in internal lateral CO2 diffusion? T Lawson (University of Essex), G Cornic (Université de Paris XI), J Morision (University of Essex) CO2 can potentially diffuse laterally in leaves, depending on the permeability of the leaf tissue and gradient in photosynthetic activity. Leaves of some species are described as heterobaric, as they have bundle sheaths which extend to the upper and lower epidermis, restricting lateral gas diffusion. In contrast, species lacking such extensions do not have the same physical barrier and might show considerable lateral diffusion. The question is how much CO2 could lateral diffusion supply to the photosynthesizing mesophyll? Chlorophyll a fluorescence imaging shows that when vertical CO2 supply is prevented using grease to block stomata, a range of species do show some lateral CO2 diffusion. The extent of the lateral diffusion depends on photosynthetic consumption along the pathway. Even in transgenic tobacco (homobaric) with reduced Rubisco and therefore reduced CO2 consumption, lateral diffusion is limited to distances of no more than 1-2 mm. Comparing images from nominally homobaric and heterobaric species reveals that there is no simple dichotomy of lateral CO2 diffusion between these leaf types. Calculations of the CO2 assimilation under patches shows that lateral CO2 can contribute significantly to photosynthesis, the amount of which depends upon the degree of air space connectivity. PS8.11 Lateral gas diffusion in the mesophyll can contribute to CO2

fixation in homobaric leaves S Jahnke (Forschungszentrum Jülich GmbH, ICG-3: Phytosphäre, 52425 Jülich, Germany), R Pieruschka (Forschungszentrum Jülich GmbH, ICG-3: Phytosphäre, 52425 Jülich, Germany; Carnegie Institution of Washington, Department of Global Ecology, 94305 Stanford, CA, USA) Lateral CO2 diffusion inside leaves is regarded to play a role on the micrometer scale e.g. between neighbouring stomata. Yet whether this diffusion can span a millimeter scale and affect CO2 assimilation is still under debate. In heterobaric leaves with mesophyll internally compartmented by bundle sheath extensions extended lateral gas diffusion is hindered. On the other hand, homobaric leaves with no (or minor) internal barriers may have adapted to take profit from lateral CO2 diffusion under certain circumstances. When stomatal conductance was reduced by drought stress and homobaric leaves were illuminated with artificial sunflecks (`lightflecks´) we found that (1) quantum efficiency of photosystem II along the boundaries of the lightflecks was higher than in their centres, (2) depending on light intensity laterally diffusing CO2 could affect photosynthesis in illuminated leaf areas over a distance up to 5 mm and (3) leaf areas illuminated with small lightflecks showed higher CO2 assimilation rates than with large lightflecks. The results also revealed that lateral gas diffusion may attenuate light stress and enhance water use efficiency (WUE). Leaf anatomy is species-specific with continuous intergradations of the continuity of the intercellular air space of the mesophyll between hetero- and homobaric leaves resulting in a wide range of lateral gas conductivities as an adaptation to variable environmental conditions. We hypothesize, that homobaric leaf anatomy may be an adaptive trait of plants which evolved in times with low atmospheric CO2 concentration under conditions of temporal water shortage accompanied by light stress.

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PS9 - CO2-Concentrating Mechanisms PS9.1 Inorganic carbon transporters in cyanobacteria. D Price (Australian National University), F Woodger (Australian National University), B Long (Australian National University), M Badger (Australian National University), S Howitt (Australian National University) Cyanobacteria have evolved a remarkable environmental adaptation, known as a CO2 concentrating mechanism (CCM), which vastly improves photosynthetic performance and survival under limiting CO2 concentrations. The CCM functions in the active uptake of inorganic carbon (Ci; HCO3- and CO2) into the cell where the accumulated HCO3pool is utilised to provide elevated CO2 concentrations around the primary CO2 fixing enzyme, ribulose bisphosphate carboxylase-oxygenase (Rubisco), which in turn is encapsulated in unique polyhedral, micro-compartments known as carboxysomes. The cyanobacterial CCM has a basal, constitutively expressed form, and mainly within the b-cyanobacteria (freshwater and estuarine strains), this can be augmented under Ci limitation by the addition of higher affinity Ci uptake systems. There are five known Ci uptake systems, two for CO2 uptake and three for HCO3- uptake, and these will be discussed in more detail. Through database analysis of some 35 complete genomic DNA sequences for cyanobacteria it is evident that considerable diversity exits in the composition of transporters employed, though in many species this diversity is yet to be confirmed by comparative physiology. There has been progress towards identifying the primary signal for transcriptional induction of the subset of CCM genes known as CO2-reponsive genes (mainly high affinity Ci uptake systems) and the importance of the transcriptional regulators CcmR (NdhR) and CmpR in the regulation of as CO2-reponsive genes will be highlighted. Finally, some prospects for introducing cyanobacterial CCM components into higher plants are considered, with the objective of engineering plants that make more efficient use of water and nitrogen.

PS9.2 Inorganic carbon transporters in the CO2-concentrating mechanism of Chlamydomonas reinhardtii. M Spalding (Iowa State University), Y Wang (Iowa State University), D Duanmu (Iowa State University) Aquatic photosynthetic microorganisms, including microalgae, have adapted to the variable and often-limiting availability of CO2, and inorganic carbon (Ci) in general, by development of inducible CO2 concentrating mechanisms (CCMs) that allow them to optimize carbon acquisition. In spite of their central role in CCM function, Ci transport systems have not been identified in microalgae. However, significant advances in understanding the Chlamydomonas reinhardtii CCM have occurred with the aid of mutational approaches and the availability of The C. reinhardtii genome sequence. As a result of these advances, an integrated picture of the functional components of the microalgal CCMs is beginning to emerge, including the identification of Ci transport candidates that might function at either the chloroplast envelope or the plasma membrane. One advance occurred with the identification of the defective gene in the Ci-transport defective mutant pmp1 as the previously identified limiting-Ci responsive gene LCIB. The unique “air-dier” phenotype, growth in high CO2 (>0.5%) and very low CO2 (<0.02%) but not in low CO2 (~0.03%), suggests that LCIB-dependent Ci transport is required specifically in low CO2. LCIB and three related

genes in C. reinhardtii compose a unique gene family that encode four closely related, apparently soluble plastid proteins with no clearly identifiable conserved motifs, making the specific role of LCIB in Ci transport unclear. Other Ci transport candidates, including HLA3/MRP1, LCI1, LCIA, CCP1 and CCP2, have varying levels of support for a potential role in Ci transport, although none have yet been definitively identified as functional Ci transporters in C. reinhardtii.

PS9.3 Progress in elucidating the structural basis of function in the carboxysome C Kerfeld (US Department of Energy), G Cannon (USM), S Heinhorst (USM), T Yeates (UCLA), M Sawaya (UCLA), F Cai (USM), S Tanaka (UCLA), Y Tsai (UCLA) Cyanobacteria and many chemoautotrophic microbes have evolved a special mechanism for dramatically enhancing CO2 fixation. In these organisms, tens of thousands of molecules of RuBisCO are packaged inside subcellular microcompartments called the carboxysome. The outer shell of the carboxysome resembles a viral capsid, being assembled from many copies of nearly identical small protein subunits. By sequestering RuBisCO together with carbonic anhydrase activity, the carboxysome provides an environment for enhanced CO2 fixation. The mechanistic details of how the carboxysome works are just beginning to emerge. Ongoing crystallographic studies on carboxysomes found in cyanobacteria and chemoautotrophs provide the first structural insights into carboxysome assembly and function, leading to new insights into how the carboxysome shell is assembled and how it serves to enhance RuBisCO activity by allowing selective transport of metabolites such as bicarbonate across the shell. The carboxysome is the best studied bacterial microcompartment, as such it provides general insights into a surprisingly widespread strategy for biological compartmentalization.

PS9.4 Knocking out three potential routes for photorespiratory glycolate turnover generates a high CO2-requiring phenotype in the cyanobacterium Synechocystis sp. PCC 6803. M Eisenhut (University of Rostock), A Kaplan (The Hebrew University of Jerusalem), H Matthijs (University of Amsterdam), H Bauwe (University of Rostock), M Hagemann (University of Rostock) Cyanobacteria are known to posses a carbon concentrating mechanism (CCM) leading to high Ci concentration in the vicinity of RubisCO, which is thought to make oxygenase activity of RubisCO in cyanobacteria unlikely. Accordingly, it is widely accepted that cyanobacteria do not perform photorespiratory glycolate turnover. Bioinformatic analyses showed the occurrence of genes for all enzymatic steps necessary for phosphoglycolate turnover in the genome of Synechocystis sp. PCC 6803 and other cyanobacterial strains. A Synechocystis-mutant impaired in glycolate dehydrogenase accumulated the characteristic photorespiratory intermediate glycolate even under high Ci conditions (5% CO2). After detailed analyses using mutants impaired in central steps of possible glycolate turnover routes identified by bioinfomatics and genome-wide DNA-microarray experiments we could postulate three different pathways of metabolizing glycolate. 1. Glycolate is degraded by a plant-like C2 cycle. 2. A route seems to exist corresponding to the bacterial glycerate pathway. 3. Additionally, there are indications for a complete decarboxylation of glycolate via oxalate and formate. A triple mutant defective in all three postulated routes

221 14th Photosynthesis Congress - PS07 showed a high-CO2-requiring phenotype as it is known from photorespiratory mutants in higher plants. Altogether, the results clearly show the occurrence of a photorespiratory glycolate metabolism in cyanobacterial cells despite of the CCM. However, compared to plants it is composed by at least three cooperating biochemical pathways. PS9.5 Relocation of photorespiratory CO2 release mesophyll to the bundle sheath of a C3 plant

from

the

N Engel (University of Rostock), N Qu (University of Rostock), H Bauwe (University of Rostock) Photorespiration was one of the selective pressures that resulted in the evolution of C4 photosynthesis. As inferred from the analysis of C3-C4 intermediate plants, the relocation of photorespiratory CO2 release by glycine decarboxylase (GDC) has been an early and very important event in this evolutionary multi-step process. We want to construct an artificial 'C3' plant where glycine is not decarboxylated in the mesophyll (the original C3 location), but only in the bundle-sheath (corresponding to the C3-C4 and C4 situation). To this end, we have isolated Arabidopsis GDC null and knockdown mutants and are now complementing these plants with GDC genes from a C3-C4 intermediate plant. In short, a complete knockout of GDC activity is lethal even under 0.9% CO2, indicating a non-replaceable function of GDC in vital non-photorespiratory processes (one-carbon metabolism). In contrast, partial knockout of GDC activity results in features as they are also typical for other photorespiratory mutants. Full complementation of one of these GDC mutants was achieved by overexpression of the corresponding bundle-sheath specific gene from a C3-C4 intermediate plant. Features of these non-complemented and complemented mutants will be described and discussed. PS9.6 Suppressors of pmp1/ad1 in Chlamydomonas reinhardtii M Spalding (Iowa State University), D Duanmu (Iowa State University), D Duanmu (Iowa State University) An active CO2 concentrating mechanism (CCM) is induced when Chlamydomonas reinhardtii acclimates to limiting Ci, either low CO2 (air level; ~0.04% CO2) or very low CO2 (~0.01% CO2) conditions. Allelic mutants pmp1 and ad1, which are defective in the limiting-CO2-inducible, plastid localized LCIB, can grow in high CO2 or very low CO2 conditions but die in low CO2, indicating a deficiency in a low-CO2-specific Ci transport system. To further understand the functions and regulation of LCIB, we used insertional mutagenesis to identify suppressors of pmp1 and ad1 that can grow in low CO2. Four independent suppressors (3 for pmp1 and 1 for ad1) were selected. Genetic analysis of 3 pmp1 suppressors identified 2 allelic, dominant suppressors (su4, su5), and one recessive suppressor (su1). Consistent with the suppression phenotype, the relative affinity of photosynthetic O2 evolution and the direct Ci uptake capacity in all 3 suppressor lines was substantially increased relative to pmp1 in low-CO2 acclimated cells. Interestingly, the relative affinity of su4/su5 was intermediate between su1 and pmp1. In contrast, the ad1 suppressor (sux) can grow well in low CO2 but not in very low CO2 conditions, indicating the suppressor mutation in this case could be an essential component involved in the CCM. The separation of at least three different pmp1/ad1 suppressors could suggest complex functions and regulation of LCIB. Identification of these suppressor genes could provide more clues about the roles played by LCIB in limiting Ci acclimation.

PS9.7 ATPase - Photosynthesis and Nitrogen N Chowhuri (SEN) The term ATpase will denote the breakdown of ATP into ADP and Pi under the formation of high energy state. I have studied comprehensively cultivation of micro-algae with various carbon and nitrogen sources under artificial conditions showing carbon control of nitrogen assimilation in photosynthesis. Photosynthesis is an oxidation-reduction reaction in which there is subtraction and addition of electron and there is a different kind of reaction, acid-base transition, in which there is subtration and addition of hydrogen ion. Acid-base transition drives such things as activation of ATpase which hydrolyzes ATP into ADP and Pi releasing energy, the energy released is utilized in the transformation of NAD into NADP. NADP is in oxidized state. Only the reduced pyridine linked enzyme is effective in light dependent assimilation of carbon-dioxide and this enzyme cannot cross the membrane but can be indirectly translocated via metabolite shuttle. PS9.8 Suppressors of pmp1/ad1 in Chlamydomonas reinhardtii D Duanmu (Iowa State University), M Spalding (Iowa State University) An active CO2 concentrating mechanism (CCM) is induced when Chlamydomonas reinhardtii acclimates to limiting Ci, either low CO2 (air level; ~0.04% CO2) or very low CO2 (~0.01% CO2) conditions. Allelic mutants pmp1 and ad1, which are defective in the limiting-CO2-inducible, plastid localized LCIB, can grow in high CO2 or very low CO2 conditions but die in low CO2, indicating a deficiency in a low-CO2-specific Ci transport system. To further understand the functions and regulation of LCIB, we used insertional mutagenesis to identify suppressors of pmp1 and ad1 that can grow in low CO2. Four independent suppressors (3 for pmp1 and 1 for ad1) were selected. Genetic analysis of 3 pmp1 suppressors identified 2 allelic, dominant suppressors (su4, su5), and one recessive suppressor (su1). Consistent with the suppression phenotype, the relative affinity of photosynthetic O2 evolution and the direct Ci uptake capacity in all 3 suppressor lines was substantially increased relative to pmp1 in low-CO2 acclimated cells. Interestingly, the relative affinity of su4/su5 was intermediate between su1 and pmp1. In contrast, the ad1 suppressor (sux) can grow well in low CO2 but not in very low CO2 conditions, indicating the suppressor mutation in this case could be an essential component involved in the CCM. The separation of at least three different pmp1/ad1 suppressors could suggest complex functions and regulation of LCIB. Identification of these suppressor genes could provide more clues about the roles played by LCIB in limiting Ci acclimation. PS9.9 LCIB functions in a multi-subunit complex essential for inorganic carbon transport in Chlamydomonas reinhardtii Y Wang (Iowa State University), M Spalding (Iowa State University) Although substantial evidence has demonstrated the existence of active inorganic carbon (Ci) transport systems in eukaryotic microalgae, only very few candidate Ci transport genes have been identified. The Chlamydomonas reinhardtii pmp1 mutant is the only mutant identified so far with a lesion directly affecting active Ci transport in eukaryotic microalgae. Recently the gene defective in pmp1 was identified by genetic characterization of its allelic insertional mutant, ad1. The PMP1/AD1 protein is encoded by LCIB, a gene previously identified as

222 14th Photosynthesis Congress - PS07 a limiting-CO2 responsive gene. Blast searches with LCIB revealed three additional homologues in the C. reinhardtii genome, LCIC, LCID and LCIE. LCIB and LCIC show similar expression patterns: very low expression in high CO2 (5%) and substantial increased expression induced by a wide range of limiting CO2 (0.15%-0.01%). LCID shows a similar CO2 expression pattern but at a much lower apparent mRNA abundance, while expression of LCIE so far has been confirmed only by a partial cDNA. Immunofluorescence examination indicated possible plastid localization of LCIB/LCIC, which is consistent with the existence of plastid signal peptides in these proteins. Western blots revealed that LCIC, even though actively transcribed, is down-regulated at the protein level by the LCIB lesion in ad1 and pmp1. Screening of LCIB-interacting proteins with the yeast two-hybrid system revealed a LCIB-LCIC interaction. We propose that LCIB is an integral component of a multi-subunit transport complex and that its role in Ci transport function relies on its interaction with other proteins, including LCIC. PS9.10 Paralogous evolution of a family of transcriptional regulators involved in inorganic carbon assimilation. S Daley (Oklahoma State University), R Burnap (Oklahoma State University) Paralogous regulators arise from gene duplication events allowing for divergence of function in one member while maintaining the original function of the other. There are 6 LysR-Type Transcriptional Regulators within Synechocystis sp. PCC6803; which appear to be the result of 5 separate gene duplication events. Two of three closely related members (sll1594, sll0030) from one duplication series control the high-affinity/low-flux inorganic carbon acquisition system, while the other (sll0998) controls an unknown but essential system, perhaps also involving carbon. Experiments aimed at identifying the ligand molecule(s) using electrophoretic mobility shift assays will be reported. Once identified the ligand molecule(s) will undergo biophysical analysis to determine the binding energies of the proteins interactions with both the DNA and ligand molecule, which will be assayed using isothermal titration calorimetry. Utilizing a bioinformatic analysis approach, a putative binding site in all three regulators has been located and mapped onto a 3-D model constructed from the known crystal structure (1IXC) of a homologous regulator. This mapping/analysis has allowed for the identification of residues located in both the DNA binding domain and regulatory domains, each of which shows conservation among the known inorganic carbon acquisition regulators (sll1594 & sll0030) and the as yet undefined sll0998. Patterns of conservation and divergence may indicate regulatory/functional differences among the different family members and have implications for the complex regulatory networks involved in inorganic/organic carbon metabolism. PS9.11 Properties of photosynthetic CO2 uptake in protoplasts isolated from thallus of green alga (Ulva pertusa) C Goh (Pusan National University), H Kim (Pusan National University), Y Eu (Pusan National University), T Han (University of Incheon), C Lee (Pusan National University) To investigate carbon accumulation mechanisms in green alga (Ulva pertusa), we enzymatically isolated the protoplasts from the thallus tissues, which have carbon contents of 0.26g/dry wtg. The enzyme solution (pH 5.5) includes 0.5% macerozyme, 1.5% cellulose RS, 0.1% pectolyase, 0.5% BSA, 0.4 M NaCl, 0.5 M sorbitol, 1 mM CaCl2, and 1 mmol/ml b-mercaptoethanol. The isolated protoplasts (99.6% purity) were 1776 mg protein/ml. From the intact tissues, the saturation of electron transport rate (ETR) was achieved at 180 mmol m-2s-1 (the half

saturation was 60 mmol m-2s-1) and declined the activity at 700 mmol m-2s-1, showing photoinhibition. The photosystem II (PS II) quantum yield, ETR, non-photochemical quenching (NPQ) were developed by dark-light transition and recovered by dark. The isolated protoplasts in 0.14 mM Mes (pH 6.0), 0.5 M sorbitol, 1 mM CaCl2, and 0. 4 M NaCl showed the similar quenching patterns as measured by a MICROSCOPY PAM. In the absence of NaCl, the PS II quantum yield of protoplasts were completely inhibited, indicating a direct involvement of Na+ for photosynthetic activity. In relation to this, we provide the properties of carbon uptake by measuring medium pH changes in blue and red light conditions. We will discuss that K+ affects the rate of carbon uptake into the algal cells as well. PS9.12 Identification of two novel genes required for growth under low CO2 conditions in Chlamydomonas reinhardtii J Moroney (Louisiana State University), R Ynalvez (Louisiana State University), S Pollock (Louisiana State University), K Cunnusamy (Louisiana State University) Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) that allows the alga to grow at very low CO2 concentrations. In order to identify genes required for a functional CCM, an extensive screening of C. reinhardtii insertional mutants was carried out. The strain D66 cw15 nit2 mt+ was transformed by electroporation with the linearized plasmid, pSP124S, which has a gene that confers resistance to Zeocin (a Bleomycin-related antibiotic). Transformants were first identified by their resistance to Zeocin and then they were screened for growth on minimal medium under high (5% CO2 in air) and low (0.01% CO2 in air) CO2 conditions. The location of the DNA insertion was then established using inverse PCR and crosses were done to determine whether the DNA insert was genetically linked to the poor growth on low CO2 phenotype. Using these methods, we have identified that two novel genes, namely CIA6 and CIA7, are required for optimal growth on low CO2 in C. reinhardtii. In both cases, disruption of the gene results in a strain that grows slowly on low CO2 and exhibits a reduced affinity for inorganic carbon. While the function of the proteins encoded by these genes is unknown, CIA6 contains a SET domain, while CIA7 shows similarity to metal binding proteins. In this presentation we will describe the molecular characterization of these genes and the physiology of the transformants. Supported by NSF Grant IOB-0516810 to JVM. PS9.13 Single-cell C4 photosynthesis in marine diatoms? R Leegood (University of Sheffield), K Roberts (University of Dundee), E Granum (University of Sheffield), J Raven (University of Dundee) Marine planktonic diatoms are responsible for 15% of primary productivity on Earth, fixing more than 10 billion tonnes of inorganic carbon (Ci) each year. Diatoms achieve this by using CO2-concentrating mechanisms (CCMs) which increase the CO2 concentration around Rubisco, diminishing photorespiration. It was generally held that diatoms have biophysical CCMs, based on direct acquisition of Ci, until evidence emerged of single-cell C4 photosynthesis, a biochemical CCM, in the marine diatom Thalassiosira weissflogii. Recent whole genome sequencing of marine phytoplankton, including the diatoms T. pseudonana and Phaeodactylum tricornutum, suggests that these organisms possess the enzymic apparatus (including phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase) to operate C4 photosynthesis. We studied short-term

223 14th Photosynthesis Congress - PS07 photosynthetic carbon fixation by 14C labelling in two diatoms, and found that T. weissflogii produced both C3 and C4 (mainly malate) compounds, while T. pseudonana produced only C3 compounds. Labelling patterns in neither diatom were altered by growth at different Ci concentrations. This lack of environmental modulation was supported by measurements (in T. pseudonana only) of metabolic gene transcripts and proteins. Growth Ci concentration had little effect on the expression of any of the genes studied except glycine decarboxylase P-protein, which was highly upregulated by low Ci, indicating induction of photorespiratory carbon oxidation. Moderately nitrogen-starved cells had reduced carbon metabolic transcript levels, but C3 and C4 carboxylase/decarboxylase gene transcripts increased rapidly when nitrate was added. The evidence from this study suggests C3-C4 intermediate photosynthesis in T. weissflogii and exclusively C3 photosynthesis in T. pseudonana, in which C4 carboxylases play key anaplerotic roles. PS9.14 A structural role for CcmM in ß-carboxysome shell formation. B Long (Research School of Biological Sciences, The Australian National University), M Badger (Research School of Biological Sciences, The Australian National University), S Whitney (Research School of Biological Sciences, The Australian National University), D Price (Research School of Biological Sciences, The Australian National University) In cyanobacteria the key enzyme for photosynthetic CO2-fixation, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is bound within proteinaceous polyhedral bodies called carboxysomes. Cyanobacteria with Form IB Rubisco produce β-carboxysomes whose putative shell proteins are encoded by the ccm-type genes. To date, very little is known of the protein-protein interactions which form the basis of β-carboxysome structure. By creating 6×His-tagged constructs of both the N and C termini of CcmM we have identified a number of carboxysomal sub-complexes which we speculate are associated with the outer shell. Notably, CcmM has been consistently observed as two forms such that N and C terminal tagging gave rise to variably tagged protein products. From these we have identified two independent CcmM complexes containing Rubisco, one of which also contains the carboxysomal carbonic anhydrase CcaA. The complex containing CcaA consists of Rubisco and the full-length 58 kDa form of CcmM (CcmM58), while the other is made up of Rubisco and a short 35 kDa form of CcmM (CcmM35) which is probably translated independently of CcmM58 via an internal ribosomal entry site within the ccmM gene. While independent L8S8 Rubisco is confined to the centre of the carboxysome, we speculate that the CcmM:CcaA:Rubisco complexes form an important assembly co-ordination within the carboxysome shell. PS9.15 Expression of inducible inorganic carbon acquisition complexes is under the control of the FtsH protease in Synechocystis sp. PCC 6803 P Zhang (Department of Biology, University of Turku), C Sicora (Department of Biology, University of Turku), N Vorontsova (Department of Biology, University of Turku), Y Allahverdiyeva (Department of Biology, University of Turku), N Battchikova (Department of Biology, University of Turku), P Nixon (Department of Biological Sciences, Imperial College London), E Aro (Department of Biology, University of Turku) Cyanobacteria possess a complex CO2-concentrating mechanism (CCM), which makes them survive and acclimate in a variety of CO2 environmental conditions. The efficiency of CCM is enhanced under low

inorganic carbon environments by expression of a set of protein complexes. To investigate the involvement of proteases in the processes of induction and degradation of the CCM complexes, we studied the FtsH2 (?Slr0228) and Deg-G (?Slr1204/?Sll1679/?Sll1427) protease mutants of Synechocystis sp. PCC 6803. WT and protease mutant cells were grown under high CO2 and then shifted to low CO2, followed by a proteome analysis of the membrane protein complexes. Interestingly, in the FtsH2 protease mutant, inducible CCM complexes were not detected upon shift to low CO2, whereas the Deg-G mutant behaved like WT. Also the transcripts of the inducible CCM genes and their regulator ndhR failed to accumulate upon shift of FtsH2 mutant cells from high to low CO2, indicating that the regulation by the FtsH2 protease is upstream of NdhR. Moreover, functional photosynthesis was shown a prerequisite for induction of CCM in WT at low CO2, possibly via generation of oxidative stress, which was shown here to enhance the expression of inducible CCM genes transiently even at high CO2 conditions. The CCM complexes were not subject to proteolytic degradation once synthesized, even when dispensable upon a shift of cells to high CO2.

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PS10 - CAM and C4 PS10.1 Post-transcriptional control of bundle sheath cell-specific Rubisco gene expression

Chromatin modifications regulate light inducibility and tissue specificity C Peterhaensel (RWTH Aachen University)

J Berry (University of Buffalo), M Patel (University of Buffalo) In leaves of most C4 plants, ribulose 1,5 bisphosphate carboxylase (Rubisco) accumulates only in bundle sheath (bs) cells that surround the vascular centers, and not in mesophyll (mp) cells. We have shown previously that control of mRNA translation and stability mediate the C4 expression patterns of genes encoding the large and small Rubisco subunits (chloroplast rbcL and nuclear RbcS, respectively). The current focus of our research is the identification and characterization of mRNA/protein interactions associated with Rubisco gene expression in C4 plants. We hypothesize that many regulatory systems occur in both C3 and C4 plants, some of which must have diverged between these two groups, through modification of pre-existing factors, or by acquisition of novel processes not present in C3 species. In support of this hypothesis we have recently identified plastid-localized mRNA binding activities specific to the 5’ UTR of rbcL mRNA, with properties that correlate with C4 Rubisco gene expression. Binding to rbcL mRNA occurs only in light, when rbcL is expressed, and at least one rbcL mRNA binding protein, p44, is present primarily in bs cell chloroplasts, where Rubisco is specifically localized in C4 leaves. In addition, analysis of Arabidopsis insertion mutants indicates that p44 is essential for the synthesis/accumulation of both Rubisco subunits in this C3 plant. The occurrence and conservation of orthologs of this protein in C4 dicots (Flaveria, Amaranth), a C4 monocot (maize), and a C3 dicot (Arabidopsis) suggests an important regulatory role in many plants, with possibly modified function in C4 species.

PS10.2 Dissecting the metabolic drivers and consequences of Crassulacean acid metabolism A Borland (University of Newcastle Upon Tyne) The photosynthetic specialization of Crassulacean acid metabolism (CAM) encompasses a complex of metabolic traits that conserve CO2 and/or water in environments that typically restrict the availability of either or both resources. By shifting part or all of net CO2 uptake to the dark period, CAM has metabolic consequences that include reciprocal day/night cycling of organic acids and carbohydrates and significant changes in the internal concentration of CO2 and O2 over the day/night cycle. The convergent evolution of CAM raises important questions regarding; 1) the extent to which general stress-responsive aspects of C3 metabolism have been recruited as part of this photosynthetic adaptation to water/CO2 limited environments and 2) which metabolic traits are a consequence of the profound day/night changes in metabolite cycling and internal gaseous composition. This paper will consider how the isolation of various CAM-deficient mutants of Mesembryanthemum crystallinum and the existence of drought-tolerant C3 and CAM species of the tropical tree genus Clusia, provide the means for tackling such questions.

PS10.3 The control of C4-specific gene expression in maize:

C4-specific gene expression in maize is regulated by light, the availability of nutrients, and leaf tissue differentiation. We are interested in the contribution of chromatin modifications to the integration of these diverse signals into a single promoter response. We analysed histone acetylation, a modification typically associated with active genes, on the C4-specific phosphoenolyruvate carboxylase (C4-PEPC) gene. Nucleosomes in both the promoter and the coding region were heavily acetylated upon illumination of etiolated leaves. Surprisingly, light-dependent hyperacetylation was also observed in bundle sheath cells, although the C4-PEPC promoter is inactive in this tissue. Furthermore, the promoter could be downregulated by nitrogen depletion in the light without any decrease in hyperacetylation. Determination of the modification state of seven individual acetylation sites on histone H3 and H4 revealed a complex pattern of leaf-specific and light-induced modifications. Light activation of transcription did not impact on the methylation state of lysine 4 on histone H3 (H3K4), although tri-methlyation at this position has been identified as one of the key marks of gene activation in genome-wide studies before. However, tri-methylation of H3K4 was clearly enriched on C4-PEPC chromatin isolated from mesophyll cells compared to bundle sheath cells where di-methylation was most abundant. The bundle sheath-specific C4-malic enzyme gene showed a reciprocal pattern. We suggest a model where individual stimuli induce distinct histone modification patterns that collectively regulate the activity of C4 promoters.

PS10.4 Phosphorylation of phosphoenolpyruvate carboxylase is not essential for high photosynthetic rates in the C4 species Flaveria bidentis. S von Caemmerer (Australian National University), T Furumoto (Hiroshima University), K Izui (Kinki University), V Quinn (Australian National University), R Furbank (CSIRO) Phosphoenolpyruvate carboxylase (EC4.1.1.31; PEPC) plays a key role during C4 photosynthesis. The enzyme is activated by metabolites such as glucose 6-phosphate and inhibited by malate. This metabolite sensitivity is modulated by the reversible phosphorylation of a conserved serine residue near the N-terminus in response to light. The phosphorylation of PEPC is modulated by a protein kinase specific to PEPC (PEPC-PK). To explore the role PEPC-PK plays in the regulation of C4 photosynthetic CO2 fixation we have transformed Flaveria bidentis (L.) Kuntze (a C4 dicot) with antisense or RNAi constructs targeted at the mRNA of this PEPC-PK. We generated several independent transgenic lines where PEPC is not phosphorylated in the light, demonstrating that this PEPC-PK is essential for the phosophorylation of PEPC in vivo. Malate sensitivity of PEPC extracted from these transgenic lines in the light was similar to the malate sensitivity of PEPC extracted from darkened wild type leaves but greater than the malate sensitivity observed in PEPC extracted from wild type leaves in the light, confirming the link between PEPC phosphorylation and the degree of malate inhibition. There were, however, no differences in the CO2 and light response of CO2 assimilation rates between wild type plants and transgenic plants with low PEPC phosphorylation showing that

225 14th Photosynthesis Congress - PS07 phosphorylation of PEPC in the light is not essential for efficient C4 photosynthesis for plant grown under standard glass house conditions. This raises the intriguing question of what role this complexly regulated reversible phosphorylation of PEPC plays in C4 photosynthesis.

PS10.5 An increase in expression of Pyruvate Pi Dikinase and its high activation energy correspond to cold-tolerant C4 photosynthesis of Miscanthus x giganteus D Wang (University of Illinois), A Portis (USDA-ARS), S Moose (University of Illinois), S Long (University of Illinois) Miscanthus x giganteus is exceptional among C4 plants in its ability to produce leaves and photosynthesize at low temperature. While the most cold-adapted Zea mays lines show loss of photosynthetic capacity when transferred to 14 oC, M. x giganteus shows no loss and can continue photosynthesis down to 8 oC. Understanding how this is achieved is critical to adapting Z. mays and other C4 crops to colder climates or allowing them to make use of solar radiation over a longer period of the year. Transgenic analysis of C4 plants has shown that two enzymes share control and are limiting to the light-saturated rate of photosynthesis: Rubisco and Pyruvate Pi Dikinase (PPDK). Previous studies have shown that PPDK is cold labile while others have shown that Rubisco activity at low temperature is likely to constrain photosynthetic rate. Analysis of the sequences for the genes coding for these enzymes in the two species has failed to reveal any differences likely to alter cold-tolerance. Kinetic analysis of the purified enzymes has similarly failed to identify any major differences. However, when Z. mays and M x giganteus are transferred from 25oC to 14oC, both light-saturated photosynthetic rate (Asat) and maximum quantum yield (F) declined by 60% in both species in the first 24-48 h. In Z. mays it continued to decline in these existing leaves over the next 7 days, but recovered in M. x giganteus. What changes underlie this difference? Within 24-hr of the temperature transition after a transient decline the PPDK protein content was seen in M x giganteus but then steadily increased a few fold over the next days while it declined in the Z. mays leaves. Rubisco levels remained constant in M. x giganteus, but declined slightly in Z. mays. RT-PCR showed that the increase in PPDK protein in the M. x giganteus leaves corresponded to a large increase in mRNA level for PPDK. The results suggest that of the two enzymes know to limit C4 photosynthesis, increase of PPDK and not Rubisco corresponds to the recovery of photosynthetic capacity during low temperature exposure of M. x giganteus. The results suggest a low temperature induction of either transcription of the PPDK gene or inhibition of breakdown of this message in M. x giganteus, but not Z. mays. PS10.6 STUDY OF THE STRUCTURE- FUNCTION RELATIONSHIP IN MAIZE PHOTOSYNTHETIC NADP- MALIC ENZYME C Andreo (CEFOBI-UNR), E Detarsio (CEFOBI-UNR), C Alvarez (CEFOBI-UNR), M Saigo (CEFOBI-UNR), M Drincovich (CEFOBI-UNR) NADP-malic enzyme catalyses the reversible oxidative decarboxylation of L-malate to yield carbon dioxide and pyruvate with the concomitant reduction of NADP. Maize presents at least three isoforms of this enzyme, one of them, the “photosynthetic isoform” is responsible for providing CO2 to the Calvin Cycle for carbon fixation. We studied different kinetic and structural aspects of this enzyme by recombinant protein engineering. Site directed mutagenesis showed that the residues A392, A387 and K435/6 contribute to the specificity of NADP over NAD as substrate of the enzyme and that the conserved basic residues K255 y R237 are implicated in substrate binding and catalysis, probably

acting as a base, accepting a proton in the malate oxidation step. The structural basis of the differences between the photosynthetic and non-photosynthetic isoforms were studied by constructing and analyzing chimeras between these isoforms. Their analysis showed that the region between residues 102 and 247 of the photosynthetic isoform is responsible for its tetramerization capacity, and the region between residues 248 and C-terminus, for the malate inhibition phenomena. This photosynthetic isoform is activated by the reduction of intramolecular disulfide bonds. The role of conserved cysteine residues, analyzed by mutagenesis, indicated that cys 192, but not cys 232, is necessary for the catalytic reaction, however, both affect the affinity for the substrate malate. PS10.7 Functional signficance of C3-C4 intermediate traits in Heliotropium L. (Boraginaceae): gas exchange perspectives P Vogan (University of Toronto), M Frohlich (Natural History Museum, London), R Sage (University of Toronto) C3-C4 intermediate traits have been identified and described in over thirty species from twelve genera. Here, we demonstrate for the first time the presence of species exhibiting C3-C4 intermediacy in Heliotropium, a genus with over 100 C3 and 150 C4 species. CO2 compensation points (G) and photosynthetic water-use efficiencies (WUE) were intermediate between C3 and C4 values in three species of Heliotropium: H. convolvulaceum, H. racemosum, and H. greggii. We also determined that H. procumbens is a weak C3-C4 intermediate based on a slight reduction in G compared to C3 Heliotropium species. The intermediate species H. convolvulaceum, H. greggii and H. racemosum exhibited over 50% enhancement of net CO2 assimilation rates at sub-ambient CO2 levels due to a reduction in photorespiration; however, no difference in stomatal conductance between C3 and C3-C4 species was observed. We also assessed the response of ? to O2 concentration for these species. The three strongly intermediate species exhibited similar curvilinear responses of G to O2, characteristic of C3-C4 photosynthesis. Recent phylogenies indicate that H. convolvulaceum, H. greggii and H. racemosum attach above the C3 species in Heliotropium section Orthostachys and at the base of the clade(s) containing C4 species. Thus, the hypothesis that the photosynthetic characteristics in these three species represent true evolutionary intermediacy is supported. These results establish Heliotropium as a valuable new system for studying the evolutionary transition from C3 to C4 photosynthesis, and may also be employed to introduce C4 anatomical and biochemical traits into important C3 crops such as rice.

PS10.8 The Identification of C3-C4 intermediates in the genus Heliotropium section Orthostachys (Boreginaceae) R Sage (Department of Ecology and Evolutionary Biology, University of Toronto), R Muhaidat (University of Toronto), M Frohlich (The British Museum of Natural History), N Dengler (University of Toronto) Both C3 and C4 photosynthesis occur in Heliotropium section Orthostachys. Here, we identify four C3-C4 intermediate species and two C3 species with Kranz-like characteristics in Heliotropium section Orthostachys. Ten species were investigated for C3-C4 intermediacy in Heliotropium using anatomical studies, activities of PEP carboxylase (PEPC) and C4-acid decarboxylases, and the intercellular distribution of Rubisco and the P-subunit of glycine decarboxylase (GDC). All species are in section Orthostachys, except H. europaeum of section Heliotropium. Heliotropium europaeum, H. calcicola, H. tenellum, and

226 14th Photosynthesis Congress - PS07 H. karwinskyi are C3 plants, while H. texanum and H. polyphyllum are fully-developed C4 species. The rest are C3-C4 intermediates, with weakly-developed Kranz anatomy in H. procumbens, and Kranz anatomy in H. convolvulaceum, H. racemosum and H. greggii. A quantitative anatomical study showed an increase in BS area and decrease in the M to BS area ratio in the four intermediate species, and the two C3 species (H. tenellum, H. karwinskyi) that are most closely related to the C4 species. In the C3-C4 H. convolvulaceum and H. greggii, labeling for Rubisco occurred in both M and BS cells, but was more pronounced in BS cells. Specific labeling for the P-subunit of GDC occurred in all photosynthetic cells of the C3 species, but exclusively in BS cells of the C3-C4 and C4 species. The C3-C4 intermediates had elevated PEPC activities compared to C3 species, but PEPC activities were much lower in the intermediates than in C4 species. These results indicate that photosynthetic efficiency is enhanced in the Heliotropium intermediates by the concentration of photorespired CO2 into the BS cells via a glycine shuttle. The presence of enlarged bundle sheath cells in close C3 relatives of the intermediates supports a hypothesis that bundle sheath enlargement is the critical initial phase in the evolution of C4 photosynthesis in Heliotropium.

PS10.9 A novel phosphoenolpyruvate carboxylase targeted to the chloroplast of rice C Masumoto (Natl. Inst. Agrobiol. Sci.), H Ohkawa (Hirosaki Univ.)), Y Taniguchi (Natl. Inst. Agrobiol. Sci.)), T Fukuda (Natl. Inst. Agrobiol. Sci.)), H Fukayama (Kobe Univ.)), M Miyao (Natl. Inst. Agrobiol. Sci.) Phosphoenolpyruvate carboxylase (PEPC) is known as a cytosolic enzyme, which catalyzes the CO2 fixation in C4 and CAM photosynthesis and has an anaplerotic function of replenishing the TCA cycle with intermediates in C3 plant leaves. Database search identified six PEPC genes in the rice genome: five encode the plant-type PEPC that has the conserved phosphorylatable serine residue (Osppc1, 2a, 2b, 3, 4) and one encodes the bacterial-type (Osppcb). All these genes are expressed in rice plants, though expression of Osppc3 and Osppcb was very low in all organs tested. One plant-type gene Osppc4 has a unique feature, an extension at the N terminus in its deduced amino acid sequence. If this extension is neglected, the encoded protein shows high similarity to other plant-type PEPCs, though it does not belong to any of C4, C3 and root types in the phylogenetic tree of PEPC proteins of Gramineae. A fusion protein of the N-terminal extension of Osppc4 with GFP expressed in dayflower epidermal cell was targeted to chloroplasts, suggesting that Osppc4 protein is a chloroplast-localized PEPC. RT-PCR analyses showed that Osppc4 is highly expressed in green organs, especially in leaf blade and leaf sheath, and promoter:GUS analyses indicated that it is specifically expressed in the mesophyll cells of these organs. Recombinant Osppc4 protein showed enzyme characteristics typical of plant-type PEPCs. Until now, genes for the chloroplastic PEPC are identified only in the genus Oryza. These results suggest that Oryza has a unique metabolism involving PEPC inside the chloroplast. PS10.10 Drought constraints on C4 photosynthesis: stomatal and metabolic limitations in C3 and C4 grasses. B Ripley (Rhodes University), M Gilbert (Rhodes University), D Ibrahim (Sheffield University), K Frole (Rhodes University), C Osborne (Sheffield University)

Grasses with the C4 photosynthetic pathway use water more efficiently than those with the C3 type, yet biogeographic analyses show a decline in the relative abundance of C4 to C3 species with decreasing rainfall. To investigate this paradox, we tested the hypothesis that the advantage of C4 over C3 photosynthesis is diminished by drought, and determined the stomatal and metabolic mechanisms underlying this response. We examined the effects of drought on C3 and C4 subspecies of the grass Alloteropsis semialata using a common garden experiment. Under mesic conditions, photosynthetic rates were significantly higher in the C4 than the C3 subspecies, but this advantage was lost during rain-free periods when unwatered plants experienced severe drought. The basis for this response was analysed using gas-exchange and chlorophyll fluorescence in a controlled-drying experiment with potted plants. These experiments demonstrated that loss of the C4 photosynthetic advantage was caused by a greater increase in metabolic limitations to C4 than C3 photosynthesis. Decreases in CO2 assimilation resulted in lower electron transport rates and decreased photochemical efficiency under drought conditions, rather than increased electron transport to alternative sinks. Further comparisons of three C3 and three NADP-ME C4 Panicoid grasses growing wild in a natural grassland showed similar losses of photosynthetic advantage. Findings suggest that the high metabolic sensitivity of photosynthesis to severe drought observed in several C4 grass species may be an inherent characteristic of the C4 pathway. The mechanism may explain the paradox of why C4 species richness declines in arid environments despite their high water-use efficiency. PS10.11 Does the C4-like pathway operate in rice leaves?: Comparison of transgenic rice plants overproducing different sets of C4 enzymes M Miyao-Tokutomi (Natl. Inst. Agrobiol. Sci.), Y Taniguchi (Natl. Inst. Agrobiol. Sci.), T Fukuda (Natl. Inst. Agrobiol. Sci.), C Masumoto (Natl. Inst. Agrobiol. Sci.), H Ohkawa (Hirosaki Univ.), H Fukayama (Kobe Univ.), H Sasaki (Univ. Tokyo) Three different double (PEPC + PPDK, PEPC + ME, MDH + ME), one triple (PEPC + PPDK + ME), and one quadruple (PEPC + PPDK + MDH + ME) transgenic rice plants were produced, and their photosynthetic characteristics and growth were compared. Overproduction of any of PPDK, MDH and ME did not affect the photosynthetic CO2 assimilation, while that of PEPC slightly suppressed it. Reduced CO2 assimilation rates caused by PEPC overproduction remained unaffected by co-overproduction of any of PPDK, ME and PPDK + ME, while it was significantly restored by co-overproduction of PPDK + ME + MDH to reach levels comparable to or even higher than that of non-transformants. These results raise the possibility of improving photosynthetic performance of C3 plants by co-overproduction of all the four C4 enzymes. The quadruple transformants, however, showed slight stunting. It was found that this stunting resulted from co-overproduction of PEPC and ME. In general, PPDK and MDH are active only in the daytime while PEPC and ME are active day and night. It is likely that the co-overproduction of PEPC and ME consumes once fixed carbon at night and under weak light illumination and thereby leads to stunting. Rice is unique in that PEPC in the leaves becomes more active at night than in the daytime. The stunting caused by the PEPC + ME co-overproduction might be less pronounced in other C3 plants than in rice. Analyses of carbon discrimination of multiple transformants are now in progress. PS10.12 Production of transgenic rice plants overproducing multiple C4 enzymes

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Y Taniguchi (Natl. Inst. Agrobiol. Sci.), T Fukuda (Natl. Inst. Agrobiol. Sci.), C Masumoto (Natl. Inst. Agrobiol. Sci.), H Ohkawa (Hirosaki Univ.), H Fukayama (Kobe Univ.), M Miyao (Natl. Inst. Agrobiol. Sci.)

PS10.14 Light dependences of the CO2 leakiness, quantum yield of CO2 fixation and activation state of key enzymes in a C4 plant, Amaranthus cruentus, grown in hi

To introduce the C4 pathway into C3 plants, at least three C4 enzymes (PEPC, PPDK, NADP-ME) have to be overproduced. Overproduction of any of the maize C4 PEPC and the maize C4 PPDK did not significantly affect photosynthesis and growth of rice plants, and double transformants overproducing PEPC and PPDK were successfully produced by crossing. On the other hand, overproduction of the maize C4 NADP-ME led to serious stunting and leaf photobleaching, which resulted from enhanced photoinhibition of photosynthesis due to an increase in the level of NADPH inside the chloroplast. Such effects of the maize ME were not mitigated by overproduction of the sorghum NADP-MDH, which acts to export the reducing power from the chloroplast. Co-overproduction of either PEPC+PPDK or PEPC+PPDK+MDH was also ineffective. Therefore, the rice C3 NADP-ME that did not show any detrimental effects was used instead of the maize enzyme. Overproduction of the sorghum MDH up to the level 40 fold that of non-transformants did not at all affect photosynthesis and growth of rice plants. Triple (PEPC+PPDK+ME) and quadruple (PEPC+PPDK+MDH+ME) transformants were produced by gene introduction into the PEPC+PPDK hybrids. Contrary to the single transformants overproducing the rice ME, the triple transformants showed stunting, which was more marked with increasing ME activities. Such stunting was largely mitigated by overproduction of the sorghum MDH in the quadruple transformants, of which MDH activities were more than tenfold that of non-transformants. These results suggest that overproduction of MDH would be prerequisite for introduction of the C4 pathway into rice.

In C4 photosynthesis, some CO2 leak from the bundle-sheath cells to the mesophyll cells or intercellular air spaces. Because the CO2 leak wastes ATP consumed in the C4 cycle, it may decrease the efficiency of C4 photosynthesis. To understand the effects of CO2 leak on the efficiency of C4 photosynthesis at low light, we investigated the light responses of the CO2 leakiness (f), the quantum yield for CO2 fixation and the activation states of key enzymes in C4 photosynthesis using Amaranthus cruentus L. (NAD-malic enzyme subtype, dicot) grown in high-light (HL) and low-light (LL). f increased with a decrease in incident photon flux density (PFD) below 150 µmol quanta m-2 s-1. In accordance with the increase in f, implying efficiency of photosynthesis, the initial part of the photosynthesis light response curve was concave rather than straight. In LL leaves, f was lower and pyruvate, orthophosphate dikinase (PPDK) activity was higher at low PFDs than in HL leaves. Perhaps, the higher PPDK activity contributed to the lower f in LL leaves. Based on the results, we discuss spurious effect of f on the estimation of the maximum quantum yield of C4 photosynthesis, which has been extensively used in ecophysiological studies.

PS10.13 Dynamic response of Crassulacean Acid Metabolism in Phalaenopsis to a warm day/cool night temperature regime

D Marshall (University of Cambridge), H Griffiths (University of Cambridge), J Hibberd (University of Cambridge)

B Pollet (Ghent University), K Steppe (Ghent University), P Dambre (Research Centre for Ornamental Plants), M Van Labeke (Ghent University; Research Centre for Ornamental Plants), R Lemeur (Ghent University) The recommended average daily temperature for production of Phalaenopsis is between 25°C and 30°C. Therefore, growing Phalaenopsis demands a large energy input. Greenhouse heating strategies that maximise the contribution of solar radiation during the day and minimise heat production during cold nights have shown their effectiveness in reducing energy costs. However, such greenhouse strategies (e.g. temperature integration) are based on empiricism and lack physiological background. During this study, Phalaenopsis ‘Isis’ and Phalaenopsis ‘Hercules’ were subjected to a warm day/cool night temperature regime (32.5/17.5°C) for 32 days while maintaining the average daily temperature at the same level of a control treatment (26/25°C). Leaf net CO2-exchange was continuously monitored and PS II photochemistry and thermal energy dissipation was assessed. Exposing Phalaenopsis to the warm day/cool night temperature regime resulted in a stronger expression of CAM. As a consequence, daily carbon gain was enhanced for the first days of the experiment. This short term increase of daily carbon gain was also reflected in PS II photochemistry and thermal energy dissipation. However, the maximum quantum efficiency of PS II suggested an increased susceptibility to photoinhibition. At the same time, monthly carbon gain was reduced. Nevertheless, daily carbon gain of P. ‘Isis’ rose above the one of the control treatment after 32 days, indicating adaptation and the start of recovery. From the dynamic response of Phalaenopsis to a combination of warm days and cool nights, it can be concluded that temperature integration, and its energy saving properties, has opportunities for commercial production of Phalaenopsis.

Y Tazoe (University of Tokyo)

PS10.15 Cleome- A new model for C4 photosynthesis

C4 plants have evolved from those using C3 photosynthesis at least 45 times in the angiosperms and involves biochemical, anatomical, and ultrastructural changes. Cleome gynandra is the C4 species phylogenetically most closely related to the C3 species Arabidopsis thaliana. The phylogenetic proximity between these two species provides the potential to translate our understanding of processes, such as the regulation of gene expression and leaf development, from Arabidopsis to a C4 plant. We have determined that C. gynandra belongs to the NAD-ME subtype, and in addition identified species within the genus that possess characteristics of C4 photosynthesis ranging from increased venation density to centripetal localisation of chloroplasts in the bundle sheath. This demonstrates important developmental flexibility within the genus and provides insights into the evolution of C4 photosynthesis. To identify whether Arabidopsis possesses trans-factors that recognise C. gynandra genes important for the C4 pathway we have generated a series of reporter constructs and placed them in Arabidopsis. The extent to which these constructs are recognised in Arabidopsis will be summarised. PS10.16 Molecular evolution of four-carbon decarboxylase genes recruited into C4 photosynthesis N Brown (University of Cambridge), J Hibberd (University of Cambridge) Evolution of efficient C4 photosynthesis required compartmentalisation of biochemistry into specific cell-types and alterations to leaf morphology, most likely through changes in gene expression. Despite this apparent complexity, it has evolved independently 45 times among

228 14th Photosynthesis Congress - PS07 the angiosperms. Although Arabidopsis (At) uses C3 photosynthesis, its vascular cells show characteristics of the C4 pathway; for example, activities of the decarboxylase enzymes NAD-ME, NADP-ME and PEPCK are highly enriched in these cells. We are interested in how the regulation of genes encoding these decarboxylases has altered in association with the evolution of C4 photosynthesis and have adopted a comparative approach using C3 and C4 plants. Promoter regions for two AtNADP-ME genes and both AtNAD-ME genes direct expression of uidA in xylem parenchyma cells and the NADP-ME 3’ regions greatly increase expression. We have identified Cleome as being a genus containing C4 species that is within the Brassicaceae and therefore closely related to Arabidopsis (TIPS, 10, 215-221). Two NAD-ME genes have been isolated from Cleome gynandra (Cg), an NAD-ME sub-type C4 species, and in situ hybridization indicates that the transcripts for both genes are localised in the bundle-sheath. We are using transgenic Arabidopsis containing CgNAD-ME2 –uidA constructs to determine whether Arabidopsis contains the trans factors required for bundle-sheath expression. We have demonstrated that 850 bp of promoter region is not sufficient for bundle-sheath expression in transgenic Arabidopsis, and the 5’ and 3’ UTRs do not mediate bundle-sheath expression by a post-transcriptional mechanism.

PS10.17 Ecological differentiation in the C3 and C4 subspecies of Alloteropsis semialata C Osborne (University of Sheffield), D Ibrahim (University of Sheffield), M Gilbert (Rhodes University), B Ripley (Rhodes University) Although half of today’s grass species utilize C4 photosynthesis, the ancient origins of this pathway in C4 grass lineages hinder direct evaluation of its ecological costs and benefits. We have addressed this problem by investigating the physiological ecology of Alloteropsis semialata, a unique grass species with co-occurring C3 and C4 subspecies. Molecular phylogenetic analyses indicate that the C3 subspecies represents an evolutionary reversion from a C4 ancestor. Controlled environment and common garden experiments have tested the consequences of this evolutionary event for plant responses to CO2, temperature, fire, water and nitrogen availability. These demonstrate a photosynthetic and growth advantage for the C4 subspecies over the C3 type at temperatures above 15 ºC. However, during drought events this advantage is lost because the C4 subspecies shows greater susceptibility to metabolic limitations imposed by water deficit. The C4 subspecies also suffers from freezing-induced mortality of its leaves during winter, which the C3 avoids via cold acclimation. Crucially, CO2, nitrogen and fire manipulation experiments suggest that photosynthetic pathway in this species is linked with ecological strategy. The C3 subspecies favours canopy development, whilst the C4 diverts greater resources to sexual reproduction and storage, allowing more rapid regrowth after fire. Together, these data suggest that C4 photosynthesis in A. semialata is advantageous in warm, high rainfall climates characterised by frequent fires, but confers a cost of greater sensitivity to frost and drought events. Higher photosynthetic rates in C4 than C3 plants may therefore allow greater flexibility in growth responses to resource limitation and disturbance. PS10.18 Diurnal synchronization between PEP carboxylase and PEP carboxykinase activities in the epiphytic CAM bromeliad Tillandsia pohliana

L Freschi (Department of Botany, University of São Paulo, 05508-900 São Paulo, SP, Brazil), M Aurineide Rodrigues (Department of Botany, University of São Paulo, 05508-900 São Paulo, SP, Brazil), H Mercier (Department of Botany, University of São Paulo, 05508-900 São Paulo, SP, Brazil) A central characteristic of the Crassulacean acid metabolism (CAM) is the temporal separation between the nocturnal synthesis of organic acids and their subsequent decarboxylation during the day. In PEPCK-CAM plants, the key enzymes required for the processes of acidification (PEPCase) and deacidification (PEPCK) are simultaneously present in the citossol of the same cells. Therefore, in order to minimize futile carbon fluxes over the diurnal cycle, there must be some efficient mechanism that synchronizes PEPCase and PEPCK activities in these plants. Since both enzymes can be regulated by reversible phosphorylation, we analyzed the importance of phosphorylation-dependent changes in the properties of PEPCase and PEPCK over the diurnal CAM cycle in the bromeliad Tillandsia pohliana. Although the maximum activities of PEPCase and PEPCK remained fairly constant throughout the day/night cycle, extensive diurnal changes were observed in the apparent phosphorylation status of these enzymes. The phosphorylation state of both PEPCase and PEPCK followed a similar pattern, with the highest levels at night, followed by a gradual decline during the first six hours of daylight and reaching the lowest levels in the middle of the day. However, since phosphorylation activates PEPCase and inactivates PEPCK, a completely opposite day/night activation pattern could be established between these enzymes, with PEPCase active at night and PEPCK most active near midday. These data showed high temporal agreement with the diurnal fluctuation of organic acids in T. pohliana and strengthen the significance of regulatory phosphorylation of PEPCase and PEPCK for the synchronization of the CAM cycle. Supported by FAPESP_Proc.05/54515-5 PS10.19 Hormonal regulation of Crassulacean acid metabolism expression in pineapple plants L Freschi (Department of Botany, University of São Paulo, 05508-900 São Paulo, SP, Brazil), M A Rodrigues (Department of Botany, University of São Paulo, 05508-900 São Paulo, SP, Brazil), D S Domingues (Department of Botany, University of São Paulo, 05508-900 São Paulo, SP, Brazil), E Purgatto (Department of Food and Experimental Nutrition, University of São Paulo, 05508-900 São Paulo, SP, Brazil), M - A Van Sluys (Department of Botany, University of São Paulo, 05508-900 São Paulo, SP, Brazil), The degree of Crassulacean acid metabolism (CAM) expression in C3-CAM facultative plants can be strongly modulated by a variety of environmental factors. Consequently, phytohormones are thought to play a crucial role in the coordination between these external stimuli and the C3-CAM transition. Since we have recently shown that young pineapple (Ananas comosus) plants can perform either C3 or CAM photosynthesis, in this work we investigated the hormonal control of CAM expression in this bromeliad. The levels of cytokinins, abscisic acid (ABA), auxins and ethylene were analyzed over 30 days in plants subjected to CAM induction by drought. Exogenous treatments with these hormones were also carried out. While the results did not show a clear effect of auxins and ethylene on CAM induction, several observations suggest that ABA and cytokinins are involved in pineapple C3-CAM transition. The elevation in PEPCase transcripts levels, which started after six days of drought, was preceded by an increase in ABA content and an equivalent reduction in cytokinins pool. Afterwards, the expression of a typical CAM cycle with increased PEPCase activity and malate accumulation was concomitant with a total decline in cytokinins and further increases in ABA content. As a result, the highest CAM expression levels and ABA/cytokinins ratios were simultaneously achieved after 15 days of

229 14th Photosynthesis Congress - PS07 drought. Additionally, ABA-treated plants were induced to CAM whereas exogenous cytokinins partially repressed this induction. Altogether, these results indicate that an antagonism between cytokinins and ABA represents a key regulatory mechanism for CAM expression in pineapple plants. Supported by FAPESP_Proc.05/54515-5 PS10.20 Consequences of photosynthetic pathway evolution for plant-climate interactions: a test using C3 and C4 subspecies of Alloteropsis semialata D Ibrahim (University of Sheffield), C Osborne (University of Sheffield), T Burke (University of Sheffield), M Gilbert (Rhodes University), B Ripley (Rhodes University) The grass Alloteropsis semialata is the only plant known to have both C3 and C4 subspecies, presenting a unique opportunity to test the ecological consequences of photosynthetic pathway evolution. We used a molecular phylogeny to examine the relationship between the subspecies, and established a common garden experiment in South Africa to investigate the role of photosynthetic pathway in determining plant-climate interactions. Sequencing of the chloroplast gene ndhF confirms the close relationship between the C3 and C4 subspecies, and suggests that the physiology of the C3 subspecies represents a reversion from the C4 pathway. Based on the temperature responses of C3 and C4 photosynthesis, we hypothesised a photosynthetic and growth advantage for the C4 over the C3 subspecies during summer and a reversal of this pattern during winter. Aboveground biomass and gas exchange were measured over two years in the common garden under both irrigated and natural rainfall treatments. The predicted summer advantages for the C4 over the C3 subspecies were observed under well-watered conditions, but were unexpectedly lost during natural drought events. During winter the C3 subspecies achieved the expected photosynthetic advantage over the C4 subspecies. However, this was not due to higher C3 photosynthetic efficiency at low temperatures, but instead because frost killed the C4 leaves while photosynthesis continued in the freezing-resistant C3 subspecies. This study demonstrates that the seasonal advantages of C3 and C4 photosynthesis predicted from theory are highly constrained by extreme climate events such as drought and frost. PS10.21 CAM photosynthesis in tropical epiphytes: an analysis of ecological and evolutionary relationships in bromeliads A Smith (University of Oxford), D Crayn (National Herbarium of New South Wales), K Winter (Smithsonian Tropical Research Institute) Epiphytic niches in tropical environments can be water-limited and microclimatically arid, even in regions with relatively high annual rainfall. The Neotropical family Bromeliaceae (ca. 3000 spp.) is a large and ecologically diverse family, containing approximately equal numbers of terrestrial and epiphytic species that can occupy a very wide range of habitats. From a survey of carbon-isotopes ratios covering nearly two-thirds of the family, we estimate that about 44 % of all bromeliad species utilize the CAM pathway as their principal mode of carbon assimilation. Phylogenetic analysis indicates that CAM photosynthesis has evolved independently a minimum of three times within the family. Ecologically, there is a tight correlation between habitat aridity and the frequency of CAM photosynthesis in these independent lineages, arguing strongly for the adaptive significance of this highly water-use-efficient mode of photosynthesis. Even within individual taxa, such as the large epiphytic genus Tillandsia, there is a close relationship between the occurrence of CAM photosynthesis, the degree of morphological reduction in plant life-form, and more xeric

habitats. Nevertheless, a number of CAM taxa can persist in less typical habitats, such as high-rainfall montane forest and at high-altitude sites in the Andes. These observations suggest that interpretation of the adaptive value of CAM photosynthesis as a functional trait must take into account both prevailing environmental conditions and the selective pressures that may have acted upon distinct lineages during their evolutionary history. PS10.22 Comparative proteomics of C3 and C4 mesophyll chloroplast envelopes and the characterization of a novel transport protein, Mep1 A Braeutigam (Heinrich-Heine-Universitaet Duesseldorf; Michigan State University), S Hoffmann-Benning (Michigan State University))), A Weber (Heinrich-Heine-Universitaet Duesseldorf; Michigan State University) Although C4 photosynthesis is a well studied pathway, most transport proteins needed for the concentration of CO2 in the bundle sheath are still unknown at the molecular level. We hypothesized that a comparative proteome analysis of Zea mays mesophyll chloroplasts and Pisum sativum chloroplasts will reveal proteins that catalyze the C4 specific metabolite fluxes because they are enriched in C4 mesophyll chloroplasts of Z. mays compared to C3 chloroplasts of P. sativum. A qualitative and semi-quantitative analysis of the proteome of Z. mays mesophyll chloroplast envelopes and P. sativum chloroplast envelopes will be presented. We also present the analysis of Mep1 (Maize envelope protein 1), one of the most abundant proteins in maize mesophyll chloroplast envelopes. Arabidopsis thaliana knock-out mutants deficient in the corresponding gene display a dwarfed bleached-leaf phenotype that is cured when plants are grown in elevated CO2. We confirmed the plastid localization with GFP fusions and determined the expression pattern with promoter::GUS fusions. Extensive metabolite analysis reveals the block of metabolism within the plants and suggests a possible role for Mep1 as monocarboxylate transporter in A. thaliana. Currently we are analyzing the transport capacities of Mep1 in whole plastids. Isolated chloroplasts from A. thaliana wild type and knock-plants and Z. mays mesophyll tissue are fed with candidate substrates and uptake is monitored with a clark-type oxygen electrode. These results together with the biochemical characterization of the heterologously expressed proteins will ultimately reveal the transport capacities of AtMep1 in A. thaliana and ZmMep1 in Z. mays. PS10.23 The extent of C4 and CAM photosynthesis in Three Grahmia species of the Portulacaceae L Guralnick (Western Oregon University ), A Cline (Western Oregon University) CAM has evolved independently in over 30 different plant families. The Portulacaceae is plant family whose members show photosynthetic diversity in that some are C3 plants; others are C4 plants which show some CAM characteristics and some members are facultative CAM plants (switching between C3 and CAM photosynthesis). Previous research has looked at the evolution and distribution of CAM in the Portulacaceae. The only genus known to have C4 photosynthetic members is the genus Portulaca. The phylogeny indicated that Portulaca evolved the C4 pathway after evolving the CAM pathway. Grahmia coahuilensis, G. frutescens, G. bracteleata are members of the Portulacaceae. Previous reports have indicated that these species and the Anancampseros genera may also contain C4 photosynthetic members. Based on the phylogeny, this would indicate multiple origins of C4 photosynthesis within the Portulacaceae. However, the placement of Grahmia as a C4 plant is not well supported in the literature. This

230 14th Photosynthesis Congress - PS07 study will study the true photosynthetic characteristics of C4 and CAM photosynthesis of the Grahmia spp. Preliminary data indicate that Grahmia may be a facultative CAM species. The carbon isotope composition of the Grahmia spp. was -24.5 o/oo which places it toward the C3 range. The titratable acidity levels are high and PEP carboxylase enzyme activity is low under well watered conditions which may mean that CAM may be induced by water stress. Leaf samples have been obtained for anatomical studies. Further studies will be needed to determine the actual photosynthetic characters of this genus. PS10.24 The effects of rubisco activase on C4 photosynthesis and metabolism at high temperature L Hendrickson (Australian National University) The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase (RA). This enzyme is thought to facilitate the release of sugar phosphate inhibitors from Rubisco catalytic sites thereby influencing carbamylation. To explore the role of RA in C4 photosynthesis, activase levels were reduced in Flaveria bidentis, a C4 dicot, by transformation with an antisense gene directed against the mRNA for RA (von Caemmerer et al.1). A range of secondary transformants were screened at 25oC and 40oC for photosynthetic rate, Rubisco activity, RA content and C3/C4 metabolites. The small isoform of Flaveria RA was purified from an E.coli-based expression system where RA was fused to poly-histidine-tagged Ubiquitin and purified using immobilised metal affinity chromatography according to Baker et al.2 The relationship between leaf activase content, CO2 assimilation rate and Rubisco carbamylation was contrasted at both temperatures. Our results at 25oC were consistent with the qualitative relationships of von Caemmerer et al.1 Our data further show that after short term treatment at 40oC, the relationship between RA and photosynthesis is non-linear indicating that at 40oC, RA is not the limiting factor for C4 photosynthesis. von Caemmerer S, Hendrickson L, Quinn V, Vella N, Millgate A, Furbank RT (2005) Reductions of Rubisco activase by antisense RNA in the C4 plant Flaveria bidentis reduces Rubisco carbamylation and leaf photosynthesis. Plant Physiology 137: 747-755. 2Baker RT, Catanzariti A-M, Karunasekara Y, Soboleva TA, Sharwood R, Whitney S, Board PG (2007) Using deubiquitylating enzymes as research tools. Methods in Enzymology 398: 540-554. 1

PS10.25 Dorso-ventral regulation of photosynthesis on the adaxial and abaxial leaf surfaces of the monocotyledonous C4 species Paspalum dilatatum A Soares (Plant Biology Department and Biological Engeneering Centre, Faculty of Sciences, University of Lisbon, Portugal), S Driscoll (Crop Performance and Improvement Division, Rothamsted Research, Harpenden, UK), E Olmos (CEBAS-CSIC, Department of Plant Physiology, Campus de Espinardo, Spain), M Arrabaça (Plant Biology Department and Biological Engeneering Centre, Faculty of Sciences, University of Lisbon, Portugal), C Foyer (Crop Performance and Improvement Division, Rothamsted Research, Har Photosynthesis is a flexible process that shows rapid responses to light and CO2 availability. Previous work with maize showed that the two leaf surfaces have different patterns of photosynthetic response to available CO2 (Driscoll et al. 2006 J Exp Bot 57:381-390). In the present study we have analysed the dorso-ventral regulation of photosynthesis in other

NADP-malic enzyme C4 species, Paspalum dilatatum Poiret cv. Raki, grown either at 350 and 700 mL L-1 CO2. Gas-exchange measurements using the infrared gas analyser were performed with adaxial or abaxial illumination to the leave in two different types of chambers, where photosynthesis was measured either on the whole leaf or on each leaf surface separately and simultaneously. Each leaf surface showed characteristic photosynthetic responses to available CO2 and light, regardless of the growth CO2 environment. In addition, there was a pronounced effect of leaf orientation towards light on the regulation of photosynthesis and stomata, whether CO2 assimilation was measured on the whole leaf or on each leaf surface separately. This differential regulation of photosynthesis was not due to variations in the content of phosphoenolpyruvate carboxylase or ribulose 1,5-bisphosphate carboxylase/oxygenase proteins, or to a differential light absorption on both leaf sides. The different sensitivities of the adaxial and abaxial stomata to light may account for the almost complete closure of stomata on the adaxial surface when the leaf receives abaxial illumination. Understanding the dorso-ventral regulation of photosynthesis in monocotyledonous leaves provides new insights into factors regulating crop productivity.

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PS11 - The C3 Cycle: limitation and regulation PS11.1 Why has evolution selected a sluggish Rubisco in higher plants? G Tcherkez (Université Paris Sud XI), G Farquhar (Australian National University), T Andrews (Australian National University)

PS11.3 New insight into the Calvin cycle regulation Glutathionylation of fructose bisphosphate aldolase in response to illumination K Ogawa (RIBS OKAYAMA), M Matsumoto (RIBS OKAYAMA)

The cornerstone of autotrophy, the CO2-fixing enzyme, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is hamstrung by slow catalysis and confusion between CO2 and O2 as substrates; an “abominably perplexing” puzzle, in Darwin’s parlance. Here we argue that these characteristics stem from difficulty in binding the featureless CO2 molecule, which forces specificity for the gaseous substrate to be determined largely or completely at the transition-state level. We hypothesize that natural selection for greater CO2/O2 specificity, in response to reducing CO2:O2 ratios in the atmosphere, has resulted in a transition state for CO2 addition in which the CO2 moiety closely resembles a carboxylate group. This maximizes the structural difference between the transition states for carboxylation and the competing oxygenation, allowing better differentiation between them. However, increasing structural similarity between the carboxylation transition state and its carboxyketone product exposes the carboxyketone to the strong binding required to stabilize the transition state and causes the carboxyketone intermediate to bind so tightly that its cleavage to products is slowed. We assert that Rubiscos from various sources may be nearly perfectly adapted to their differing CO2/O2 environments, optimizing this compromise between CO2/O2 specificity and the maximum rate of catalytic turnover. Our hypothesis explains the feeble rate enhancement displayed by Rubisco in processing exogenously supplied carboxyketone intermediate, compared to its non-enzymatic hydrolysis, and the positive correlation between Sc/o and 12C/13C fractionation. It further predicts that, because a more product-like transition state is more ordered (decreased entropy), the effectiveness of this strategy will deteriorate with increasing temperature.

PS11.2 Novel aspects of redox regulation of the Calvin cycle C Raines (University of Essex) Regulation of the Calvin cycle is complex and although many details of the regulatory features of individual enzymes are well documented the importance of these processes in vivo is not well understood. For example it has long been known that changes in the redox state of thioredoxin regulates the activity of a number of Calvin cycle enzymes and it has been assumed that this is the mechanism that allows the activity of the Calvin cycle to be balanced with the availability of ATP and NADPH produced in the light reactions. However, the situation is more complex than this and data will be presented demonstrating that the co-ordinate and transient modulation of Calvin cycle activity in response to changes in light intensity is mediated by a multiprotein complex formed between phosphoribulokinase (PRK), glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and the small protein, CP12. A model proposing that thioredoxin mediates activation of PRK and GAPDH by two mechanisms; directly through reduction of disulphide bonds within these enzymes and, indirectly via interactions with a small chloroplast protein, CP12 will be presented. A wider role for CP12 in mediating thioredoxin regulation of chloroplast carbon metabolism will be presented.

Since the Calvin cycle is redox regulated by thioredoxins using NADPH and reduced ferredoxin produced by the light reactions, it is subjected to oxidative inhibition when the absorbed light energy exceeds the capacity of photosynthesis and the photoprotective mechanisms are overwhelmed. It is thought that glutathionylation of proteins (the covalent biding of the glutathione moiety to the target protein through the disulfide bridge) contributes to the suppression of irreversible inactivation of redox-regulated enzymes. However, we found that the Calvin cycle enzyme aldolase is activated via glutathionylation and inactivated by thioredoxin. Here we will show that glutathionylation/deglutathionylation of aldolase is regulated in response to illumination and facilitates the Calvin cycle.

PS11.4 The non-essential role of peroxisomal malate dehydrogenase during photorespiration. A Cousins (The Australian National University), I Pracharoenwattana (The University of Western Australia), S Smith (The University of Western Australia), M Badger (The Australian National University) Peroxisomes are important in maintaining rates of photosynthesis by recycling carbon and nitrogen that would otherwise be lost during photorespiration. The reduction of hydroxypyruvate to glycerate catalyzed by hydroxpyruvate reductase (HPR) in the peroxisomes is thought to be facilitated by the production of NADH by the peroxisomal malate dehydrogenases (pMDH). pMDH, which has two isoforms, reduces NAD+ to NADH via the oxidation of malate to oxaloacetate supplied from the cytoplasm. A double mutant lacking the expression of both pMDH genes was viable in air and had rates of photosynthesis only slightly lower than wild-type (WT). This is in contrast to the classical photorespiratory mutants which had severely reduced rates of photosynthesis and required high CO2 to grow1. The pMDH mutant had a higher O2 dependent CO2 compensation point than WT implying that either Rubisco specificity had changed or that the rate of CO2 released per Rubisco oxygenation was increased in the pMDH plants. Rates of gross O2 evolution and uptake were similar in the pMDH and WT plants indicating chloroplast linear electron transport and photorespiratory O2 uptake were similar between genotypes. The CO2 post illumination burst and the rate of CO2 released during photorespiration were both greater in the pMDH mutants compared to WT, suggesting the ratio of photorespiratory CO2 release to Rubisco oxygenation was altered in the pMDH mutants. Glyoxylate decarboxylation is being investigated. In summary, pMDH is not essential for maintaining rates of photorespiration implying there is an additional mechanism for supplying reductant to the peroxisome HPR reaction. 1Somerville CR, Ogren WL (1982) Genetic-Modification of Photo-Respiration. Trends in Biochemical Sciences 7: 171-174

PS11.5 Evolutionary Potential of RuBisCO-like Protein in Bacillus

232 14th Photosynthesis Congress - PS07 subtilis: Interaction RuBisCO.

with

Transition-State

Analogue

of

Y Saito (Nara Institute of Science and Technology (NAIST), Graduate School of Biological Sciences, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan), H Ashida (Nara Institute of Science and Technology (NAIST), Graduate School of Biological Sciences, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan), A Sekowska (Genetics of Bacterial Genomes, Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France), A Danchin (Genetics of Bacterial Genomes, Institut Pasteur, 28, rue du Docteur Roux, 75724

association of cpn60-ß with activase was dependent on the duration and intensity of the heat stress and decreased following recovery. Taken together, these and other data suggest a possible role for cpn60-ß in acclimating photosynthesis to heat stress, possibly by protecting activase from thermal denaturation. PS11.7 Improvement of cyanobacterial RuBisCO by introducing the latch structure involved in high affinity for CO2 in red algal RuBisCO.

RuBisCO-like protein (RLP) from Bacillus subtilis catalyzes the 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) enolase reaction in the methionine salvage pathway. This reaction resembles that of the first step in the RuBisCO reaction; enolization of ribulose-1,5-bisphasphate (RuBP). The chemical structure of DK-MTP-1-P is very similar to that of RuBP. However, the positions of the proton abstracted in enolization reaction are different between the two enzymes. We are interested in the relationship reactions catalyzed by RuBisCO and RLP, and analyzed here the general enzymatic properties of the RLP using the recombinant B. subtilis enzyme. B. subtilis RLP was homodimer and had the Michaelis constant for DK-MTP-1-P of 19.3 mM and the maximum reaction velocity of 112.5 mmol min-1 (mg protein) -1. The optimum temperature and pH for the reaction were 35 degrees Celsius and 8.2, respectively. B. subtilis RLP required a divalent cation for catalysis, and was specific for Mg2+. Interestingly, the reaction-intermediate analog 2-carboxyarabinitol-1,5-bisphosphate and the reaction-product phosphoglycerate of RuBisCO exhibited competitive inhibition with respect to DK-MTP-1-P, but 2-carboxyribitol-1,5-bisphosphate did not. B. subtilis RLP conserves essential residues, K175, K201, D203 and E204, for enolization reaction of RuBisCO. Mutational analysis showed that these residues also played key roles in RLP. The present study revealed that bona fide RuBisCO and B. subtilis RLP resemble each other not only in their primary sequences of the proteins and catalytic reaction but also active site structure and catalytic mechanism. Thus, the gene for Bacillus RLP may be functionally an origin of the gene for photosynthetic RuBisCO.

RuBisCO catalyzes both carboxylation and oxygenation reactions. The specificity for carboxylation relative to oxygenation is represented by Srel. We expect that the creation of RuBisCO with high Srel enables plants to acquire high photosynthetic efficiency.RuBisCOs of algae and higher plants are classified into two groups, the green and red-like RuBisCO subclasses. Srel values of red-like RuBisCOs are 2 to 3-times higher than those of plant RuBisCOs.We have revealed that one of the red-like RuBisCO, Red alga Galdieria partita RuBisCO, shows the highest Srel of 238 among the RuBisCOs examined so far. Large subunit of Galdieria RuBisCO has a unique “latch structure”. The latch structure is a hydrogen bond between the main chain oxygen of Val332 and the side chain amide nitrogen of Gln386. All red-like RuBisCOs conserved amino acid residues to form the latch structure. On the other hand, green-like RuBisCOs cannot form the latch structure because Gln386 is replaced with histidine. Therefore we assume that the latch structure is involved in high Srel in red-like RuBisCOs.To clarify the relationship between Srel and the latch structure, we introduced the latch structure into Synechococcus sp.PCC7002 RuBisCO by mutagenesis. The wild type and the mutant RuBisCO were expressed in Escherichia coli and purified to analyze enzymatic properties. The mutant RuBisCO shows a 16% increase in Srel with a 29 % decrease in Vmax(CO2) and 35% improvement in a Km(CO2) compared to the wild type.We would like to discuss about relationship between the latch structure and Srel in this presentation.

PS11.6 Association of Activase with cpn60 during Heat Stress: Possible Mechanism for Photosynthetic Acclimation

PS11.8 Increased Rubisco content in transgenic rice transformed with “sense” rbcS gene

M Salvucci (USDA-ARS)

Y Suzuki (Tohoku University), M Ohkubo (Tohoku University), H Hatakeyama (Tohoku University), K Ohashi (Tohoku University), R Yoshizawa (Tohoku University), S Kojima (Tohoku University), T Hayakawa (Tohoku University), T Yamaya (Tohoku University), T Mae (Tohoku University), A Makino (Tohoku University)

Previous studies have shown that inhibition of photosynthesis by moderate heat stress is a consequence of Rubisco deactivation, caused in part by thermal instability of activase. This involvement of activase was confirmed in recovery experiments using transgenic plants with altered activase:Rubisco ratios. Compared with control plants, photosynthesis and Rubisco activation were more thermotolerant and recovered faster from heat stress in tobacco plants with reduced levels of Rubisco (i.e., higher activase:Rubisco) and were less thermotolerant and recovered more slowly in Arabidopsis plants with reduced levels of activase (i.e., lower activase:Rubisco). Transgenic Arabidopsis plants that expressed a modified form of activase containing an eight amino acid Strep-tactin binding sequence were used to identify factors that might be involved in the acclimation of photosynthesis to heat stress. Affinity adsorption and elution of activase identified a 58 kD polypeptide that co-eluted with activase in leaf extracts prepared from heat stressed, but not control plants. Sequence analysis identified this polypeptide as the ß-subunit of chaperonin 60 (cpn60-ß), the chloroplast GroEL homolog. Immunoblot analysis following separation by non-denaturing gel electrophoresis indicated that the activase and cpn60-ß that co-eluted from the affinity column were associated in a high molecular mass complex. Immunoblot analysis also established that the apparent

N Ninomiya (NAIST), H Ashida (NAIST), A Yokota (NAIST)

Rice (Oryza sativa L.) plants with substantially increased Rubisco content were obtained by Agrobacterium-mediated transformation with the rice rbcS sense gene under the control of the rice rbcS promoter. The primary transformants were screened for the ratio of Rubisco to leaf-N content, and the transformants with above 120% wild-type levels of Rubisco were selected. In the progeny of the selected lines of the transformants, the mRNA levels of one member of the rbcS gene family were increased from 3.9- to 6.2-fold, whereas those of other members of the rbcS gene family were unchanged. The total levels of rbcS mRNA were increased from 2.1- to 2.8-fold. The levels of rbcL mRNA were increased from 1.2- to 1.9-fold. Rubisco protein content was significantly increased by 30% on a leaf area basis. The ratio of Rubisco-N to leaf-N was also increased by 10 to 20%, irrespective of N treatment. The specific activity of Rubisco per unit of enzyme protein was not different. However, light-saturated photosynthesis was not enhanced even when the rate was measured at low [CO2] where Rubisco becomes limiting for photosynthesis. Some lines showed lower

233 14th Photosynthesis Congress - PS07 photosynthesis at high [CO2] (above 60 Pa). We conclude that introduction of additional sense rbcS leads to overexpression of rbcS and that this overexpression slightly up-regulates the gene expression of rbcL at a transcript level and enhances the amount of Rubisco holoenzyme. However, overproduction of Rubisco protein does not improve photosynthesis. PS11.9 Rubisco activation state and its relationship to photosystem I in leaves H Eichelmann (Tartu University, Institute of Molecular and Cell Biology), E Talts (Tartu University, Institute of Molecular and Cell Biology), V Oja (Tartu University, Institute of Molecular and Cell Biology), E Padu (Tartu University, Institute of Molecular and Cell Biology), A Laisk (Tartu University, Institute of Molecular and Cell Biology) In planta CO2 response kinetic curves of Rubisco were measured with a fast-response gas exchange measurement system at constant ribulose 1,5-bisphosphate concentration. Maximum RuBP pool was accumulated in the leaf during the steady-state photosynthesis at saturating light at 200 mmol CO2 mol-1 and 20 mmol O2 mol-1. Rapid transitions to different CO2 concentrations up to 1500 mmol mol-1 were made within 0.5 s and the following rate of CO2 uptake by the leaf was measured with time resolution of 1.5 s. The initial rate of CO2 uptake was found extrapolating the measured rate to the moment when CO2 concentration was changed. CO2 solubilisation was considered by subtracting the similar transient measured in the dark. Rubisco-site CO2 concentration was calculated considering the stomatal and mesophyll diffusion resistances. This way reliable CO2 response curves of Rubisco corresponding to the maximum RuBP concentration were obtained up to the CO2 concentrations of 3 Km, from which Vm was extrapolated. Leaf Rubisco concentration was measured by quantitative SDS electrophoresis, calibrated with purified sunflower Rubisco gravimetrically (by weight). kcat was calculated from Vm and Rubisco content. Measurements were carried out with leaves of laboratory-grown potato and wild type and Rubisco-deficient transgenic tobacco, as well as with leaves of field-growing birch. Biological variation was significant, kcat being reciprocally related to the Rubisco content. The kcat values were generally low, from 1.5 to 3.5 s-1 in the wild type plants containing much Rubisco, but reached about 5 s-1 at 22.5 C in some transgenic leaves with low Rubisco content. There was a general tendency that kcat was higher in leaves containing more PSI and less Rubisco. The data are interpreted to show that carbamylated Rubisco sites may have variable catalytic turnover rates in planta, regulated by Rubisco Activase in cooperation with a PSI-related factor. PS11.10 Does Rubisco indeed limit the rate of photosynthesis? Y Marcus (Tel Aviv University), A Snir (Tel Aviv University), H Altman-Gueta (Tel Aviv University), M Gurevitz (Tel Aviv University) The universal carboxylation enzyme Rubisco is considered the main rate-limiting factor of photosynthesis under saturating illumination and limiting CO2 concentrations. We examined the extent of limitation imposed by Rubisco on photosynthesis of the amphibious plant Nuphar lutea and the cyanobacterium Synechocystis PCC 6803. In Nuphar aerial leaves, but not in its submerged leaves, we found a daily rhythm in the light-saturated rate of photosynthesis resulting from synchronous alterations in stomatal opening and Rubisco activity, the factors that co-limit the rate of photosynthesis in this plant. In Synechocystis

PCC6803 we found that point mutations (including three at the catalytic site), which reduced the catalytic turnover of Rubisco (Kcat) up to 10% of its value in WT and increased the Km(RuBP) had little to moderate effect on the rate of photosynthesis and hardly affected the photoautotrophic growth. In some mutants the inferior kinetic parameters of the enzyme were compensated by massive increase in its content. By correlating the photosynthesis rate at saturating substrate concentrations (Pmax) and Rubisco content with the Kcat of the enzyme in various Rubisco mutants we found that unlike in higher plants, the cyanobacterial photosynthesis is hardly limited by Rubisco activity. As a result, a decrease of up to 2/3 in Rubisco Kcat has no effect on its Pmax and content. Since Rubisco content and Pmax are inversely related (Rubisco content increases as the Pmax decreases), we now examine whether Rubisco content is regulated by the level of Calvin cycle intermediates or their derivatives. PS11.11 The differential role of Pi binding sites in modulation of Rubisco activity Y Marcus (Tel Aviv University), H Altman-Gueta (Tel Aviv University), M Gurevitz (Tel Aviv University) Orthophosphate (Pi) has an antagonistic effect on Rubisco activity: it inhibits catalysis by competing with RuBP (ribulose-1,5-bisphosphate), but it also stimulates Rubisco activation. The crystal structure of Rubisco in complex with Pi revealed three Pi binding sites, two within the catalytic site (where 1P and 5P of RuBP bind), and the third at the 'latch site' (a positively-charged pocket involved in active-site closure during catalysis by interaction with Asp473 at the C-tail). The role of the three Pi binding sites in activation and catalysis of Rubisco from the cyanobacterium Synechocystis PCC6803 was examined by mutagenesis and kinetic assays. Mutations at the 'latch site' as well as D473A at the C-tail, which hindered the catalytic site closure, decreased the Kcat of the enzyme, slightly increased its Km(RuBP), but hardly affected the Km(CO2). Although Arg134 at the 'latch site' is distant from the activation site, it seems to stabilize the carbamate as evident from the accelerated rate of Rubisco deactivation in mutant R134A. We found that Pi stimulated the activation in a 'bump kinetics' manifested as a saturation curve with an intermediary plateau. Whereas substitutions at the three Pi-binding sites inhibited the Pi-stimulatory effect, only substitution H327Q at the 5P RuBP-binding site influenced the Pi-inhibitory effect on catalysis. Unlike a previous model suggesting that Pi binding to a single site stimulates the activation and inhibits the catalysis, our results demonstrate that only partial overlapping exists between the stimulatory and inhibitory sites and that the stimulatory effect occurs by multiple Pi-binding sites.

PS11.12 The Temperature Response of Photosynthesis in Tobacco with Reduced Amounts of Rubisco D Kubien (University of New Brunswick), R Sage (University of Toronto) The reasons for the decline in net CO2 assimilation (A) at high temperatures are controversial. We tested the hypothesis that increasing the ratio of Rubisco activase to Rubisco catalytic sites would enhance both the activation state of Rubisco and A at high temperatures, using wild-type (WT) and two transgenic (anti-RbcS) Nicotiana tabacum genotypes with reduced amounts of Rubisco. We measured photosynthetic gas-exchange, in vivo electron transport (J) and the activation state of Rubisco between 15oC and 45oC. Transgenic plants had lower A than WT at all measurement temperatures, but had a similar thermal optimum for photosynthesis. Both A and J declined 20% to 30%

234 14th Photosynthesis Congress - PS07 between 35oC and 42oC in all three genotypes. This decline was fully-reversible with a 20-minute recovery at 35oC. In WT plants Rubisco was fully-activated at 32oC, but the activation state declined to 64% at 42oC. By contrast, the activation state of Rubisco was above 90% in an anti-RbcS line with 30% of WT levels of Rubisco at all temperatures between 15oC and 42oC. The proportional reductions in A, J, and Rubisco activation state above the thermal optimum of A were of similar magnitude in wild type plants. By contrast, the reduction in A above the thermal optimum was not associated with changes in the activation state of Rubisco in anti-RbcS plants with 30% of WT levels of Rubisco. PS11.13 A novel strategy to rapidly purify cyanobacteria Rubiscos from E. coli H Kane (The Australian National University), D Orr (The Australian National University), S Bala (The Australian National University), S Whitney (The Australian National University) The large (L) and small (S) subunits of Form I Rubiscos from different cyanobacteria can be correctly assembled into functional hexadecamers (L8S8) in Escherichia coli, though the levels of expression are generally low (at most 1-5% of the soluble cellular protein). We have replaced the time consuming traditional purification techniques with an alternative 1 hour purification protocol that utilises a 6x-histidine-ubiquitin (H6-Ub) affinity tag that, following purification by immobilised metal affinity chromatography (IMAC), can be removed from the Rubisco without leaving addition residual amino acid fusions that might alter the Rubisco kinetics. Using a two plasmid expression system, the Rubisco operon (rbcL-rbcS) from a cyanobacterium is cloned downstream of the T7 promoter in plasmid pET30 and the rbcS gene cloned in frame to the 3' end of the H6-Ub gene located downstream of the trc promoter engineered into the compatible plasmid pACYC184. Both plasmids are transformed into E. coli BL21(DE3) and expression of L, S and H6-UbS induced with IPTG. Both S and H6-UbS assemble with the L8-cores producing hexadecamers that incorporate one or more H6-UbS subunits facilitating purification by IMAC. The H6-Ub sequence is subsequently removed with a 6x-histidine-ubiquitin protease (H6-UbP) producing bona fide L8S8. The contaminating H6-Ub and H6-UbP can then be removed by IMAC. We have used the technique to purify the Synechococcus sp. PCC7942, Synechococcus sp. WH8102 and Prochlorococcus marinus MED4 Rubiscos. A biochemical critique of the purification process and the kinetics of the purified Rubiscos will be presented. PS11.14 Using artificial evolution to uncover novel structure-function interactions remote from the Rubisco active site. O Mueller-Cajar (The Australian National University), S Whitney (The Australian National University) Despite decades of intensive research our understanding of the photosynthetic CO2-fixing enzyme Rubisco remains incomplete. In particular the role of residues outside the immediate vicinity of the active site remain obscure. We have developed and applied an evolution system based on metabolically engineering Escherichia coli towards Rubisco dependency. Evolution of both Form I and Form II Rubiscos towards increased fitness in this novel physiological role have yielded a large number of mutants that have interesting catalytic phenotypes, albeit none have been directed towards improving carboxylation efficiency or selectivity for CO2 over O-2. The evolved variants of the dimeric Form II enzyme from Rhodospirillum rubrum all showed similar kinetics and highlighted a hydrogen bond outside of the active site between the

conserved His-44 and Asp-117 residues that appears critical for maintaining CO2/O2 specificity. Using similar selection conditions numerous, but reproducibly selected, evolved variants of the Form I Synechococcus PCC6301 Rubisco have been isolated that show differing kinetics with respect to their affinities for substrate ribulose-P2 and sugar phosphate inhibitors. The versatility of selecting for evolved Rubisco variants in E. coli is proving to be a useful tool for highlighting catalytically relevant residues that would otherwise have remained obscure. PS11.15 Improving the throughput for generating and kinetically screening mutated tobacco Rubiscos R Sharwood (Australian National University), S Whitney (Australian National University) Rubisco plays a critical, frequently rate-limiting, role in photosynthesis. Rubiscos CO2-assimilation capacity is marred by its confused fixation of O2 and its slow catalytic performance compared to other enzymes of mainstream carbon and energy metabolism. Biotechnological efforts therefore continue to be devoted to improving the catalytic properties of Rubisco and engineer such improvements into crop plants. Higher plant Rubisco comprises eight large (L, coded by the plastome rbcL gene in the plastome) and eight small subunits (S, coded by multiple nuclear RbcS genes). The inability to assemble Rubisco from eukaryotes within Escherichia coli or Chlamydomonas reinhardtii frustrates mutagenic studies on higher plant Rubisco, necessitating the use of homologous plastome transformation methods to manipulate rbcL in planta (typically tobacco). Unfortunately obtaining mature tobacco transformants with only mutated rbcL copies (homoplasmic transformants) is a protracted process taking >5 months. Also the efficiency of obtaining homoplasmic lines is marred by frequent recombination events between wild-type and mutated rbcL copies in heteroplasmic tissue that normally removes the mutation while maintaining the selective marker gene (aadA). We have circumvented these problems by generating aadA-free tobacco-rubrum plants where tobacco rbcL has been replaced with the non-homologous rbcM gene from Rhodospirillum rubrum that codes a 100 kDa homodimeric Rubisco (L2). The throughput for producing and kinetically characterizing tobacco Rubisco mutants is augmented by the 5-fold improved transformation efficiency of tobacco-rubrum and an ability to selectively purify L8S8 Rubisco (550 kDa) from L2 thereby negating the need for obtaining homoplasmic plants before undertaking kinetic analyses. PS11.16 Installation of a Formaldehyde-Fixation Pathway of Methylotroph as a Bypass of Carboxylation Step of the Calvin Cycle in Higher Plants L Chen (Kunming University of Science and Technology), Z Song (Kunming University of Science and Technology ), F Yin (Kunming University of Science and Technology ), Z Pan (Kunming University of Science and Technology ), K Li (Kunming University of Science and Technology ), I Orita (Kyoto University), H Yurimoto (Kyoto University ), N Kato (Kyoto University ), Y Sakai (Kyoto University ), K Izui (Kinki University) Plants are now known to emit not only methanol (MeOH) but also methane (CH4) to atmosphere. Since CH4 is an eminent global warming gas, and MeOH and formaldehyde (HCHO) are environmental pollutants, it may be worthwhile if we could confer plants an ability of assimilating these compounds photosynthetically. As a first step to this end, we have been trying to install a bacterial ribulose monophosphate pathway by which HCHO is fixed on ribulose 5-phosphate to yield fructose 6-phosphate in plants. Because CH4 and MeOH are assimilated

235 14th Photosynthesis Congress - PS07 after being converted to HCHO in methano- and methylotrophs, respectively. Previously we succeeded in expressing the two bacterial enzymes of this pathway, namely hexulose 6-phosphate synthase (HPS) and 6-phosphohexulose isomerase (PHI), in the chloroplasts of Arabidopsis thaliana and tobacco. The resulting double transformants showed an enhanced capacity of HCHO assimilation and were endowed with strong tolerance to HCHO. To establish applicability of this strategy to wider range of plants, ornamental plants such as petunia and pelargonium were transformed with the fused HPS/PHI gene from Mycobacterium gastri MB19 in the present study. The transgenic ornamental plants displayed enhanced tolerance to both aqueous and gaseous HCHO. A 30-40% increase in the HCHO assimilation rate was also observed. The results indicate validity of the strategy for ornamentals and that the use of the fused HPS/PHI gene might be a quick way to produce functional ornamental plants which might be useful for phytoremediation of HCHO pollution. PS11.17 CA1P-Phosphatase: Regulation?

A

Ubiquitous

Role

in

Rubisco

M Parry (Rothamsted Research), P Madgwick (Rothamsted Research), J Andralojc (Rothamsted Research), J Loveland (Rothamsted Research), A Keys (Rothamsted Research), P Lee (Lancaster University), B Forde (Lancaster University), S Gutteridge (DuPont) The gene for CA1P-phosphatase (CA1Pase) from wheat has been cloned and expressed in E. coli, with an N-terminal his-tag. The resulting enzyme had a pH optimum, catalytic rate and substrate preference similar to the CA1Pase purified from P. vulgaris but with a higher affinity for the Rubisco inhibitor, CA1P. A high affinity of this enzyme for other inhibitors of Rubisco became apparent when Rubsico activity progressively increased in the presence of the CA1Pase, owing to the removal of a contaminant (pentodiulose bisphosphate, PDBP) in the RuBP. Since PDBP may also give rise to the phenomenon of Rubisco fallover, we propose that CA1Pase has a ubiquitous role in the removal of this inhibitor. The CA1Pase also promoted the reactivation of RuBP-inhibited and CA1P-inhibited Rubisco. Furthermore, whilst the CA1Pase activity of the his-tagged enzyme was unaffected by redox reagents (DTT, GSH or GSSG), forms of the enzyme in which the his-tag was either absent or separated from the true N-terminus by a linker region, were highly responsive to redox regulation. PS11.18 A new isoform of fructose-1,6-bisphosphate in chloroplasts A Serrato Recio (Estación Experimental del Zaidín), L Sandalio González (Estación Experimental del Zaidín), E Yubero Serrano (Universitario de Rabanales), J Muñoz Blanco (Universitario de Rabanales), J Caballero (Universitario de Rabanales), A Sancho (Estación Experimental del Zaidín), M Sahrawy Barragán (Estación Experimental del Zaidín) FBPase catalyses the breakdown of fructose-1,6-bisphosphate to fructose-6-phosphate and Pi [1]. Up until now two types of FBPase have been described in plant cells. In addition to the cytosolic enzyme (cyFBPase), which participates in gluconeogenesis and sucrose synthesis [2], another chloroplastidial isoform (cpFBPase) is also present in photosynthetic eukaryotes which displays a similar tertiary structure with the exception of an extra sequence of 20-30 amino acids holding three cysteines able to form disulfide bonds that can be reduced by plastidial thioredoxins [3][4]. Three FBPase cDNAs have been isolated from Fragaria x ananassa (strawberry). Two of the deduced aminoacid sequences showed a high identity to the already described cytosolic and chloroplastic isoforms but the third deduced polypeptide displayed similarity to a putative

chloroplastic FBPase but without the regulatory domain of the cpFBPase. One outstanding fact of this unknown isoform is the presence in its active site of a histidine residue instead of a highly conserved lysine. Enzymatic assays clearly evidenced a FBPase activity and specific polyclonal antibodies inmunodetected this new FBPase, that we called cpFBPaseII, in mesophyll chloroplasts. PS11.19 Assembly of supramolecular complexea if calvin cycle enzymes as mediated by the intrinsically unstructured protein CP12 F Sparla (University of Bologna), L Marri (University of Bologna), P Trost (University of Bologna), X Trivelli (University of Lille), L Gonnelli (University of Florence) CP12 is a small regulatory protein universally distributed in oxygenic photosynthetic organisms. In Arabidopsis thaliana, CP12 (isoform 2) is localized in chloroplasts where it leads to the assembly of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), two non-consecutive enzymes of the Calvin cycle. As a consequence of complex formation, both enzymes alter their kinetic properties according to the general regulation of photosynthetic carbon assimilation (Marri et al. (2005) Plant Phys 139: 1433-1443) In spite of four cysteine residues able to form two intramolecular disulfide bridges with different midpoint redox potentials (Em,7.9 -326±2 and -352±6 mV), CP12-2 is a member of the IUP (intrinsically unstructured proteins) family. NMR studies show that recombinant CP12-2 lacks a well defined three-dimensional structure, even under oxidizing conditions. In the presence of NAD and under oxidizing conditions CP12-2 and A4-GAPDH interact with a KD of 0.18 µM to form a binary complex with a (A4-GAPDH)-(CP12-2)2 stoichiometry, as determined by isothermal titration calorimetry and multiangle light scattering. Site specific mutants indicate that only the C-terminal disulfide of CP12-2 is involved in the interaction with A4-GAPDH, while the N-terminal disulfide is required for the formation of the ternary complex. Indeed, PRK interacts with the preformed binary complex (KD 0.17 µM) giving rise to a 500 kDa ternary complex with a [(A4-GAPDH)-(CP12-2)2–(PRK)]2 stoichiometry. The protein supercomplex is suggested to have a toric structure with A4-GAPDH tetramers bearing two binding sites for free CP12-2, and each PRK dimer bearing two binding sites for (A4-GAPDH)-(CP12-2)2 binary complexes. PS11.20 Structural basis of thioredoxin regulation in photosynthetic A2B2-glyceraldehyde-3-phosphate dehydrogenase F Sparla (University of Bologna), S Fermani (University of Bologna), G Falini (University of Bologna), P Martelli (University of Bologna), R Casadio (University of Bologna), P Pupillo (University of Bologna), A Ripamonti (University of Bologna), P Trost (University of Bologna) The major isozyme of chloroplast glyceraldehyde-3-phosphate dehydrogenase (AB-GAPDH) is a light-regulated, NAD(P)H-dependent enzyme involved in photosynthetic carbon reduction. The mechanism of AB-GAPDH regulation has been revealed at the molecular level by combining crystallographic data, site-specific mutagenesis and computational modelling. A and B-subunits of AB-GAPDH are 80% identical in amino acid sequence, except for an additional extension of 30 amino acids at the C-terminus of subunits B (CTE), which includes a

236 14th Photosynthesis Congress - PS07 pair of redox-active cysteines responsible for thioredoxin-mediated regulation. In fully active enzymes the pair of cysteines of the CTE are reduced and the CTE has no effect on enzyme activity. These active tetramers assume a distinctive crystallographic conformation with the 2’-phosphate group of bound NADP interacting with Arg77 and Ser188, and sulfate ions (deriving from the crystallization medium) occupying specific sites (P-sites) of the catalytic domain. Under oxidizing conditions a disulfide forms within the CTE, favoring the docking of the CTE in a cleft delimited by A and B-subunits through interactions between negative residues of the CTE and positive charges on the surface of the cleft. In this conformation, Arg77 of B-subunits is displaced away from the 2’-phosphate of NADP by interactions with anionic residues in the vicinity of the disulfide bond. As a consequence, the overall conformation of the tetramer is changed, occupation of P-sites by sulfate ions is different and the enzyme is inactivated.

PS11.21 Photosynthesis in veinal cells of C3 plants controls leaf morphology S Janacek (University of Cambridge), B Palmer (University of Sheffield), P Quick (University of Sheffield), J Hibberd (University of Cambridge) Photosynthetic cells surrounding the vascular bundle have been well characterised in C4 plants, due to the important function of bundle sheath cells in the C4 cycle. However, the role of photosynthesis in equivalent cells of C3 plants is less well understood. To investigate this, we have generated Arabidopsis lines in which photosynthesis has been removed specifically from cells adjacent to the vascular bundle. This has been achieved by combining an RNAi approach with the use of enhancer traps. Several lines with varying degrees of silencing have been generated. In all lines, removing photosynthesis from the veins leads to a change in leaf development such that a layer of palisade mesophyll cells is lost. In addition to this, the rate of plant growth is reduced and seed production is compromised. These data reveal that there is a critical role for photosynthesis in vascular cells of C3 as well as C4 species.

237 14th Photosynthesis Congress - PS07

PS12 - Starch and Sucrose PS12.1 Control of Sucrose and Starch Synthesis in Leaves. J Lunn (Max Planck Institute of Molecular Plant Physiology) Sucrose and starch are the main products of photosynthesis in most plants. Sucrose is synthesized in the cytosol and mostly exported from the leaves to supply sink organs with carbon and energy, whereas starch is synthesised in the chloroplasts and retained in the leaf to support respiration and sucrose export at night. The rates of sucrose and starch synthesis are coordinated with the rate of photosynthetic CO2 fixation by a relatively well characterized regulatory network, which involves allosteric control, protein phosphorylation and redox modulation of key enzymes in the pathway. The partitioning of photoassimilate between sucrose and starch can also respond to any imbalance between carbohydrate supply and demand in sink organs that leads to accumulation of sugars in the leaves, but the mechanisms involved are less well understood. It has recently been proposed that trehalose 6-phosphate (Tre6P) is involved in such feedback regulation, acting as a signal of sugar status in the cytosol that influences the rate of starch synthesis in the chloroplasts via redox modulation of ADPglucose pyrophosphorylase. Evidence is now emerging that Tre6P could also play an important role in control of metabolic and developmental processes in meristematic tissues, perhaps as a signal of sugar availability. Progress towards understanding the functions of Tre6P in control of photoassimilate partitioning in leaves and source-sink interactions will be presented.

PS12.2 The pathways and control of starch breakdown in Arabidopsis thaliana S Zeeman (ETH Zürich), J Chen (Institute of Molecular Biology, Academia Sinica), J Gatehouse (Durham University), A Smith (John Innes Centre), S Smith (University of Western Australia) Recent results provide new insight into the mechanisms of starch breakdown in Arabidopsis leaves at night. Surprisingly, a-amylase – the endoamylase usually associated with the initiation of starch breakdown is not required. Plants lacking a-amylase metabolise starch normally. However, multiple ß-amylases and debranching enzymes are involved in starch degradation. Mutations affecting the respective genes decrease starch degradation, causing elevated starch levels. The actions of ß-amylase and debranching enzymes yield maltose and maltotriose, consistent with previous observations that maltose is exported from the chloroplast via the chloroplast envelope maltose transporter (MEX1) and that the disproportionating enzyme metabolises maltotriose at night. ß-Amylase and debranching enzymes may act at the surface of the starch granule and be dependent on prior glucan phosphorylation by enzymes of the glucan, water dikinase class – known to be required for starch breakdown. Interestingly, mutants deficient in debranching enzymes accumulate soluble branched oligosaccharides – typical products of a-amylolysis. This suggests that a-amylase, though not required, can participate in starch degradation under some circumstances. The loss of a chloroplastic, glucan-binding phosphatase, SEX4, also causes excess starch accumulation. The targets of SEX4 may be proteins (e.g. starch catabolic enzymes) or the glucan itself. In this case, SEX4 would remove phosphates added by glucan, water dikinases. SEX4 is related to Laforin, a protein that controls glycogen metabolism in animals. In the absence of Laforin, starch-like polyglucosan bodies are formed. The similarities between the animal and plant mutant phenotypes suggest a common regulatory mechanism, which may be widely conserved or resulting from convergent evolution.

PS12.3 Glucan, water dikinase activity stimulates breakdown of granular starch by chloroplastic ß-amylases G Ritte (Plant Physiology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, Building 20, 14476 Potsdam-Golm, Germany), C Edner (Plant Physiology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, Building 20, 14476 Potsdam-Golm, Germany), J Li (ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, WA 6009, Australia), S Smith (ARC Centre of Excellence in Plant Energy Biology, Un Phosphate monoesterified to the C6- or the C3-position of glucosyl residues is a minor constituent of most starches. Glucan, water dikinase (GWD, formerly known as R1, EC 2.7.9.4) specifically phosphorylates the C6-position. The biosynthesis of the less frequent C3-phosphate esters is catalyzed by phosphoglucan, water dikinase (PWD, EC 2.7.9.5). Activity of PWD strictly relies on a preceding starch phosphorylation by GWD. The catalytic mechanism of both dikinases includes autophosphorylation of the enzyme. The β-P of ATP is firstly transferred to a conserved histidine residue and then to the (phospho)glucan. Transgenic or mutant plants in which the activity of GWD or PWD is reduced, display increased starch levels in their leaves, with the phenotype of the GWD-deficient plants, such as the Arabidopsis mutant sex1-3, being more severe. Obviously, the glucan phosphorylating enzymes are required for normal starch breakdown in leaves. We hypothesized that the activities of certain starch degrading enzymes rely on the presence of phosphate esters in starch and we aimed to purify such enzymes from Arabidopsis leaf extracts. By this approach, in combinantion with the analysis of purified recombinant enzymes, we show that breakdown of granular starch by plastidic β-amylases strongly increases if the starch is simultaneously phoshorylated by GWD. β-amylolytic attack in turn stimulates glucan phosphorylation by GWD. The interdependence between the activities of GWD and chloroplastic β-amylases offers an explanation for the starch excess phenotype of the GWD-deficient mutants.

PS12.4 The requirement for debranching enzymes in starch biosynthesis S Streb (Institute of Plant Sciences, ETH Zürich), T Delatte (Institute of Plant Sciences, ETH Zürich), S Zeeman (Institute of Plant Sciences, ETH Zürich) Starch is composed of branched and linear polymers of glucose. In amylopectin, the major polymer, a-1,4-linked glucose chains are connected by a-1,6-bonds (branches) to form a tree-like structure. Branches are arranged in way that allows the formation of double helices between chains. These pack into concentric layers, resulting in semi-crystalline starch granules. Three classes of enzymes synthesise amylopectin: starch synthases, branching enzymes and, surprisingly, debranching enzymes. Two genes (ISA1 and ISA2) encode subunits of a single isoamylase-type debranching enzyme. Mutation of either gene in Arabidopsis results in the partial replacement of starch with phytoglycogen – a highly branched soluble glucan with properties similar to glycogen. Isoamylase may remove branch points that prevent crystallisation during synthesis. However, some cells still make starch granules in these mutants, suggesting that other factors - possibly the

238 14th Photosynthesis Congress - PS07 other debranching enzymes, ISA3 and limit dextrinase - enable amylopectin synthesis. We made and analysed all double-, triple- and quadruple-mutant combinations. With all debranching enzyme activities missing, starch granules are no longer visible. However, insoluble glucans are still measurable and small particles are visible alongside phytoglycogen inside chloroplasts using TEM. These glucans are abnormal, branched glucans, which are only partially mobilised during the night, suggesting that degradation is blocked by the presence of branch points. These results support the idea that debranching enzymes play a key role in glucan biosynthesis but leave the possibility that glucans synthesized in their absence can still form insoluble particles. Analysis of these insoluble glucans is ongoing. PS12.5 Association mapping within Arabidopsis accessions to identify regulation networks in the primary metabolism. Development of a rapid and sensitive non-radioactive assay for ribulose-1,5-bisphosphate carboxylase R Sulpice (Max Planck Institute of Molecular Plant Physiology), Y Gibon (Max Planck Institute of Molecular Plant Physiology), H Tschoep (Max Planck Institute of Molecular Plant Physiology), B Usadel (Max Planck Institute of Molecular Plant Physiology), M Steinhauser (Max Planck Institute of Molecular Plant Physiology), M Höhne (Max Planck Institute of Molecular Plant Physiology), M Guenther (Max Planck Institute of Molecular Plant Physiology), H Wituka-Wall (Institute of Biochemistry and Biology) To unravel regulatory networks controlling the primary metabolism, we use natural variation as a starting point, and combines cutting edge genotyping tools with the use of a phenotyping platform for primary metabolism. The analysis was performed with 130 lines grown in short days and low light (C-limiting) growth conditions. Plants were analysed for growth, central metabolites, major structural components and 39 enzyme activities in primary metabolism. Considering the number of determinations required for such a study, a new non-radioactive microplate-based assay, which sensitivity and convenience compare favourably with radioisotopic assays, was developed. One aim of the study was to search for across this wide population for correlations between different physiological and metabolic parameters. This could give insights into the regulatory networks controlling metabolism and growth, as well as inter-connections between compartments and pathways. One of the emerging results is that many metabolites (especially starch and amino acids) show a negative trend with growth, whereas some enzymes show a weak positive relation with growth. This indicates that one key component of the ‘fast growth’ character is increased fluxes and consumption of resources, which may in part be driven by increased activities of enzymes in central metabolism. Another aim was to investigate whether fine mapping of QTLs would be possible using association mapping. Association tests performed yielded 34 marker effects significant at at Bonferoni corrected p < 0.05. Some QTLs for growth, enzymes or metabolites co-associated, indicating they may identify genes with higher order regulatory roles. PS12.6 Effects of cytosolic FBPase on photosynthetic carbon metabolism under high CO2 conditions M Tamoi (Dept. Adv. Biosci., Grad. Sch. Agr., Kinki Univ., CREST, JST), Y Hiramatsu (Dept. Adv. Biosci, Grad. Sch. Agr. , Kinki Univ.), S Nedachi (Dept. Adv. Biosci, Grad. Sch. Agr. , Kinki Univ.), T Tabuchi (CREST, JST), K Otori (CREST, JST), S Shigeoka (Dept. Adv. Biosci., Grad. Sch. Agr., Kinki Univ., CREST, JST) Carbon partitioning of photosynthetic products is thought to be regulated

by capacity of sucrose synthesis. Sucrose can either be utilized by glycolysis or be translocated to sink organ and converted to starch for storage and used at a later time. The key regulatory steps of sucrose biosynthesis are supposed to be the dephosphorylation of fructose 1,6-bisphosphate catalyzed by fructose-1,6-bisphosphatase (FBPase) and/or the synthesis of sucrose-6-phosphate from UDP-glucose and fructose 6-phosphate catalyzed by sucrosephosphate synthase (SPS). To clarify the contribution of cytosolic fructose-1,6-bisphosphatase to the photosynthetic carbon metabolism, we generated transgenic tobacco plants expressing cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in cytosol (TcFS) and then analyzed photosynthetic characteristics under normal and high CO2 conditions. In TcFS plants, lateral shoot, leaf number and fresh weight were increased compared with those in the wild-type plants under high CO2 (1200 ppm) conditions. TcFS showed increased photosynthetic activity compared with that in the wild type. The levels of hexose of wild type accumulated in upper leaves, while levels of sucrose and starch of TcFS plants accumulated in lower leaves and lateral shoots. These findings suggest that the cytosolic FBPase contributes to efficient conversion of hexose into sucrose, and that the change of the carbon partitioning affects photosynthetic capacity, hormone biosynthesis and morphogenesis. PS12.7 Sugar regulation of the redox state in chloroplast. J Barajas-López (Estación Experimental del Zaidín), A Serrato (Estación Experimental del Zaidín), A Chueca (Estación Experimental del Zaidín), M Sahrawy Barragán (Estación Experimental del Zaidín) f and m are known to regulate several Calvin cycle enzymes controlling the sugar metabolism (Miginiac-Maslow, M., et al. 2000, Physiol. Plantarum 3: 322-329). It has also been shown that plastidial Trxs are present in heterotrophic tissues where the Trxs may function in different processes. Moreover, redox regulation is emerging as another important post-translational mechanism in sugar control of plant metabolism (Tiessen, A., et al. 2003, Plant J. 4: 490-500). In this work we study the sugar dependent regulation of Trxs by analysing the expression pattern of these proteins when pea plants are fed with different sugars. We show that sucrose induce a lower photosynthesis rate as well as a decrease of mRNA and protein level for both Trxs. The sequence of Trx f and m promoters shows several Dof-like elements that are likely related to the transcriptional regulation of enzymes involved in carbon metabolism. PS12.8 Protein phophatases required for normal starch degradation in leaves A Smith (John Innes Centre), S Comparot-Moss (John Innes Centre), O Kötting (John Innes Centre), S Zeeman (ETH Zürich, Universitätstrasse 2) Starch degradation in leaves at night provides a continuing supply of sucrose to the rest of the plant. Although the understanding of the pathway is growing, little is known about its regulation. Mutants unable to degrade part or all their starch at night provide a valuable tool for tackling the problem. We have recently described a regulatory protein encoded by the gene at the SEX4 (Starch Excess 4) locus. This protein encodes a putative dual-specificity protein phosphatase and has a domain resembling the starch-binding domain CBM_20. We have now identified a gene encoding a protein very similar to SEX4 which also appears to be required for starch degradation by night. This protein is localised in the chloroplast and we present here a characterization of its knock-out mutant.

239 14th Photosynthesis Congress - PS07 The identification of regulatory proteins having an impact on the degradation of starch in Arabidopsis leaves raises exciting possibilities for understanding the tight control of this mechanism. We discuss here the possible role of these putative dual-specificity protein phosphatases in starch degradation in Arabidopsis leaves.

240 14th Photosynthesis Congress - PS07

PS13 - Interactions between Electron Transport and Stromal Reactions PS13.1 Coupled Regulation of Cyclic Electron Flow around PSI with Photosynthesis - Its Contribution to Non-photochemical quenching Evidenced with Transplastomic Tobacco Plants over-expressing Ferredoxin in Chloroplasts

not resemble damages. We conclude that a novel type of PSI modification, caused by double reduction of A1, acts to prevent oxidative damages. The possibility that the high redox pressure between PSII and PSI induces an extreme form of light acclimation in the mutant plants is discussed.

C Miyake (Research Institute of Innovative Technology for the Earth) We tested the hypothesis that ferredoxin (Fd) limits the activity of cyclic electron flow around PSI (CEF-PSI) in vivo and the relief of its limitation causes non-photochemical quenching (NPQ) of Chl fluorescence to be enhanced. To prove this hypothesis, we made a transplastomic plant expressing Fd, originated from Arabidopsis thaliana, in chloroplasts of tobacco leaves. In trasplastomic plants, minimum yield of Chl fluorescence (Fo) was higher than that in wild-type plants. Its increase in Fo was suppressed to the level of wild-type plants on the illumination of far-red light to leaves of transplastomic plants. Furthermore, oxidation rate of P700 by far-red light was suppressed, compared to wild-type. These results implied the enhanced donation of electrons by Fd-quinone oxidoreductase (FQR) from stroma in chloroplasts to plastoquinone in transplastomic plants. That is, it is assumed that transplastomic plants had a higher activity of CEF-PSI more than wild-type plants did. In fact, the activity of CEF-PSI in transplastomic plants became higher at the limited activity of photosynthetic linear electron flow in chloroplasts than wild-type plants. Furthermore, NPQ of Chl fluorescence was enhanced in transplastomic plants, compared to wild-type plants. On the other hand, pool size of xanthophylls-cycle pigments and the amount of PsbS protein were almost the same each other between them. These results support our hypothesis strongly. Strengthening NPQ of Chl fluorescence by the enhancement of CEF-PSI activity can be a strategy for making plants to have tolerance against abiotic stress, where light utilization-efficiency as observed in F(PSII) is low.

PS13.2 High light acclimation in transgenic potato (Solanum tuberosum L. var. Desirée) plants with decreased leaf-ferredoxin content. J Backhausen (Universität Osnabrück), I Voss (Universität Osnabrük), S Holtgrefe (Universität Osnabrück), R Scheibe (Universität Osnabrück) The effect of increasing light intensity was analyzed in transgenic potato plants with reduced leaf-ferredoxin content. After a transfer of low-light acclimated plants into high light, the Fd-underexpressing mutants rapidly developed an elevated PSII-back pressure, but the stroma remained largely oxidized. The CO2-assimilation rate declined steadily until after one week negative values were reached. Due to the lack of ferredoxin in the transgenic plants, a high rate of O2 reduction was expected. However, by applying various methods, no indications for oxidative stress were found. Measurements of the near-infrared absorbance changes indicated that the ability to oxidize P700 disappears in parallel with the loss of CO2 fixation, while the composition of PSI remained unchanged. The major differences between wildtype and mutant thylakoids during the initial stages of the high-light treatment were the portion of ferredoxin-NADP+ oxidoreductase bound to the cyt b/f complex, and the PSII-phosphorylation pattern. With prolonged treatment, the plants continuously lost Chl a and Chl b, and LHCII shifted from a trimeric into a monomeric state. Otherwise, the thylakoid composition was unchanged, and the apparent loss of Chl was caused by a decromposition of complete thylakoids. All changes in the ferredoxin-antisense plants can be divided into subsequent stages, and do

PS13.3 Characterization of the ternary complex formed by ferredoxin:thioredoxin reductase, ferredoxin and thioredoxin m. D Knaff (Texas Tech University), X Xu (Leiden University), M Ubbink (Leiden University), P Schürmann (University of Neuchatel), S Kim (Texas Tech University) Ferredoxin:thioredoxin reductase (FTR), catalyzes the two-electron reduction of thioredoxins in chloroplasts and cyanobacteria (thioredoxins f and m and perhaps other thioredoxins are substrates), using reduced ferredoxin as the electron donor. Reduced thioredoxins then play important roles in redox regulation. FTR, a heterodimer with a unique [4Fe-4S] cluster as its sole prosthetic group, has a single binding site for ferredoxin and a separate single binding site for thioredoxin. NMR spectroscopy was used to map the binding site on ferredoxin for FTR in a 1:1 complex of the two proteins. A mono-gallium analog of this [2Fe-2S] ferredoxin was obtained by reconstituting apo-ferredoxin in a gallium-containing refolding buffer. The use of this diamagnetic Ga structural analog eliminates the paramagnetic broadening of NMR resonances of amino acids in the vicinity of the [2Fe-2S] cluster in native ferredoxin. This has allowed the first complete mapping of the interaction interface of a [2Fe-2S] ferredoxin for a target enzyme. NMR spectroscopy was also used to map the interaction domain for FTR on thioredoxin m in a 1:1 complex of the two proteins. Both similarities and differences are seen in the thioredoxin m interaction domain for FTR in the non-covalent complex examined by NMR and in a disulfide-linked covalent complex of FTR and thioredoxin m for which an X-ray crystal structure has been obtained. NMR has also been used to characterize a ternary complex between ferredoxin, FTR and thioredoxin m in solution, confirming the presence of separate binding sites on FTR for its two substrates.

PS13.4 The Impact of Ferredoxin and Ferredoxin:NADP(H) Oxidoreductase on Photosynthetic Electron Channeling G Hanke (Osaka University, Japan), Y Kimata-Ariga (Osaka University, Japan), T Hase (Osaka University, Japan) IN metabolism.In the last step of the photosynthetic electron chain, the soluble electron transfer protein ferredoxin (Fd) receives electrons from PSI and donates them to the enzyme Fd:NADP(H) reductase (FNR). The reaction catalysed by FNR therefore determines whether electrons will be available to stromal metabolism as reduced Fd, or as NADPH. Both Fd and FNR are represented in higher plant genomes and EST collections as multiple iso-proteins. Many stromal enzymes are dependent on Fd as a source of reducing power and we have previously shown that Fd iso-proteins from maize and Arabidopsis have variable affinity for different Fd-dependent enzymes. In this talk we will present further results of work on maize and Arabidopsis using recombinant proteins, and mutant and transgenic plants, which reveal Fd and FNR iso-protein specific channeling of photosynthetic electrons into different

241 14th Photosynthesis Congress - PS07 areas of metabolism. PS13.5 The role of PGR5 in redox poising of photosynthetic electron transport B Nandha (Uuniversity of Manchester) The pgr5 mutant of Arabidopsis thaliana has been described as being deficient in cyclic electron flow around photosystem I, however the precise role of the PGR5 protein remains unknown. In this study the electron transport of pgr5 was examined in intact leaves and found to perform cyclic electron flow, at a rate similar to the wild type. PGR5 is not therefore, essential for cyclic flow. Exposure of both wild type and pgr5 leaves to high light or low CO2 concentrations resulted in enhancement of cyclic electron flow. At low CO2, pgr5 and wt leaves were similarly capable of performing high energy state quenching, a pH dependent process linked to cyclic flow. The pgr5 mutant is therefore, affected in the redox poising of the chloroplast stroma. This results in defective feedback regulation of photosynthetic electron transport and in reduced efficiency of cyclic electron flow. PS13.6 Functional analysis of the luminal thioredoxin-like protein, HCF164. K MOTOHASHI (CRL, Tokyo Tech., ATP system, ERATO, JST), M YOSHIDA (CRL, Tokyo Tech., ATP system, ERATO, JST), T HISABORI (CRL, Tokyo Tech., ATP system, ERATO, JST) In higher plant chloroplasts, reducing equivalents are produced by the photosynthetic electron transport system and predominantly accumulated in the stroma mainly as NADPH. However, a part of them are used for the redox regulation of the thiol enzymes, which are located in the stroma side. Interestingly, in the chloroplasts, there is a thioredoxin like protein, HCF164, which is a membrane anchored thioredoxin-like protein faced to thylakoid lumen side. In this study, we found that chloroplast thioredoxin-m is the source of reducing equivalents for reduction of HCF164. This finding provides strong evidence that higher plant chloroplasts possess a trans-membrane reducing equivalent transfer system similar to that found in bacteria. We then screened the reducing equivalent acceptor proteins from HCF164 using a resin-immobilized HCF164-single cysteine mutant, and successfully identified the candidate proteins in the thylakoids. We found PSI-N subunit of photosystem I as a target candidate protein, and confirmed its reduction by HCF164 both in vitro and in isolated thylakoids. In addition, two components of the cytochrome b6f complex, the cytochrome f and Rieske FeS proteins, were also captured as novel potential target proteins by our affinity chromatography method. Based on these findings, we discuss the reducing equivalent transfer mechanism to HCF164 from the stroma side across the thylakoid membrane and the physiological role of HCF164 in the thylakoid lumen as well. PS13.7 Structural and functional characterization of leaf-type ferredoxin-NADP+-oxidoreductase isoforms in Arabidopsis thaliana P Mulo (University of Turku), M Lintala (University of Turku) Ferredoxin-NADP+-oxidoreductase (FNR) is an enzyme catalysing the final step of linear electron transfer reducing NADP+ to NADPH. In Arabidopsis thaliana, the chloroplast targeted FNR enzyme exists as two isoforms, AtLFNR1 and AtLFNR2, encoded by two distinct nuclear genes. Both isoforms were evenly distributed between the thylakoids and

soluble stroma, and they were separated by 2-D electrophoresis in four distinct spots, suggesting post-translational modification. We have characterized knock-out mutants of both isoforms in order to reveal their functional specificity. Absence of either one isoform resulted in reduced size of the rosette with pale green leaves, which was accompanied by a low chlorophyll and LHC protein content. Also the PSI/PSII ratio was significantly lower in the mutants, although the PSII activity, measured as FV/FM ratio, remained nearly unchanged. Slow re-reduction rate of P700 measured in the mutant plants suggests that both isoforms are involved in PSI-dependent cyclic electron flow. Impaired function of FNR also resulted in decreased capacity of carbon fixation whereas nitrogen metabolism was up-regulated, detected as changes in the levels of nitrate transporter and nitrate reductase transcripts and as increased accumulation of nitrite in the leaves. In the absence of AtLFNR1, we found AtLFNR2 exclusively in the stroma, suggesting that AtLFNR1 is required for membrane attachment of FNR. Structural modeling supports the formation of AtLFNR1-AtLFNR2 heterodimer that would mediate the membrane attachment of AtLFNR2. Dimer formation, in turn, might regulate the distribution of electrons between the cyclic and linear electron transfer pathways according to environmental cues. PS13.8 Properties and physiological function of a super complex of NDH-1 in the Cyanobacterium Synechocystis sp. Strain PCC 6803 H Mi (National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) Weimin Ma, Yong Deng, Teruo Ogawa and Hualing Mi * National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, SIBS, 300 Fenglin Road, Shanghai, 200032, PR China In cyanobacteria, the type-1 NAD(P)H dehydrogenase (NDH-1) contains at least 15 subunits (NdhA-O; Herranen et al. 2004, Prommeenate et al. 2004, Zhang et al. 2004, Battchikova et al. 2005), which are encoded by genes homologous to the chloroplast and mitochondrial ndh genes (Ohyama et al. 1986, Kaneko et al. 1996). A hydrophilic subcomplex with a molecular mass of about 380 kDa active in NADPH oxidation has been isolated from the cyanobacterium Synechocystis PCC 6803 by chromatography of the cell homogenate (Matsuo et al. 1998). By improving their method, Deng et al. (2003c) isolated a low CO2-inducible NDH-1 complex of about 380 kDa that contains the NdhA subunit and is active in NADPH oxidation. Recently, three NDH-1 complexes have been identified in Synechocystis 6803 (Herranen et al. 2004, Prommeenate et al. 2004, Zhang et al. 2004) and Thermosynechococcus elongatus (Zhang et al. 2005) by blue native (BN)-PAGE. The NDH-1 complex with a molecular mass of about 550 kDa in maize chloroplasts forms dimers of 1,000–1,100 kDa and splits into 300 and 250 kDa subcomplexes as analyzed by BN-PAGE combined with mass spectrometry (Darie et al. 2005). However, none of these complexes in cyanobacteria and chloroplasts showed NADPH oxidation activity. In this work, we identified NDH-1 complexes of Synechocystis 6803 active in NADPH oxidation. We compare the wild-type (WT) and mutant strains of Synechocystis 6803 for the presence of these active complexes and the response of their activity to high or low CO2. A supercomplex of NDH-1 (about 1,000 kDa) was first identified and was suppressed under low CO2. A possible physiological function for active NDH-1 complexes is discussed.

242 14th Photosynthesis Congress - PS07 PS13.9 Overexpression of Ferredoxin in Tobacco Chloroplasts stimulates Cyclic Electron Flow around Photosystem I (CEF-PSI) and enhances Non-Photochemical Quenching (NPQ) of Chl Fluorescence. H Yamamoto (Research Institute of Innovative Technology for the Earth), C Miyake (Research Institute of Innovative Technology for the Earth) Recent studies indicated that under stressful conditions, CEF-PSI contributes to the formation of ΔpH across the thylakoid membranes, and induces NPQ of Chl fluorescence to dissipate excess photon energy as heat. In CEF-PSI, electron flow from PSI to plastoquinone was proposed to be mediated by two enzymes; NADPH dehydrogenase and ferredoxin:quinone oxidoreductase (FQR). However, a molecular mechanism of CEF-PSI has not been fully understood. In this study, we tested the hypothesis that ferredoxin (Fd) limits the activity of CEF-PSI in vivo and that the relief of this limitation promotes the NPQ of Chl fluorescence. In transplastomic tobacco expressing Fd from Arabidopsis in its chloroplasts, the minimum yield of Chl fluorescence (Fo) was higher than in the wild type. Fo was suppressed to the wild-type level upon illumination with far-red light, implying that the backflow of electrons by FQR from the chloroplast stroma to plastoquinone was enhanced in transplastomic plants. The activity of CEF-PSI became higher in transplastomic than in wild-type plants under conditions limiting photosynthetic linear electron flow. Similarly, NPQ of Chl fluorescence was enhanced in transplastomic plants. On the other hand, pool sizes of the pigments of the xanthophyll cycle and the amounts of PsbS protein were the same in all plants. Furthermore, stimulation of CEF-PSI did not affect the rate of photosynthesis and growth of plants. All these results supported the hypothesis strongly. PS13.10 A genetic screen for the identification of factors involved in non-photochemical PQ reduction in Chlamydomonas reinhardtii. B Ghysels (CEA Cadarache/ CNRS/ Aix Marseille II University), I Reiter (Cadarache/ CNRS/ Aix Marseille II University), M Havaux (Cadarache/ CNRS/ Aix Marseille II University), S Cuiné (Cadarache/ CNRS/ Aix Marseille II University), N Bécuwe (Cadarache/ CNRS/ Aix Marseille II University), B Genty (Cadarache/ CNRS/ Aix Marseille II University), L Cournac (Cadarache/ CNRS/ Aix Marseille II University), G Peltier (Cadarache/ CNRS/ Aix Marseille II University) Alternative electron transfer pathways such as chlororespiration and cyclic electron transfer around photosystem I (PSI) rely on non-photochemical reduction of plastoquinones (PQ) by stromal electron donors. In higher plant chloroplasts, PQ reduction is mediated by the NDH complex, but the chloroplast of Chlamydomonas reinhardtii lacks such a complex. Nevertheless, this alga shows significant cyclic electron transport activity and is able to produce H2 from stromal reductants by a PSI-dependent process, thus indicating the existence of non-photochemical PQ reduction. In order to identify genes encoding the principal actors involved in this reaction, we screened for C. reinhardtii insertion mutants with altered chlorophyll a fluorescence signature during a light-to-dark transition. In analogy with higher plants, we observed in C. reinhardtii a transient rise of (F0) fluorescence right after actinic illumination was switched off, which is linked with (non-photochemical) reduction of PQs in the dark . Using a fluorescence imaging system, we isolated 15 photo-autotrophic mutants with altered fluorescence signature, which were further classified into 4 different groups. One of these groups consists of mutants with an altered pattern of the F0 rise upon light to dark transition and includes a mutant in which no F0 rise could be detected. Further characterization of this mutant showed that it performed linear photosynthetic electron transfer like the

WT but that cyclic electron transfer around PSI and NADH-dependent PQ reduction were affected. Molecular and genetic characterization of the mutants is ongoing. PS13.11 Excess light-induced molecular responses of chloroplast Rieske FeS protein S Sanda (NAIST), K Yoshida (NAIST), Y Nakajima Munekage (NAIST), K Akashi (NAIST), A Yokota (NAIST) In higher plants, photosynthetic conversion of light energy into chemical energy is achieved through the electron transport chain on the thylakoid membranes of chloroplasts. Under drought/strong light stress, limited entry of CO2 leaves causes the suppression of photosynthetic carbon fixation, and the electron transport chain is prone to be over-reduced, leading to the oxidative damages in photosynthetic apparatus. To elucidate the regulation of photochemistry under such conditions, we have analyzed the change in the proteome of the membrane fraction of wild watermelon leaves in response to the drought stress. Two-dimentional electrophoresis and Western blot analysis have revealed that there were multiple spots corresponding to the chloroplast Rieske protein, a subunit of the cytochrome b6f complex, with very similar molecular weights but with different isoelectric points. Furthermore, some new spots with more acidic pI appeared under both drought and strong light stress, but they disappeared upon resuming irrigation or shifting to normal light condition. Southern blot analysis showed that the chloroplast Rieske protein is encoded by a single copy gene in wild watermelon genome. In addition, the amount of the Rieske protein in wild watermelon leaf did not change under the stress. These results suggest that the Rieske protein is post-translationally modified when absorption and utilization of light energy are imbalanced. We discuss the involvement of the modified Rieske protein in the regulation of electron transport under excess light stress. PS13.12 Interaction between electron transport and stromal reactions G Hanke (Osaka University), Y Kimata-Ariga (Osaka University), T Hase (Osaka University) In the last step of the photosynthetic electron chain, the soluble electron transfer protein ferredoxin (Fd) receives electrons from PSI and donates them to the enzyme Fd:NADP(H) reductase (FNR). The reaction catalysed by FNR therefore determines whether electrons will be available to stromal metabolism as reduced Fd, or as NADPH. Both Fd and FNR are represented in higher plant genomes and EST collections as multiple iso-proteins. Many stromal enzymes are dependent on Fd as a source of reducing power, and we have previously shown that Fd iso-proteins from maize and Arabidopsis have variable affinity for different Fd-dependent enzymes. In this talk we will present further results of work on maize and Arabidopsis using recombinant proteins, and mutant and transgenic plants, which reveal Fd and FNR iso-protein specific channeling of photosynthetic electrons into different areas of metabolism. PS13.13 Regulation of alternative electron transfer pathways in Arabidopsis chloroplasts studied in vivo by the ‘afterglow’ chlorophyll luminescence A C Cazalé (CEA Cadarache), D Rumeau (CEA Cadarache), M Havaux (CEA Cadarache) Alternative electron transfer pathways, such as chlororespiration and

243 14th Photosynthesis Congress - PS07 cyclic electron transport around PSI, catalyze the non-photochemical reduction of plastoquinones from stromal electron donors via two different routes. One of them, the ferredoxin-plastoquinone oxidoreductase (FQR) activity, is sensitive to antimycin. The second one, insensitive to this chemical, is mediated by NAD(P)H and involves the chloroplastic NAD(P)H-dehydrogenase complex (NDH). We used a chlorophyll luminescence signal, called afterglow luminescence (AG), to measure both pathways in Arabidopsis leaves and to study their regulation by the environmental conditions. The AG is a long-lived luminescence induced in plants by far-red light, which results from a back-flow of electrons from stromal reductants to oxidized PSII centers, leading to the light-emitting S2/3 QB- states. This signal can be conveniently measured in leaf discs by thermoluminescence as a band peaking at around 40°C, thus providing a unique tool to probe in vivo cyclic/chlororespiratory electron flows in plants. This technique is particularly useful in C3 plants whose cyclic PSI activity is difficult to assess in vivo by conventional methods. We observed that non-photochemical plastoquinone reduction is subjected to complex regulations under changing environmental conditions, which are reflected by modifications of the amplitude and/or the peak temperature of the AG band. We are currently investigating those regulatory processes by combining this biophysical approach with genetical and pharmacological tools. Interestingly, we found that the relative contributions of the FQR and NDH pathways to plastoquinone reduction vary markedly with the environmental light conditions. PS13.14 Protection of photosystem I against photoinhibition by NADP+/NADPH ratio

synthesised, and how quickly, the individual chloroplast or mitochondrion can ensure the most effective operation of its electron transport chain (Allen et al. 2005). The results shown, indicate that if the redox poise of isolated chloroplasts and mitochondria is altered, giving reducing or oxidising conditions, the proteins synthesised are different subsets of the whole.The redox state of specific electron carriers may therefore regulate expression of specific genes in chloroplasts and mitochondria, which is consistent with the original hypothesis It is also shown that phosphorylation of proteins is also influenced by the redox potential of the organelles. Under reducing or oxidising conditions, different proteins are phosphorylated, indicating levels of post translational control.Some proteins are only phosphorylated under extremely oxidising conditions, and some under reducing, indicating a response to redox stress. Components of the intra-chloroplast redox signalling pathway may themselves be chloroplast phosphoproteins. Allen J. F. (1993) Control of gene expression by redox potential and the requirement for chloroplast and mitochondrial genomes. J Theor Biol 165: 609-631 Allen, J. F., Puthiyaveetil, S., Ström, J. and Allen, C. A. (2005) Energy transduction anchors genes in organelles. BioEssays 27: 426-435 PS13.16 Cyclic electron transport of photosystem I in plants under heat stress probed by “after-glow” luminescence A Gauthier (Université du Québec à Trois-Rivières), R Carpentier (Université du Québec à Trois-Rivières) )

chilling-induced

D Joly (Université du Québec à Trois-Rivières), S Govindachary (Université du Québec à Trois-Rivières), C Bigras (Université du Québec à Trois-Rivières), J Harnois (Université du Québec à Trois-Rivières), R Carpentier (Université du Québec à Trois-Rivières) This study evidences the enhanced electron flow from the stromal compartment of the photosynthetic membranes to P700+ via the cytochrome b6/f complex (Cyt b6/f) in leaves of Cucumis sativus L. submitted to chilling-induced photoinhibition. The above is deduced from the P700 oxidation-reduction kinetics studied in the absence of the control by linear electron transport from water to NADP+, cyclic electron transfer mediated through Q-cycle of the Cyt b6/f and charge recombination in photosystem I (PSI). A protective mechanism is proposed by integrating a recent model of the cyclic electron flow in C3 plants based on the Cyt b6/f structural data and photoprotective functions elicited by low NADP+/NADPH ratio. Over-reduction of the photosynthetic apparatus results in the accumulation of NADPH in vivo to prevent NADP+ induced reversible conformational changes in PSI and its extensive damage. The excess electrons from the NADPH pool are routed via the Ndh complex in a slow process to maintain moderate reduction of the plastoquinone pool and redox poise required for the operation of ferredoxin:plastoquinone reductase mediated cyclic flow. PS13.15 Redox Effects on Chloroplast Protein Synthesis and Phosphorylation C Allen (Queen Mary, University of London) It has been proposed (Allen 1993) that the reason that chloroplasts and mitochondria have retained their own genetic system is to be able to respond quickly to changes in their internal redox state. This is necessary to minimise the production of free radicals, which have the potential to damage and even destroy the cell. By controlling which proteins are

During photosynthesis, light energy is converted into ATP and NADPH through coordinated function of photosystem I (PSI) and II (PSII). Under physiological conditions, this electron transport (ET) from water to NADP+ is catalyzed by ferredoxin-NADP+-reductase (FNR). Alternative ET pathways includes the cyclic ET to plastoquinone (PQ) from reduced Fd mediated by ferredoxin-plastoquinone reductase (FQR) or NAD(P)H dehydrogenase (Ndh) complex involving PSI alone. Despite the roles of cyclic pathways are partially understood, it is suggested that they confer protection to photosynthetic apparatus under physiological stress. This is studied here in leaves of Helianthus annuus L. and Cucumis sativus L., exposed to heat stress by monitoring far-red light (FR-light) induced afterglow (AG) luminescence. Upon pre-illumination of leaves with FR-light, thermoluminescence curves showed a major peak at 45oC (AG-band) ascribed for the electron flow from stromal reductants to PQ with a faint shoulder at 25-27oC (B-band) arising from recombination of S2+3QB- pairs. With heat treatment (60 s), the magnitudes of these bands declined and their peaking temperatures downshifted to ~20oC and ~41oC, respectively due to the loss of PSII donor side partners for QB as the S-state advancement is severely impeded. The activation energy (EA) for B-, and AG-band are also significantly decreased by about 18% and 40%. Besides, the ratio of AG/B band emission remarkably increased to ~2.91 from ~1.99. This corroborates the accelerated donation of electrons mediated by Ndh or antimycin-sensitive FQR pathway. These results will be discussed in the present communication. PS13.17 Modelling photosynthesis irradiance curves J Harbinson (Wageningen University) The modeling and simulation of photosynthesis/irradiance responses is poorly developed in comparison to photosynthesis/CO2 concentration responses. Often the strategy adopted is to use the non-mechanistic rectangular hyperbola construction of Thornley and Johnston. It would be better if a model existed that had mechanistic foundations. It is relatively easy to build a model electron transport through photosystem I, and some years ago we described such a model for FPSI

244 14th Photosynthesis Congress - PS07 irradiance curves. This model was composed of parameters that reperesented the rate constant for P700 excitation and oxidation per unit irradiance, the rate constant for P700+ reduction, the size of high potential donor pool per unit P700, and the equilibrium constant between the reduced high potential pool and P700+. It has been experimentally shown that FPSI and the relative rate of PSI electron transport is usually closely related the corresponding parameter for PSII or CO2 fixation. It therefore seemed likely that a PSI based model could be adapted for use in simulating or fitting PSII and CO2 irradiance responses. Adapting the PSI model for PSII is simple and allows measured PSII irradiance responses to be fitted and, within certain limits, to be simulated. Even CO2 fixation/irradiance responses, which in principle can have complicated relationships to photosynthetic electron transport, can often be accurately fitted by a simple variant of the basic PSI electron transport model. The integration of these models with the well established biochemically based models of C3 photosynthesis will hopefully yield an improved means for modeling and simulating photosynthesis.

comparative analysis of wt strains and mitochondrial mutants of tobacco (CMS2, lacking complex I) and of chlamydomonas (dum22, lacking both complex I and III). In both organisms, inhibition of mitochondrial activity enhances the stromal reducing pressure in the dark. This is accompanied by a slowing down of CO2 assimilation at the onset of illumination, which is paralleled by a transient but substantial rerouting of electrons towards alternative sinks, and by an increased ?pH generation. Interestingly, the same changes in photosynthesis have different effects on the photoprotective response (NPQ) of the two organisms. In plant mutants, the increased ?pH mainly drives a strategy based o n the stabilisation of qE, which is otherwise rapidly reversible in the wt. Conversely only a small reversible qE is seen in Chlamydomonas mutants, where most of the NPQ is driven by redox-mediated changes in the PSII antenna size (state transitions).

PS13.18 Chl a fluorescence and 820 nm transmission changes occurring during a dark-to-light transition in pine needles and pea leaves: a comparison

D Rumeau (CEA/CNRS Cadarache), G Peltier (CEA/CNRS Cadarache), M Havaux (CEA/CNRS Cadarache)

G Schansker (University of Geneva), Y Yuan (Agricultural University of Hebei), R Strasser (University of Geneva) The more primitive photosynthetic organisms (e.g. cyanobacteria/green algae containing organisms like lichens and corals but also Ginkgo biloba) are characterized by a fast activation of their PSI-acceptor side that is most likely due to a fast activation of the enzyme FNR in the light. This activation is accompanied by a loss of the slowest fluorescence rise phase (IP-phase). Here, we show, that gymnosperms also show a fast PSI-acceptor side activation. In addition, in pine trees inactivation of FNR in the dark following a pre-illumination is a very slow process needing 40-60 min to complete. A single 0.5 s pulse of light is enough to make the IP-phase disappear. To see if this difference between gymnosperms and angiosperms had an effect on the kinetics of a dark-to-light transition, a comparison was made between pine needles and pea leaves. OJIP-fluorescence and 820 nm transmission transients were measured simultaneously at fixed times following illumination with a pulse of red light (360 µmol photons m-2 s-1, length: 10-900 s). Where 1 s was enough for pine trees, in the case of the pea leaves it took about 250 s of illumination before the amplitude of the IP-phase reached its minimum value. Based on the experimental data we propose that during a dark-to-light transition pea plants control electron flow by gradually removing the limitation at the acceptor side of PSI. In needles this mechanism is lacking and a faster formation of non-photochemical quenching (level determined 100 s after lights off) is observed. PS13.19 Chlamydomonas reinhardtii. G Finazzi (IBPC), P Cardol (Université Liège), R de Paepe (IBP), F Franck (Université Liège) Since Warburg (1963) stated that photosynthesis was strictly dependent on respiration, this relationship has been a subject of controversy. Although it is established that isolated intact chloroplasts are capable of high rates of CO2 assimilation, data obtained both in plants and algae suggest that a strong interaction between the photosynthetic and respiratory metabolisms may take place in vivo. To study how the interplay between the two metabolisms affects photosynthesis during a dark-light transition, we performed a

PS13.20 Characterization of the chloroplast NAD(P)H plastoquinone oxidoreductase (NDH) complex subunits. A story to be continued…

The chloroplast homologue of the respiratory complex I, the NDH complex reduces plastoquinones and is involved in cyclic electron flow around photosystem I supplying extra-ATP for photosynthesis under environmental stress conditions. Despite many efforts devoted to determine its structure the polypeptide composition of the NDH complex has not been elucidated. The subunits forming the electron-input module which are not encoded by the plastid genome have not been identified yet leading to controversy over substrate specificity. Previously, in order to identify the “missing” subunits, we purified NDH complex from transplastomic plants expressing a polyhistidine-tagged ndhH gene using Ni2+ affinity chromatography. Among isolated polypeptides, we identified three new NDH subunits (NDH-L, M and N; Plant Cell, 2005, 17, 219-232). Recently, we have identified other polypeptides that surprisingly look like oxygen-evolving complex (OEC) subunits. Their functional characterization in Arabidopsis confirmed that they are new NDH subunits. The possible role of these subunits in NDH catalytic mechanism will be discussed. PS13.21 Several carbonic anhydrases in higher plants thylakoids N Rudenko (IBBP RAS), L Ignatova (IBBP RAS), B Ivanov (IBBP RAS), M Khristin (IBBP RAS) Carbonic anhydrase (CA) is the enzyme catalyzing the interconversion between CO2 and bicarbonate. This conversion is essential for photosynthesis since CO2 is the substrate of the main reaction of Calvin cycle, and moreover, an inorganic carbon is important for electron transport reactions, what was distinctly shown for the reactions in Photosystem II. Thus, CA is most obvious candidate to be the interlink between the carbon metabolism and the energy transformation in thylakoids. Nevertheless, the properties and structure of the thylakoid CAs remain insufficiently studied. We have studied distribution of CAs in thylakoids. Three membrane-bound CAs were found. One of them was situated close to PSI, and the other was situated close to PSII. They differed in thermostability, the effects of detergents and inhibitors. After electrophoresis of thylakoid membranes enriched either with PSI or with PSII, these CAs were found in lipid-protein complexes with apparent molecular masses about 20 kD and 50 kD, accordingly. The binding of the component(s) of PSII core-complex to the specific inhibitor of CA, mafenide, revealed the presence of the third membrane-bound CA. It is possible that the source of this CA activity is not the separate protein. The fourth CA was identified using native electrophoresis of thylakoids

245 14th Photosynthesis Congress - PS07 incubated with Triton X-100. It was soluble protein, about 260 kD coming probably from the lumen. It differed from the known soluble CAs. Thus, poorly characterized until now CA activity of higher plants thylakoids is originated from the presence of at least four CAs, three membrane-bound and one soluble. PS13.22 Rates and roles of cyclic and alternative electron flow in leaves (of potato with altered content of chloroplast NADP-malate dehydrogenase) A Laisk (Tartu University, Institute of Molecular and Cell Biology), V Oja (Tartu University, Institute of Molecular and Cell Biology), H Eichelmann (Tartu University, Institute of Molecular and Cell Biology), E Talts (Tartu University, Institute of Molecular and Cell Biology), R Scheibe (Universität Osnabrück, Pflanzenphysiologie, FB5) Measurements of 810 nm transmittance changes in leaves, simultaneously with Chl fluorescence, CO2 uptake and O2 evolution, were carried out on potato (Solanum tuberosum L.) leaves with altered expression of plastidic NADP-dependent malate dehydrogenase. Electron transport rates were calculated, JC from the CO2 uptake rate considering RuBP carboxylation and oxygenation, JO from the O2 evolution rate, JF from Chl fluorescence parameters and JI from the post-illumination re-reduction speed of PSI donors. In the absence of external O2, JO equalled (1.005 ±0.003) JC, independent of the transgenic treatment, light intensity and CO2 concentration. This showed nitrite and oxaloacetate reduction rates were very slow. The Mehler type O2 reduction was evaluated from the rate of electron accumulation at PSI after O2 concentration was decreased from 210 to 20 mmol mol-1, and resulted in less than 1% of the linear flow. JF and JI did not differ from JC while photosynthesis was light-limited, but considerably increased at saturating light. Then typically JF = 1.2 JC and JI = 1.3 JC, and JF –JC and JI –JC little depended on CO2 and O2 concentrations. The results showed, the alternative and cyclic electron flow necessary to compensate variations in the ATP/NADPH ratio were less than a few per cent of the linear flow. The data do not support the requirement of 14H+/3ATP by the chloroplast ATP synthase. We suggest that the fast PSI cyclic electron flow JI – JC, as well as the fast JF – JC are energy-dissipating cycles around PSI and PSII at light saturation. PS13.23 How impairment of PGR5-dependent cyclic electron transport around photosystem I impacts on photosynthesis and growth of Arabidopsis thaliana ? Y Munekage (NAIST, CEA Cadarache), B Genty (CEA Cadarache), G Peltier (CEA Cadarache) PGR5 has been reported as an essential factor for the activity of the ferredoxin-dependent cyclic electron transport around photosystem I. To elucidate the role of PGR5 on C3 photosynthesis, we characterize photosynthetic electron transport rate (ETR), CO2 assimilation and growth in the Arabidopsis thaliana pgr5 mutant in various irradiance and CO2 regimes. In low light grown pgr5, CO2 assimilation rate and ETR were similar to the wild-type at low irradiance, but decreased at saturating irradiance under photorespiratory conditions as well as non-photorespiratory conditions. Although NPQ was not induced in pgr5 under steady-state photosynthesis, we show that it was induced under dark to light transition at low CO2 concentration, and reversed more rapidly in the dark than in the wild-type. Under low light conditions in air, pgr5 showed same growth as the wild-type but a significant growth reduction compared to the wild-type above 150 μmol photons m-2 s-1.

This growth impairment was largely suppressed under high CO2 concentration. Based on the CO2 concentration dependency of CO2 assimilation, ETR and P700 oxidation measurements, we conclude that reduction of photosynthesis and growth result from 1) ATP deficiency and 2) inactivation of photosystem I. We discuss these data in relation to the role of PGR5-dependent regulatory mechanisms in tuning the ATP/NADPH ratio and preventing inactivation of photosystem I, especially under conditions of high irradiance or enhanced photorespiration.

246 14th Photosynthesis Congress - PS07

PS14 - Metabolic Integration PS14.1 Regulation transport

of

photosynthesis

via

PSI

cyclic

electron

T Shikanai (Kyushu University) In higher plants PSI cyclic electron transport consists of PGR5-dependent and NAD(P)H dehydrogenase (NDH)-dependent pathways. Characterization of the Arabidopsis mutants defective in the pathways indicate that PSI cyclic electron transport is essential for both protecting chloroplasts from photo-oxidative damage and supplying ATP for photosynthesis. Although the physiological function of PSI cyclic electron is becoming clearer, the exact routes taken by electrons are still unclear. To clarify the function of PGR5 protein, we characterized the Arabidopsis plants over-accumulating PGR5. We found that the level of PGR5 is closely related to the rate of PSI cyclic electron transport. The PGR5 level should be controlled for maintaining the redox balance in chloroplasts during fluctuating light conditions. The chloroplast NDH complex is required for another route of electrons in PSI cyclic electron transport. Although the complex is similar to the bacterial NDH-1, the subunits involved in electron donor-binding is unclear in chloroplasts. Consequently, the electron donor to the complex is still unclear. Using the chlorophyll fluorescence imaging, we identified several mutants specifically defective in NDH activity. Whereas some genes are involved in the expression of chloroplast ndh genes encoding the Ndh subunits, some encodes the candidates for the novel subunits.

that links photosynthetic metabolism in guard cells to stomatal conductance (gs) remains not available although the concentration of ATP has been suggested as a candidate signal. Stomata also respond to internal environmental factors such as ABA and zeathantin concentrations. We hypothesize that the gs is controlled by ATP powered osmotic changes, which can be modified [ABA]. To test this hypothesis, we linked a complete dynamic model of leaf photosynthesis with a detailed model of stomata control. This whole model includes the complete description of the light reactions, the detailed biochemical reactions involved in the Calvin cycle, photorespiratory pathway, sucrose synthesis, starch synthesis, and xanthophylls cycle. Furthermore, the regulation of activities of enzymes in these pathways via Ferrodoxin-Thioredoxin system, Mg2+, lumenal and stromal pH and Rubisco activase were also included. The hydromechanical and biochemical model of gs of Buckley et al. (2003; Plant Cell Env., 26: 1767) was linked to the photosynthesis model using ATP as the signal linking photosynthesis and gs. The ABA concentration modulates the ability of ATP to support osmotic gradient across guard cell plastomembrane. The results showed that the model, despite its complexity is numerically stable and realistically simulated the dynamic responses of photosynthesis and gs to changes in external [CO2], [O2], and light.

PS14.4 Physiological, biochemical and molecular analysis of the coordinated up-regulation of photosynthetic, respiratory and biosynthetic metabolism in soybean leaves under Free-Air CO2 Enrichment

PS14.2 Mitochondrial uncoupling protein is required for efficient photosynthesis

A Leakey (University of Illinois), F Xu (University of Illinois), K Gillespie (University of Illinois), E Ainsworth (University of Illinois), S Long (University of Illinois), D Ort (University of Illinois)

A Fernie (MPI-MPP)

Atmospheric [CO2] is rising, with significant consequences for plant function in natural and managed ecosystems. Currently we cannot fully explain the effects of elevated [CO2] on vegetation under field conditions, where interactions with abiotic and biotic factors are important. To better understand plant responses to elevated [CO2] we have combined genomic, biochemical, physiological and ecological investigation of soybean grown in the field at the SOYbean Free-Air Concentration Enrichment (SOYFACE) facility at the University of Illinois. Soybean was grown in four plots at ambient [CO2] (~380 ppm) and four plots at elevated [CO2] (~550 ppm), from sowing until harvest. This provided a model system, where low genetic and environmental variability between experimental units increased the ability to detect subtle treatment effects. The impact of elevated [CO2] on dark respiration is a controversial subject, with prior studies variously reporting stimulation, inhibition or no change in CO2 efflux. The principal molecular response of soybean to elevated [CO2] was increased gene expression for many components of respiratory metabolism, including glycolysis, the TCA cycle and mitochondrial electron transport. These molecular responses were reflected in greater pool sizes of key carbon metabolites and greater rates of respiratory oxygen uptake and carbon efflux. The integrated genomic, biochemical and physiological responses provide unique evidence for stimulated respiration at elevated [CO2]. Greater respiration will partially offset the stimulation of photosynthesis by elevated [CO2] at whole-plant and ecosystem scales, while also generating additional energy and carbon-skeletons. Gene expression for some associated biosynthetic pathways was altered. Gene expression for cellulose synthesis was greater at elevated [CO2], but gene expression for lignin synthesis did

Uncoupling proteins (UCPs) occur in the inner mitochoand regulation of plant UCPs have been described, the physiological purpose of UCP in plants has not been established. Here,urpose of UCP in plants has not been established. Here, biochemical and physiological analyses of an insertional knockout of one of the Arabidoposis UCP genes (AtUCP1) are presented that resolve this issue. Absence of UCP1 results in localized oxidative stress but does not impair the ability of the plant to withstand a wide range of abiotic stresses. However, absence of UCP1 results in a photosynthetic phenotype. Specifically there is a restriction in photorespiration with a decrease in the rate of oxidation of photorespiratory glycine in the mitochondrion. This change leads to an associated reduced photosynthetic carbon assimilation rate. Collectively, these results suggest that the main physiological role of UCP1 in Arabidopsis leaves is related to maintaining the redox poise of the mitochondrial electron transport chain to facilitate photosynthetic metabolism.

PS14.3 A unifying model of photosynthesis and stomatal responses to environmental factors – control of stomatal conductance by ATP and ABA X Zhu (University of Illinois), S Long (University of Illinois) Stomata respond to a large array of external environmental factors such as light, relative humidity, CO2 and O2 concentration. The mechanism

247 14th Photosynthesis Congress - PS07 not change. Greater cellulose to lignin ratios can alter the rate of leaf litter decomposition. In summary, microarray analysis revealed previously unknown changes in gene expression which underlie key physiological and ecological responses of soybean to elevated [CO2].

PS14.5 Malate and fumarate emerge as key players in primary metabolism: Arabidopsis thaliana overexpressing C4-NADP-ME offer a way to manipulate the levels of malate and to analyse the physiological consequences. V Maurino (Institute of Botany, University of Cologne, Germany), H Fahnenstich (Institute of Botany, University of Cologne, Germany), M Saigo (CEFOBI, University of Rosario, Argentina), M Drincovich (CEFOBI, University of Rosario, Argentina), M Zanor (Max Planck Institute for Molecular Plant Physiology), A Fernie (Max Planck Institute for Molecular Plant Physiology), U Flügge (Institute of Botany, University of Cologne, Germany) Maize C4 NADP-malic enzyme was expressed under the control of the CaMV 35S promoter in Arabidopsis thaliana. An increase in the plastidic NADP-ME activity led to a pale green phenotype in plants growing in short days. Under these conditions, MEm plants have a decreased fresh weight/area ratio and thinner leaf sections. Measurements of chlorophyll content and chlorophyll fluorescence indicated an altered photosynthetic metabolism. While no differences in morphology and development were evident in long-day growth conditions, a parallel analysis of metabolite levels of rosettes from transgenic plants grown in different photoperiods revealed that plants grown in both long and short days showed a disturbed metabolic profile. Dark-induced senescence of intact plants progressed more rapidly in MEm plants compared to the wild-type. Interestingly, a retardation of senescence in the transgenic lines was gained by exogenous supply of glucose, sucrose and malate, suggesting that the lack of a rapid energy source is likely to be the initial factor leading to the induction of senescence in these plants. A fairly complete picture of primary metabolism assessed by GC-MS and the in vitro metabolic complementation assays allow us to conclude that MEm transgenic plants entered dark induced senescence more rapidly due to an accelerated starvation. Comparison of the data obtained for the MEm transformants and the wild-type indicated that extremely low levels of malate and fumarate are responsible for the accelerated dark-induced senescence encountered in the MEm plants. Reinforcing previous results, our data indicate that malate and fumarate are key players in the primary metabolism of Arabidopsis thaliana. PS14.6 The role of ferritin family proteins in iron homoeostasis in the cyanobacterium Synechocystis sp. PCC6803 N Keren (Hebrew University) Mechanisms for iron storage play a central role in maintaining primary productivity in iron limited aquatic environment. Major iron storage complexes in photosynthetic organism include ferritins, bacterioferritins and DPS proteins. In order to study the function of these proteins we have inactivated the bfrA, bfrB and mrgA genes in the cyanobacterium Synechocystis sp. PCC6803. The bfr genes code for the two subunits of the bacterioferritin complex. The mrgA protein of the cyanobacterium is a member of the DPS Fe storage protein family. The physiological role of this protein was studied by measuring intercellular Fe quotas, 77K chlorophyll fluorescence and growth rates. It was found that inactivation of any of one of these three genes results in a significant slowdown of growth on Fe limited media. However, significant differences in the internal iron quota were measured. The iron quota of bfr mutants was reduced approximately 50% that of the wild type, while the iron quota of the mrgA mutant was 10% higher than that of the wild type. Based on

these results, we suggest that mrgA plays an important role in the transport of intracellular Fe from storage (within bacterioferritins) to biosynthesis of metal cofactors throughout the cell's growth. The reduction of the internal iron quota had a direct effect on the photosynthetic palpates as observed by a decrease in the number of PSI subunits per cell and the induction of the isiA antenna system even under iron sufficient conditions. PS14.7 Balancing the energy from photons to new biomass in different species and under different growth conditions reveals new insights in the interplay between photosynthesis and metabolic flux control C Wilhelm (University of Leipzig), T Jakob (University of Leipzig), H Wagner (University of Leipzig), K Stehfest (University of Leipzig), U Langner (University of Leipzig) Fluorescence-based measurements of electron transport rates are convenient to assess photo-physiological fitness but data are not directly related to newly formed biomass. In this study we have determined the energy balance from photon to biomass in a diatom and a green alga under dynamic light conditions, nitrogen (N) limitation and different external pH. For the diatom Phaeodactylum tricornutum we found that under high nutrient conditions the quantum efficiency of carbon-related biomass production (FC) and electron requirement for carbon (C) incorporation into biomolecules were found to be strongly controlled by the light climate. Under N-limited conditions the light climate was less important for FC. It became evident that N-limitation induced pronounced changes in the composition of macromolecular compounds and, thus, influenced the degree of reduction of the biomass as well as the metabolic costs of C-incorporation. Since the metabolic costs are not predictable from the photosynthesis rates, electron transport rates as well as gas exchange measurements during the light phase can severely mismatch the true energy storage in the biomass especially under high-nutrient conditions in combination with non-fluctuating light climate. A similar result was obtained for Chlamydomonas acidophila grown under neutral and acidic pH, when C-becomes severely limiting. PS14.8 Upregulation of Photosynthetic Performance and Growth as a Consequence of a Decreased TCA Cycle Enzymes Activity in Genetic Manipulated Tomato Plants A Nunes-Nesi (Max-Planck-Institut für Molekulare Pflanzenphysiologie, 14476 Golm, Germany) As part of an ongoing project to determine the function of the TCA cycle in the illuminated leaf, we have comprehensively phenotyped the tomato wild species (Solanum pennellii) mutant Aco1, deficient in aconitase (isoform 1) expression. Decreased metabolite flux through the TCA cycle supports the decreased levels of TCA cycle intermediates measured by GC-MS. This was accompanied by increased adenylate levels and rate of CO2 assimilation. In a different study transgenic antisense tomato (Solanum lycopersicum) plants with lower mitochondrial malate dehydrogenase (mMDH) activity exhibited enhanced photosynthetic activity and aerial growth under atmospheric conditions. In comparison to wild-type plants, CO2 assimilation rates and total plant dry matter were up to 11% and 19% higher in the transgenics, when assessed on a whole-plant basis. Carbohydrates and redox-related compounds such as ascorbate were also elevated. Experiments in which ascorbate was fed to isolated leaf discs also resulted in increased rates of photosynthesis providing strong indication for an ascorbate-mediated

248 14th Photosynthesis Congress - PS07 link between respiration and photosynthesis. In addition, new array data for Aco1 and mMDH antisense plants revealed elevated levels of photosynthesis associated transcripts. Taken together these lines of evidence suggest that the repression of these mitochondrial localized enzymes can improve both carbon assimilation and aerial growth in a crop species. PS14.9 Structure-function relationship studies of the Arabidopsis thaliana NADP-malic enzyme isoforms

four

M Drincovich (CEFOBI - Biochemistry Faculty, Rosario National University; Argentina), M Gerrard Wheeler (CEFOBI - Biochemistry Faculty, Rosario National University; Argentina), C Arias (CEFOBI Biochemistry Faculty, Rosario National University; Argentina), V Maurino (Botanisches Institut - Universität zu Köln, Germany), C Andreo (CEFOBI - Biochemistry Faculty, Rosario National University; Argentina)

and more acidic versions of the enzyme (as confirmed by isoelectric focussing). Analysis of the wheat chloroplast proteome using two-dimensional electrophoresis has identified pFNR species in vivo equivalent to the four over-expressed pFNR proteins. The linear gradient of cells in the developing primary wheat leaf (from heterotrophic basal cells to photosynthetic cells at the leaf tip) provides a suitable model system for us to investigate whether the expression of pFNR isoforms changes with metabolic needs. The effect on pFNR isoform expression of altering the metabolic capacity of the leaf by inducing nitrate assimilation has also been studied. The implications of these results in terms of distinct metabolic roles for pFNR isoforms will be discussed – along with the potential for manipulation to effect improvements in photosynthetic capacity under high light, oxidative stress tolerance, or nitrate use. PS14.11 Photosynthesis and Growth: GoFORSYS - A Systems Biology-based Approach J Rupprecht (Max Planck Institute of Molecular Plant Physiology)

The Arabidopsis thaliana genome contains four genes encoding NADP-malic enzymes (NADP-ME1-4). NADP-ME4 is localized to plastids whereas the other three isoforms are cytosolic. NADP-ME2 and 4 are constitutively expressed in mature organs, while NADP-ME1 and 3 are restricted to secondary roots and to trichomes and pollen, respectively. Although the four isoforms share a high degree of identity, the recombinant NADP-ME1 to 4 show well-distinct kinetic and structural properties. NADP-ME2 exhibits the highest specific activity, while NADP-ME3 and 4 present the highest catalytic efficiency for NADP and malate, respectively. When analyzing the activity of each isoform in the presence of possible metabolic effectors, the results obtained indicate that NADP-ME2 is the most highly regulated isoform both by activation and inhibition. The four isoforms behave differently in terms of reversibility, presenting NADP-ME3 the highest ratio between the reverse -carboxylation and reduction of pyruvate- to the forward reaction. In order to identify residues or segments of the primary structure of each NADP-ME isoform that could be involved in the differences in kinetic and regulatory properties among the isoforms, NADP-ME2 mutants and deletions were constructed and analysed. The results obtained show that R115 is involved in fumarate activation, while the regions involved in aspartate and CoA modulation are located at the amino-terminal part of the protein. On overall, these studies show that minimal structural changes are responsible for the different kinetic behaviour of each AtNADP-ME isoform. Moreover, these differences are in correlation to the biological function of each isoform in vivo

GoFORSYS proposes a Systems Biology approach towards the study of photosynthesis and its enhancement in crop plants. GoFORSYS is a collaborative effort of the Institute of Biochemistry and Biology at the University of Potsdam, the Max Planck Institutes of Molecular Plant Physiology and Colloids and Interfaces. The key objective of the initiative is the comprehensive systems analysis of photosynthesis and its regulation in response to selected environmental factors in a model algal system, Chlamydomonas reinhardtii, and integration of the obtained insight with research on higher plants. Photosynthesis is a critical determinant of growth and productivity as well as a central process for the biosphere as a whole that also strongly influences global climate. Photosynthetic efficiency has also moved into focus as part of increasing efforts to use plants for bio-fuels production while meeting demands for increased food supply at the same time. There are three main reasons for performing the initial systems analysis in the unicellular organism C. reinhardtii. Firstly, its accessibility to a systems approach. In contrast to higher plant in which complex developmental factors and different cell types influence photosynthetic rates, unicellular algae represent simpler systems while still exhibiting key characteristics of the photosynthetic process. Secondly, their cultivation in a tailor-made photo-bioreactor provides the tools to control and monitor growth parameters more precisely than in higher plant systems. Thirdly, the high degree of conservation of the central processes of photosynthesis across species allows the transfer of the insight obtained to higher organisms.

PS14.10 Multiple isoforms of ferredoxin NADP+ oxidoreductase (FNR) and the integration of photosynthesis, carbon fixation and nitrogen assimilation

PS14.12 Identification of genes and characterization of proteins involved in polyphosphate metabolism in photosynthetic organisms

A Moolna (University of Manchester), J Gummadova (University of Manchester), G Hanke (Institute for Protein Research, Osaka, Japan), T Hase (Institute for Protein Research, Osaka, Japan)

T Alb (Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla), A Serrano (Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla)

The primary function of FNR in the chloroplast is to couple photosynthesis with carbon fixation by generating NADPH. FNR is also involved in other metabolic roles, including cyclic electron flow and resistance to oxidative stress. In addition, FNR can generate an alternative heterotrophic source of the reduced ferredoxin necessary to support metabolic processes including nitrate assimilation. Wheat has two photosynthetic FNR (pFNR) genes, each of which translates into two proteins with different N-terminal processing. We have over-expressed these four pFNR isoforms in Escherichia coli. Biochemical analysis of the purified proteins demonstrates significant differences in enzyme kinetics. In common with other plant species studied, the pFNR1 and pFNR2 genes encode, respectively, more basic

Inorganic polyphosphate (polyP) are linear polymers of orthophosphates (Pi) residues linked by high energy, phosphoanhydride bonds. The ubiquitous occurence of polyPs suggests that they may have important regulatory and stress-tolerance roles that are fundamental to life. We have performed the first systematic survey of genes of polyP metabolism in photosynthetic organisms, both prokaryotes (anoxygenic photobacteria, cyanobacteria) and eukaryotes (microalgae, mosses, and higher plants). All main photobacterial groups exhibit genes encoding polyP-kinases (PPK, EC 2.7.4.1) and Ppx-GppA exopolyphosphatases (PPX, EC 3.6.1.11), and in some species (i.e., Chlorobia) paralogous genes are found. Noteworthy, the cyanobacteria exhibit as a group feature multi-domain PPXs containing an extra HD-phosphohydrolase

249 14th Photosynthesis Congress - PS07 domain, and heterocystous strain possess in addition polyP-glucokinase (PPGK, EC 2.7.1.63) encoding genes. These genes have been cloned and validated by heterologous expression and protein characterization. Interestingly, orthologs of cyanobacterial PPK and PPX genes are present and expressed in photosynthetic protists (green and red algae), the moss Physcomitrella patens, tallophytic red algae and some higher plants, indicating that they have been functionally preserved during the evolution of the photosynthetic lineage. Some of these algal and plant genes (i.e. from Thellungiella halophila) were cloned and heterologously expressed. The molecular phylogeny of these proteins and their relationships with orthologs from non-photosynthetic organisms is discussed. Supported by BFU2004-843 (MEC) and PAIDI group CVI 261 (JA) grants. PS14.13 Chloroplastic NADPH thioredoxin reductase: modulator of plant development and acclimation

a

novel

E Rintamäki (University of Turku), A Lepisto (University of Turku), S Kangasjarvi (University of Turku), B Ruokamo (University of Turku), N Sipari (University of Joensuu), M Keinanen (University of Joensuu) A chloroplastic thioredoxin reductase utilizing NADPH as reducing power (NTRC) has recently been described in Arapidopsis. This novel NTRC is encoded by a nuclear single copy gene, and carries a unique C-terminal thioredoxin-like domain in addition to the thioredoxin reductase sequence. We have characterized Arabidopsis SALK T-DNA insertion lines of the NTRC gene under various growth conditions. Besides retarded growth, ntrc plants showed distinct developmental disorders when grown under short photoperiod. Mesophyll cells had irregular shape and small size, and possessed lowered number of chloroplasts per cell. Moreover, flowering and senescence were significantly delayed. Interestingly, by the time the wild type plants turned senescent, ntrc leaves developed a reticulate phenotype with dark green cells flanking the vascular tissue. A transient reticulate phase of greening was even more pronounced when plants were grown under low temperature. Both the developmental disorders and the conditional phenotypes of ntrc suggest that NTRC regulates basic plant metabolism in chloroplasts. Indeed, amino acid analysis showed distinctly different profiles of plastid-synthesized amino acids between wild type and ntrc plants. For example, the levels of aromatic amino acids, which serve as precursors for secondary metabolites and the growth hormone auxin were significantly different. Intriguingly, the content of auxin was also remarkably diminished in ntrc plants. These metabolic changes also correlated with the conditional phenotype of ntrc. Finally, growth of ntrc plants on a medium supplemented with auxin, aromatic amino acids or isoleucine restored the cell size and the number of chloroplasts per cell in the leaves of ntrc. PS14.14 Complexities in the regulation of chloroplast carbon metabolism revealed by over-expression of plastid transketolase S Lefebvre (University of Essex), J Lloyd (University of Essex), C Raines (University of Essex) Transketolase is an amphibolic enzyme that catalyses reversible reactions in both the photosynthetic carbon reduction (Calvin) cycle and in the non-oxidative branch of the oxidative pentose phosphate pathway. The reactions catalysed by TK are particularly important as they provide key precursors for biosynthesis of aromatic amino acids (erythrose 4-phosphate), biosynthesis of sucrose and precursors of glycolysis (glyceraldehydes 3-phosphate), starch biosynthesis (fructose 6-phosphate) and nucleotide biosynthesis (ribose 5-phosphate). It has been shown previously using antisense technology, that TK has a high flux control coefficient over photosynthetic CO2 assimilation and the

shikimic acid pathway. This finding suggests that it could be possible to increase carbon assimilation rates, aromatic amino acids and phenylpropanoids content by increasing TK activity in transgenic plants. Transgenic lines in which the wild type TK content has been increased by expression of an Arabidopsis thaliana TK cDNA have been produced in order to test this hypothesis. Unexpectedly, the transgenic tobacco lines with increased TK activity develop a severe bleaching in the interveinal mesophyll cells but the areas adjacent to the veins remain green. This chlorotic phenotype suggests that increased TK activity has perturbed the flow of carbon in the plastid and this is supported by data from the analysis of photosynthesis and metabolites in these plants. These results reveal that our understanding of the mechanisms regulating the flow of products from the Calvin cycle to the chloroplast biosynthetic pathways is limited.

250 14th Photosynthesis Congress - PS07

PS15 - Regulation of Light Harvesting PS15.1 Molecular Basis of Photoprotection in Higher Plants B Robert (Institute of Biology and Technology CEA Saclay) Through their light-harvesting antenna, plants frequently absorb more solar energy than they can use in photosynthesis. This excess energy has the potential to cause cell damage, such as pigment bleaching and protein inactivation. To minimise photodamage, excitation quenchers rapidly appear in the plant photosynthetic membrane when exposed to high illumination conditions. Until recently, this mechanism was still poorly understood. In the last years, combined use of advanced spectroscopic methods, applied either on isolated light-harvesting complexes or on whole leaves, have yielded a precise picture of the molecular events which underlie photoprotection in higher plants. It was in particular shown that LHCII, the main light harvesting complex of higher plant chloroplasts, has the capability to undergo transformation into a dissipative state by conformational change, and that such transformation does occur in vivo, at an extent compatible with the extent of photoprotection observed. Adaptation of plants to their light environment thus occurs via changes in conformation of LHCII, which control the balance between utilisation and dissipation of light. This conformational change promotes the appearance of quencher molecules, the chemical nature of which was very recently identified.

PS15.2 Identification of gene products involved in feed-back de-excitation (qE) in vivo and in-vitro. L Dall'Osto (Universita di Verona) Oxygenic photosynthesis drives electron transport and ATP synthesis with light. When absorbed light exceeds the capacity of downstream processes, reactive intermediates (ROS) are formed, that damage the photosynthetic apparatus. The feedback de-excitation mechanism (NPQ) avoids over-excitation reaction centers by dissipating excess energy into heat. It can be measured as a non-photochemical quenching of chlorophyll fluorescence and has a rapidly reversible component (qE), and a slower component (qI). The xanthophyll zeaxanthin is a key element in the regulation of light harvesting: Zea binds to Lhc complexes yielding into chlorophyll fluorescence quenching; CP26 (Lhcb5) undergoes a pI shift upon binding to Zea accompanied by fluorescence lifetime decrease. Thus, Zea acts as an allosteric modulator by switching CP26 between two conformations, the Zea binding/short living one being characteristic of qI state. Zeaxanthin is also involved in modulating ΔpH-dependent quenching component (qE). Femtosecond time-scale measurements (Holt et al. Science 2005 vol. 307, 433) associated qE to the formation of a carotenoid radical cation, triggered by zeaxanthin accumulation. In order to localize the antenna subunits involved in the carotenoid cation formation, xanthophyll-binding Lhcb subunits, either binding violaxanthin or zeaxanthin were analyzed for zeaxanthin-chlorophyll radical pair formation (see poster by Avenson et al., PS3 session) finding that minor antennae binding Zea, but not LHCII, exhibited carotenoid radical cation signal. The role of the minor light-harvesting complexes of PSII in the regulation of non-photochemical fluorescence quenching in vivo was then investigated using a series of Arabidopsis knock-out mutants lacking the target proteins CP24, CP26 and CP29.

PS15.3 A non-photochemical quenching mutant of Chlamydomonas

reveals a role for LI818/LHCSR proteins G Peers (University of California, Berkeley), T Truong (University of California, Berkeley), D Elrad (Stanford University), A Grossman (Stanford University), K Niyogi (University of California, Berkeley) Plants and algae are protected from excess light energy, on the time scale of seconds to minutes, by feedback de-excitation of singlet excited chlorophyll molecules in photosystem II. This is measured as the rapid component of non-photochemical quenching of chlorophyll fluorescence (qE), and it results in the thermal dissipation of surplus light energy. Chlamydomonas, the model green alga, is able to generate qE, but it is unknown which protein components of this process are shared between algae and plants. A qE-deficient mutant of Chlamydomonas, npq4, has deletions in two of the three genes encoding LHCSR (formerly called LI818). This relatively unstudied protein is a distant relative of the LHC family with homologues in a broad group of photosynthetic eukaryotes. The accumulation of these proteins in wild-type cells correlates with qE capacity in both steady-state and high light perturbation experiments. Additionally, LHCSR mRNA accumulates in response to high light and nutrient stress. Although the Chlamydomonas genome contains two genes encoding PsbS, so far we have no experimental evidence for its role in qE. The recently sequenced genomes of several Ostreococcus species also encode PsbS and LHCSR orthologs and we find that expression of the latter is also influenced by high light. We will discuss the diversity of qE mechanisms that appear to exist among algae and plants.

PS15.4 Achieving better light harvesting in the shade: Accumulation of lutein epoxide increases light-harvesting efficiency in shade leaves of Inga species S Matsubara (Forschungszentrum Jülich), T Morosinotto (Università di Padova), H Krause (Heinrich-Heine Universität Düsseldorf), M Seltmann (Heinrich-Heine Universität Düsseldorf), K Winter (Smithsonian Tropical Research Institute), B Osmond (Australian National University), P Jahns (Heinrich-Heine Universität Düsseldorf), R Bassi (Università di Verona) Substantial accumulation of lutein-5,6-epoxide (Lx), an epoxidised lutein (L), has been reported for shade leaves of different higher-plant species. In these plants two xanthophyll cycles operate in parallel: the common cycle with violaxanthin, antheraxanthin and zeaxanthin (V cycle) and another cycle with Lx and L (Lx cycle). While the importance of the V cycle for photoprotection has been well established, the physiological role of the Lx cycle is still largely unknown. Here, we propose a model in which the presence of the Lx cycle extends the xanthophyll-mediated modulation of light harvesting and photoprotection in Lhcs towards higher light-harvesting efficiency. This model is based on the following observations: (1) in-vitro reconstitution of recombinant Lhcb5 with Lx significantly increased the fluorescence yield, presumably by increasing the energy transfer efficiency between Chl a molecules and from xanthophylls to Chl a, (2) in-vivo measurements of fast fluorescence induction in Inga leaves indicated more rapid fluorescence rise in the photochemical phase at higher Lx/L ratio, (3) modulation of light-harvesting efficiency seemed to involve Lx-L exchange in L2 site, and (4) extremely slow recovery of Lx at the expense of L required a few days of shade acclimation in Inga leaves, ensuring that high Lx occurs only in the shade. The ability to accumulate large amounts of Lx, which can be quickly converted to L upon light exposure and recovers only slowly in the shade, may provide an additional photoacclimatory tool for species having leaves that encounter

251 14th Photosynthesis Congress - PS07 both sun and shade environments during long life span. PS15.5 Excitation energy transfer within the PSI-LHCI/II supercomplex from Chlamydomonas reinhardtii cells in state 2 J Minagawa (Hokkaido University), M Yokono (Hokkaido University), M Iwai (Hokkaido University), S Akimoto (Kobe University) State transition in photosynthesis is a short-term balancing mechanism of energy distribution between photosystem I (PSI) and photosystem II (PSII). When PSII is preferentially excited (state 2), a pool of mobile light-harvesting complex II (LHCII) antenna proteins migrates from PSII to PSI. We previously identified three of those mobile proteins (CP26, CP29, and LhcbM5); they were associated with the PSI-LHCI supercomplex isolated from the green alga Chlamydomonas reinhardtii placed in state 2. Here, we demonstrated the functional interaction between the chlorophylls in the mobile LHCII proteins and those in the PSI-LHCI supercomplex. An absorption spectrum of the PSI-LHCI supercomplex with mobile LHCII proteins was well reproduced as a combination of those of the PSI-LHCI supercomplex and the isolated mobile LHCII proteins, indicating that the attachment of the LHCII proteins did not introduce any strong interaction between the Chls. We did observe, however, marked effects of the attachment on excitation relaxation dynamics. Simulation of the time-resolved fluorescence spectra of the PSI-LHCI/II supercomplex with those of PSI-LHCI supercomplex and the isolated mobile LHCII proteins indicated that excitation energy was redistributed between LHCII monomers and the PSI-LHCI supercomplex through multiple pathways. PS15.6 Is qE always the major component of non-photochemical quenching? N DAmbrosio (University of Naples Federico II, Department of Structural and Functional Biology), C Guadagno (University of Naples Federico II, Department of Structural and Functional Biology), A Virzo De Santo (University of Naples Federico II, Department of Structural and Functional Biology) The light energy absorbed in excess to that utilized in photosynthetic electron transport can be potentially harmful for the photosynthetic apparatus of plants. The thermal dissipation of light energy represents an important photoprotective strategy, by which excess excitation energy is safely dissipated as heat. Thermal dissipation is commonly assessed as non-photochemical quenching (NPQ) of chlorophyll fluorescence because it decreases the emission of chlorophyll fluorescence. According to current knowledge three basic mechanisms contribute to NPQ: 1) the ?pH-dependent process across the thylakoid membrane or high energy state (qE); 2) state-transitions (mechanism balancing excitation of PSII and PSI by reversible phosphorylation-related migration of a light harvesting complex II pool between the two photosystems, defined as qT); 3) photoinhibition of photosynthesis (qI). It is widely reported in literature that the qE mechanism represents the major and most important component of NPQ under most environmental conditions. We have defined the three components of NPQ, by their different relaxation kinetics in darkness after a period of illumination (40 minutes), in intact leaves from wild-type and mutants plants of Arabidopsis thaliana with different extent of NPQ and exposed to various photon flux densities (85, 175, 420, 750, 1200 µmol m-2 s-1). First data suggest that qE mechanism is not the main component of NPQ under most of our experimental conditions. The partitioning of the three components of NPQ under different light intensities will be discussed.

PS15.7 The occurrence of PsbS gene product and its correlation with energy quenching in different photosynthetic organisms of the green lineage S Caffarri (Université Aix/Marseille II), G Bonente (Université Aix/Marseille II), F Passarini (University of Verona), S Cazzaniga (University of Verona), M Buia (Stn Zool Anton Dohrn Naples), R Bassi (University of Verona) Mechanisms for dissipating excess energy are needed in all photosynthetic organisms operating in a fluctuating light/temperature environment to avoid photoinhibition. Plants dissipate excess energy by quenching chlorophyll excited states upon a decrease in lumenal pH sensed by the PsbS protein. The green alga Chlamydomonas reinhardtii has a gene sequence for a putative PsbS protein and EST clones were found. We have analysed the accumulation of the PsbS protein in algae and plants belonging to the green lineage using a polyclonal antibody with high affinity for the PsbS recombinant protein of both higher plants and algae. PsbS is not detectable in Chlamydomonas under a large variety of growth conditions. Use of a recombinant crPsbS protein shows that antigen concentration down to 1/100 with respect to higher plants would be easily detected and that, when over-expressed, the protein could not be addressed to the chloroplast compartment. All unicellular green algae tested lack PsbS-reactive bands, while some multicellular algae exhibit PsbS-immunoreactive bands similar to land plants. No correlation was observed between the presence of PsbS-immunoreactive bands and the presence/amplitude of light induced/nigericine-sensitive energy quenching. Such quenching was actually observed at high amplitude in species lacking PsbS immunoreactive bands. Immunoblotting with crLi818 antibody, also a Lhc-like protein, shows that green algae with high qE have Li818-immunoreactive bands. We conclude that the PsbS protein, although indispensable for qE in higher plants, is not necessary for this process in all green algae, where Li818 or other Lhc-like proteins could accomplish its function in qE PS15.8 Chloroplast specialization in situ revealed with temporally and spectrally resolved multimodal nonlinear excitation microscopy R Cisek (University of Toronto and Institute for Optical Sciences), A Tuer (University of Toronto and Institute for Optical Sciences), A Major (University of Toronto and Institute for Optical Sciences), N Prent (University of Toronto and Institute for Optical Sciences), V Barzda (University of Toronto and Institute for Optical Sciences) The variation of spectral and temporal properties of fluorescence from individual chloroplasts within fresh Pea leaves, Pisum sativum, are investigated with spectrally and temporally resolved nonlinear excitation laser scanning microscopy. The heterogeneity of chloroplasts within one cell as well as between the adaxial and abaxial sides of the leaf has been observed. The spectral properties of individual chloroplasts were measured with a two-photon excitation laser scanning microscope featuring time correlated single photon counting detection. Each fluorescence photon was dispersed into different color channels and fluorescence decays were recorded for each channel giving multidimensional discrimination of chloroplasts according to their spectroscopic properties. By fitting multi-exponential functions to the fluorescence decays, 2D fluorescence lifetime maps were built for a range of emission wavelengths. Several 2D lifetime maps have been constructed for each imaged chloroplast, revealing fluorescence lifetime distributions from different pigment-protein complexes associated with Photosystem I and Photosystem II. A very fast fluorescence lifetime component found in each chloroplast was attributed to exciton-exciton annihilation, and varied significantly between different chloroplasts. The observed variation in spectroscopic properties is attributed to different

252 14th Photosynthesis Congress - PS07 levels of light adaptation in individual chloroplasts. This study presents evidence that regulation of light harvesting is individually tuned by each chloroplast present in the leaf. PS15.9 Structural organization of individual chloroplasts investigated with harmonic generation microscope V Barzda (University of Toronto), R Cisek (University of Toronto), A Tuer (University of Toronto), N Prent (University of Toronto), A Major (University of Toronto) The efficiency of light-harvesting is influenced by the macro-organization of pigment-protein complexes in thylakoid membranes of chloroplasts. It has been shown that LHCII, the major light-harvesting chlorophyll a/b pigment-protein complex associated with the photosystem II, is largely responsible for the chiral macroorganization of the grana. Furthermore, light-induced changes in the macrochirality have been correlated with the changes in non-photochemical quenching of chlorophyll fluorescence. In this work, the orientation dependent structural organization of the grana has been studied with the second and third harmonic generation, and multiphoton excitation fluorescence laser scanning microscope. The generation of harmonics and two-photon excitation fluorescence of carotenoids was provided by the 300fs, 14.7MHz repetition rate Yb:KGW laser operating at 1040nm. The second harmonic was generated in the grana and showed the orientation dependency with respect to the linear polarization of the laser. The second harmonic was sensitive to the chiral organization of the stacked thylakoid membranes. The third harmonic was also efficiently generated in chloroplasts from a multilamellar structure of the grana. Simultaneous second and third harmonic generation imaging gives complementary structural information about the macroorganization of the grana. Combination of harmonic generation with the multiphoton excitation fluorescence enables to perform simultaneous measurements of the structural changes in the grana and variations of the fluorescence quenching in individual chloroplasts. PS15.10 Dissociation of Light-harvesting Complex II from Photosystem II Supercomplex during State Transitions in Chlamydomonas reinhardtii M Iwai (Hokkaido University), J Minagawa (Hokkaido University) State transition is a plant photoacclimation mechanism to regulate the equilibrium of the light-driven excitation of photosystem I (PSI) and photosystem II (PSII). It has been considered that redistribution of light-harvesting complex (LHC) II polypeptides adjusts the absorption-cross section of each photosystem. Recently, we have isolated a giant protein complex comprised of PSI, LHCI, and LHCII polypeptides, so-called PSI-LHCI/II supercomplex. Our finding is of importance because LHCII polypeptides, not only major trimeric ones but also minor monomeric ones, act as light-harvesting antenna for both PSII and PSI, providing evidence that the association of LHCII polypeptides with PSI could take place during state transitions. Nevertheless, there has been no evidence to show those LHCII polypeptides bound to PSI-LHCI/II supercomplex migrate truly from PSII. In this study, we designed a new protocol to isolate a protein supercomplex composed of PSII and LHCII, so-called PSII-LHCII supercomplex, to determine the changes of its polypeptide composition during state transitions. The results indicated that a certain member of LHCII polypeptides was not found in PSII-LHCII supercomplex when PSI-LHCI/II supercomplex was formed. Thus we concluded that the displacement of LHCII polypeptides from PSII-LHCII supercomplex surely occurred, and those LHCII polypeptides could migrate to PSI to form PSI-LHCI/II supercomplex.

PS15.11 Suppression of CP29 causes an absence of the PSI-LHCI/II supercomplex of Chlamydomonas reinhardtii under state 2 conditions R Tokutsu (Hokkaido University), J Minagawa (Hokkaido University) State transition is a mechanism that plants acclimate to variable light conditions. During state transitions, the light harvesting-complexes II (LHCIIs) reversibly migrate between photosystem I (PSI) and photosystem II (PSII). However, the molecular mechanisms was not clearly determined. Recently, we presented biochemical evidence which indicates three monomeric LHCIIs (CP29, CP26 and LhcbM5) could associate with PSI and formed PSI-LHCI/II supercomplex in state 2. In this study, we generated knock-down transformants of CP29 (CP29-RNAi) to investigate the specific role of this monomeric LHCII in state transitions in a unicellular green alga Chlamydomonas reinhardtii. Although the transformants showed a variable extent of mRNA reduction, one of the transformants exhibited an almost complete suppression of the expression of CP29 protein. The results of sucrose density gradient ultracentrifugation showed that no PSI-LHCI/II supercomplex existed in thylakoid membranes of this transformant in state 2. It is thus possible that an absence of CP29, presumably acting as a main linker between major trimeric LHCIIs and PSI, inhibits formation of PSI-LHCI/II supercomplex. PS15.12 A short sequence in the N-terminal domain of chlorophyllide a oxygenase regulates its accumulation Y Sakuraba (Hokkaido University), R Tanaka (Hokkaido University), A Tanaka (Hokkaido University) Chlorophyllide a oxygenase (CAO) is a rieske-type monooxygenase that catalyzes conversion of chlorophyll a to chlorophyll b. Since the antenna size of a photosystem is partly determined by the amount of chlorophyll b in the light-harvesting complexes (LHC), it is important to understand the regulatory mechanism of chlorophyll b biosynthesis. CAO protein consists of three domains, namely, A, B and C domains. Previously, we demonstrated that C domain alone has a catalytic function, B-domain is a linker, and A domain alone is involved in the regulatory mechanism of the CAO protein level, in which mechanism the CAO protein level is maintained below a certain level. Furthermore, we demonstrated that chlorophyll b and Clp protease are involved in the regulation. To identify the amino acid sequences within the A domain which are involved in the regulation, we deleted amino acids sequence of A domain by 10 amino acids from its N terminus, and overexpressed these sequences in Arabidopsis thaliana to examine their effects on the CAO protein levels of the transgenic plants. We found that CAO accumulated excessively by deletion of a specific amino acid sequence in the middle of A domain. Subsequently, we overexpressed this sequence fused with the B and C domains of CAO, and found that this sequence was almost sufficient to maintain the CAO protein level. It is possible that this sequence is recognized by Clp protease or other chloroplast proteases in a chlorophyll b dependent manner. PS15.13 Drought-induced ultra-fast fluorescence quenching in photosystem II in lichens revealed by picosecond time-resolved fluorescence spectrophotometry M Komura (Nagoya Univ.), I Iwasaki (Akita Prefectual Univ.), S Itoh (Nagoya Univ.) Lichens survive under the extreme drought environments. It has been suggested that dried lichens convert excess light energy into heat by

253 14th Photosynthesis Congress - PS07 unknown mechanism to prevent the accumulation of the harmful photoproducts. We studied 18 lichen species as for their steady-state fluorescence spectra, PAM and picosecond time-resolved fluorescence decay profiles at 4-300 K. Quantitative analysis of the decay profiles was applied as shown in previous study [1]. We obtained following results: (1) All dried lichens showed low intensity of PS II fluorescence. (2) The picosecond decays of PS II fluorescence were significantly fast (<10 ps) in most of dry lichens. (3) Excitation energy transfer from LHC II to CP43/CP47 was still active. (4) The lifetime of the PS I fluorescence was little affected. (5) These changes were fully reversed within 1 min after the re-hydration. (6) Some lichens showed no fast decay of PS II fluorescence. We noticed two different types of drought-induced energy dissipation mechanisms: Most of lichens dissipated almost all the excitation energy in a few picoseconds by an unknown quencher; some lichens decreased the antenna size of PS II by the state transition mechanism. New type of the quencher found in this study seems to be situated in the core antenna, and different from the well-known non-photochemical quenching mechanism. [1] M. Komura, Y. Shibata, S. Itoh, A new fluorescence band F689 in photosystem II revealed by picosecond analysis at 4-77 K: Function of two terminal energy sinks F689 and F695 in PS II, BBA 1757 (2006) 1657-1668. PS15.14 The effect of pH of the stromal part of thylakoids on the nonphotochemical quenching of chlorophyll fluorescence Y Eu (Pusan National University, Korea), S Mishra (Pusan National University, Korea), I Zulfugarov (Pusan National University, Korea), C Lee (Pusan National University, Korea) Nonphotochemical quenching (NPQ) is important in the dissipation of excess light energy under high light condition. Energy dependent nonphotochemical quenching (qE) is the major component of NPQ and is triggered, enhanced and sensed by the acidification of thylakoid lumen, the presence of PsbS protein. To determine whether the pH of the stromal part of thylakoid membrane affects the development of NPQ, we examined the chlorophyll fluorescence induction curve at various pH by measuring thylakoid samples simultaneously with imaging system. The maximum fluorescence (Fm) showed bell-shaped curve with peak from pH 7 to 8. The minimum fluorescence (Fo) was not affected by low pH, but increased by high pH. The pH pattern of photochemical efficiency was found to be inverse of that for Fo and changed little at low pH, but decreased at high pH. The effect of pH on electron transport activity from photosystem II to photosystem I was similar to that on Fm. Maximum fluorescence under actinic light, Fm`, was not recovered after turning off actinic light at pH lower than 6.5, resulting in negative qE. The variation of qE with the pH of thylakoid suspension medium showed bell-shaped curve with pH maxima at 7.5 – 8. These results suggest that the sensor of thylakoid membranes detects the relative proton gradient between lumen and stroma of thylakoids, but not absolute pH of lumen. PS15.15 Aggregation-free conformational switch in LHCII complexes of higher plants C ILIOAIA (Institut de Biologie et Technologies de Saclay (iBiTecS), CEA-Saclay, 91191 Gif-sur-Yvette, Cedex, France), (Sheffield University), P Horton (Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK), A Ruban (School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UK) The process of light-harvesting in higher plants and algae is regulated to optimise energy input within the chloroplast membrane. Under

conditions of high light illumination light-harvesting antennae are switched into a photoprotective state, where the excess absorbed energy is dissipated as heat, a process known as non-photochemical quenching (NPQ). In vitro, aggregation of the isolated antenna protein LHCII results in a switch to a dissipative state, and this switch was suggested to mimic the NPQ process in vivo. However it has also been suggested that this in vitro quenching may be a result of artefactual chlorophyll-chlorophyll interactions between adjacent trimers in an aggregate. We have analysed the spectral properties of the LHCII complexes incorporated into a solid gel matrix, with the proposition that when the detergent is removed from the system, protein would me immobilised and could not form aggregates. This approach demonstrates that the dissipative mode of LHCII does not require protein-protein interaction, being rather an intrinsic feature of the trimeric subunits. We show that two pigment-binding domains are affected during this transition - namely neoxanthin and the lutein 1 domain. PS15.16 Tla1, a novel protein functions in the regulation of the chlorophyll antenna size in Chlamydomonas reinhardtii M Mitra (University of California, Berkeley), H Kirst (University of California, Berkeley), S Titali (University of California, Davis), A Melis (University of California, Berkeley) The Chlamydomonas reinhardtii tla1 mutant was generated upon DNA insertional mutagenesis and shown to specifically possess a smaller than wild type chlorophyll antenna size in both photosystems. Molecular analysis revealed plasmid DNA insertion at the end of the 5’UTR and just prior to the ATG start codon of a nuclear gene (termed Tla1), which encodes a protein of 213 amino acids. The Tla1 gene in the mutant is transcribed with a new 5’UTR sequence, derived from the 3’end of the transforming plasmid. Replacement of the native 5’UTR and promoter regions resulted in enhanced transcription of the tla1 gene in the mutant but inhibition in the translation of the respective tla1 mRNA. Transformation of the tla1 mutant with wild type Tla1 genomic DNA successfully rescued the mutant. These results are evidence that expression of the Tla1 gene is a prerequisite for the development/assembly of the full Chl antenna size in C. reinhardtii. A BLAST search revealed that the Tla1 protein is highly conserved in many eukaryotes and has homology to a protein of unknown function in Arabidopsis thaliana, Drosophila melanogaster and Homo sapiens. The Tla1 gene expression is controlled by light irradiance and apparently regulates genes that determine the Chl antenna size in the photosynthetic apparatus of C. reinhardtii. Current efforts focus on immunolocalization of the Tla1 protein in C. reinhardtii and apply Tla1 over-expression or down-regulation approaches to study the functional role of the Tla1 protein. Applications of Tla1 gene in microalgal productivity under mass culture conditions will be discussed. PS15.17 Distrubution of antenna complexes between photosystems I and II in cyanobacteria, prochlorophytes, cryptophytes, and red algae I Stadnichuk (A.N.Bakh Institute of Biochemistry Russian Academy of Sciences), V Boichenko (A.N.Bakh Institute of Biochemistry Russian Academy of Sciences) Action spectra of PSII-driven oxygen evolution and of PSI-mediated H2 photoproduction or photoinhibition of respiratory oxygen uptake were used to determine the distribution of antennal pigment complexes between the two photosystems in different groups of oxygenic photosynthtetics. The integral pigment sizes of the two photosystems and their ratio were obtained from the light saturation curves of relevant photoreactions. Calculation of phycobiliproteins to chlorophyll ratio from each type of the action spectra shows that hemidiscoidal

254 14th Photosynthesis Congress - PS07 phycobilisomes in cyanobacteria and red algae are docked to the dimers of PSII and the trimers of PSI. Our data make unlikely the energy migration from phycobilisomes to PSI through the mediation of PSII. Phycobilisome is the form of phycobiliprotein supramolecular organization that allows these pigments to deliver the absorbed energy to the both photosystems. Contrary to the red algae and cyanobacteria, phycobiliproteins of cryptophytes do not form phycobilisomes and transfer the energy solely to PSII. The second type of antenna presented in this algal group by Chl a/c-proteins is equally distributed between the PSI and the PSII. Absence of phycobiliproteins in prochlorophytic cyanobacteria does not disturb formation of PSII core dimers and PSI core trimers that were shown to be surrounded by Chl a/b-polypeptide complexes in plane of the thylakoid membrane. The obtained different photosystem ratios of (2-5):1 in favor of PSI in phycobilisome-containing and prochlorophytic cyanobacteria and of 4:1 in favor of PSII in cryptophyte algae correlate with the different integral antenna sizes of the two photosystems in these groups of photosynthetics. PS15.18 Interactions between the Orange-Carotenoid-Protein (OCP) and the phycobilisomes in Synechocystis PCC 6803 C BOULAY (CEA, CNRS), A WILSON (CEA, CNRS), D KIRILOVSKY (CEA, CNRS) The OCP is a cyanobacterial 35kD soluble protein that binds a carotenoïd molecule (hydroxyechinenone). We have demonstrated that the OCP is essential for a mechanism of photoprotection involving non-photochemical fluorescence quenching (NPQ) in cyanobacteria. This mechanism decreases the energy arriving from the phycobilisomes (PBS), the extrinsic cyanobacterial antenna complex, at the reaction centres of Photosystem II under strong illumination in cells grown under Fe-replete or Fe-starvation conditions. We have also shown that the OCP is mainly associated to the stromal side (phycobilisome side) of the thylakoids. Moreover, almost all the OCP was present in the membrane-phycobilisome fraction showing a strong interaction with the thylakoids. The exact role(s) and function(s) of OCP still remain to be elucidated. We constructed mutants containing His-tagged OCP and checked that they presented a blue-light-induced NPQ. OCP has been purified from one of this mutants using affinity Ni-chromatography and loaded on DEAE cellulose column to separate it from other histidine-rich proteins. Studies on OCP-PBS interactions have been performed. The OCP did not co-isolate with phycobilisomes when Triton was used during their purification. However, when purified OCP was incubated with whole phycobilisomes isolated from WT cells or from a mutant lacking phycocyanin, the OCP co-migrates with the phycobilisomes in a sucrose gradient suggesting OCP-PBS relationships. Also when the phycobilisomes were dissociated and then reconstructed in the presence of OCP, the OCP co-migrates with the phycobilisomes. Experiments are in process in order to better characterize OCP-PBS interactions. The progress of these studies will be described in the presentation. PS15.19 Seasonal changes in relative abundance and phosphorylation status ofindividual light harvesting and reaction center proteins that correlate withsustained energy dissipation in the overwintering evergreens Pinus strobus and Abies balsamea A Verhoeven (University of St. Thomas), A Osmolak (University of St. Thomas), P Morales (University of St. Thomas), J Crow (University of St. Thomas) During winter, the light harvesting complexes (LHCs) of evergreen plants functionally change from energy harvesting to energy dissipating

centers. The goal of this study was to determine if there are physical changes in the composition of the photosynthetic apparatus that accompany these functional changes. Seasonal changes in chlorophyll fluorescence, pigment content, and relative abundance and phosphorylation status of light harvesting and reaction center proteins in the evergreens Pinus strobus (growing in the sun) and Abies balsamea (growing in sun and shade environments) in the seasonally cold climate of Minnesota were examined. Measurements and collections were performed every two months from January of 2005 until January of 2006. Results indicate typical seasonal changes in chlorophyll fluorescence and pigment content, with sustained reduced Fv/Fm during winter, accompanied by retention of zeaxanthin and antheraxanthin. Seasonal increases in xanthophyll cycle pigments and lutein, and seasonal decreases in a-carotene, were observed. The majority of LHCs decreased in relative abundance during winter in sun plants, although Lhcb2 and PsbS decreased less than other LHCs. Shade plants showed smaller changes in LHC abundance, with some showing relative increases during winter, including Lhcb2, PsbS and Lhca2. Thylakoid protein phosphorylation status suggests increases in phosphorylation of D1 and PsbH during winter, with shade plants also showing increases in phosphorylation of D2 and CP43 during winter. The results support a model of reorganization of light harvesting complexes during winter accompanied by increased relative abundance of Lhcb2 and PsbS, and are suggestive of novel functional differences between individual light harvesting proteins. PS15.20 Carotenoid-induced non-photochemical fluorescence quenching in phycobilisomes of the cyanobacterium Synechocystis sp. PCC 6803 M Rakhimberdieva (A.N. Bakh Institute of Biochemistry), Y Bolychevtseva (A.N. Bakh Institute of Biochemistry), I Elanskaya (M.V. Lomonosov Moscow State University), N Karapetyan (A.N. Bakh Institute of Biochemistry) A new pathway of dissipation of excess energy was recently found in cyanobacteria. In green plants, the radiation-less discharge of excessive absorbed light energy takes place via non-photochemical quenching (NPQ), observed as a decrease in chlorophyll a (Chl) fluorescence of the LHCII. We have shown that NPQ in cyanobacteria is triggered by intense blue light, with the action spectrum resembling the absorption spectrum of a carotenoid. The blue-light-induced NPQ quenches fluorescence of the allophycocyanin (APC) core of phycobilisomes (PBS) in a PSII-deficient mutant, and of both APC and Chl in the wild strain of Synechocystis sp. A water-soluble orange carotenoid protein (OCP), with an absorption spectrum that matches the NPQ action spectrum, was previously found to be critical for the development of blue light-induced quenching of PBS fluorescence in the cyanobacterium (Wilson et al., 2006). Our inquiry into the effect of temperature on the NPQ in the PSII-deficient mutant has lead to identification of two temperature-dependent processes: one is responsible for the quenching rate, and one determines the yield of PBS fluorescence. Non-Arrhenius behavior of the light-on quenching rate suggests that carotenoid-absorbed light triggers a process that bears a strong resemblance to soluble protein folding, showing temperature-dependent enthalpy of activated complex formation. The response of PBS fluorescence yield to hydration changing additives and to passing of the membrane lipid phase transition point indicates that the pool size of phycobilisomes subject to quenching depends on the state of some membrane component. This work was supported by the RFBR grant 05-04-48526. PS15.21 Violaxanthin deepoxidation in barley leaves treated with methyl viologen is stimulated by moderate light from a

255 14th Photosynthesis Congress - PS07 fluorescent tube but not by light of the same intensity coming from a tungsten lamp

between diatom species.

P Ilík (Laboratory of Biophysics, Faculty of Science, Palacký University, Olomouc, Czech Republic), H Kyseláková (Laboratory of Biophysics, Faculty of Science, Palacký University, Olomouc, Czech Republic), E Kotabová (Laboratory of Biophysics, Faculty of Science, Palacký University, Olomouc, Czech Republic), J Nauš (Laboratory of Biophysics, Faculty of Science, Palacký University, Olomouc, Czech Republic)

PS15.23 Mechanisms of photosynthetic apparatus acclimation of C4 plants to different irradiance

On the contrary to untreated leaf segments, the irradiation of MV-treated segments with a fluorescent tube (100 µmol photons m-2 s-1 at 400 – 700 nm, growth conditions) induced an extra enhancement of nonphotochemical quenching and a pronounced conversion of violaxanthin to zeaxanthin. However, no such effect was observed when MV-treated segments were irradiated with a tungsten lamp of the same light intensity. It has been proposed that light participate in trans-cis isomerization of violaxanthin in light-harvesting complexes of photosystem II. This isomerization was postulated as a basic mechanism leading to the release of violaxanthin into lipid phase, making it accessible for enzymatic deepoxidation (Niedzwiedzki et al. 2005: J. Photochem. Photobiol. B – Biol. 78, 109-114). Considering this mechanism and the spectral characteristics of light sources used in this work, we speculate that some spectral lines in the Soret region stimulate violaxanthin deepoxidation in MV-treated leaf segments. Acknowledgements: We thank the Grant Agency of Czech Republic (grant No. 552/06/0979) and the Ministry of Education, Youth and Sports of Czech Republic (grant No. MSM 6198959215) for financial support. PS15.22 Evidence for the existence of a fast xanthophyll cycle-independent NPQ component in diatoms I Grouneva (Institut für Biologie I, Pflanzenphysiologie, Universität Leipzig, Johannisallee 21-23, 04103 Leipzig, Germany), T Jakob (Institut für Biologie I, Pflanzenphysiologie, Universität Leipzig, Johannisallee 21-23, 04103 Leipzig, Germany), C Wilhelm (Institut für Biologie I, Pflanzenphysiologie, Universität Leipzig, Johannisallee 21-23, 04103 Leipzig, Germany), R Goss (Institut für Biologie I, Pflanzenphysiologie, Universität Leipzig, Johannisallee 21-23, 04103 Leipzig, Germany) For the diatom Phaeodactylum tricornutum it was reported that non photochemical quenching (NPQ) strictly depends on the de-epoxidation state of the xanthophyll cycle pigment pool. In our present experiments we show that in the marine diatom Cyclotella meneghiniana both the build-up and the relaxation of the diatoxanthin (Dtx)-dependent NPQ are accompanied by a fast component which is not related to the xanthophyll cycle. The contribution of this fast NPQ component to the overall extent of NPQ is of quantitative significance. Application of the de-epoxidase inhibitor DTT has revealed that Dtx-independent NPQ displays a fast rise (within a time range of seconds) upon the onset of illumination followed by a gradual relaxation to a steady-state level in the light as well as a strong dependence on actinic light intensity. This fast NPQ component relies on the presence of the proton gradient and can be inhibited by addition of the uncoupler nigericin. Further, a comparative study between C. meneghiniana and P. tricornutum was carried out in order to underline the considerable differences between these two diatoms regarding Dtx-independent NPQ. The mechanism underlying the fast NPQ component is discussed with respect to a possible transient NPQ originating in the reaction centre of PS II or a redistribution of excitation energy between the two photosystems upon illumination. The present findings show that in diatoms the mechanisms of NPQ are more complex than previously thought and may also vary considerably

E Romanowska (Department of Plant Physiology, Univeristy of Warsaw, Poland), A Drozak (Department of Plant Physiology, Univeristy of Warsaw, Poland), M Powikrowska (Department of Plant Physiology, Univeristy of Warsaw, Poland), M Zienkiewicz (Department of Plant Physiology, Univeristy of Warsaw, Poland), B Pokorska (Department of Plant Physiology, Univeristy of Warsaw, Poland) Plants can adjust photosynthetic apparatus to wide daily and seasonal fluctuations in light conditions. Acclimation to different light intensities is manifested by changes in fluorescence parameters, pigments’ content, chloroplast ultrastructure, efficiency of photosystems and relative levels of thylakoid components. It is known that in photosynthesis of C4 plants are involved the mesophyll and bundle sheath chloroplasts, which differ structurally and functionally, but up to date little is known about acclimation of two types of C4 chloroplasts to light intensity. The three species representing C4 subtypes: maize (Zea mays, NADP-ME), millet (Panicum miliaceum, NAD-ME) and guinea grass (Panicum maximum, PEP-CK) were grown under low (50 µmol m-2 s-1), moderate (350 µmol m-2 s-1) and high light (800 µmol m-2 s-1). We noted different strategies in acclimation of these plants to irradiance. Although in both Panicum species there were not any significant differences in content of leaf chlorophyll and the amount of LHCII proteins at all light intensities, there were noticed changes in amount and size of grana. In maize the level of LHCII rised with decreased irradiance (even in agranal bundle sheath chloroplasts) without an influence on chloroplast ultrastructure. Moreover, different light intensity during growth moduleted the amount of reaction centre proteins of PSII, whereas transcription of psbA was similar in response to irradiances. Thus this regulation is propably achieved on post-transcriptional level. The results show that acclimation mechanisms can vary between plants and types of chloroplasts (balance between level of LHC complexes and chloroplast ultrastructure). PS15.24 Non-photochemical-quenching mechanisms in the cyanobacterium Thermosynecococcus L Abasova (Biological Research Center), C Boulay (CEA, CNRS), I Vass (Biological Research Center), D Kirilovsky (CEA, CNRS) Synechocystis PCC 6803 grown under iron replete or iron deplete conditions, blue light induces a photoprotective Non-Photochemical-Quenching (NPQ) mechanism. This energy dissipation mechanism involves the phycobilisomes and the Orange Carotenoid Protein (OCP) encoded by the slr1963 gene. In the thermophyle cyanobacterium Termosynechococcus elongatus there is no OCP-like genes instead two adjacent genes encode for the N-terminal and C-terminal domains of an OCP-like gene. The goal of our study is to elucidate the possible role of these genes in a NPQ mechanism. T elongatus cells grown under low-light and high-light conditions in the presence or absence of Fe were used in this study. During iron starvation, the induction of IsiA synthesis was observed like in Synechocystis and other cyanobacteria strains. The presence of IsiA was confirmed by the blue shift in the room temperature chlorophyll a absorbance peak and an increase in the 77K fluorescence peak at 685nm. While in iron starved Synechoscystis cells the decrease of chlorophyll was more rapid than that of phycobiliproteins, in iron starved T elongatus cells the decrease of phycobiliproteins was faster than that of chlorophyll and no accumulation of disconnected phycobilisomes was

256 14th Photosynthesis Congress - PS07 observed. Blue light (at any intensity) was not able to induce any quenching of fluorescence in T elongatus cells grown in the presence or absence of Fe. In contrast, normal state transitions were observed in T elongatus cells. Data from other cyanobacteria strains containing or not OCP will be presented. PS15.25 Novel insights into state transitions of plant chloroplasts: mechanism and physiological significance M Tikkanen (University of Turku), M Nurmi (University of Turku), M Suorsa (University of Turku), F Mamedov (Uppsala University), R Danielsson (Lund University), S Styring (Uppsala University), E Aro (University of Turku) State 1 – state 2 transition of the photosynthetic machinery involves a reversible association of a fraction of the light harvesting complex II (LHCII) between photosystem II (state 1) and photosystem I (state 2). Traditionally, the phosphorylatable fraction of LHCII has been assigned as the functional moving unit of state transition. Here, a mechanical fractionation of thylakoid membrane of Spinacia oleracea leaves in state 1 and in state 2 was performed in order to compare the dynamic protein movements with the excitation changes occurring in the two photosystems upon state transitions. Despite the fact that the amount of the Lhcb1, Lhcb2, Lhcb3 and Lhcb5 proteins increase in stroma lamellae upon transition to state 2, no increase occurs in the 77 K fluorescence emitted from PSI in this membrane fraction. On the contrary, such an increase in fluorescence occurs in the grana margin fraction, and the functionally important mobile unit is the PSI-LHCI complex. A new model for state transitions thus emphasises an increase in PSI absorption cross-section occurring in grana margins upon transition to state 2 and resulting from the movement of PSI-LHCI complexes from stroma lamellae and subsequent co-operation with the P-LHCII antenna from the grana. By using a state transition mutant of Arabidopsis thaliana (stn7), it is demonstrated that the benefit from state transitions becomes apparent only when plants are grown under fluctuating light and elevated CO2, the combination of environmental conditions that strongly challenges the capacity and dynamics of the photosynthetic light reactions. PS15.26 Reorganization of LHC II complexes in the thylakoid membrane in response to high light adaptation (NPQ) Y Miloslavina (Max-Planck-Institut für Bioanorganische Chemie, Stiftstr. 34-36 D-45470 Mülheim a.d. Ruhr, Germany), M Nilkens (Institut für Biochemie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany), P Jahns (Institut für Biochemie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany), A Holzwarth (Max-Planck-Institut für Bioanorganische Chemie, Stiftstr. 34-36 D-45470 Mülheim a.d. Ruhr, Germany) Regulation of photosynthetic light harvesting is required for the adaptation of plants to varying light conditions. At low-light intensities the light energy is efficiently harvested by the antenna systems of both photosystems. At high-light intensities, however, much more light is absorbed in the antennae than can be utilized in photosynthetic electron transport. The dissipation of this excess light energy as heat represents an efficient photoprotective mechanism to minimize photooxidative damage under such unfavourable conditions. The generation of this non-photochemical quenching of light energy, often termed as NPQ or qE, is believed to be strictly dependent on the PsbS protein and caused by quenching processes located in the antenna of photosystem (PS)II. They have been shown to be regulated by the pH of the thylakoid lumen. We report here that, under physiological conditions, the fast component qE of NPQ represents a state transition, contributing about 30% to the

total reduction in PSII fluorescence. The remaining 70% is caused by quenching processes in the antenna of PSII and occurs to be largely independent from psbS. We will compare results from various mutants modified in the psbS content or the carotenoid composition. These results provide an entirely new understanding of the role of psbS in the NPQ phenomenon. PS15.27 Occurrence of lutein epoxide in the photosynthetic pigment system of tropical plants H Krause (University of Duesseldorf), S Matsubara (Phytosphere Institute), A Gallé (University of Bern), M Seltmann (Phytosphere Institute), T Kurasr (University of Utah), P Jahns (Heinrich Heine University Duesseldorf), K Winter (Smithsonian Tropical Research Institute) Lutein epoxide (Lx) has been found to be a major pigment component of the light-harvesting complex1 in shade leaves of some higher plant species1-3. Its reversible de-epoxidation to lutein in high light (‘Lx cycle’) resembles the well-known violaxanthin cycle. We report the occurrence of high Lx levels in leaves of several neotropical tree species. Shade and sun leaves of 28 species from 13 plant families were collected in Panama and maintained under low-light conditions for several hours. Nine Inga spp. (Fabaceae) tested contained high pools of Lx in shade leaves (18-39 mmol mol-1 Chl a+b). Lx levels in sun leaves were substantially lower (4-9 mmol mol-1 Chl). Small amounts of Lx, mostly below 5 mmol mol-1 Chl, were found in shade and sun leaves of 16 further species tested. However, in three species of Virola (Myristicaceae), both sun and shade leaves contained high amounts of Lx (22-68 mmol mol-1 Chl). Lx levels in canopy sun leaves of V. surinamensis and V. elongata were the highest so far reported for sun leaves and exceeded the pool size of violaxanthin-cycle pigments. We hypothesize that Lx contributes to optimise light harvesting in the shade4, and that conversion of Lx to lutein in high light participates in photoprotection. The taxonomic distribution of Lx remains enigmatic. PS15.28 TSP9 phosphoprotein as a regulator of light harvesting in Arabidopsis thaliana. R Fristedt (Division of Cell Biology, Linköping University, Linköping, Sweden), M Hansson (Division of Cell Biology, Linköping University, Linköping, Sweden), I Carlberg (Department of Biochemistry and Biophysics, Stockholm University, Sweden.), A Zygadlo (Department of Plant Biology, Faculty of Life Sciences, University of Copenhagen.), A Vener (Division of Cell Biology, Linköping University, Linköping, Sweden) We are characterizing a small thylakoid protein known as the thylakoid soluble phosphoprotein of 9kDa (TSP9, At3g47070) in Arabidopsis thaliana. TSP9 has been identified in spinach thylakoids as a basic protein that becomes triply phosphorylated in response to light. Genes for homologous proteins were found in 49 other plant species but not from any other organism, making TSP9 a plant specific protein. To understand which of the kinases is responsible for TSP9 phosphorylation we selected mutant lines from SALK collection with T-DNA insertions in the genes for STN7 and STN8 kinases. The light-induced phosphorylation of recombinant TSP9 by either wild-type thylakoids or thylakoids isolated from STN7 and STN8 knockout plants were studied in vitro. TSP9 was found phosphorylated by the thylakoids from the wild type and STN8-lacking mutant, but not when the thylakoids from STN7-defficient plants were used in the reactions. Upon illumination the phosphorylated form of spinach TSP9 is released from the photosynthetic membranes, and recent data have shown association of

257 14th Photosynthesis Congress - PS07 TSP9 with LHCII as well as the peripheries of the photosystems. Fluorescence measurements showed that TSP9 knockout mutant was less efficient compared to wild type in short-term regulation of light harvesting. This was shown by measurements of both the state transitions and non-photochemical energy dissipation. We conclude that TSP9 is involved in the regulation of state transition process as well as in modulation of light harvesting in Arabidopsis thaliana. PS15.29 Different roles of alpha- and beta- branch xanthophylls in photosystem assembly and photoprotection L Dall'Osto (Università di Verona), A Fiore (Ente per la Nuove tecnologie), S Cazzaniga (Università di Verona), G Giuliano (Centro Ricerche Casaccia), R Bassi (Università di Verona) Xanthophylls (oxygenated carotenoids) are essential components of the plant photosynthetic apparatus, where they act in photosystem assembly, light harvesting and photoprotection. Nevertheless, the specific function of individual xanthophyll species in the alpha- and beta- branch awaits complete elucidation. In this work we analyze the photosynthetic phenotypes of two newly isolated Arabidopsis mutants in carotenoid biosynthesis containing respectively either alpha-branch xanthophyll (chy1chy2lut5) or beta branch xanthophylls (chy1chy2lut2). Both mutants show complete elimination of qE, the rapidly reversible component of NPQ and high levels of photoinhibition and lipid peroxidation under photooxidative stress. Both mutants are much more photosensitive than npq1lut2, which contains high levels of viola- and neoxanthin, suggesting that the latter xanthophylls play an important role in photoprotection. In addition, chy1chy2lut5, which has lutein as the only xanthophyll, shows unprecedented photosensitivity even in low light conditions, reduced electron transport rate, enhanced photobleaching of isolated LHCII complexes and a selective loss of CP26, while chy1chy2lut2 shows loss of LHCII trimers. The stronger PSII photoinhibition of both mutants correlates with the higher rate of singlet oxygen production from isolated light-harvesting complexes, while carotenoid composition of PSII core complex does not affect generation of oxidizing molecules from it. In-depth analysis of the mutant phenotypes suggests that alpha-branch (lutein) and beta-branch (zeaxanthin, violaxanthin, neoxanthin) have quite distinct roles in photosystem assembly and photoprotection.

PS15.30 Dynamics of the truncated lutein epoxide cycle in avocado; implications for efficiency of light harvesting B Osmond (The Australian National University), B Förster (The Australian National University), B Pogson (The Australian National University) Leaves of avocado (Persea americana) accumulate the a-xanthophyll lutein-epoxide (Lx) in excess of violaxanthin (V) in shaded habitats. On transfer to strong light both Lx and V are de-epoxidised by violaxanthin de-epoxidase (VDE) to lutein (L) and to antheraxanthin (A) and zeaxanthin (Z) respectively, with about the same kinetics. However, whereas Z and A are rapidly epoxidised to form V by zeaxanthin epoxidase (ZE) when light exposure is followed by shade or darkness, lutein is not reverted to Lx, and hence little Lx is present in sun leaves. The slow accumulation of Lx in the shade in avocado, and in other plants such as laurel and Inga spp., may indicate that in these species ZE has mutated, acquiring a lower but finite affinity for L than for A and Z (Matsubara et al. 2003; Planta 217, 868-879). Moreover, since Lx accumulating shade leaves are rarely exposed to light intensities high enough to facilitate de-epoxidation, there may be little selection pressure for rapid L epoxidation. We investigated relationships between

xanthophyll pool sizes and PSII efficiency (dark adapted Fv/Fm) in avocado leaves and discuss implications for the regulation of light harvesting efficiency arising from the differing kinetics of these two co-located xanthophyll cycles (Matsubara et al 2007; Plant Physiology, in press).

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PS16 - Metabolite Transport and Intracellular Interactions PS16.1 Redox and energy photosynthesis

flux in roots and the dynamics of vacuolar sugar flux. relations

between

respiration

and

P Gardeström (Umeå University) Photosynthesis is not a process only concerning chloroplasts. Rather, at the cellular level efficient photosynthesis depends on coordinated functions by all cell compartments. In particular mitochondrial functions in the light are closely related to chloroplasts and photosynthesis. For example, mitochondrial activity is essential for effectively switching from respiratory meta-bolism in the dark to photosynthetic metabolism in the light by facilitating redox communication. Mitochondria also contribute to changes in photosynthetic rate when light intensity suddenly increases, possibly by decreasing effects of phosphate limitation on stromal reactions. Many of the interactions between chloroplasts and mitochondria in the light are coupled to photorespiration. In fact, a major function for leaf mitochondria in the light is linked to oxidation of glycine in the photorespiratory glycolate pathway. The flux through this is considerable and the NADH formed during mitochondrial glycine oxidation and the consumption of NADH in the peroxisomal reduction of hydroxy pyruvate strongly affect the redox situation of the cell. The redox states of different sub cellular compartments are connected by translocators and shuttles such as the "malate valve". Thereby also chloroplasts are affected by changes of redox state in other compart-ments. Examples will bw given that suggest that an important function for leaf mitochondria in the light is connected to redox homeo-stasis of the photosynthetic cell. In this way the mitochondria support CO2 fixation in the chloroplasts by creating a favourable redox environment for the photosynthetic reactions.

PS16.2 Carb Fluxomics in Plants F Hörmann (Carnegie Institute), B Chauduri (Carnegie Institute), K Deuschle (Carnegie Institute), I Lager (Carnegie Institute), S Okumoto (Carnegie Institute), S Lalonde (Carnegie Institute), W Frommer (Carnegie Institute) Besides their role in cellular carbon metabolism and energy metabolism, sugars also play an important role as signaling molecules to modulate growth, development and stress response. Plants use sucrose as the predominant sugar for long distance transport whereas mammals use glucose for circulation in the vascular system. Plant cells also contain free glucose, although glucose is typically phosphorylated and does not occur not as a direct product of photosynthesis, but results from sucrose and starch metabolism. Sugar metabolism is dynamic and metabolic flux changes dramatically depending on light, nutrient availability and biotic or abiotic stress, as well as during development. In our lab we have developed genetically encoded nanosensors to directly visualize glucose and sucrose flux in living tissues in real time with minimal invasion, cellular and subcellular resolution. The nanosensors consist of a recognition element, typically a periplasmic binding protein (PBP), fused with Cyan (CFP) and Yellow Fluorescent Proteins (YFP) in various configurations. Changes in the conformation of the PBP upon interaction with glucose or sucrose lead to a change in fluorescence resonance energy transfer efficiency between the fluorophores. A set of nanosensors with varying affinities allowed us to quantify the cytosolic sugar concentration in leaves and roots. We could show that in the absence of external sugar supply, cytosolic glucose levels dropped below 100nM within 1-2 minutes. Similar experiments using a set of sucrose nanosensors is underway. By expressing the sensors in Arabidopsis mutants, we can address questions regarding transport and metabolic

PS16.3 Multiple Factors Mediate the Cross Talk Between Mitochondrial Metabolism and Photosynthetic Carbon Assimilation: Important Roles of Ascorbate and Photorespiratory CO2 A Raghavendra (University of Hyderabad), K Riazunnisa (University of Hyderabad), L Padmavathi (University of Hyderabad), B Sunil (University of Hyderabad) The biochemical interactions of chloroplasts, mitochondria, peroxisomes and cytoplasm within the plant cell are essential to optimize photosynthesis. Our earlier work demonstrated that the redox state and relative levels of metabolites (triose-P/PGA and malate/oxalacetate) were modulated during such interactions. Elevation of Asc (by L-galactone,1-4-lactone) made leaf discs of pea to sustain high rates of photosynthesis at supraoptimal light, and alleviated the inhibition of photosynthesis by inhibitors of mitochondrial metabolism. The deficiency of Asc (vtc1 mutants of Arabidopsis) resulted in a significant decrease in photosynthesis at high light. Photosynthesis in vtc1 mutants, particularly at high light, was extremely sensitive to SHAM. The ratio of reduced to total Asc appears to be more important than the absolute levels of Asc, in protecting photosynthesis against photoinhibition. Mesophyll protoplasts of pea required much less CO2 (74.1 mM) for maximal photosynthesis, than their chloroplasts (588 mM CO2). Such low requirement for CO2 could be due to internal carbon source and/or CO2 concentrating mechanism in mesophyll protoplasts. The marked sensitivity of protoplast photosynthesis to photorespiratory inhibitors (aminoacetonitrile or glycine hydroxamate) and ethoxyzolamide, an inhibitor of carbonic anhydrase, indicated that photorespiration could be a significant source of CO2 for photosynthesis in mesophyll protoplasts at limiting CO2 and atmospheric levels of oxygen. Sustained supply of CO2 through mitochondrial reactions of photorespiration appeared to optimize the photosynthetic carbon assimilation. We therefore suggest that ascorbate (Asc) and recycling of photorespiratory CO2 can be additional factors involved in the beneficial interactions of mitochondria and chloroplasts.

PS16.4 Screening for solute transporters in plant photosynthetic membranes C Spetea Wiklund (Division of Cell Biology, Linköping University, Linköping, Sweden), L Ruiz Pavón (Division of Cell Biology, Linköping University, Linköping, Sweden), F Lundh (School of Pure and Applied Natural Sciences, Kalmar University, Kalmar, Sweden), S Thuswaldner (Division of Cell Biology, Linköping University, Linköping, Sweden), A Mishra (Division of Cell Biology, Linköping University, Linköping, Sweden), B L Persson (School of Pure and Applied Natural Sciences, Kalmar University, Kalm) As compared to chloroplast envelope transporters, the field of thylakoid transporters is largely unexplored. There is evidence for several transport activities in the plant thylakoid membrane, but only a copper P-type ATPase and an ATP/ADP carrier have been so far identified at the gene level in Arabidopsis thaliana (1, 2). Using in silico analyses, we have predicted the existence of approx. fifteen thylakoid transporters, including phosphate transporters and cation channels. For experimental

259 14th Photosynthesis Congress - PS07 validation, we have used peptide-specific antibodies and functional analyses in heterologous system. These novel data are highly relevant to understand the transport network of the thylakoid membrane and its role in photosynthesis and stress adaptation. (1) Abdel-Ghany, S. E., Muller-Moule, P., Niyogi, K. K., Pilon, M., and Shikanai, T. (2005) Two P-type ATPases are required for copper delivery in Arabidopsis thaliana chloroplasts. Plant Cell 17, 1233-1251. (2) Thuswaldner, S., Lagerstedt, J.O., Rojas-Stütz, M., Bouhidel, K., Leborgne-Castel, N., Mishra, A., Marty, F., Schoefs, B., Adamska, I., Persson, B.L. and Spetea, C. (2007) Identification, expression and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis, J. Biol. Chem. 282, 8848-8859. PS16.5 Comparative biochemical characterization of cytosolic tetrapyrrole binding proteins of Arabidopsis thaliana. S Takahashi (University of Tokyo), T Masuda (University of Tokyo) In plant cells, heme is synthesized in plastids and distributed to various organelles, where it binds to apoproteins forming active holoenzymes. In addition, tetrapyrrole intermediates are reported as plastid-derived signals that regulate the expression of nuclear photosynthetic genes. However, mechanisms of distribution and transportation of tetrapyrroles are poorly understood. Since tetrapyrroles are hydrophobic, they are presumed to be distributed by binding to carrier proteins. In this study, to clarify this mechanism, we characterized genes of Arabidopsis thaliana, which exhibited homology to tetrapyrrole binding protein family, namely p22HBP/SOUL, that is predicted to be involved in tetrapyrrole transportation in animal cells. In Arabidopsis thaliana, 6 gene products were identified as homologs of p22HBP/SOUL family and two of which possessed putative N-terminal signal peptides. One gene (At1g78450) contained 10-base deletion in ORF suggesting a pseudogene. The other three gene products without signal peptide were presumably localized in cytoplasm. Actually, fluorescence of GFP fusion proteins of At1g17100 was detected in cytoplasm. RT-PCR analysis showed that At1g17100 predominantly expressed in leaves, while At2g37970 is mainly expressed in roots. Using purified recombinant proteins of At1g17100 and At2g37970, we performed binding analysis to metal porphyrins. Both proteins showed specific binding to heme and Mg-protoporphyrin IX dimethyl ester. Upon binding to heme, both proteins showed distinct oligomeric forms. Furthermore, the heme-binding form of each protein was able to reconstitute a peroxidase activity, when mixed with apoprotein of horseradish peroxidase. These results suggested that both proteins function as cytosolic tetrapyrrole carrier proteins in distinct tissues of plant cells. PS16.6 The role of the mitochondrial electron transport chain in photosynthesis, stress responses, and the integration of carbon-nitrogen metabolism G Noctor (Université Paris), C Dutilleul (Université Paris)), C Lelarge (Université Paris)), J Prioul (Université Paris)), R De Paepe (Université Paris)), C Foyer (University of Newcastle Upon Tyne) Leaf mitochondria play important roles in photosynthetic processes like photorespiration and the coordination of carbon and nitrogen metabolism. We have investigated the significance of mitochondrial electron transport status in leaf function by exploiting a Nicotiana sylvestris mutant, CMS, that lacks complex I and grows by respiring through alternative dehydrogenases. Complex I deficiency does not decrease dark respiration: both oxygen consumption and CO2 evolution are increased in CMS compared to WT leaves. In contrast, complex I appears to play a role in optimizing photosynthesis, since CO2 assimilation in air is slower in CMS leaves than in the WT, even though

photosynthetic capacity is unchanged [1]. Decreased photosynthesis relative to respiration is associated with increased resistance to abiotic and biotic stress, as well as alterations in both the expression levels and day/night rhythms of antioxidative enzymes [2]. This occurs without significant changes in leaf ascorbate, glutathione, or NADP, but is associated with increased NAD+ and NADH. Accumulation of NADH could occur to satisfy the high KMNADH of the alternative mitochondrial dehydrogenases, while accompanying changes in NAD+ may reflect mechanisms operating to promote redox homeostasis. Associated with increases in NAD pools are accumulation of nitrogen-rich amino acids and depletion of carbohydrates [3]. The results point to a potentially crucial role for mitochondrial-driven changes in NAD status in determining stress resistance and influencing metabolic coordination in plants. [1] Dutilleul et al. (2003) Plant Physiology 133: 264-275 [2] Dutilluel et al. (2003) Plant Cell 15: 1212-1226 [3] Dutilleul et al. (2005) Plant Physiology 139: 64-78 PS16.7 Interactions between photosynthesis and respiration during elicitor-induced cell death in tobacco C Foyer (Newcastle University), M Garmier (Institut de Biotechnologie des Plantes (IBP), P Priault (Université Paris Sud), G Vidal (Institut de Biotechnologie des Plantes (IBP) ), S Driscoll (Newcastle University), R Djebbar (Université Pierre et Marie Curie), M Boccara (Université Pierre et Marie Curie), C Mathieu (Institut de Biotechnologie des Plantes (IBP) ), R De Paepe (Newcastle University) Light and oxygen are considered to be important in the execution of programmed cell death (PCD) in plants, principally because they are involved in the generation of reactive oxygen species (ROS). We compared the hypersensitive response (HR)-like PCD process in N. sylvestris leaves that is elicited by harpin Nea,, under light/dark and atmospheric/very low oxygen (VL ox: < 0,1%). PCD was induced under all conditions, but it was delayed in the dark and accelerated under VL ox. Rapid inhibition of photosynthesis and an acceleration of respiration are characteristic of light–elicited leaves. The harpin-induced collapse of CO2 assimilation and photosystem II activity was delayed under VL. Moreover, the burst of CO2 release caused by enhanced mitochondrial respiration in dark-elicited leaves was inhibited under VL ox. Superoxide and hydrogen peroxide accumulation inside the chloroplasts was observed in both the light and the dark, but not under VL ox. The accumulation of transcripts encoding cytosolic antioxidant genes was also greatly affected by oxygen deprivation. These results indicate that extra-mitochondrial ROS are not key signals to harpin-induced cell death. Rather, the acceleration of the HR-like process under VL ox is associated with the perturbation of respiratory pathways and possibly enhanced mitochondrial ROS production.

PS16.8 Functional analysis of mitochondrial respiratory chain as a dissipation system of excess light energy K Yoshida (The University of Tokyo), I Terashima (The University of Tokyo), K Noguchi (The University of Tokyo) In illuminated leaves, mitochondrial respiration is closely correlated with photosynthesis. It is known that excess reducing equivalents generated in the chloroplasts can be transported to mitochondria and be dissipated by the respiratory chain. Plant mitochondria possess alternative oxidase (AOX), an unique terminal oxidase in the respiratory chain. Although AOX catalyzes energy-wasteful respiration, it may prevent chloroplast over-reduction through the efficient dissipation of excess reducing

260 14th Photosynthesis Congress - PS07 equivalents. We examined this possibility in a physiological context. Firstly, we inhibited AOX in broad bean leaves and monitored the redox state of photosynthetic electron transport. Even under low-light, the inhibition of AOX (but not the cytochrome pathway) caused a decrease in photosynthetic rate and an imbalance between the operating efficiencies in the two photosystems. Secondly, we used Arabidopsis mutants defective in cyclic electron flow around PSI (pgr5, crr2-2) and performed integral analyses of the accumulation of reducing equivalents in the chloroplasts, the transport activity between organelles, and the property of mitochondrial respiration. AOX capacity in the mutants was higher even in low-light growth conditions. AOX capacity, even in the wild-type, was preferentially up-regulated by high-light treatment, which is concomitant with the accumulation of reducing equivalents in the chloroplasts and an increase in the activities of enzymes needed to transport reducing equivalents. These results suggest that AOX can dissipate excess reducing equivalents transported from the chloroplasts, and serve in efficient photosynthesis. We will also discuss about factors for AOX up-regulation under the light. PS16.9 Photosynthetic activity of two Desmodesmus armatus (Chlorophyta) strains during light-induced cell cycle Z Tukaj (University of Gdansk), K Matusiak-Mikulin (University of Gdansk) Chlorococcal green algae such as Desmodesmus divide by multiple fission. In synchronized by light:dark (14/10 h) regime cultures each cell of Desmodesmus divided into 8 autospores. This means that each cell must reach three commitment points (G1/S) to initiate three successive cell divisions, preceded by the appropriate growth phases (G1). The distinct differences between both strains were observed as regards the intensity and the efficiency of photosynthesis. The strain B1-76 was characterized by about 2 times higher intensity of oxygen evolution than 276-4d strain. Similarly, the photosynthetic capacity and efficiency calculated from the light response curves were higher in the B1-76 strain cells, especially in the first 3 hours of the cell cycle. On the contrary, the values of ΦPSII, FV'/FM' and qP characterized by chlorophyll fluorescence measurements (PAM method) was about 15% higher in the 276-4d strain. The values of NPQ dropped rapidly during the first 3 h in both strains and then declined more gradually to 14 h of cycle. The major component of NPQ in B1-76 strain was qE, whereas the similar values of qE and qI were observed during the cycle of 276-4d. OJIP-tests revealed the characteristic course of φE0 changes during the cell cycle. The some relationship between the φE0 and the phase of cell cycle was observed. In particular, the maxima φE0 were well correlated with successive G1 phases of the cell cycle, whereas the minima obtained at the G1/S commitment points.

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PS17 - Biogenesis of Photosynthetic Apparatus PS17.1 Chloroplast biogenesis and the trafficking of proteins and metabolites F Kessler (University of Neuchatel) During biogenesis chloroplasts import several hundred different proteins from the cytosol. Translocon complexes at the outer (Toc) and inner membrane of the chloroplast (Tic) accomplish protein transport across the envelope. Arabidopsis mutant phenotypes ranging from pale green to albino and embryo lethal have demonstrated the essential role of Toc and Tic components in chloroplast biogenesis. We will present new data on the functional mechanisms of the Toc-complex. Once across the envelope many proteins are sorted further to the thylakoid membrane system. But not only proteins also small organic molecules are channeled from the envelope membranes to the thylakoids. We will demonstrate that plastid lipid droplets (plastoglobules) participate in metabolism, storage and supply of tocopherol, phylloquinone and plastoquinone from the envelope to the thylakoid membrane and photosystems.

PS17.2 Transport of proteins across chloroplast membranes S Theg (University of California-Davis)

PS17.3 Structure and function of the low molecular mass proteins PsbTn and PsbX in the PSII supra-complex W Schröder (Department of Chemistry), R Horn (Department of Chemistry), J García-Cerdán (Department of Chemistry) The Photosystem II (PSII) complex of higher plants, algae and cyanobacteria drives the water oxidation process of photosynthesis. Electron and X-ray crystallography analyses have revealed that the PSII core complex contains between 34 and 36 transmembrane a-helices, depending on the organism studied. Of these helices at least 12-14 are attributed to low molecular mass proteins (< 10 kDa), at least 18 of them are expected to reside in the entire PSII super-complex [reviewed in Shi and Schröder 2004, BBA1608:75-96]. Most of these small proteins contain a single transmembrane span and their protein sequences are conserved among photosynthetic organisms. The PsbTn protein is nuclear encoded by two genes PsbTn1 and PsbTn2. The mature protein has a molecular mass of 3.2 kDa in Arabidopsis. T-DNA knockout of PsbTn1 shows a 90% reduction of the total PsbTn protein based on immunological studies. We were able to confirm the predicted luminal localisation of this protein using site-directed antibodies. Interestingly however, our data suggest that the protein is attached to PSII, equally strong as is the extrinsic PsbO protein. Further analyses to locate the protein and characterize its function will be presented. Further on another small protein of PSII, the PsbX protein, has been analysed by characterisation of T-DNA knock out Arabidopsis mutants. While these plants do not show a distinct phenotype under normal growth conditions, thermoluminescence studies indicate a destabilisation of the QB pocket of PSII in the absence of PsbX.

PS17.4 The Cysteine Desulfurase CpNifS is Essential for Iron-Sulfur

Cluster Biogenesis in Chloroplasts E Pilon-Smits (Colorado State University), D Van Hoewyk (Colorado State University), C Cohu (Colorado State University), M Pilon (Colorado State University) CpNifS is a chloroplastic NifS-like protein in Arabidopsis thaliana that can catalyze the conversion of cysteine (Cys) into alanine and elemental sulfur (S), and of selenocysteine into alanine and elemental selenium (Se). Earlier, overexpression of CpNifS was shown to enhance plant Se tolerance and Se and S accumulation (Van Hoewyk et al. (2005) Plant Physiol 139: 1518-1528). To study the role of CpNifS in Fe-S cluster biogenesis in vivo, CpNifS was silenced via an inducible RNAi system (Van Hoewyk et al. (2007) Proc Natl Acad Sci USA 104: 5686-5691). The resulting transgenics with reduced CpNifS levels showed chlorosis, abnormal chloroplast structure, stunted growth, and eventually died. Photosynthetic electron transport and carbon dioxide uptake were significantly reduced in the RNAi transgenics. The observed phenotype associated with CpNifS silencing appeared to be caused by impaired plastidic Fe-S cluster biogenesis: the RNAi plants had lower leaf concentrations of all chloroplastic Fe-S proteins tested, representing all five Fe-S cluster types. Mitochondrial Fe-S protein abundance and respiration were not affected, suggesting that mitochondrial and chloroplastic Fe-S assembly operate independently. We conclude that CpNifS is necessary for the maturation of all plastidic Fe-S proteins, and as such is essential for plant growth. The activity of CpNifS toward Cys is likely stimulated (50-fold) and regulated by various CpSufE proteins in vivo (Ye et al. (2006) J Biol Chem 281: 8958-8969; Murthy et al. (2007) J Biol Chem, in press).

PS17.5 Cyanobacterial NADPH:protochlorophyllide oxidoreductase (POR) compensates for a knockdown mutation of PORA in Arabidopsis thaliana S Masuda (Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology), R Ikeda (Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology), T Masuda (Graduate School of Arts and Science, The University of Tokyo), T Tsuchiya (Hall of Global Environmental Research, Kyoto University), M Mimuro (Hall of Global Environmental Research, Kyoto University), H Ohta (Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology), K Takamiya (Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology) NADPH-dependent protochlorophyllide oxidoreductase (POR) catalyzes a light-dependent reduction of protochlorophyllide a that is a key regulatory step in the biosynthesis of chlorophyll in all oxygenic phototrophic organisms. In eukaryotic phototrophs, PORs are localized in plastid, suggesting that it was originated by symbiosis of an ancestor of cyanobacterium. In light of the evolutionary considerations, we studied the POR from the cyanobacterium Gloeobacter violaceus that has been suggested to retain the ancestral properties of cyanobacteria since on the phylogenetic tree based on 16S rRNA sequences, it branched off at the earliest stage within the cyanobacterial linage. We found that the recombinant POR of Gloeobacter expressed in E. coli possessed a light-dependent activity of protochlorophyllide reduction. The kinetic properties of the protein, such as Km value to protochlorophyllide, are similar to those of PORs from higher plants rather than those of another cyanobacterium Synechocystis sp. PCC6803. We introduced cyanobacterial POR genes in a por (porA) knockdown mutant of Arabidopsis thaliana in trans. Gloeobacter POR fully complemented the phenotype of the mutant line, although Synechocystis

262 14th Photosynthesis Congress - PS07 POR only partially, but distinctly, restored it. These results indicated that Gloeobacter POR is fully functional even in plastid of Arabidopsis. Further evolutionary implications of the enzyme will be discussed based on the comparative genetic and biochemical analyses of PORs from cyanobacteria and higher plants. PS17.6 A novel pathway of cytochrome c biogenesis is involved in the assembly of the cytochrome b6f complex in Arabidopsis chloroplasts L Lezhneva (Physiologie Membranaire et Moleculaire du Chloroplaste UMR7141 (CNRS/UPMC), Institut de Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie, 75005 Paris, France), R Kuras (Physiologie Membranaire et Moleculaire du Chloroplaste UMR7141 (CNRS/UPMC), Institut de Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie, 75005 Paris, France), G Ephritikhine (Institut des Sciences du Vegetal, UPR 2355, CNRS, 1 Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France), C de Vitry (Physiologie Membranaire et Moleculaire du Chloroplaste UMR7141 (CNRS/UPMC), Institut de Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie, 75005 Paris, France) Analysis of the cytochrome b6f complex structure at 3 angstrom resolution revealed a novel heme covalently bound to cytochrome b6. Compared to the other members of the c-type family, this heme designated ci i) possesses unusual structural properties: a single thioether bond to the apoprotein and the pentacoordinated iron and ii) resides on the n-side (negatively charged) of the membrane corresponding to the chloroplast stroma. To extend the knowledge of c-type cytochrome biogenesis in plants, we analysed the functions of the three Arabidopsis orthologs of nuclear CCB genes which are specifically required for heme attachment to apocytochrome b6 in the unicellular green alga Chlamydomonas reinhardtii. In Chlamydomonas, the four CCB proteins constitute a novel pathway of the c-type cytochrome maturation1,2. Applying a reverse genetics approach, we characterized Arabidopsis T-DNA lines carrying insertions in the CCB genes. Disruption of CCB1, CCB2 and CCB4 prevents binding of heme ci to cytochrome b6 and consequently accumulation of the cytochrome b6f complex leading to impairment in photosynthesis. We conclude that the CCB pathway is a general pathway involved in the holocytochrome b6 assembly in chloroplasts. 1. Kuras R, de Vitry C, Choquet Y, Girard-Bascou J, Culler D, Buschlen S, Merchant S and Wollman FA (1997) Molecular genetic identification of a pathway for heme binding to cytochrome b6. J. Biol. Chem. 272, 32427-32435. 2. Kuras R, Saint-Marcoux D, Wollman, FA and de Vitry C (2007) A novel c-type cytochrome maturation system is required for oxygenic photosynthesis. Proc. Natl. Acad. Sci. USA, submitted.

PS17.7 Analysis of the effect of elevated cytokinin content on the photosynthetic apparatus using Blue Native PAGE A Cortleven (Centre for Environmental Sciences), R Valcke (Centre for Environmental Sciences) Two-dimensional gel electrophoresis is a very powerful tool for the analysis of a large number of proteins. Unfortunately, this technique is not very useful for the separation of highly hydrophobic membrane proteins and the isolation of native protein complexes. Blue-native gel electrophoresis offers a very good alternative. Previous analyses have shown that cytokinins affect the photosynthetic apparatus. To study the

effects of cytokinins on the composition of the complexes in the photosynthetic apparatus, transgenic tobacco plants with increased cytokinin content are used. Those are compared to wild-type plants. The transgenic plants (Pssu-ipt) were transformed by the Agrobacterium tumefaciens transformation system and contain the ipt-gene encoding for isopentenyl transferase which is a key enzyme of the cytokinin biosynthetic pathway. This gene is coupled to the light-inducible promoter of the small subunit of the RubisCO-enzyme of Pisum sativum. After isolation of intact chloroplasts of transgenic and wild-type plants, digitonin (2,5%) and ß-dodecyl-maltoside (1%) are used to obtain the native conformation of the photosynthetic complexes. A separation in the first dimension under native conditions, Blue Native PAGE, is combined with SDS-PAGE for separation of the protein complexes into their subunits. This provides a better insight in the network of protein complexes of the photosynthetic apparatus and the physiological changes due to elevated cytokinin content.

PS17.8 Insertional Mutagenesis, Genomic Complementation and Biophysical Phenotyping in Chlamydomonas reinhardtii X Johnson (IBPC CNRS UMR7141), R Kuras (IBPC CNRS UMR7141), F Wollman (IBPC CNRS UMR7141), O Vallon (IBPC CNRS UMR7141) The unicellular green alga, Chlamydomonas reinhardtii, has advantages for photosynthetic gene discovery as cells grown heterotrophically on acetate maintain normal chloroplast development even when one of the photosynthetic complexes is missing. In order to identify novel genes involved in the photosynthetic processes we have produced a targetted and varied collection of mutants to be characterised by genomic complementation. By electroporating C. reinhardtii strain arg7 cw15 with antibiotic resistance cassettes, selecting on a range of antibiotic concentrations and using a selective enrichment procedure, acetate requiring phenotypes could be recovered at a frequency of 14% of insertional mutants. Thirty-four mutants were further investigated. Fluorescence induction on dark adapted cells revealed the presence of b6f, PSI, PSII and ATPase deficient mutants. A small set of mutants with WT fluorescence induction kinetics were further characterised using spectroscopic techniques and revealed unexpected features. Eight b6f mutants were subjected to a primary screen using nuclear genes known to encode structural proteins or regulatory factors of the b6f complex, lack of complementation revealing that these mutants were unique. Mutants with similar phenotypic traits were pooled into groups and subjected simultaneously to complementation with an indexed cosmid library. Cosmid pools containing the gene of interest were identified by restoration of photosynthetic growth. Characterisation of new photosynthetic mutants and technical progress in the pursuit of a fast and reliable method to identify genes in Chlamydomonas will be presented. PS17.9 Excitation pressure modulates the extent of variegation through the regulation of chloroplast biogenesis in the immutans mutant of Arabidopsis thaliana D Rosso (University of Western Ontario), D Saccon (University of Western Ontario), L Schillaci (University of Western Ontario), S Wang (University of Western Ontario), W Li (University of Western Ontario), S Rodermel (Iowa State University), D Maxwell (University of Western Ontario), N Huner (University of Western Ontario) The immutans variegation mutant of Arabidopsis thaliana contains green and white sectored leaves. White sectors result from a blockage in carotenoid biosynthesis. IMMUTANS protein regulates carotenoid biosynthesis and is presumed to act as the plastid terminal oxidase in the

263 14th Photosynthesis Congress - PS07 oxidation of reduced plastoquinone through chlororespiratory electron transport. Excitation pressure, measured by chlorophyll fluorescence as 1-qP, is an estimate of the redox state of the PQ pool and can be modulated by both light and temperature. We hypothesized that the redox state of the PQ pool regulates the extent of variegation in immutans. To test this hypothesis, immutans was grown at either 25ºC or 12ºC with increasing light intensities of 50, 150 and 450 µmol photons m-2s-1 under either a short day photoperiod (8/16 day/night) or under continuous light. An imaging technique was developed to quantify the kinetics of leaf variegation with concomitant measurements of excitation pressure during steady-state photosynthesis. As the growth light intensity increased at 25ºC, the extent of variegation increased from no detectable variegation at 50 µmol photons m-2s-1 to a maximum of 65% variegation at 450 µmol photons m-2s-1. In contrast, immutans grown at 12ºC exhibited significant variegation even when grown at low light under either a short day photoperiod or continuous light conditions. Since the development of white sectors was correlated with excitation pressure, it appears that the redox state of the PQ pool regulates the extent of variegation. Greening experiments confirm that the lack of IMMUTANS impairs chloroplast biogenesis during growth under high excitation pressure. PS17.10 Functional analysis of two PsbP-like (PPL) proteins in Arabidopsis thaliana S Ishihara (Graduate school of Biostudies, Kyoto University), A Takabayashi (Graduate school of Biostudies, Kyoto University), T Endo (Graduate school of Biostudies, Kyoto University), K Ifuku (Graduate school of Biostudies, Kyoto University), F Sato (Graduate school of Biostudies, Kyoto University) PsbP, an extrinsic subunit of photosystem II (PSII), has been reported to be present only in plants and green algae. Our recent studies suggest that PsbP is an indispensable regulator of PSII in higher plants. On the other hand, genomic and proteomic studies have demonstrated the existence of the PsbP homolog in cyanobacteria (cyanoP), whereas it was reported that cyanoP was rarely associated with PSII. These reports suggest that cyanoP would not be an ortholog of PsbP. In fact, higher plants have additional PsbP-like (PPL) proteins that have higher sequence similarity to cyanoP than to PsbP; however, the function of PPL proteins has not yet been elucidated. In Arabidopsis thaliana, there are two PPL proteins, AtPPL1 and AtPPL2. Searching genome and EST databases by the tBlastN program identified orthologs of AtPPL1 and AtPPL2 in all of the plant species we checked, and their amino acid sequences were highly conserved. On the other hand, no PPL protein ortholog was found in Chlamydomonas reinhardtii, which contains PsbP. In Cyanidioschyzon merolae, we found two PsbP homologs, whose sequences are much closer to cyanoP than to PPL proteins in higher plants. These results suggest that both PPL1 and PPL2 are well-conserved unique proteins in higher plants, but not in chlorophytes or rhodophytes. High conservation of PPLs indicates the importance of their function in higher plants. To elucidate their functions, characterization of Arabidopsis mutants lacking either of two PPLs, ppl1 and ppl2, is on going.

utilising chlorophyll d (Chl d) as its major photosynthetic pigment. A. marina cells contain 90-99% Chl d with minor amounts of chlorophyll a and a chlorophyll c-like pigment. These unusual characteristics make it an excellent candidate to study various aspects of photosynthesis driven by Chl d. However, little is known about the pathway of Chl d biosynthesis. We specifically designed HPLC methods to analyse pigment compositions of A. marina. This enabled us to detect intermediate products of the chlorophyll biosynthesis. We identified Mg-Protoporphyrin (MgP, MgPMe) and Mg-3,8-divinyl pheoporphyrin (MgDVP) as well as environmental factors influencing their concentration levels. HPLC-facilitated analysis of pigments from A. marina cells cultured under various light quantities was performed; light stress conditions induced an increase in the ratio of MgDVP to Chl d. Pigment analysis of A. marina cells grown under oxygen-stressed conditions demonstrated a decrease in MgDVP levels. We propose that the Chl d biosynthesis pathway favours an aerobic environment despite the fact that A. marina cells can survive under anaerobic conditions. PS17.12 Chlorophyll a biosynthesis under anaerobic environments in the cyanobacterium Synechocystis sp. PCC 6803 Y Fujita (Nagoya University), T Goto (Nagoya University), K Minamizaki (Nagoya University) There are two oxygen-dependent reactions, coproporphyrinogen III oxidation and the E-ring formation, in chlorophyll a biosynthesis. Many strains of cyanobacteria are capable of growing photosynthetically under anaerobic environments. Here we show how these oxygen-dependent reactions proceed in cyanobacteria. We searched candidate genes for these steps from the genome sequence of Synechocystis sp. PCC 6803 and found hemF and two hemN (hemN1 and hemN2) genes for oxygen-dependent and oxygen-independent coproporphyrinogen III oxidases (CPOs), respectively, and two acsF genes (acsF1 and acsF2) encoding Mg-protoporphyrin IX monomethylester (MPE) cyclase. A series of mutant lacking one of these genes was isolated. Two mutants lacking hemF and acsF1 showed an oxygen-sensitive phenotype with anomalous accumulation of coproporhyrin and MPE, respectively, suggesting that both genes are essential for chlorophyll biosynthesis under aerobic conditions. While no detectable phenotype was observed in a hemN2-lacking mutant, a hemN1 mutant did not grow under anaerobic conditions. It is suggested that hemN1 encodes oxygen-independent CPO that operates under anaerobic conditions. An acsF2-lacking mutant accumulated MPE with slight growth retardation under anaerobic conditions. Interestingly, hemN1 and acsF2 appear to form a small operon. RT-PCR indicated that both hemN1 and acsF2 are induced under anaerobic conditions in contrast to the constitutive expression of hemF and acsF1. Taken together, it is suggested that under anaerobic conditions the cyanobacterium proceeds coproporphyrinogen III oxidation by oxygen-independent CPO, HemN1, and E-ring formation by AcsF2, the second homolog of AcsF, which could utilize a low level of oxygen evolved endogenously from photosystem II.

PS17.11 The function of MgDVP in a chlorophyll d-containing organism

PS17.13 Biochemical analysis of two catalytic components of nitrogenase-like enzymes protochlorophyllide reductase and chlorophyllide a reductase from Rhodobacter capsulatus

M Schliep (School of Biological Sciences, University of Sydney), M Chen (School of Biological Sciences, University of Sydney), T Larkum (School of Biological Sciences, University of Sydney), R Quinnell (School of Biological Sciences, University of Sydney)

J Nomata (Nagoya University), M Kitashima (Kanagawa University), T Ogawa (University of Tokyo), K Inoue (Kanagawa University), Y Fujita (Nagoya University)

The cyanobacterium Acaryochloris marina is an exceptional organism

Bacteriochlorophyll a has bacteriochlorin ring structure that is formed from porphyrin ring by the sequential actions of two nitrogenase-like

264 14th Photosynthesis Congress - PS07 enzymes, dark-operative protochlorophyllide reductase (DPOR) catalyzing the D-ring reduction and chlorophyllide a reductase (COR) catalyzing the B-ring reduction. We have previously demonstrated that DPOR consists of L-protein and NB-protein and COR also does of X-protein and YZ-protein. The structural relatedness of these components to nitrogenase Fe-protein and MoFe-protein suggests that L-protein and X-protein are the reductase components and NB-protein and YZ-protein are the catalytic components. The catalytic components provide a molecular basis for the substrate specificity, which determines the order of reduction of D- and B-ring in bacteriochlorophyll biosynthesis. However, they have been largely uncharacterized. Here we report comparative biochemical characterization of NB-protein and YZ-protein. NB-protein and YZ-protein overexpressed in Rhodobacter capsulatus were purified as the substrate-bound forms, supporting that they are the catalytic components. Overexpression of the two components in a bchH-mutant of R. capsulatus allowed purifying them as substrate-free "naked" forms. Absorption spectra of the naked forms suggested that both are Fe-S proteins. Dithionite-reduced YZ-protein showed an EPR signal indicative of [4Fe-4S] cluster. While dithionite-reduced NB-protein was EPR silent, an EPR signal typical for [4Fe-4S] cluster was observed in the presence of L-protein and ATP. Metal content supported the presence of a pair of [4Fe-4S] cluster in NB-protein. These results suggested that both catalytic components carry simple [4Fe-4S] clusters with different properties rather than the complex clusters, P-cluster and FeMoco, of MoFe protein. PS17.14 Assembly of the light harvesting apparatus in Rhodobacter sphaeroides. J Tucker (University of Sheffield), N Hunter (University of Sheffield) The photosynthetic apparatus of the purple non-sulphur bacterium Rhodobacter sphaeroides consists of three types of membrane-bound pigment protein complex: the reaction centre (RC), which is the site of charge separation across the membrane, and two different light harvesting complexes, LH1 and LH2 which are associated with the RC. These complexes are located in the highly invaginated intracytoplasmic membrane (ICM). Current models, based on detailed structures presented for bacterial pigment protein complexes, propose that the RC is enclosed within the LH1 complex, which is in turn surrounded by many LH2 complexes of . Assembly of pigment-protein complexes has been shown to be partially dependent on factors encoded for by open reading frames (ORFs) in the photosynthetic gene cluster (PGC) of Rba. sphaeroides and other related photosynthetic bacteria. LhaA is the gene product of one such ORF in Rhodobacter capsulatus and has been shown to influence the assembly of LH1 complexes in this organism (C.S.Young et al. (1988) J. Bacteriol., 180 1759-1765). The PGC of Rba. sphaeroides contains a direct homologue of LhaA. In both organisms these ORFs are sandwiched between the genes for the RC-H subunit puhA and the chlorophyll biosynthesis gene bchM. This poster will discuss evidence that LhaA in R.sphaeroides is a functional homologue of that in Rba. Capsulatus. Disruption of LhaA leads to obliteration of LH1 assembly in the ICM; possible roles for LhaA at the confluence of biosynthesis and assembly are suggested. PS17.15 Immunophilins and prolyl isomerase activity in the thylakoid lumen of Arabidopsis A Shapiguzov (Division of Cell Biology, Linkoping University, Sweden), A Vener (Division of Cell Biology, Linkoping University, Sweden) The diversity of immunophilins in the thylakoid lumen of higher plants is one of the challenging mysteries of chloroplast proteomics. These ubiquitous enzymes are known to catalyze cis/trans isomerization of the

peptide bond formed by a proline residue – a reaction that dramatically alters protein topology and often represents the rate-limiting step in protein folding. Generally recognized as protein folding catalysts, immunophilins, however, demonstrate a highly complicated range of functions and substrate specificities. Thylakoid immunophilins are likely to be involved in regulation of photosynthetic processes, but their functions and protein partners are virtually unknown. The study of prolyl isomerase activity towards the set of synthetic peptide substrates in the thylakoid lumen of Arabidopsis revealed only two active enzymes: AtFKBP13 (At5g45680) and AtCYP20-2 (At5g13120) [1]. The remainder of up to 14 lumenal immunophilins had no detectable isomerase activity, which agrees with poor conservation of the crucial amino acid residues in their sequences. Our results suggest that the majority of the lumenal immunophilins evolved high specificities for unique interacting partners while AtFKBP13 and AtCYP20-2 retained broader substrate specificity, possibly facilitating general protein folding catalysis in the thylakoid lumen. The ongoing studies on AtFKBP13 and AtCYP20-2 knockout Arabidopsis lines indicate that these isomerases are redundant and that their activity is the subject of stringent regulation. The functions of thylakoid immunophilins and the physiological role of their isomerase activity await further investigation, particularly by characterization of AtFKBP13/AtCYP20-2 double mutant Arabidopsis lines. 1. Shapiguzov, A., Edvardsson, A., and Vener, A.V. (2006) FEBS Lett 580, 3671-3676 PS17.16 Isolation and characterization of genes necessary for achievement of RuBisCO accumulation in Arabidopsis thaliana. T Ogawa (NAIST), K Nishimura ( NAIST)), H Ashida ( NAIST)), A Yokota ( NAIST) Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO) is the most abundant protein in plant leaves, and catalyzes the initial reactions in the Calvin cycle and photorespiration. An accumulation of RuBisCO is properly regulated by various environmental conditions and developmental stages. However, the molecular mechanisms for biosynthesis and maintenance of RuBisCO remain to be understood. In order to identify genes involved in these processes, we have established a positive screening method for nara (genes necessary for achievement of RuBisCO accumulation) mutants with low amounts of RuBisCO using methionine sulfoximine (MSX). MSX is a specific inhibitor of glutamine synthetase required for re-assimilation of photorespiratory ammonia. On the selection medium containing MSX, The wild-type accumulates ammonia drastically, and cannot survive in normal air, but can grow in 5% CO2. Meanwhile, The rca mutant lacking RuBisCO activase does not accumulate ammonia, and exhibits resistance to MSX in normal air. These results suggest that the effect of MSX depends on the oxygenase reaction of RuBisCO. Therefore we assumed that this medium can be used for screening of nara mutants. 6 lines of recessive nara mutants with 15% - 60% wild-type levels of RuBisCO were screened from approximately 20,000 EMS-mutagenized seeds of Arabidopsis. In three nara mutants, genetic mapping have revealed that mutations are found in already identified genes related to photorespiration or chloroplast RNA metabolisms. In contrast, mutations have been found in genes encoding unknown proteins with predicted plastid-targeting signal in other nara mutants. We would like to discuss about the relation between nara phenotype and these genes.

PS17.17 Slr1923 of Synechocystis 6803 is related to chlorophyll a metabolism

265 14th Photosynthesis Congress - PS07

M Islam (University of Hyogo), Y Kashino (University of Hyogo), K Satoh (University of Hyogo), H Koike (University of Hyogo) The deduced amino acid sequence of a slr1923 gene product is a water soluble protein carrying a 4Fe-4S cluster and is homologous to F420H2 oxidoreductase which acts as an electron input device to NAD(P)H dehydrogenase complex in archaebacteria. However, the function of the gene product is remained to be identified. A mutant in which the gene slr1923 was inactivated by insertion of spectinomycin-resistant cassette has been created in Synechocystis sp. PCC 6803. The mutant showed responses against growth conditions different from those of wild type (WT) under various stresses. Under normal condition (3% CO2 + 100 μE m-2s-1), it showed slower growth rate compared with WT. It grew rather slowly under low CO2 (0.03%), low light (30 μE m-2s-1) and high salt stress (0.5M NaCl) conditions. The mutant did not grow under high light (300 μE m-2s-1) condition. Absorption spectra of the intact cells indicate that chlorophyll a content of the cell has been reduced drastically in the mutant. It was also of note that Soret band was shifted toward longer wavelength by about 6 nm, while red band peak position was not changed. The retention time of chlorophyll a was shorter than that of wild type by HPLC analysis, suggesting the existence of a modified chlorophyll a species in the mutant. A high fluorescence intensity at 77K peaking at about 685 nm might be an indication of miss-connection and/or decreased efficiency of energy transfer from phycobilisomes to PSII. Other characteristic features will also be reported.

(Kyoto Prefecture Univ.), R OHNIWA (Kyoto Univ.), T SHIINA (Kyoto Prefecture Univ.), K TAKEYASU (Kyoto Univ.) P-loop GTPases and related ATPases represent an abundant and remarkable group of proteins in bacteria, which perform essential functions in all kingdoms of life. The Obg and Era super family comprises a group of ancient GTPases belonging to the TRAFAC (for translation factors) class. The Era/Obg family proteins have been reported to be involved in several diverse bacterial functions, including ribosome assembly, DNA repair, sporulation, and morphological development. GTPases of the Era/Obg family are also conserved in eukaryotes, including mammals and plants. However, little is known about the biological role of these GTPases in eukaryotic cells. In this study, intensive search on gene and protein databases revealed that Arabidopsis thaliana have 10 (ten) proteins belong to the Obg super family and 8 (eight) proteins belong to the TrmE-Era super family. We found that most bacterial homologues were targeted to chloroplasts (eight) or mitochondria (three) or both (two), whereas other Archaea related proteins were mainly targeted to nucleus (two) or retained in cytoplasm (two) or targeted to cytoplasm and nucleus. Furthermore, most of the Arabidopsis Era/Obg family genes were found to be essential for plant development in Arabidopsis. These results suggest that members of the GTP-binding Era/Obg family play crucial roles in the biogenesis and/or the functioning of plant organelles, including chloroplasts. PS17.20

PS17.18 Identification of a chloroplast-localized SAP domain containing protein in Arabidopsis thaliana.

A dominant nuclear mutation in Chlamydomonas identifies a factor controlling chloroplast mRNA stability by acting on the coding region of the petA transcript

Y Yagi (Kyoto prefecture univ.), Y Ishizaki (Kyoto prefecture univ.)), T Shiina (Kyoto prefecture univ.)

A Boulouis (CNRS-UPMC Paris 6), Y Choquet (CNRS-UPMC Paris 6), J Girard-Bascou (CNRS-UPMC Paris 6), K Wostrikoff (Boyce Thompson Institute for Plant Research, Cornell University, Ithaka, NY), F Wollman (CNRS-UPMC Paris 6)

Plastids are plant-specific organelles that originated from cyanobacteria, and possess their own genome and generic system. In higher plants, plastids may differentiate into several forms, including chloroplasts, amyloplasts and chromoplasts. Unlike cyanobacteria, plastid genes in higher plants are transcribed by two distinct RNA polymerases: plastid encoded eubacterial-type RNA Polymerase (PEP) and nuclear encoded phage-type RNA Polymerase (NEP). It has been established that plastid transcription plays an important role in plastid differentiation. However, molecular mechanisms underlying plastid differentiation is still unclear. To identify genes involved in nuclear control of plastid differentiation, we screened for albino mutants in RIKEN phenome database and found 12 candidates. Among them, we focused on At3g04260, since it is predicted to be localized in chloroplasts and contains a SAP motif that is involved in DNA binding in eukaryotic matrix attachment region binding proteins. Transient expression of the GFP fusion protein in Arabidopsis protoplasts suggested that N-terminal sequences of At3g04260 act as a plastid transit peptide. In order to understand the role of At3g04260 in plastid differentiation, we isolated T-DNA insertion mutant of At3g04260. As expected, the mutant showed albino phenotype. The tilling macroarray analysis showed that expression of All PEP-dependent genes including photosynthesis and rRNA genes was significantly reduced in the mutant plants. In contrast, the mutant plants showed increased accumulation of NEP–dependent transcripts including rpoA,B,C encoding PEP core subunits. These results suggested that plastid localized protein, At3g04260 is involved in the regulation of plastid gene expression during chloroplast development. PS17.19 Localization of Obg-Hflx and TrmE-Era super family small GTPases in various organelles in plant cells. I SUWASTIKA (Kyoto Univ.), M DENAWA (Kyoto Univ.), A HATA

We isolated a mutant strain of Chlamydomonas reinhardtii, Su0, which is affected in the expression cytochrome b6f complex. It shows an 10-fold decreased amount of petA transcripts, when compared to the wild type. Crosses of the Su0 mutant with wild type demonstrated the nuclear origin of the mutation. The study of su0/+ diploid strains showed that su0 is a dominant mutation. Using chimeric genes comprising different parts of the petA gene introduced by transformation in the chloroplast genome of the Su0 strain, we showed that the gene product mutated in strain Su0 acts on the petA coding sequence, more precisely in the 3’ half of the coding region. We discuss the possible functional roles of this nucleus-encoded factor on chloroplast transcripts metabolism. We compare the phenotype of this mutant with that of the previously described strain mda1-ncc1. PS17.21 Role of thioredoxin interactions of the luminal chloroplast proteome of Arabidopsis thaliana T Kieselbach (Dep. of Chemistry, Umeå University, 90187 Umeå, Sweden), M Hall (Dep. of Chemistry, Umeå University, 90187 Umeå, Sweden) Redox signalling via thioredoxins plays central roles in the light-mediated regulation of metabolic pathways of the chloroplast. Recent observations indicate strongly that thio-transduction pathways not only take place in the chloroplast stroma but also regulate functions of the chloroplast lumen [1, 2, 3, 4]. Thioredoxin signalling is probably an intrinsic characteristic of the entire chloroplast. Central questions are, if there are thioredoxins or related proteins that reduce luminal proteins. What are their sources of regeneration and their target proteins? Are

266 14th Photosynthesis Congress - PS07 there links to other luminal pathways and how is redox-regulated luminal signal transduction coupled to the function of photosynthesis and signalling in the chloroplast stroma? This study aims to identify luminal thioredoxin targets and their biochemical functions. The initial experimental set-up using the E. coli thioredoxin/thioredoxin reductase system and fluorescence electrophoresis was able to clearly detect the known prevalent luminal thioredoxin targets that include PsbO1, PsbO2, TL17 and FKBP13. In addition, novel thioredoxin interactions were observed for the xanthophyll cycle enzyme violaxanthin de-eopoxidase and three other luminal chloroplast proteins. The experimental approach is currently further developed to enable detection of low abundance luminal thioredoxin targets targets and to study the role of thioredoxin interactions for the regulation of the xanthophyll cycle. References [1] Gopalan G, He Z, et al. (2004) PNAS 101: 13945-13950 [2] Kieselbach T and Schröder WP (2003) Photosynth Res 78: 249–264 [3] Marchand C, Le Marechal P, et al. (2004) Proteomics 4: 2696-2706 [4] Marchand C, Le Marechal P, et al. (2006) Proteomics 6: 6528-6537 PS17.22 A new c-type cytochrome maturation system is required for oxygenic photosynthesis D Saint-Marcoux (IBPC-CNRS), R Kuras (IBPC-CNRS), C de Vitry (IBPC-CNRS), F Wollman (IBPC-CNRS)

activity. In parallel, bioinformatics searches for unknown genes that co-regulate with known chlorophyll biosynthetic genes have been performed to obtain candidates for the missing cyclase subunits. One hit is a chloroplast localized NADPH-dependent reductase with similarities to NADPH-thioredoxin reductases, and for which the Arabidopsis T-DNA mutant shows a chlorotic phenotype. Feeding d-aminolevulinic acid to mutant Arabidopsis plants deficient either in the diiron component of the cyclase or the putative reductase results in accumulation of the same chlorophyll precursors: Mg-protoporphyrin IX and MgP-monomethyl ester. This suggests that both proteins might be involved in the same reaction. Both the diiron protein and the putative reductase are being expressed in E. coli. Currently the proteins are tested for activity both alone and in combination with plastid components using the in vitro cyclase assay. PS17.24 Phosphoprotein CaS is associated with the thylakoid membrane of plant chloroplasts J Vainonen (Turku University), Y Sakuragi (University of Copenhagen), S Stael (University of Turku), V Paakkarinen (University of Turku), E Aro (University of Turku), M Suorsa (University of Turku), M Tikkanen (University of Turku), Y Allahverdiyeva (University of Turku), H Vibe Scheller (University of Copenhagen), A Vener (University of Linkoping), E Aro (University of Turku)

c-type cytochromes are generally found on the p-side of membranes in many bioenergetic systems. Depending on the organism and/or on the type of organelle, three different pathways have been identified for the covalent attachment of the c-haem to its apocytochrome target. Cytochrome b6 - a key subunit of cytochrome b6f complexes from oxygenic photosynthesis – contains a very unusual c’ haem located next to the n-side of the thylakoid membranes. This c’- haem is attached to cytochrome b6 by a single thioether bond. Using a genetic approach in Chlamydomonas reinhardtii, we identified and characterized four proteins, that we named CCBs, that control binding of this ci’ haem during cytochrome b6 biogenesis. ccb mutants are non photosynthetic and loose the whole cytochrome b6f complex because of impaired binding of haem ci’. The four CCB products are encoded by the nuclear genome, targeted to the chloroplast where they insert into the thylakoid membrane as transmembrane proteins. We provide evidence that CCB2 and CCB4 are homologous subunits of a heterodimer complex. Site-directed mutagenesis points to some conserved tryptophan residues on the n-side of the membrane that are critical for the function of CCB2 and CCB3. These four proteins are present in all procaryotic and eucaryotic organisms performing oxygenic photosynthesis, whose genomes are currently available. They define a novel maturation system of c-type haems that works on the n-side of the membrane.

Exposure of Arabidopsis thaliana plants to high light revealed specific phosphorylation of 40 kDa protein in photosynthetic thylakoid membranes. The protein was identified by mass spectrometry as CaS (extracellular calcium sensing receptor), previously reported to be located in the plasma membrane (1). Localization of CaS to the chloroplasts was confirmed by confocal microscopy with YFP-fusion protein. Both the content of CaS in the thylakoid membrane and its phosphorylation level strongly responded to light intensity and further thylakoid fractionation showed the enrichment of CaS in stroma thylakoids. The phosphorylation site was mapped to the stroma-exposed threonine residue 380 located in motifs for interaction with 14-3-3 proteins and forkhead-associated domain, which suggests the involvement of CaS in stress responses and signaling pathways. Phosphorylation of CaS was abolished in the mutants lacking light-dependent thylakoid protein kinase STN8. The Cas knock-out Arabidopsis line revealed retarded growth demonstrating the importance of CaS for plant development. The lack of CaS caused significant reduction in the level of thylakoid lumen proteins implying the importance of CaS for proper protein import across the thylakoid membrane.

PS17.23 The aerobic cyclase complex in chlorophyll biosynthesis

O Vallon (Institut de Biologie Physico-Chimique), B Derrien (Institut de Biologie Physico-Chimique), W Majeran (Cornell University), K van Wijk (Cornell University), F Wollman (Institut de Biologie Physico-Chimique)

A Stenbæk (University of Copenhagen), A Hansson (University of Copenhagen), P Jensen (University of Copenhagen) The aerobic cyclase system catalyses the formation of the fifth ring found in all chlorophylls. The reaction catalysed by the cyclase is a six electron oxidation, proposed to occur in three sequential steps, resulting in the incorporation of a single oxygen atom and the formation of a carbon-carbon bond. The cyclase has been shown to consist of at least two and possibly three protein components. One of the cyclase components has been shown to be a membrane bound diiron carboxylate protein (CRD1 or CHL27). In analogy to other membrane bound diiron carboxylate proteins it has been suggested that a reductase and a substrate binding protein or regulatory subunit might constitute the unknown subunits. We have developed an in vitro assay for cyclase

PS17.25 The Chlamydomonas ClpP complex : slicing the cutter

The chloroplast ClpP complex has been implicated in the degradation of both soluble and membrane proteins, in particular under stress conditions. It is a hetero-oligomer associating the chloroplast-encoded ClpP1 protein and a series of nuclear-encoded ClpP (active) and ClpR (inactive) subunits. In Chlamydomonas reinhardtii, ClpP1 presents an insertion sequence (IS1) that has been postulated to serve as a protein intron (1). Immunodetection studies have shown that a full-size ClpP1H protein is found in the 540 kDa ClpP complex, together with a low MW band ClpP1L (2). Here, we show, using C-terminally and internally-tagged clpP1 transformants, that ClpP1L is not the product of a splicing event. Rather, it represents the N-terminal immunoreactive

267 14th Photosynthesis Congress - PS07 fragment generated by endoproteolytic maturation of ClpP1. Two C-terminal fragments can be detected within the complex, resulting from cleavage in the C-terminal part of IS1 after residues 316 and 332. The ClpP complex therefore presents the large additional IS1 domain on at least one of its apical surfaces, likely interfering with binding of the ClpC chaperone that is necessary for ATP-dependent proteolytic activity. We have affinity-purified the ClpP complex and will present its preliminary characterization, in particular the presence of new ClpS3 and ClpS4 subunits. The ClpR1 protein shows cleavage of its large N-terminal domain, emphasizing the importance of post-translational maturation in the biogenesis of this major chloroplast protease.

cyt-bf-biogenesis is irreversibly down-regulated, once the photosynthetic apparatus is assembled in mature leaves. To identify the molecular basis of the ontogenetic switch-off of cyt-bf biogenesis, we determined gene expression patterns of the subunits of the cyt-bf. No leaf-age related differences in mRNA accumulation could be observed, strongly indicating that the ontogenetic down-regulation is due to alterations in translation or assembly, which is now analysed using polysome analyses. To determine the signals, which initiate the developmental depression of cyt-bf biogenesis, we analysed the effects of altered sink-source-balance and altered phytohormone contents in mature leaves. Both cytokinin application and increased sink demand can re-induce cyt-bf-biogenesis, indicating that the ontogenetic switch-off in mature leaves is due to systemic signals, integrating the developmental state of the plant.

PS17.26 The 5' UTR targets atpA transcripts towards translation or non-polysomic ribonucleoproteic complexes in the chloroplast of Chlamydomonas reinhardtii

PS17.28 Coordinated functions of plastid sigma factors in chloroplast development and gene expression

S Eberhard (Institut de Biologie Physico-Chimique), C Loiselay (Institut de Biologie Physico-Chimique), S Bujaldon (Institut de Biologie Physico-Chimique), D Drapier (Institut de Biologie Physico-Chimique), Y Choquet (Institut de Biologie Physico-Chimique), F Wollman (Institut de Biologie Physico-Chimique)

T Shiina (Kyoto Prefectural University), Y Ishizaki (Kyoto Prefectural University), K Ozono (Kyoto Prefectural University), C Takenaka (Kyoto Prefectural University), M Hanaoka (University of Tokyo), K Kanamaru (Kobe University), K Tanaka (University of Tokyo), Y Nakahira (Kyoto Prefectural University)

We show that the 5’ UTR-driven initiation of translation of the chloroplast atpA transcript is dependent on the nucleus-encoded TDA1 factor. Using the current procedures for polysome purification, we investigated the relative distribution of chloroplast transcripts in a WT strain and in two mutant strains defective for translation of the chloroplast atpA gene. The first strain, tda1-F54, bears a nuclear mutation in the gene for TDA1, that prevents translation of the atpA transcript, while the second strain bears a mutagenized atpA gene, where the initiation codon has been mutated to a stop-codon. In both cases, non-translated atpA transcripts were detected in high-density fractions of the sucrose gradient that were partly EDTA-insensitive. Our results suggest that atpA transcripts can be either associated with polysomes, in a TDA1-dependent manner, or be found in non polysomic ribonucleoproteic complexes, that would store non-translated transcripts in the chloroplast. Using chimeric genes, we show that the 5’UTR of the atpA transcript is sufficient to adress unrelated mRNA sequences to these non polysomic complexes. We discuss the physiological significance of these ribonucleoprotein complexes in light of our previous reports that mRNA levels are not limiting for translation in the chloroplast.

Higher plant chloroplasts contain the prokaryotic type plastid encoded RNA polymerase (PEP) and additional nucleus-encoded phage-type RNA polymerases (NEP). PEP and NEP serve different sets of genes in plastids, and PEP is predominantly involved in the transcription of photosynthesis genes. Promoter specificity to PEP is conferred by nuclear-encoded sigma factors. Six distinct sigma factor genes (SIG1 to SIG6) have been identified in higher plants. Arabidopsis mutants deficient in one of the six sigma factors have revealed that each sigma factor is responsible for transcription of specific genes. However, plastid sigma factors may act redundantly in plastid gene expression and provide compensatory functions during plant development. To analyze interactive functions of plastid sigma factors, we produced several double mutants by crossing sig2, sig5 and sig6 mutants. The sig5sig6 double mutants showed additive phenotypes, indicating that SIG5 and SIG6 function independently of each other. On the other hand, seedlings of sig2 and sig6 mutants exhibited a partial defect in chloroplast developments, whereas chloroplast development was completely arrested and photosynthesis gene expression was greatly reduced in sig2sig6 double mutants. These results indicate that SIG2 and SIG6 act in a highly redundant manner to control chloroplast development. Interestingly, the psbA transcript levels were specifically and greatly reduced in sig2sig5 double mutants, suggesting that SIG2 and SIG5 genes are required for the expression of psbA gene encoding photosystem II D1 protein. These results suggested that chloroplast sigma factors coordinately orchestrate the transcription and/or gene expression program of chloroplast development.

PS17.27 Ontogenetic changes in the biogenesis of the Cytochrome-b6f complex C Flügel (Max-Planck-Institut für Molekulare Pflanzenphysiologie), W Thiele (Max-Planck-Institut für Molekulare Pflanzenphysiologie), R Bock (Max-Planck-Institut für Molekulare Pflanzenphysiologie), M Schottler (Max-Planck-Institut für Molekulare Pflanzenphysiologie) The cytochrome-b6f complex (cyt-bf) is the predominant point of photosynthetic flux control and functions in the adjustment of photosynthetic electron transport to the metabolic ATP and NADPH demand. However, almost nothing is known about the regulation of its assembly, its stability and lifetime. Investigations of a chloroplast transformant lacking a non-essential subunit of the cyt-bf (DpetL) revealed that the cyt-bf is highly stable with a lifetime in the range of several days to weeks (Schöttler et al. (2007): J. Biol. Chem. 282, 976-985): Loss of the L-subunit results in a destabilization of the complex, which in mature and old leaves cannot be compensated by de novo biogenesis, leading to an accelerated loss of both cyt-bf and assimilation capacity. We conclude that the capacity for

PS17.29 Functional analyses of the PsbW and PsbY subunits of Photosystem II in higher plants J García-Cerdán (Umeå University) Photosystem II (PSII) is the enzyme responsible for the light-driven water oxidation with concomitant production of oxygen and the reduction of the plastoquinone pool in photosynthetic organisms. PSII is a multi-subunit complex of about 700 kDa, almost half of its components are low molecular mass proteins (< 10 kDa). Despite of being highly conserved in all photosynthetic organisms, there is still not much known about the function and location of these small proteins within the PSII complex. Here we have analyzed the PsbY and the PsbW proteins using T-DNA mutagenesis. The psbY gene encodes the only known chloroplast-targeted poly-protein i.e. the PsbY precursor consists

268 14th Photosynthesis Congress - PS07 of two subunits of ~4 kDa with high homology, PsbY-A1 and PsbY-A2. In this way two proteins are imported into the chloroplast for the “cost” of one. Analysis of the psbY knock-out mutant revealed no distinct phenotype. However, compared to the wild type the mutant exhibits a different pattern in the 77K fluorescence emission spectrum with an additional peak. The psbW gene codes for a subunit with molecular mass of about 6 kDa that has been shown to be involved in the stabilization of PSII super-complexes. Analysis of the T-DNA mutant revealed an alteration in state transition, similar to the one observed in mutant with altered CP24 (Kovacs et al. (2006) Plant Cell 18, 3106). Characterization of the functional importance of these two mutants will be presented. PS17.30 The balance between chloroplast protein synthesis and degradation as an important factor of chloroplast biogenesis and maintenance W Sakamoto (Okayama University) We have been studying the Arabidopsis leaf-variegated mutant var2 that results from the loss of FtsH2, a major component of the chloroplast FtsH complex. FtsH is an ATP-dependent metalloprotease in thylakoid membranes and degrade several chloroplastic proteins. In order to understand the role of proteolysis by FtsH and the mechanisms leading to leaf variegation, we characterized a second-site recessive mutation fu-gaeri1 (fug1) that suppressed leaf variegation of var2. We revealed by map-based cloning that FUG1 encodes a protein homologous to prokaryotic translation initiation factor 2 (cpIF2). We show evidence that cpIF2 indeed functions in chloroplast protein synthesis in vivo. Suppression of leaf variegation by fug1 is observed not only in var2 but also in var1 (lacking FtsH5) and var1 var2. Thus, the suppression of leaf variegation caused by the loss of FtsHs was most likely attributed to reduced protein synthesis in chloroplasts. This hypothesis was further supported by the observation that another viable mutation in chloroplast translation elongation factor G also suppressed the leaf variegation of var2. Based upon these results, we propose that the balance between protein synthesis and degradation is one of the determining factors leading to the variegated phenotype in Arabidopsis leaves. Influence of this balance in photodamage is also discussed, since FtsH plays a major role in the repair cycle of Photosystem II. Reference Miura, E., Kato, Y., Matsushima, R., Albrecht, V., Laalami, S., and Sakamoto, W. (2007) The balance between protein synthesis and degradation in chloroplasts determines leaf variegation in Arabidopsis yellow variegated mutants. Plant Cell, in press. PS17.31 A pentatricopeptide repeat (PPR) protein regulates chloroplast biogenesis at the early growth stage W Zhou (Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, 200032 Shanghai, People's Republic of China) Wenbin Zhou and Jirong Huang National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, 200032 Shanghai, People's Republic of China Chloroplast biogenesis is dependent on the coordinate gene expression between nuclear and plastidic genomes. A mutant designated ys1(yellow seedling 1) were identified in a screen for defects in chloroplast development at the early growth stage from T-DNA insertion lines of Arabidopsis. The ys1 mutant exhibited a yellow seedling phenotype. However, it was interesting that the ys1 plants was able to gradually recover to the same growing state as wild-type plants in terms of chlorophyll contents and other photosynthetic parameters about one

month after seeds were germinated. The ultrastructure of chloroplasts by electronic transmission microscopy revealed that the thylakoid membrane formation was apparently impaired in ys1. Immunoblot analysis showed proteins composed of photosynthetic complexes were significantly reduced in the ys1 leaves than those in wild-type. The flanking sequence showed that YS1 encodes a pentatricopeptide repeat (PPR) protein. A putative chloroplast transit peptide was predicted at the N-terminal region of the YS1 protein. This prediction was testified by fusion of YS1 or the predicted transit peptide with GFP. The molecular mechanism how YS1 mutation results in the defect in chloroplast development is in progress.

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PS18 - Origin and Evolution of Photosynthetic Systems PS18.1 Inorganic complexes in the onset of life and oxygenic photosynthesis M Russell (JPL; California Institute of Technology), J Allen (Queen Mary, University of London), E Milner-White (University of Glasgow) Hydrothermal convection currents pumped materials and chemical energy to emerging and burgeoning life: convection currents in the Earth’s mantle ― acting on the oceanic crust ― conveyed living systems to further nutrients and to chemical and photochemical sources of energy [1]. Certain minerals, acting as host, hatchery, catalyst and shelter to metabolizing systems, enabled chemosynthesis and oxygenic photosynthesis to emerge, Amongst these were canaphite (CaNa2P2O7.4H2O), mackinawite ([Fe>>Ni)S]), greigite (NiS2[Fe4S4]S2Fe) and a tunnel manganite (CaMn4O8) similar in structure to hollandite [1-3]. The inorganic complexes contributing to the growth of such minerals (e.g., HP2O73-; [FeS2Fe]4H2O; [Fe4S4]0/+; [Fe3S4] +/2+; NiFe5S8 and CaMn4O8) were later sequestered by small organic molecules (initially polypeptides or carboxyl groups) as active centres of the enzyme precursors that initially catalyzed the primary reactions of energy conversion and nutrient cycling [4,5]. Examples of such adventitious cooption would produce (i) pyrophosphate (or acetyl phosphate) in successive main chain NH+ + peptide nests; (ii) protoferredoxins as thiolated eggs in peptide nests; (iii) precursors to CODH/ACS by Ni-peptide and a thiolated egg in peptide nests and (iv) the active centre of the OEC [2,6] by periplasmic carboxyl groups [2,5,6]. 1. Russell, M.J., Hall, A.J. (2006) Geological Society of America, Memoir 198, p. 1-32. 2. Yano, J. et al., (2006) Where water is oxidized to dioxygen. Science 314, 821-825. 3. Barrier, N. et al. (2005) Journal Material Chemistry 15, 386-393. 4. Baymann, F. et al. (2003) Phil. Trans R. Soc. London, 358B, 267-274. 5. Milner-White, E.J., Russell M.J. (2005) Origins Life Evolution Biosphere 35, 19-27. 6. Allen, J.F., Martin W. (2007) Nature 445, 610-612.

PS18.2 The cyanobacterial genome core and the origin of photosynthesis A Mulkidjanian (University of Osnabrück), E Koonin (NCBI, NLM, National Institutes of Health), K Makarova (NCBI, NLM, National Institutes of Health), R Haselkorn (University of Chicago), M Galperin (NCBI, NLM, National Institutes of Health) The comparative analysis of 15 complete cyanobacterial genome sequences has revealed 84 protein families (Cyanobacterial clusters of Orthologous Groups of proteins, or CyOGs) that are exclusively shared by cyanobacteria and plants. The majority of them could be attributed to photosynthetic enzymes, the remaining 35 families of uncharacterized proteins, by analogy, are likely to be previously unrecognized components of the photosynthetic machinery. In contrast, only a few photosynthetic CyOGs are represented in the genomes of anoxygenic phototrophic bacteria Chlorobium tepidum, Rhodopseudomonas palustris, Chloroflexus aurantiacus or Heliobacillus mobilis. Based on this analysis, a concomitant consideration of the available biochemical and geological data as well as of the physical constrains that were

imposed by the environment of the primordial Earth, we suggest that photosynthesis originated in the cyanobacterial lineage under the selective pressure of UV light and depletion of electron donors. The results of our analysis corroborate the view that the first phototrophs were anaerobic ancestors of cyanobacteria ('procyanobacteria') that conducted anoxygenic photosynthesis using a photosystem I-like photosynthetic reaction center. From procyanobacteria, photosynthesis spread to other phyla via lateral gene transfer.

PS18.3 A bacterial-type sensor kinase couples electron transport to gene expression in chloroplasts S Puthiyaveetil (Queen Mary, University of London), J Allen (Queen Mary, University of London) Two-component systems, comprising sensor histidine kinases and response regulators, are ubiquitous signal transducers in bacteria. Chloroplasts, despite having a bacterial (cyano) ancestry, do not appear to possess two-component systems as signal transducers. Apart from a few reported cases of two-component systems of the red algal chloroplasts, it is generally believed that the two-component systems of the ancestral symbiont were lost or recruited in various locations of the host cell other than chloroplasts. Here we report a typical bacterial-type sensor kinase in chloroplasts. The gene for this kinase is found in cyanobacteria, from which chloroplasts evolved, and has moved, in evolution, to the nuclear genomes of algae and green plants. The gene encoding this Chloroplast Sensor Kinase (CSK), when inactivated in Arabidopsis results in plants that are disabled in photosynthetic control of chloroplast gene transcription. This CSK-dependent process requires a sensor of electron transport between chloroplast photosystems I and II. Thus CSK is involved in a redox regulatory mechanism that couples photosynthesis to chloroplast gene expression. Sequence similarity searches find homologues of CSK to be present in many different lineages of algae and plants and to be related phylogenetically to the known plastid two-component systems of red algae. The persistence of this ancient signalling system of cyanobacteria in chloroplasts and its function in coupling photosynthesis to chloroplast gene expression bears directly to the premise that chloroplasts retain genes whose expression must be regulated by photosynthetic electron transport and that the mechanism of regulation has been conserved from the prokaryotic, ancestral endosymbiont.

PS18.4 Candidatus Chloracidobacterium thermophilum: an aerobic phototrophic Acidobacterium with chlorosomes and type 1 reaction centers D Bryant (The Pennsylvania State University), A Garcia Costas (The Pennsylvania State University), J Heidelberg (University of Southern California), D Ward (Montana State University) Chlorophototrophs, organisms which use chlorophyll (Chl) to convert light energy into chemical energy, have not yet been demonstrated in the domain Archaea, and until now examples were known in only five phyla of the Bacteria: Cyanobacteria, Proteobacteria, Chlorobi, Chloroflexi, and Firmicutes. Phototrophic microbial mats of alkaline siliceous hot springs in Yellowstone National Park have been extensively characterized and are known to contain thermophilic cyanobacteria and two members of the phylum Chloroflexi, Chloroflexus aurantiacus and Roseiflexus sp. (whose genome was recently completely sequenced). The

270 14th Photosynthesis Congress - PS07 availability of extensive metagenome data for this phototrophic microbial community provided an opportunity to examine whether other previously unknown chlorophototrophs might occur in this community. Analyses of the metagenomic sequence data suggested the presence of a population of green sulfur bacteria (Chlorobi) as well as the existence of a novel bacteriochlorophyll (BChl)-synthesizing bacterium with Type 1 reaction centers. A highly enriched culture of this bacterium was obtained from the mats. Growth of the organism under heterotrophic conditions was strongly stimulated by light. The bacterium synthesizes BChls a and c under oxic conditions, and it produces type 1 reaction centers, FMO, and chlorosomes. The BChl c is methylated at C-82 and C-121, and isolated chlorosomes are similar in size and appearance to those of Chlorobium tepidum. Sequencing of a 282-kb region of the genome conclusively demonstrated that this bacterium belongs to the poorly characterized phylum, Acidobacteria. “Candidatus Chloracidobacterium thermophilum” is the first chlorophototroph from the phylum Acidobacteria, only the sixth bacterial phylum that contains members with Chl-based phototrophy. PS18.5 The structure of plant photosystem I at 3.4 Å resolution – 3.5 billion years of perfection N Nelson (Tel Aviv University), A Amunts (Tel Aviv University), O Drory (Tel Aviv University) Photosystem I (PSI) emerged as a homodimeric structure containing several chlorophyll molecules over 3.5 billion years ago, and has perfected its photoelectric properties ever since. Recently we solved the structure of PSI supercomplex at 3.4 Å resolution (Amunts, A., Drory, O. and Nelson, N. The structure of a plant photosystem I supercomplex at 3.4 Å resolution. Nature in press). The PSI model at 3.4 Å resolution reveales 17 protein subunits including PsaN. The reaction center is highly homologous to that of the cyanobacterial PSI and maintains the position of most transmembrane helices and chlorophylls during the last 1.5 years of separate evolution. The assignment of 5 carotenoids and parts of the phytol chain of 65 chlorophyll molecules allow dipper look at the evolution of PSI and other reaction centers. It also provided better understanding of the supercomplexes formed during electron transfer between plastocyanin and ferredoxin as well as a potential LHCII binding site. The structure provided a first glimpse at the fine architecture of the most efficient nano-photochemical machine in nature and it tells a tale on the evolution of photosynthetic reaction centers. PS18.6 The chloroplast genome – terminal decline or faded grandeur? C Howe (University of Cambridge), A Barbrook (University of Cambridge), E Nisbet (University of Cambridge), S Purton (University College London) During the billion or so years since chloroplasts originated by endosymbiosis, their genome has undergone sustained reduction. In most photosynthetic organisms, typically around 100 – 150 genes remain. The peridinin-containing dinoflagellate algae, an important group of organisms responsible for a range of processes such as coral reef formation (as symbiotic zooxanthellae within the coral organisms) and red tides, have taken this process of reduction to an extreme, with fewer than 20 genes retained. More bizarrely, the retained genes are located on small circles, most of which carry only a single gene. We have found that, in addition, the rRNA genes have been trimmed down to a minimum, reminiscent of the rRNA genes found in some mitochondrial genomes. A few tRNA genes are also retained, including one for fMet-tRNA.

BR> Whether chloroplast genomes are in terminal decline and will eventually disappear, or are resolutely retaining a core of essential genes is an interesting question. We discuss possible reasons for the continuing existence of a chloroplast genome. The proposal that genes are retained for redox regulation is attractive, but less obviously applicable to non-photosynthetic organisms, such as Plasmodium. We suggest other reasons for retention of a chloroplast genome in these cases. PS18.7 Conversion of Chl a into Chl d by heat-treated papain S Fukuyo (University of Tsukuba), S Ohashi (University of Tsukuba), K Iwamoto (University of Tsukuba), Y Shiraiwa (University of Tsukuba), M Kobayashi (University of Tsukuba) In 1943, Chl d was first reported by Manning and Strain as a minor pigment in several red macroalgae. In 1996, a novel cyanobacterium, Acaryochloris marina, was isolated from colonial ascidians, and A. marina was found to contain Chl d as the dominant chlorophyll. However, the biosynthesis pathway of Chl d has not been clarified yet. Chl d is thought to be synthesized from Chl a, like Chl b from Chl a, while no experimental evidence was given. In 2004, we reported the formation of Chl d from Chl a in acetone or ethanol containing 10 % water in the presence of a crude papain (EC 3.4.22.2) at room temperature in the dark. Chl d was not formed when papain was absent. Here we report the conversion of Chl a into Chl d in aqueous acetone at room tempearature in the presence of papain treated at 96 for 20 min. The yield of Chl d from Chl a did not change, when heat-treated papain was used. The result indicates that conversion of Chl a into Chl d in aqueous organic solvents did not happened enzymatically, but was caused by something contained in commercially available papain. PS18.8 Succession of co-factors in photosystem I S Ohashi (University of Tsukuba), T Tsuchiya (Kyoto University), K Iwamoto (University of Tokyo), H Miyashita (Kyoto University), T Watanabe (University of Tokyo), Y Shiraiwa (University of Tsukuba), M Mimuro (Kyoto University), M Kobayashi (University of Tsukuba) Succession of co-factors in PS I-type reaction centers (RCs) from anoxygenic photosynthesis into oxygenic photosynthesis was considered from the viewpoint of the molecular structures. In the PS I-type RCs, BChl g in anoxygenic photosynthesis succeeded to Chl a in oxygenic photosynthesis; BChl g, but not BChl a, can be an ancestral pigment of Chl a, because the conversion from BChl g into Chl a easily took place by isomerization (intramolecular hydrogen transfer) under a relatively mild condition. The epimers of BChl g and Chl a, namely, BChl g' and Chl a', function as the primary electron donors in respective organisms. In the PS I RC of Acaryochloris marina, Chl d' function as the primary electron donor. The primary electron acceptors of PS I have been found to be Chl a derivatives even in anoxygenic photosynthetic organisms, suggesting a consistency in the binding site. The secondary electron acceptors in the PS I-type RCs are naphtoquinone molecules, and side chains are modified after a birth of cyanobacteria, leading to succession from menaquinone to phylloquinone in oxygenic organisms. It is notable that changes in co-factors did not occur in accordance with the birth of oxygenic photosynthesis, but an intermediary organisms, such as a cyanobacterium Gloeobacter violaceus PCC 7421, would give an idea for succession of co-factors in PS I. PS18.9 In between algae and plants: analysis of the LHC multigene family of the moss Physcomitrella patens allows identification of light harvesting proteins associated to

271 14th Photosynthesis Congress - PS07 adaptation to sub-aerial environments T Morosinotto (University of Padova), A Alboresi (Université de la Méditerranée), S Caffarri (Université de la Méditerranée), R Bassi (Universitá di Verona) The antenna system in photosynthetic eukaryotes is composed by members of a light harvesting complex (Lhc) multigenic family. These proteins play a key role in photosynthesis, being involved both in light harvesting and photoprotection. The moss Physcomitrella patens is emerging as a new model organism of photosynthetic eukaryotes: since it diverged from higher plants later with respect to green algae and early after land colonization, its study allows obtaining information on the evolution of the green lineage and adaptation to aerial environment. In this work we used the available polypeptides sequences of Physcomitrella patens to compare its antenna system of this organism with green algae and higher plants. We identified antenna polypeptides of both Photosystem I and II (Lhca1, Lhca2, Lhca3 and Lhcb4, Lhcb5), which evolved early in the green lineage and constitute the nucleus of antenna system in Viridiplantae. Other members of the family, on the contrary, evolved later and are suggested to play a role in the adaptation to the sub-aerial environment: in Photosystem II, Lhcb3 and Lhcb6 were shown to be present only in land organisms. Land plants increased their content in low energy-absorbing chlorophylls, as demonstrated by the differences in Photosystem I antenna polypeptides. A new subunit, named Lhcb9, was shown to be peculiar of Physcomitrella and absent in any other known photosynthetic organism: its preliminary characterization demonstrated that this protein has individual properties different from any other known antenna. Sequence analyses were integrated with a biochemical and functional characterization of this multigenic family in Physcomitrella. PS18.10 Tracing the Evolution of the Light-Harvesting Antennae in Chlorophyll a/b-Containing Organisms

PS18.11 Evolution of chloroplast

the

light

responsive

psbD

promoter

in

S Shimmura (Kyoto Prefectural University), M Nozoe (Kyoto Prefectural University), T Shiina (Kyoto Prefectural University) Transcription in plastids is driven by two distinct RNA polymerases: a bacteria-type multisubunit enzyme (PEP) and a phage-type single subunit enzyme (NEP). Typical PEP-dependent promoters have bacteria-like -35 and -10 promoter elements. The psbD-psbC operon encoding the D2 and CP43 proteins of photosystem II is transcribed from a unique light and stress-responsive PEP-dependent promoter (psbD LRP). Unlike typical PEP promoters, the psbD LRP contains a conserved upstream transcription activating element (AAG-box), but lacks a functional -35 element. The psbD LRP is highly conserved in both dicots and cereals. However, light-induced transcription in the psbD-C operon in lower plants is largely uncharacterized. In order to study the evolution of light-regulated psbD LRP, we identified the primary transcription initiation sites of the psbD-C operon, and further examined the light-dependent transcription of the psbD-C operon in various land plants, including the mosses, ferns, gymnosperms and angiosperms. The psbD LRP sequences are found in gymnosperms, including Cycadophyta, Ginkgophyta and Pinophyta, but not in ferns, indicating that the psbD LRP emerged during the early evolution of the gymnosperms. Interestingly, the psbD LRP has been lost during the evolution of Gnetophyta of gymnosperm. Furthermore, we found that the psbD genes are constitutively expressed irrespective of light conditions in Adiantum (fern), Cycad and Black pine (gymnosperm), and Bay laurel (angiosperm), whereas psbD gene expression is induced by light in Tabacco and Arabidopsis (angiosperm). These results suggest that the evolution of the psbD LRP sequences preceded the establishment of the light-responsive psbD transcription system. PS18.12

D Durnford (University of New Brunswick), A Koziol (University of New Brunswick), T Borza (Dalhousie University), K Ishida (University of Tsukuba), P Keeling (University of British Columbia), R Lee (Dalhousie University)

Comparative analysis of partially sequenced chloroplast DNA of Cicer arietinum

The Light-harvesting complexes (LHCs) of land plants and green algae have essential roles in light capture and photoprotection. Though the functional diversity of the individual LHC proteins are well described in many land plants, the extent of this family in the majority of green algal groups is unknown. In order to examine the evolution of the chlorophyll a/b antennae system and to infer its ancestral state, we initiated several expressed sequence tag (EST) projects from a taxonomically broad range of chlorophyll a/b-containing protists. This included representatives from the Ulvophyceae (Acetabularia acetabulum), the Mesostigmatophyceae (Mesostigma viride) and the Prasinophyceae (Micromonas sp.), as well as one representative from each of the Euglenozoa (Euglena gracilis) and Chlorarachniophyta (Bigelowiella natans), whose plastids evolved secondarily from green alga. It is clear that the core antenna system was well developed prior to green algal diversification and likely consisted of the CP29 (Lhcb4) and CP26 (Lhcb5) proteins associated with PSII plus a PSI antenna composed of proteins encoded by at least Lhca3 and two green algal-specific proteins encoded by the Lhca2 and 9 genes. In organisms containing secondary plastids, we found no evidence for orthologs to the plant/algal antennae with the exception of CP29. We also identified PsbS homologs in the Ulvophyceae and the Prasinophyceae, indicating that this distinctive protein appeared prior to green algal diversification. This analysis provides a snapshot of the antenna systems in diverse green algae, and allows us to infer the changing complexity of the antenna system during green algal evolution.

Two arbitrarily selected Bam HI fragments of ~4 and ~6 kb from Cicer arietinum chloroplast DNA were hydrolyzed by restrictase enzymes HindIII and EcoRI and the obtained sub-fragments were partially sequenced (totally, 2032 bp). Using the complete plastid genome sequences of dicotyledonous tobacco and arabidopsis and monocotyledonous rice and maize, a comparative computer analysis of the nucleotide and translated amino acid sequences has been performed. It revealed the existence of the almost whole tRNAAsn ? psaI genes, as well as 3/ -portions of petA, cemA, 4,5S rrn ? accD genes in the 2 partly overlapping Bam HI fragments. It was found that the hypothetical ORF 23 gene with the intact CDS, annotated in the plastid genomes of monocots, was not preserved in C. arietinum chloroplast genome. It was also shown that 2 group of genes (petA, cemA, accD, 4,5S rrn and tRNAAsn), located at the different parts (separated by ~40 kb) of plastid genomes of tobacco, arabidopsis, rice and maize, are included by the same region of chick pea plastid DNA. These findings suggest that during the evolution substantial structural changes occurred in the chickpea plastid genome. .

Z Suleymanova (Botany), I Shahmuradov (Botany), J Aliyev (Botany)

PS18.13 Establishing potential chloroplast function through phylogenomics S Merchant (Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095), S Prochnik (DOE Joint Genome Institute, 2800

272 14th Photosynthesis Congress - PS07 Mitchell Drive, Walnut Creek, CA94598), S Karpowicz (Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095), D Rokhsar (Center for Integrative Genomics, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720), A Grossman (The Carnegie Institution, Department of Plant Biology, Stanford, CA 94305) Chloroplasts from green algae and land plants are strikingly well conserved, especially with respect to components of the photosynthetic apparatus and the factors involved in its assembly and maintenance. The putative orthologs of Chlamydomonas genes from organisms with full genome sequences (Arabidopsis, Physcomitrella, diatoms, Ostreococcus spp., cyanobacteria, nematode, slime mold, human, Neurospora, Phytophthora, archaea, and non-photosynthetic bacteria) were determined based on a mutual best hits approach with WU-blast (Version 2). Next, we attempted to add only close paralogs or “inparalogs” to pairs of orthologs, while excluding ‘outparalogs’. The combination of paralogs and orthologs generated families of proteins that were presumably represented in the ancestor by a single gene. We were then able to ask for those that contained proteins from certain combinations of organisms, enabling us to generate a series of lists of proteins conserved in different green organisms but not present in non-photosynthetic eukaryotes or prokaryotes from the protein clusters. A working list consisting of 349 Chlamydomonas proteins consisting of proteins conserved in Arabidopsis, Chlamydomonas, Physcomitrella and Ostreococcus was analyzed in detail for a) known or predicted protein functions, b) predicted or experimentally-determined protein localization and c) pattern of expression of the Arabidopsis homolog. Most of the proteins on the list are indeed plastid localized or predicted to be so, and in most cases the pattern of expression is compatible with a function in photosynthesis or other anabolic pathways. Our conservative phylogenomics strategy is likely to have identified many novel proteins involved in photosynthesis, with few false hits. PS18.14 The origin and distribution of C3 and C4 photosynthetic species of the Centrospermeae along an altitudinal gradient in western Kenya. S Fedha (Maseno University, Kenya) Dicot species of the Centrospermeae were collected at different altitudes along the semi-arid and arid gradient in western Kenya. Climatic factors along the gradient were obtained from existing meteorological research stations. Species were screened for C3 and C4 photosynthetic systems using the leaf kranz syndrome, d13C values and carbon dioxide compensation points. C4 photosynthesis is restricted to advanced members of dicotyledoneae and exhibits multiple evolutionary origins. C4 pathways have evolved independently multiple times and within a family, it often occurs within one to several genera and then often only within two to three species. The current abundance and distribution of C4 dicots is related to high temperature, low precipitation and altitude, and high potential rate of evaporation (i.e. aridity). The low d 13C ratio negative value of C4 species is a potential aridity indicator and C4-syndrome marker unlike the C3 syndrome occurring in more moist and low temperature regimes. d13C value is a good predicator for spatial diversity and shift of the species along the altitudinal gradient of environmental factors interplay. A few C4 dicot species occurring at high altitudes (3000m – 4000) include Sagina gallica, Silene abyssinica and Melandrium nordiflorum. The transition zone between C3 and C4-dicot is rather abrupt and within altitudinal range of 1500m – 1700m, less behind that recorded for the monocots at 2000m – 2200m altitude. C4-dicot species are abundant at low altitude in contrast to the C3 dicot high altitude. The general pattern of 13c/12c isotopic ratio values distribution along altitudinal differentiation show that the values of

–10.60 to –16.55, -17.75 to –18.87, and –18.89 to –32.42 occurs at low altitude (0 – 1500m), intermediate altitude (1550m – 1700m) and high altitudes (1800m - 4200m), respectively. The low altitudes are associated with water stress, high temperatures and low relative humidity. C4 dicot species can be intercropped to increase bioproductivity for the betterment of the flora and fauna in the semi-arid and arid ecosystem. C4-species are potential candidates for exploitation in the agroforestry systems especially for long-term management programmes.The study is highly relevant to better understanding of global change on the photosynthetic pathways ,herbivory and vegetation dynamics and ecosystem bioproductivity. PS18.15 Redox switches and evolutionary transitions J Allen (Queen Mary, University of London), C Allen (Queen Mary, University of London), S Puthiyaveetil (Queen Mary, University of London)

PS18.16 The Origin of Plastids: A Shopping Bag Model A Larkum (University of Sydney), P Lockhart (Massey University), C Howe (University of Cambridge) The origin plastids is explained by the endosymbiotic hypothesis in which an early cyanobacterium was taken up by an early eukaryotic organism. This may have occurred as a single event (monophyly) and the bulk of the current evidence supports this hypothesis. Nevertheless, the evidence is by no means beyond criticism and the alternative hypothesis, that more than one endosymbiotic event led to different lines of plastids (polyphyly), is a possibility. Both hypotheses may be an oversimplification, and we have put forward a different scheme, which involves gene sharing at an early stage of symbiosis. In this model some of the early cyanobacterial symbionts lost genes to the nucleus of the host eukaryotes and then went out of symbiosis, only to be followed by different types of early cyanobacteria. In this way genes such as those for light-harvesting pigments and proteins were quickly shared across a range of early photosynthetic eukaryotes. After this period of shopping around for suitable genes, endosymbiosis led, over time, to the present lines of primary and secondary plastids. PS18.17 Phylogeny of Prochlorococcus marinus: how is this species related to the rest of the Cyanobacteria? A Larkum (University of Sydney), C Ip (University of Sydney), M Chen (University of Sydney), L Jermiin (University of Sydney) The recently discovered marine nano-phytoplankton species Prochlorococcus marinus is so widespread and abundant that it may account for up to half of all marine primary production in the oceans. It also utilises an unusual light-harvesting protein, Pcb, binding divinyl Chl a and divinyl Chl b. As a result of the intense interest generated by this species many whole genome studies are now available. Different types of Prochlorococcus marinus form a well-defined clade distinct from the rest of the cyanobacteria. However, some marine Synechococcus fall within the same clade as P. marinus, even though the marine Synechococcus possess a classical phycobilisome light-harvesting system rather than the Pcb light-harvesting system used by P. marinus. Furthermore, the Pcb family of light harvesting proteins is found in a number of other cyanobacteria, which are broadly spread throughout the cyanobacterial radiation (P. marinus , Prochloron didemni,

273 14th Photosynthesis Congress - PS07 Prochlorothrix hollandica, Acaryochloris marina). We address the two problems: a) how the Pcb light harvesting system evolved and b) how the clade of P. marinus and marine Synechococcus species evolved, using phylogenetic sequence analysis. PS18.18 The Evolution of Oxygenic Photosynthesis – A Scenario K Sauer (University of California) Photosynthesis as a mechanism for converting light into chemical energy originated in an anoxic, strongly reducing and harsh environment. Solar radiation 3-4 Ga ago was unfiltered of near and far UV by oxygen and ozone in the early Earth atmosphere. In the primordial ocean, chemical reductants such as Fe(II)(aq), Mn(II)(aq), H2S and H2 were abundant, especially near thermal vents. In sufficiently sheltered locations, these substances provided reducing power for the development of the first anoxygenic photosynthesis. As ions like Fe(II) and Mn(II) become oxidized, they become less soluble in a weakly acidic or basic aqueous environment. Mn(III) formed at certain type II reaction centers could consequently have become bound at or near the site of electron donation. A similar step in modern photosynthetic organisms is the first of a series that leads to the formation of the Mn4Ca water-oxidation cluster. Further reaction center activity could result in the accumulation of additional Mn and/or higher oxidation states capable of oxidizing water at least to the level of hydrogen peroxide. Associated catalase activity could then lead to the formation of dioxygen prior to the appearance of a fully evolved cyanobacterial PS II. The ability to use abundant water as a source of reducing power would provide a strong force for driving this evolutionary path. This hypothesis proposes a step-by-step process leading ultimately to the Mn4Ca cluster that we find in all oxygenic photosynthetic organisms today.

274 14th Photosynthesis Congress - PS07

PS19 - Organelle Communication PS19.1 Coordination of expression of nuclear light-harvesting chlorophyll proteins with synthesis in the chloroplast

genes for chlorophyll

A Smith (University of Cambridge), M Moulin (University of Cambridge), A Lichtfuss (University of Cambridge), B Schmid (University of Cambridge), H Ottenhof (University of Cambridge) During greening of etiolated seedlings there is a need for the coordinated synthesis of chlorophyll with that of the cognate apoproteins. The major proteins are the light-harvesting chlorophyll proteins (LHCs), which are encoded in the nucleus. Their synthesis is controlled not just by light, but also by the circadian clock and by retrograde signals from the plastid to the nucleus that report the physiological status of the chloroplast. Arabidopsis mutants defective in plastid-nuclear signaling (gun mutants for genomes uncoupled) have been identified by the fact that they still express LHC genes even when the chloroplast is severely compromised, for example by treatment with the bleaching herbicide norflurazon. Many of these gun mutants have been found to have lesions in the enzymes of the tetrapyrrole synthesis pathway, and a model has been proposed in which one of intermediates of the pathway, Mg-protoporphyrin IX, is responsible for plastid-nuclear signaling. We have been investigating the relationship between chlorophyll synthesis and the expression of LHC genes during the greening of etiolated seedlings of gun mutants, using microarray and metabolite analysis. The pattern of expression of the LHC genes in the different gun mutants indicates that the plastid signal is unlikely to be a single tetrapyrrole metabolite. Morevoer, using a sensitive LC/MS method we have developed to measure chlorophyll synthesis intermediates, we do not find any accumulation of Mg-protoporphyrin IX under conditions in which the LHC genes are repressed. Conversely, feeding the tetrapyrrole precursor, 5-aminolaaevulinic acid to raise levels of Mg-protoporphyrin IX artificially does not cause repression of LHC gene expression.

PS19.2 Dynamic Morphology of Plastids and Stromules in Angiosperm Plastids M Hanson (Cornell University), E Kwok (School of Medicine and Biomedical Science, SUNY-Buffalo), D Reisen (Bitplane AG), H Ishida (Tohoku University), A Holzinger (Institut of Botany), R Köhler (Harvard Medical School) Labeling of plastids with fluorescent proteins expressed from nuclear or chloroplast transgenes has completely revised our notion of plastid morphology and dynamics. The tubular extensions of chloroplasts that were repeatedly discovered, and then forgotten, prior to 1970, are now definitely established as features of chloroplasts and other plastids. These so-called “stromules” (stroma-filled tubules) are common on plastids in some types of cells but are only sporadic in mesophyll cells. Stromules extending from the main plastid body are frequently found in many different types of non-green plant cells. Stromules often appear to contact the plasma membrane and nuclear envelope, surround other organelles, and sometimes even pass through nuclei. Proteins move between plastids connected by stromules; however, genetic evidence indicates chloroplast genomes do not traffic between plastids, though RNAs and small DNAs may be transmitted through stromules. Like chloroplasts, stromules exhibit motility mediated by the actin cytoskeleton. Except for the necessity of actin microfilaments, how stromules form and move is not known, nor do we understand their roles within the plant cell. Among the possible functions of stromules are (1)

to increase the envelope surface area to facilitate import and export (2) to transfer molecules between different plastids (3) to channel materials and signals to appropriate locations (4) to reduce diffusion distance between plastids and other subcellular compartments and (5) to facilitate autophagy during senescence. Still images and time-lapse movies obtained through confocal and epifluorescence microscopy illuminate the diverse activities of stromules within the plant cell.

PS19.3 Chloroplast localization of nonmevalonate pathway enzymes and regulation of mitochondrial genes in ispD and ispE albino mutants in Arabidopsis M Hsieh (Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan), C Chang (Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan), S Hsu (Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan), J Chen (Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan) Plant isoprenoids are derived from two independent pathways, the cytosolic mevalonate pathway and the plastid nonmevalonate (nonMVA) pathway. Here we use green fluorescent fusion protein assays to demonstrate that the entire Arabidopsis nonMVA pathway enzymes are localized in the chloroplast. We have also characterized three Arabidopsis albino mutants, ispD-1, ispD-2 and ispE-1, which have T-DNA insertions in the IspD and IspE genes of the nonMVA pathway. Levels of photosynthetic pigments are almost undetectable in these albino mutants. Instead of thylakoids, the ispD and ispE mutant chloroplasts are filled with large vesicles. Impairments in chloroplast development and functions may signal changes in the expression of nuclear, chloroplast and mitochondrial genes. We used northern blot analysis to examine the expression of photosynthetic and respiratory genes in the ispD and ispE albino mutants. Steady-state mRNA levels of nucleus- and chloroplast-encoded photosynthetic genes are significantly decreased in the albino mutants. In contrast, transcript levels of nuclear and mitochondrial genes encoding subunits of the mitochondrial electron transport chain are increased or not affected in these mutants. For instance, levels of cob and cox1 mRNA are significantly increased in the ispD and ispE albino plants. Genomic Southern blot analysis revealed that the DNA amounts of mitochondrial genes cob and cox1 are not enhanced in the ispD and ispE albino mutants. Our results support the notion that inter-organellar crosstalk may exist between chloroplasts and mitochondria. The functional state of chloroplasts may affect the expression of nuclear and mitochondrial genes.

PS19.4 Systemic and Intracellular responses to Photo-oxidative stress in Arabidopsis B Pogson (The Australian National University), D Hussain (The Australian National University), P Wilson (The Australian National University), J Rossel (The Australian National University), K Howell (University of Western Australia), J Whelan (University of Western Australia), K Kazan (CSIRO Plant Industry) Excess sunlight results in oxidative damage, reduced photosynthesis and yield penalties. As the sun’s angle changes, adjustments to minimise oxidative damage requires mutually beneficial responses in different subcellular compartments and, potentially, in distal shaded leaves that will be subsequently exposed to high light (HL). We studied the effect of modulating the expression of an Arabidopsis zinc finger transcription factor, ZAT10, on oxidative stress tolerance in the chloroplast, cytosol

275 14th Photosynthesis Congress - PS07 and vacuole. ZAT10 overexpression resulted in reduced oxidative damage, increased mRNA expression of antioxidative genes including APX2, elevated anthocyanins and increased photosynthetic rates in high light. Decreased ZAT10 expression resulted in the converse. Second, a rapid, systemic response to localised HL induced the expression of 301 genes in distal, shaded leaves, which included ZAT10, ZAT12, COR15A, IP3 KINASE and members of the DREB family. The majority of the distally-induced genes were co-regulated in the HL-exposed leaves (70 90%). The 10 fold upregulated genes are responsive to ABA, methyjasmonate and/or hydrogen peroxide, but are largely not responsive to other hormones, including salicylic acid. Thus, HL-induced systemic acquired acclimation (SAA) is distinct from pathogen-induced systemic acquired resistance (SAR). The SAA response was detectable, but possibly attenuated by mutations in ABA synthesis and perception. SAA was detected in vasculature of the floral bolt, cauline leaves and rosette within 15-30 minutes and apparently involves a novel signal or combination of signals. PS19.5 Importance of chloroplastic/mitochondrial interactions during elicitor-induced hypersentive response in tobacco leaves: acceleration of the cell death process by light and anoxia R De Paepe (Institut de Biotechnologie des Plantes), M Garmier (Institut de Biotechnologie des Plantes), P Priault (UMR-CNRS-Universite Paris-Sud), G Vidal (Institut de Biotechnologie des Plantes), S Driscoll (Rothamsted Research), R Djebbar (Universite des Sciences et de la Techmologie Houari Boumedienne), M Boccara (Universite Pierre et Marie Curie), C Mathieu (Institut de Biotechnologie des Plantes), C Foyer (Rothamsted Research) Light and oxygen are considered important in the execution of plant programmed cell death (PCD), principally because they are involved in the generation of reactive oxygen species (ROS). We compared the hypersensitive response (HR)-like PCD process in N. sylvestris leaves that is elicited by harpin Nea in the light and in darkness, in air and under very low oxygen (VL ox: 0,1%). PCD was induced under all conditions, but was delayed in the dark compared to the light and accelerated under anoxia. Photosynthetic and respiratory signatures, involving a collapse of photosynthesis and an acceleration of respiration that are characteristic of light-elicited leaves were altered under VL ox. Furthermore, the dark-induced CO2 respiratory burst was inhibited by anoxia. Harpin-induced accumulation of superoxide and hydrogen peroxide was observed inside chloroplasts in both the light and the dark, but not under VL ox. These results indicate that apoplastic and chloroplast localized ROS are not crucial signals in the orchestration of harpin-induced PCD. In contrast, the expression of enzymes encoding cytosolic antioxidant enzymes depended both on light and oxygen. The acceleration of the elicitor-induced cell death under anoxia and in the light implicates coordinated changes in chloroplastic and mitochondrial mitochondrial metabolisms in the harpin-induced cell suicide program PS19.6 The structure and function of Gun4 and its interactions with the chlorophyll biosynthetic enzyme magnesium chelatase P Davison (Sheffield University), A Dinsdale (Sheffield University), D Canniffe (Sheffield University), N Hunter (Sheffield University) The gun (genome uncoupled) mutants in Arabidopsis thaliana show a disruption in signalling between the nucleus and chloroplast resulting in the expression of nuclear-encoded photosynthetic genes that are normally switched off in wild-type plants in the absence of chloroplasts. Gun mutants also exhibit a pale phenotype and four of the genes encode proteins that function in tetrapyrrole biosynthesis; haem oxygenase (gun2) and biliverdin reductase (gun3) from the haem catabolic pathway,

the porphyrin binding H subunit of magnesium chelatase (gun5) and a protein (gun4) from the chlorophyll biosynthetic pathway. Mg chelatase consists of three subunits (H, I, and D) and catalyses the conversion of protoporphyrin IX to magnesium protoporphyrin IX, the first committed step of chlorophyll biosynthesis. Kinetic analyses using an in vitro assay containing purified Synechocystis wild-type H, I and D Mg chelatase and Gun4 subunits show that Gun4 dramatically increases the efficiency of transformation of porphyrin substrate to metalloporphyrin product, and it also reduces the threshold Mg2+ concentration required for activity at low porphyrin concentration. The exact sites of interaction between Gun4 and Mg chelatase are as yet unknown although it is known to interact with the H subunit. The structure of Thermosynecocchus elongatus Gun4 has been determined to 1.5Å and several surface residues that may interact with Mg chelatase have been mutated to cysteines for use in chemical cross-linking experiments with APDP. This modifies cysteine –SH groups in the dark which can then be chemically cross-linked to neighbouring molecules when irradiated with UV light. PS19.7 PPR proteins function as a trans-factor in chloroplast RNA editing K Okuda (Graduate School of Agriculture, Kyushu University), T Shikanai (Graduate School of Agriculture, Kyushu University) In higher plants, RNA editing is a post-transcriptional process of altering a specific C nucleotide to U in an RNA molecule in mitochondria and plastids. About 30 editing sites have been detected in plastids and over 400 in mitochondria. For site-specific RNA editing, a cis-element is essential and consists of fewer than thirty nucleotides surrounding the editing site. This cis-element is considered to be a binding site for trans-factor that may include the editing activity converting C to U. We have identified Arabidopsis crr4 mutants which are specifically defective in RNA editing that creates the translational initial codon of the plastid ndhD gene. The CRR4 gene encodes a member of the pentatrico-peptide repeat (PPR) protein family, which is extraordinarily large in higher plants. We also showed that the recombinant CRR4 binds to the sequence surrounding the editing site. Taken together with the genetic evidence, we conclude that the trans-factor required in plastid RNA editing is a PPR protein. We recently discovered Arabidopsis crr21 mutants that are specifically impaired in the RNA editing of the site 2 of ndhD. Both CRR21 and CRR4 belong to the E+ group in the PLS subfamily that is characterized by the presence of a conserved C-terminal region. This region is highly conserved and exchangeable between CRR21 and CRR4, although it is not essential for the RNA binding. Our results suggest that the C-terminal region in the E and E+ groups might have a common function among trans-factors of RNA editing in plastids. PS19.8 Visualization of Rubisco-containing bodies derived from chloroplasts in living cells of Arabidopsis H Ishida (Tohoku University), K Yoshimoto (National Institute for Basic Biology), D Reisen (Cornell University), A Makino (Tohoku University), Y Ohsumi (National Institute for Basic Biology ), M Hanson (Cornell University ), T Mae (Tohoku University) During senescence and times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Previously by immuno-electron microscopy (IEM), we demonstrated that Rubisco is released from chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves (Chiba et al. 2003, Plant Cell Physiol. 44, 914-921). In this study, we visualized RCBs in living cells of transgenic

276 14th Photosynthesis Congress - PS07 Arabidopsis plants containing stroma-targeted green fluorescent protein (GFP). When leaves of transgenic Arabidopsis plants were incubated under starvation conditions with a vacuolar-ATPase inhibitor and/or a cysteine-protease inhibitor, spherical bodies exhibiting GFP fluorescence without chlorophyll fluorescence were observed. Spherical bodies were not observed when leaves were provided with a sugar and nutrient solution. Double-labeled IEM with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. These results strongly support the hypothesis that stromal proteins can be degraded by possibly autophagy before chloroplast destruction. PS19.9 The metabolic and proteomic analysis of plastids isolated from different cell types of Arabidopsis thaliana leaves C Bowsher (The University of Manchester), P Chovanec (The University of Manchester), D Thornton (The University of Manchester), A Tobin (University of St Andrews) Plastids are the organelles that distinguish the plant cell from that of all other eukaryotes. The most clearly defined role of leaf mesophyll cell plastids (chloroplasts) is in photosynthesis. In contrast the plastids of roots, tubers, flowers and developing seeds are generally classed as non-photosynthetic plastids, and usually distinguished by their roles in storage and/or producing major metabolic products, such as starch, lipids and pigments. Although extensive research has been carried out into the metabolic properties of chloroplasts, little is known about the existence and metabolic capacity of other plastid types in leaves. Much of our knowledge of leaf plastids, other than the chloroplasts, has come from microscopy studies. Nevertheless, plastids in non-photosynthetic leaf cells are a significant part of the population. Information will be presented on protocols developed for isolating plastids from distinct cell types within Arabidopsis leaves. Results of proteome analysis and metabolic analysis will be presented. The functional implications of these results in terms of their impact on leaf metabolism and the necessary communication between different plastid sub-populations will be considered. PS19.10 Effect of chloroplast maturation on the expression of w3 fatty-acid desaturases in soybean plants V Andreu (EEAD-CSIC), M Alfonso (EEAD-CSIC) The ?3 fatty-acid desaturases are responsible for trienoic fatty-acid production in plants. These desaturases are encoded by nuclear genes and are localized in two different cell compartments: FAD3 is specific of the endoplasmic reticulum while FAD7 and its cold-inducible isozyme FAD8 are plastid specific. We have studied the effect of the chloroplast maturation on the expression of ?3 fatty-acid desaturases in soybean plants. During leaf development, there is an increase of linolenic acid (18:3) levels. Interestingly, the contribution of the ?3 desaturases to 18:3 synthesis during this process seems to be different. Thus, in young leaves (0-3 days of leaf formation), when chloroplasts are in the early maturation phase, GmFAD3 seems to be the major ?3 desaturase responsible for 18:3 synthesis. On the contrary, during leaf development (7-18 days), when chloroplasts are getting into the later stage of maturation, there is a specific increase of GmFAD7 mRNA and GmFAD7 protein levels. This increase was coincident with the decrease of GmFAD3 transcript levels. These results indicate the existence of a strong coordination between chloroplast and the nucleus for 18:3 production. Retrograde signals from the chloroplast could be involved in this coordination. PS19.11

GUN1 (GENOMES UNCOUPLED1) encodes a pentatricopeptide repeat (PPR) protein involved in plastid protein synthesis-responsive retrograde signaling to the nucleus A J Cottage (University of Cambridge), E K Mott (University of Cambridge), J-H Wang (University of Cambridge), J A Sullivan (University of Cambridge), D MacLean (University of Cambridge), L Tran (University of Cambridge), M-K Choy (University of Cambridge), C A Newell (University of Cambridge), T A Kavanagh (University of Cambridge), S Aspinall (University of Cambridge), J C Gray (University of Cambridge) Plastid-to-nucleus signaling coordinates the expression of nuclear and plastid genes required for the assembly of functional chloroplasts. We have isolated new alleles of gun1 (genomes uncoupled1) by screening EMS and X-ray mutagenised lines of Arabidopsis thaliana containing the GFP reporter gene under the control of a tobacco RbcS promoter for GFP expression in the presence of norflurazon, a carotenoid biosynthesis inhibitor, or lincomycin, an inhibitor of plastid translation. gun1 mutants were able to express photosynthesis-related nuclear genes in the presence of lincomycin, unlike other gun (gun2-gun5) mutants. Microarray analysis identified CA1 (CARBONIC ANHYDRASE1) as the gene most responsive to lincomycin in 7-day-old wild-type seedlings, and a CA1 promoter::GFP reporter gene was used to map the gun1 mutation to a 224 kb region on chromosome 2. Sequencing of candidate genes identified GUN1 as At2g31400, encoding a pentatricopeptide repeat (PPR) protein of 918 amino acid residues, with a putative N-terminal plastid-targeting sequence of 41 amino acid residues, 10 copies of the PPR motif and an SMR (small MutS-related) domain with possible nuclease activity near the C-terminus. gun1-100 contained a point mutation creating a stop codon at amino acid 56, whereas gun1-1 contained a point mutation resulting in an Ala259Val change near the first PPR motif. The identification of GUN1 should facilitate further characterization of additional components of the plastid protein-synthesis-responsive signaling pathway.

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PS20 - Photosynthesis: a fundamental tool for modern agriculture and forestry PS20.1 Will forest ecosystems continue to remove CO2 from the atmosphere as the climate changes ? P Jarvis (Univ. of Edinburgh, School of GeoSciences, The Kings Buildings, Edinburgh, EH9 3JN, Scotland, UK), S Linder (Faculty of Forest Science, Swedish University of Agricultural Sciences, Box 49, SE-230 53 Alnarp, Sweden) In almost every forest we look today we find a net removal of CO2 from the atmosphere and carbon is accumulating in the soil and in the trees. We define the net primary production (NPP) as the difference between the gross photosynthetic production (GPP) and the losses of carbon resulting from respiration associated with growth and maintenance of live biomass, the autotrophic respiration (RA), i.e., NPP = GPP – RA. Thus the net ecosystem production (NEP) is the difference between the net primary production (NPP) and the respiration associated with mineralisation of organic matter in the soil, the heterotrophic respiration (RH), i.e., NEP = NPP - RH. For a forest at equilibrium, we would expect NEP to be zero (i.e., NPP = RH). Conversely, when we measure NEP > 0, NPP must exceed RH. This lack of equilibrium in forests today is the reason why they are a large global carbon sink, currently removing close to 40% of CO2 emissions derived from fossil fuels from the atmosphere. It is commonly assumed, and has been shown by some models, including GCMs, that as atmospheric [CO2] and surface temperature increase, this current lack of equilibrium will reverse (i.e., RH > NPP). Recent projections by models linking the carbon and nitrogen cycles show NPP for forests increasing over the next 100 years in parallel with RH, as atmospheric [CO2] and surface temperature increase. Thus the ongoing removal of CO2 from the atmosphere by forests (NPP > RH) is likely to be maintained.

PS20.2 Photosynthesis and water use efficiency: improving plant productivity in semi-arid environments H Medrano (Instituto Mediterráneo de Estudios Avanzados), J Galmes (Institute Mediterráneo), M Ribas-Carbó (Institute Mediterráneo), J Gulias (Instituto Mediterráneo), J Bota (Instituto Mediterráneo), A Pou (Instituto Mediterráneo), M Moreno (Instituto Mediterráneo), J Cifre (Instituto Mediterráneo), J Flexas (Instituto Mediterráneo) Water is an important limiting factor for plant and crop productivity and, according to the last predictions of climate change, it will be even scarcer in wide areas where competition between human and agricultural water use is increasing. Therefore, improving water use efficiency (WUE) is becoming crucial and, now the decision of irrigation schedule based on physiological criteria is a promising research field. The relationship between net photosynthesis and stomatal conductance (AN vs. gs) opens two main targets for improving water use efficiency: diminishing stomatal conductance, and consequently water consumption, or increasing photosynthesis. For most plants, stomatal conductance can be reduced by half with only 10-20% losses on photosynthesis. New plant water status indicators, some of them based on photosynthetic parameters, can be very useful tools. Recent results of our group show interesting achievements applicable to field use. On the other hand, improving photosynthesis without increasing stomatal conductance will suppose an improvement of WUE. Recently, we have found in Limonium gibertii, a Mediterranean species of arid habitats, the highest Rubisco specificity factor known for a C3 plant. This opens the

possibility to explore future transformed plants with higher WUE. Finally, the important role of mesophyll conductance variations in the photosynthesis rate, which is being widely studied in our group, also indicate that any possible increase of mesophyll conductance would lead to concomitant increases of photosynthesis over the same stomatal conductance bringing a new way to explore for increasing the WUE of plants.

PS20.3 Arabidopsis thaliana functional genomics. R Furbank (CSIRO Plant Industry, GPO Box 1600 Canberra ACT 2601 AUSTRALIA), H Fallahi (CSIRO Plant Industry, GPO Box 1600 Canberra ACT 2601 AUSTRALIA), M Badger (Research School of Biological Sciences, ANU, GPO Box 475 Canberra ACT 2601 AUSTRALIA), G Estavillo (Dept Biochemistry and Molecular Biology, ANU Canberra ACT 0200 AUSTRALIA), B Pogson (Dept Biochemistry and Molecular Biology, ANU Canberra ACT 0200 AUSTRALIA), A Walter (Research Center Jülich, ICG-III Phytosphere, 52425 Jülich, Germany), Reverse genetics in Arabidopsis, using knock out lines or gene suppression techniques such as RNAi, is a powerful tool for plant functional genomics. However, phenotypes are often in one of two classes. Plants either show no visible phenotype (either due to gene redundancy or subtle changes in plant function) or show gross morphological effects which are difficult to quantify. A useful phenotypic screen for the study of such plant material is growth analysis. The classical, destructive method involves tissue harvesting and estimation of different plant physical parameters such as leaf area and weight at regular intervals. This method is time consuming and because of its destructive nature, is not amenable to continuous high throughput monitoring of growth when only small populations of plants are available. An alternative method is the use of non invasive techniques based in image recording and software analyses that allows for periodical sampling in a non destructive way. Here we report the use of non-destructive imaging of plant growth and photosynthetic performance to study mutants altered in both primary metabolism and in genes of no known function and highlight the potential of phenomic screening for agricultural traits.

PS20.4 Photosynthesis and the new challenges in agriculture M Pinto (Facultad de Ciencias Agronómicas, Universidad de Chile, Casilla 1004, Santiago, Chile), P Horton (University of Sheffield, Dept of Molecular Biology and Biotechnology, Firth Court, Sheffield S10 2TN, UK), E Murchie (University of Nottingham, Sutton Bonington campus, Division of Agricultural and Environmental Sciences, LE12 5RD, UK) The changing demands being placed on modern agriculture present new challenges for plant biologists and agronomists. Greater production is needed from less land and in the face of greater climatic uncertainty. There are requirements for increased nutritional value of crop products and increased demand for non-food crops, including biofuels. Future improvements in yield will rely on the capacity of the plant itself to express yield potential in diverse environmental conditions. Crop plants must sense and respond appropriately. The chloroplast plays an important role in the mechanism of optimisation - in sensing both the

278 14th Photosynthesis Congress - PS07 external environment and the energetic and metabolic status of the cell. Here we discuss a new approach where the photosynthetic system is not only a provider of biomass but also a sensor-processor that integrates external signals from a continuously changing environment with the internal metabolic and developmental constraints to determine yield. For example, the chloroplast can signal sink size, predicting future grain-filling potential and conversely can trigger appropriate developmental responses when photosynthesis cannot match predicted grain development. Photosynthetic capacity itself is determined in part by a balance between incident irradiance and metabolic demand. This is a complex phenomenon since imposed upon it are the effects of climatic and other environmental factors and the temporal dimension that includes developmental programmes and circadian rhythms. At any given time, photosynthetic rate is an integration of all of these factors. An increased knowledge of the relationship between photosynthesis and crop yield will play a vital role in the future. PS20.5 The intensity of CO2 assimilation, photorespiration and productivity of wheat genotypes (Triticum L.) J Aliyev (Institute of Botany, Azerbaijan National Academy of Sciences) The created genefund which comprises several thousands of wheat genotypes with contrast photosynthetic traits and grain yield ranging 3-9 t×ha-1 makes possible to elucidate the relationship between CO2 assimilation, photorespiration and productivity. High productive genotypes possess higher intensity of CO2 assimilation in leaf ontogenesis. High rate of CO2 assimilation is not accompanied with low intensity of photorespiration. As a result of versatile measurements it was established that along with high intensity of photosynthesis, high values of photorespiration are characteristic for high productive genotypes. The ratio of true photosynthesis to photorespiration in genotypes with different productivity is rather constant and equal on the average to 3:1. A value of photorespiration constitutes 28-35% of intensity of photosynthesis in contrast genotypes. Therefore, on contrary to earlier conception on wastefulness of photorespiration [1, 2], it was proved that photorespiration is useful process in plants and the attempts to reduce its intensity by various ways with the purpose of increasing the crop productivity are inconsistent. The genetic code of key enzyme of photorespiration - phosphoglycolate phosphatase was determined [3]. In order to better understand the correlation between intensity of CO2 assimilation, photorespiration and productivity, consideration of basic parameters of architectonics of plants is also essential. [1] Zelitch, Photosynthesis, photorespiration and plant productivity. Acad. Press, New York, 1971. [2] Long et al. Plant, Cell and Environment, 2006, 29, 315-330. [3] Mamedov et al. J.Biol.Chem., 2001, 276, 45573-45579. PS20.6 Developmental changes in leaf photosynthesis and Nitrogen use efficiency in rice cultivated in Venezuela. Historical trends over the past 50 years A Pieters (IVIC), S Irazabal (IVIC), E Graterol (Fundación DANAC), M Benitez (IVIC) Photosynthesis represents a major target to increase rice yield potential as leaf area index and harvest index are already high. However, data on the relation between saturated rate of photosynthesis (Pmax), growth and grain production are frequently conflicting. We analysed the photosynthetic performance and nitrogen use efficiency at different development stages in field grown rice of the 14 most cultivated rice genotypes in Venezuela, released since 1951 until 2007. The genotypes used differed in plant architecture, leaf duration, orientation, plant height and development time. The experiment was conducted during the dry

season 2006-2007 in Guárico state (10 30 0N and 66 50 60W), Venezuela. Transplanted rice of the 14 cultivars was managed according to current agricultural practices of fertilizer application, pest and disease controls. At the maximum tillering stage, 60 days after transplant (DAT), Pmax of the youngest fully expanded leaf (penultimate leaf) ranged from 23.9 to 28.9 micromolCO2m-2s-1. Ninety DAT, Pmax of fully expanded flag leaves was 20-30% lower than in the penultimate leaf except in two cultivars in which the decrease was ca. 10%. The reduction of Pmax continued until grain maturation in all the cultivars. Leaf nitrogen (N) paralleled the behaviour of Pmax being maximal in the penultimate leaf 60 DAT and decreasing steadily in the flag leaf. Thus a negative relation between panicle weight, Pmax and leaf N was found. There was no relation between Pmax and year of release. The implications of these results for the improvement of rice yield potential will be discussed. PS20.7 Parameters of reflectance spectra of Norway spruce needles and their relation with photosynthetic pigment content M Navrátil (Ostrava University), M Vašková (University of South Bohemia), A Ac (Institute of Systems Biology and Ecology), V Špunda (Ostrava University) Spectral optical properties of leaves indicate their interaction with incident radiation and therefore can be used for non-invasive estimation of plant physiological status. The diurnal course of spectral reflectance, pigment content and selected photosynthetic parameters were examined for 3 different year-class of Norway spruce [Picea abies (L.) Karst.] needles during several sunny days at the Experimental ecological site Bílý Kříž in the Beskydy Mts. (Czech Republic). The applicability of reflectance spectroscopy to examine the diurnal and needle-age dependent variability of pigment content and photosynthetic efficiency was tested. In addition, some improvements of needle spectral reflectance measurement with integrating sphere were introduced. Among the reflectance indices designated to estimate chlorophyll content, some of Chl a+b indices correlated with chlorophylls content whereas no correlations were found with carotenoid content. The best correlation with pigment content was found for the reflectance indices based on combination of reflectance at 750, 550 and 445 nm. Spatial variability of the reflectance over the exposed part of the tree crown (mainly due to the contribution of the different needle year-classes) dominates over its diurnal changes. Both influences should be taken into account for reliable comparison of the pigment contents of Norway spruce stands using remote sensing reflectance imaging. Photosynthetic parameters (actual rate of CO2 assimilation and quantum yield of PSII photochemistry) revealed characteristic midday depression, but they loosely correlated with photosynthetic reflectance index (PRI). PS20.8 Application of photosynthetic parameters in screening of wheat genotypes for improved tolerance to drought and high temperature M Zivcak (Slovak Agricultural University in Nitra), M Brestic (Slovak Agricultural University in Nitra), K Olsovska (Slovak Agricultural University in Nitra) Application of physiological criteria in screening process is very helpful for improvement of wheat breeding efficiency. We assessed several traits and methods for drought and high temperature tolerance screening in vegetation pot trials with collection of winter bread wheat genotypes of different provenance. Although methods of chlorophyll fluorescence measurement are not in direct relation to yield, fast chlorophyll a fluorescence kinetics measurements appeared as useful tool for monitoring of drought and heat stress effects on photosynthesis. We elaborated the high temperature test with leaf segments by which we measured a lot of material in short time. We observed significant

279 14th Photosynthesis Congress - PS07 differences in sensitivity of different genotypes expressed by fluorescence parameters. Comparison of drought effects using chlorophyll fluorescence in observed genotypes was feasible using Performance Index (PI) as the most sensitive fluorescence parameter recording drought stress effects and genotypic differences in drought susceptibility. Direct gasometric measurements of photosynthesis showed that decrease of net assimilation rate was mainly due to stomatal closure. The non-stomatal effect was observed only in severely stressed leaves with very low values of net assimilation rate. The varieties differed in sensitivity of stomatal closure and hence in net assimilation rate, too. Delayed stomatal closure with higher values of net assimilation was correlated with higher grain yield in drought conditions. Similarly, non-stressed values of transpiration efficiency were also connected with better drought tolerance. PS20.9 Avenues for genetic gains in leaf photosynthesis and canopy radiation-use efficiency in cereals J Foulkes (University of Nottingham, Division of Agricultural and Environmental Science, School of Biosciences, Sutton Bonington campus, LE12 5RD, UK) One of the aims of future genetic improvement in cereals should be the increase of biomass production while maintaining the present values of biomass partitioning to the grain. Radiation-use efficiency (dry matter production per unit radiation interception; RUE) can potentially be improved by manipulating traits at the biochemical, cellular, leaf or canopy levels of organization. One approach is to seek a more efficient Rubisco and to use it or knowledge of its structure to improve crop plants. Rubisco has been the subject of intense analysis, and results with regard to genetic improvement in cereal plants are briefly discussed. There are prospects of introducing Rubisco into crop plants with higher catalytic rates or greater specificity for CO2, thereby reducing photorespiration. To increase photosynthetic rate per unit leaf incident radiation in cereal plants is not a trivial task. There appears to be limited variation in the light-saturated rate of leaf photosynthesis in the gene pools of cereal species. Nevertheless, there is evidence that in the last decades cereal breeders in some countries have succeeding in selecting for higher RUE. Interestingly, modern wheat cultivars in the UK had both smaller flag leaves and greater specific leaf dry weight (ratio of dry weight to green leaf area), and there were statistically significant correlations with RUE. In this review the prospects of future genetic improvement in traits underlying leaf photosynthesis and/or canopy radiation-use efficiency are considered, as well as the extrapolation from leaf photosynthesis to whole canopy photosynthesis. PS20.10 Identification of novel light intensity dependent genes and promoters for crop improvement using gene expression profiling S Harris (Ceres, Inc.), N Rampal (Ceres, Inc.), S Kwok (Ceres, Inc.) Light is an essential component of photosynthesis and therefore vital for the survival of plants. Although, the quantity of light driving photosynthesis can be limiting in a dense canopy, it can also exceed what can be utilized in photochemistry in direct sunlight and lead to the inactivation of photosynthetic functions. Therefore, gene expression must be controlled by light intensity to allow plants to adapt rapidly to large fluctuations in light quantity. It has been shown that some genes such as CAB and RBCS are expressed at high levels upon exposure to light while PHYA and genes encoding asparagine synthetase are negatively regulated by light (Chattopadhyay et al., 1998). To identify additional novel genes and promoters that are differentially expressed in a light intensity dependent manner, we performed a series of genome-wide transcription profiling experiments in Arabidopsis

thaliana. Differentially expressed genes were selected for over-expression studies and some produced novel light responsive phenotypes. In addition, novel light dependent promoters specific to either high or low light intensity were discovered through GFP induction analysis. The goal of these studies is to discover the optimal promoter-gene combinations that can be used to improve the productivity of crop species by maximizing the utilization of light for photosynthesis. PS20.11 Geotechnical impact of heavy engines used during urban street renovation on trees vitality M Eyletters (Université Libre de Bruxelles), V Decoux (Ministère de la Région Bruxelles Capitale), A Willems (Université Libre de Bruxelles), B Buntinx (ALIWEN sa Avenur Georges Lemaître 57), J C Verbugge (Ministère de la Région Bruxelles Capitale) Urban trees are submitted to combined abiotic stresses which influence their vitality. In parallel to classical stress linked to climate (temperature, relative humidity, modified light intensity) and unfavourable conditions to growth (dry and poor soil, no space to develop the root system..), trees are usually submitted to aggressions during work of street renovation. Roots, branchs, trunks are injuried. In some cases, root system could be influenced by an increase of soil compacity. Then, the root – soil interaction is completely disturbed which influence the mineral nutrition of tree. In the framework of a street restoration consisting to install tramway very closed to old plane trees, we analysed by quantitative way the impact of pressure done by the tramway on soil compacity (measurement of Aterberg limit). We compared also the trees vitality based on the fast kinetic of chlorophyll-a fluorescence measurement before and after work restoration. It seems that the pressure on soil acted by tramway doesn’t modify irreversibly the mesh of soil and doesn’t influence the trees vitality. The mineral status of leaves is not modified. But an attention must be focused on heavy engines which participate to renovation work and which induce very high pressure to soil. If the irreversible limit of soil compacity is reached, the mesh of soil is distorted and the root system could not evolve in favourable conditions to make efficient mineral nutrition. PS20.12 Evaluation of Bean [Phaseolus vulgaris] Seeds Inoculation with Rhizobium phaseoli and Plant Growth Promoting Rhizobacteria (PGPR) on Yield and Yield Components. M Yadegari (University) Mehrab Yadegari1, Ghorban Noormohammadi2, Hadi Asadi Rahmani3 , Amir Aeeneband4,Abdollah. Ghasemi Pirbalouti1, 1: Scientific members, Islamic Azad University of SHAHREKORD Unit, ShahreKord, Iran. 2: Assist Prof. Dept. of Agronomy, Olum & Tahghighat, Azad University, Tehran, Iran 3: Assist Prof. Soil and Water Research Institute, Tehran, Iran. 4: Scientific member, Chamran University, Ahvaz, Iran. In order to evaluate seeds inoculation effects on bean (Phaseolus vulgaris L.) Iranian cultivars with different strains of legominosarum biovar phaseoli on yield, N2 fixation rate and determining the best bacteria * cultivar’s combination, a spilt plot experiment in randomized complete block design was conducted in the field at SHAHREKORD, Iran on 2006 and 2007. The factors were six bacterial strains Rb-133,

280 14th Photosynthesis Congress - PS07 Rb-133* Azospirillum, Rb-133* Pseudomonas, Rb-136, Rb-136* Azospirillum, Rb-136* Pseudomonas and non-inoculated controls including application of nitrogen fertilizer treatment (100 kg N ha -1) and without application of nitrogen fertilizer in main plots. Three bean cultivars Sayad, Akhtar and Goli were assigned as factor B to subplots. The results revealed that there were significant differences among factor A in grain yield, number of pods per plants, number of seeds per pods, number and weight nod at early flowering (45-50 days after emergence), shoot nitrogen content and N2 fixation rate. Treatment of inoculated seeds with strain bacteria* Pseudomonas and non-inoculated control (without N – fertilizer application) demonstrated the highest and lowest grain yield, respectively. The interaction between strains * cultivars effects in seed yield were no significant difference, but the results of mean comparison by DMRT (at 5% level) showed the highest and lowest seed yield were associated to Akhtar * N fertilizer (non-inoculated strain) and Goli and Sayad * control. However, Akhtar * Rb-133* Azospirillum, Akhtar * Rb-133* Pseudomonas, Akhtar * Rb-136* Pseudomonas, Akhtar* Rb-Rb-136* Azospirillum combinations similar were observed in seed yield with N fertilizer treatment (non-inoculated strain). The results showed that all treatments were capable of nodulation, however, strains Rb-133 gave highest nodule dry weight, N total (shoot) and percent of fixed N2. An efficient symbiotic was achieved with strain Rb-133, since total N content of plants inoculated with these strains and N content in soil after harvesting in main plots were similar to that of plants supplied with N –fertilizer treatment. Key words: Bean (Phaseolus vulgaris L.), Cultivars, Seeds Inoculation, Strains of Rhizobium legominosarum biovar phaseoli, Yield and N2 fixation rate. PS20.13 Low-Light Irradiation at the Beginning or the End of the Daily Dark Period Accelerates Leaf Expansion and Growth in Spinacia oleracea L. K OHASHI (The University of Tokyo), M TAKASE (The University of Tokyo), K KURATA (The University of Tokyo) Thirty minutes irradiation of red light at the beginning of dark period (RB) or the blue light at the end of dark period (BE) at a PPFD less than 20% of a PPFD of light period promoted biomass production in spinach (Hanyu and Shoji, 2000). We supposed this promotion of growth was related to the leaf area enlargement or the promotion of photosynthesis in leaves and investigated how RB or BE affected the leaf expansion at first. Total dry weight and leaf area of spinach grown under RB were 1.6 and 1.4 times greater than those grown under control (no supplemental irradiation), and total dry weight and leaf area of spinach grown under BE were 1.2 and 1.5 times greater than those grown under control, respectively. Microscopic analysis of the cross-sections of the leaves revealed that RB increased the elongation of the leaf cell in horizontal direction, while it wasn’t affected by the BE. These results suggested that RB accelerated the leaf expansion by the increase of leaf cell size and BE accelerated leaf expansion by the increase of leaf cell number, which would contribute to the promotion of the photosynthesis of the whole plant and growth in spinach. BE increased the leaf thickness accompanied by the elongation of the palisade cells in vertical direction and the thickness of the spongy tissues layer, which was considered to affect photosynthetic rate per leaf area. We are currently analyzing the effect of RB and BE on photosynthetic characteristics of leaves. PS20.14 Preservation of photosynthetic pigments under limiting working conditions J Garciá-Plazaola (Universidad del País Vasco), I Fleck (Universitat de Barcelona), M Pintó-Marijuan (Universitat de Barcelona), J Becerril

(Universidad del País Vasco), R Esteban (Universidad del País Vasco), L Balaguer (Universidad Complutense de Madrid), R Rubio de Casas (Universidad Complutense de Madrid), E Manrique (CCMA-CSIC), R Ochoa (CCMA-CSIC) Most methods for pigment analyses involve freezing leaf samples in liquid nitrogen or/and storage at –80°C until extraction. These conditions warrantee the absence of chemical transformations or pigment degradation, but they limit experimental designs to conditions in which liquid nitrogen, a freezer, an HPLC and/or dry ice are available. These requirements greatly restrict field investigation in remote areas, limiting the possibilities of ecophysiological studies. Although the best option is to freeze leaf samples in liquid nitrogen, we evaluate in the present work several alternatives for sampling, storage, transport and extraction. Our aim was to validate and describe the limitations of an array of techniques that do not require liquid nitrogen for freezing, electrical power for storage or dry ice for transportation. Passive extraction and storage in acetone, desiccation with silica and lyophylization were analysed. Two main artefacts were observed when comparing with the use of liquid nitrogen: cis-trans carotenoid isomerization and formation of chlorophyll (Chl) enantiomers. The parameters used to characterise the feasibility of each method were Chl degradation, xanthophyll cycle (VAZ) epoxidation state, changes in carotenoid/Chl ratios, and carotenoid isomerization rate. Extraction and conservation in acetone was adequate for the study of VAZ while desiccation provided a way for long-term preservation of plant material with slight modifications of the VAZ epoxidation state. Liophilization represented an efficient system for conservation and transport but restricted for its technical requirements. We also standardized the conditions and effect of several oxidation preservatives during liophilization. The addition of oxidation preservatives did not improve results.

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PS21 - Artificial Photosynthesis Symposium PS21.1 The de novo synthetic protein approach to artificial photosynthesis design. C Moser (University of Pennsylvania), R Koder (University of Pennsylvania), B Lichtenstein (University of Pennsylvania), J Cerda (University of Pennsylvania), L Dutton (University of Pennsylvania) Extensive exploration of natural photosynthesis has provided us with the basic engineering principles about how to design robust systems that capture light and guide electron transfer through electron tunneling chains. De novo protein design and synthesis allows us to create a simplified, adaptable and robust protein framework that secures, on the tens of angstrom scale, light activated and other redox cofactors at the appropriate distances for energy conversion. Design of synthetic proteins also simplifies cofactor self-assembly as well as patterning of the protein exterior for self-assembly at surfaces and electrodes. Clustering multiple redox centers and multi-redox electron centers within the protein framework for bond making and breaking in light activated catalysis is the next challenge.

PS21.2 Charge separation and charge compensation in Artificial Photosynthetic Complexes. A Magnusson (Uppsala Universitet), M Anderlund (Uppsala Universitet), R Lomoth (Uppsala Universitet), O Johansson (Uppsala Universitet), P Huang (Uppsala Universitet), N Shaikh (Uppsala Universitet), M Borgström (Uppsala Universitet), S Styring (Uppsala Universitet), L Hammarström (Uppsala Universitet) We aim at constructing a molecular system for utilizing solar energy for fuel production. Our approach is to design and synthesize supramolecular complexes that mimick structural and functional aspects of natural photosynthetic systems. In natural oxygenic photosynthesis, Photosystem II (PSII) uses sunlight as driving force to extract electrons from water, utilizing a tetranuclear manganese complex in the water oxidizing active site. To mimick essential structural and functional parts of the water oxidizing complex, we couple a photoactive [Ru(bpy)3]2+ (tris-bipyridine ruthenium(II)) complex to redox active manganese and tyrosine donors and to organic acceptors. Thus, we have synthesized several multinuclear ruthenium-manganese complexes, that undergo light-induced electron transfers. We have demonstrated that electron transfer from Mn to Ru is intramolecular, and that the same Mn moiety can transfer several electrons to the Ru sensitizer, in the presence of an exogenous electron acceptor. We could also demonstrate sequential, three-step oxidation of a manganese dimer covalently linked to the chromophore, bringing it from Mn2II,II to Mn2III,IV, in experiments where a sacrificial electron acceptor in aqueous solution was used. Many donor-chromophore-acceptor triad models have earlier been designed to mimic charge separation in photosynthetic reaction centers, using [Ru(bpy)3]2+ as chromophore. To successfully mimic PS II, very long-lived intermediate charge-separated states, stable on a time scale of seconds, are required. We have studied the photoinduced electron transfer reactions in the triad molecule Mn2(II,II)-Ru(II)(bpy)3-NDI, by time resolved optical and EPR spectroscopy. We demonstrate that photoexcitation of the triad results in a very long-lived charge-separated state.

PS21.3 Designing Artificial Photosynthesis: Engineering bacterioferritin for light-activated electron transfer. B Conlan (Australian National University), T Wydrzynski (Australian National University), W Hillier (Australian National University) Natural photosynthesis involves the conversion of light into chemical energy through a series of electron transfer events within membrane-bound pigment/protein complexes. In the unique Photosystem II complex these electron transfer events result in the oxidation of water to molecular oxygen. The exact nature of the water oxidizing process remains poorly understood and the aim of this work is to ultimately produce an in vitro model to help elucidate this mechanism. As the starting point for the development of an artificial light-activated, metal-binding protein, we have used the naturally occurring bacterioferritin protein (BFR) from E.coli. This protein is not light-active but has many design features which are useful in engineering a functional electron transport chain. BFR is an 18.5 kDa protein which dimerises to form a heme binding pocket with axial ligands to the heme iron from each monomer. The amino acid sequence was modified to prevent non-specific binding of Zn-chlorin, allowing bound heme to be removed and replaced with photoactive Zn-chlorin. BFR also naturally contains a di-iron metal binding site which has very similar ligands to those found in several manganese binding proteins. Using EPR we have shown that when the di-iron is removed, manganese can be bound to the metal site in a redox active form and can act as an electron donor to the Zn-chlorin. We will report on the functionality for light-induced electron transfer from the bound Mn to the electron acceptors in solution in this artificial protein as a model of Photosystem II electron transfer. Funded by the Australian Research Council (DP0450421)

PS21.4 Synthesis and characterisation of Ruthenium Based Artificial Photosynthesis Mimics C Herrero (UMR, CNRS & CEA Saclay), A Quaranta (CEA Saclay), W Leibl (CEA Saclay), A Rutherford (CEA Saclay), A Aukauloo (CEA Saclay) We design, synthesise and characterise molecules that mimic the reactions observed in Photosystem II. These molecules are able to undergo light-induced charge separation, electron transfer, proton transfer and accumulation of oxidizing/reducing equivalents, mimicking processes occurring in natural systems. These supramolecular systems contain a photoactive component covalently linked through a spacer to a cavity where a metal ion or cluster is located. The photosensitizer used is a [Ru(bipy)3]2+ (bipy = 2,2’-bipyridine) analogue, a counterpart to P680, which absorbs light in the visible region and triggers an electron transfer process. The resulting RuIII species has a reversible oxidation potential of 1.3 eV vs. SCE, similar to that of P680, and is capable in principle of oxidizing a Mn cluster and water. The Mn systems studied are those with terpyridine coordination cavities since Mn di m-oxo Mn dimers of this kind have been reported to catalyze the oxidation of water into molecular oxygen. In the literature this reaction is driven by external oxidants, in contrast it is our aim to use light instead of the chemical oxidant. Here we present new high valence Ruthenium-Manganese complexes of this kind and we describe the current status of our efforts to use them for light driven self-assembling processes and water

282 14th Photosynthesis Congress - PS07 oxidation. PS21.5 De Novo peptides modelling the binding sites of [4Fe-4S] clusters in Photosystem I M Antonkine (Max-Planck-Institut fuer Bioanorganische Chemie; Freie Universitaet Berlin ), C Breitenstein (Max-Planck-Institut fuer Bioanorganische Chemie), B Epel (Max-Planck-Institut fuer Bioanorganische Chemie), E Bill (Max-Planck-Institut fuer Bioanorganische Chemie), W Gärtner (Max-Planck-Institut fuer Bioanorganische Chemie), W Lubitz (Max-Planck-Institut fuer Bioanorganische Chemie) Photosystem I (PS I) converts the energy of light into chemical energy. The terminal electron transfer cofactors in PS I are three iron clusters named FX, FA and FB. The PsaC subunit of PS I harbors binding sites of the FA and FB clusters. We modeled the binding sites of the [4Fe-4S] clusters FA and FB of PsaC by preparing two peptides (maquettes) sixteen amino acid each by using solid state peptide synthesis. These model peptides incorporate the consensus iron-sulfur binding motif along with amino acids from the immediate environment of the respective iron-sulfur cluster. The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by their optical absorbance, EPR and Mössbauer spectra. Comparison of continuous wave and pulsed EPR, Electron-Nuclear DOuble Resonance (ENDOR) and Mössbauer spectra of the model [4Fe-4S] clusters with the respective spectra of FA and FB shows that the environment of the clusters is very similar. The oxidation-reduction potential of the iron-sulfur clusters in the model peptides is close to that of FA and FB in PsaC at room temperature and is considerably lower than was observed in other [4Fe-4S] model systems described earlier1. 1Mulholland, S.E., Gibney, B.R., Rabanal, F., Dutton, P.L. (1998) J. Am. Chem. Soc., 120, 10296-10302

PS21.6 The design of a hybrid photoelectrochemical cell used to generate reducing and oxidizing potential M Hambourger (Arizona State University), G Moore (Arizona State University), P Liddell (Arizona State University), D Gust (Arizona State University), A Moore (Arizona State University), T Moore (Arizona State University) A biohybrid photoelectrochemical cell employing the photoanode architecture of a dye-sensitized solar cell has been assembled and used as a test platform for the generation of reducing and oxidizing equivalents that could be coupled to the solar production of fuels. In one version, a porphyrin dye sensitizes a TiO2 semiconductor over most of the visible range. Photoinduced charge separation at the dye-TiO2 interface results in electron migration to a cathode, while the holes generated on surface bound dyes drive the oxidation of soluble electron mediators. The increased [Ox]/[Red] ratio of the mediator facilitates the solution-based enzymatic oxidation of appropriate substrates. The accumulation of anodic and cathodic products may be expected to limit cell performance. A highly oxidized anodic solution and a highly reduced cathodic solution were prepared and the impact on the cell short-circuit current was assessed. The NAD+/NADH and benzoquinone/hydroquinone redox couples were studied as sacrificial electron donors in the absence of appropriate enzymes or substrates. Comparatively poor cell performance was observed using the benzoquinone/hydroquinone couple. This effect is explained in terms of rapid charge recombination by electron donation from the electrode to benzoquinone in solution, as compared to much less recombination with

NAD+. With the NAD+/NADH couple the cell performance is relatively independent of the redox poise of the anode solution, but limited by accumulation of reduction products in the cathodic compartment. Highly oxidizing tetrapyrrole sensitizers and bioinspired high potential mediators have been designed for the oxidation of water once a suitable catalyst becomes available. PS21.7 Visible light induced water oxidation with photosynthesis organ from green plant Y Amao (Oita University) Photosynthesis reaction in green plant is consists of two photochemical reactions; one is the photosystem I (PSI) is photoreduction of NADP+ and the other is the photosystem II (PSII) is photoinduced water oxidation. In oxygen evolved type photosynthesis, water acts as an electron donor. The oxygen-evolved complex in PSII consists of cluster structure by four manganese ions. In this study, photosynthesis organ (a Grana part) including PSI and PSII is isolated from spinach with surfactant β-octylglucoside (OG) micelle and photoinduced water oxidation system is developed with Grana and 2,6-dichloroindophenol (DCIP) as an electron acceptor. The effect of manganese (II) or (III) ion on the photoinduced water oxidation system is investigated. PS21.8 Improvement of nitrogenase-based photobiological hydrogen production by cyanobacteria by gene engineering -hydrogenases and homocitrate synthaseH Sakurai (Waseda Univ), H Masukawa (Kanagawa Univ)), X Zhang (Waseda Univ)), H Ikeda (Waseda Univ) We are improving cyanobacteria by gene engineering in order to realize large-scale photobiological hydrogen production, and have created several hydrogenase and homocitrate synthase mutants. From Nostoc sp. PCC 7120: disrupted in uptake hydrogenase gene (?hupL), bidirectional hydrogenase gene (?hoxH), and the both of them, and from Nostoc sp. PCC 7422: disrupted in uptake hydrogenase gene (?hupL). Disruption of hupL was proven to be very effective in improving the hydrogen productivity. Notably, PCC 7422 ?hupL was able to accumulate hydrogen to 20-30% (v/v) in 3 to 8 days, and the efficiency of light energy conversion into hydrogen was 3.7% vs visible light or about 1.7% vs. total radiation. PCC 7120 has two homocitrate synthase genes nifV1 and nifV2, and homocitrate bound to Fe-Mo cofactor of dinitrogenase is considered to be important for efficient nitrogen fixation, but not for hydrogen production. We have created mutants disrupted in one of the genes and the both of them with ?hupL as the parent. In the presence of nitrogen gas, the hydrogen production activities of the mutants ?nifV1/?hupL and ?nifV1/?nifV2/ ?hupL were less susceptible to inhibition by nitrogen. In the presence of nitrogen gas, the hydrogen production and nitrogenase activities of the parent ?hupL were quickly decreased in about 10 hrs after the maxima were attained, but the activities of nifV mutants were sustained longer. Modulation of homocitrate synthase activity can be a valuable tool for improving hydrogen productivity of cyanobacteria. PS21.9 Syntheses and photochemical properties of trimanganese complexes containing naphtyl residues S YANO (Nara Women's University) Since the manganese cluster in the biological photosystem II, is believed

283 14th Photosynthesis Congress - PS07 to consist of four manganese atoms and one calcium atom, development of manganese cluster complexes is an important target for artificial photosynthesis systems. However, there is a few manganese complex having photofunctional moieties. We wish to report the synthesis and characterization of trinuclear manganese complexes, (1) and [Mn3(μ3-O)(μ-O2CCH2-1-naph)6(py)3] [Mn3(μ3-O)(μ-O2CCH2-2-naph)6(py)3] (2) (1 or 2-naph-CH2CO2H = 1 or 2-naphtylacetic acid, py = pyridine) as the structural OEC model complexes having a manganese cluster. These complexes were characterized by elemental analysis, EXAFS analysis, X-ray crystallography and magnetic susceptibility. The electrochemical and photochemical behaviors were investigated by cyclic voltammetry (CV), electron absorption spectroscopy, emission spectroscopy and fluorescence decay. The X-ray crystallography and magnetic susceptibility showed that the complex 1 has the trinuclear complex containing a [Mn3O-(O2CR)6] core with mixed-valent Mn3- (II,III,III) centers. The EXAFS analysis data suggested that the complex 2 has the same structure as 1. CV exhibited two reduction peaks which are ascribed to Mn2(II)Mn(III)/Mn(II)Mn2(III) and Mn3(II)/Mn2(II)Mn(III) in CH3CN. This result shows that the solid-state structures of complexes 1 and 2 remain in the CH3CN solution. The fluorescence intensities of complexes 1 and 2 become smaller than those of naphtylacetic acids, depending on the dielectric constant of solvent. These results suggested that there is an electron transfer between manganese centers and naphtyl moieties. PS21.10 Proton/electron concerted processes useful for splitting reaction using Cu(II/III) and Ni(II/III) systems.

water

I Kinoshita (Osaka-City University), H Hashimoto (Osaka-city University), N Kuwamura (Osaka-City University), K Kitano (Osaka-City University), T Nicshioka (Osaka-City University) Water splitting reaction is one of the most important processes during the artificial photosynthesis. Even though, it is still challenging to find the useful proton/electron concerted reaction managed by the conventional metal ions. From the viewpoint of sustainable feature, it is required to find the processes especially by using conventional first transition metal ions. For this purpose, we are constructing the processes using a novel tripodal system with stabilized carbanion, e.g. tris-pyridylthiomethyl (tptm) system. [NiII(tptm)X]2 performs two electron oxidation process producing stable [NiIII(tptm)X]22+ as a result. Using OH for X, the system should act as the promising material suitable for the water splitting reaction. The preliminary process equivalent for hydrogen production have been achieved by use of [Cu(tptm)F] as the strong H-Bonded structure maker. The photochemical conversion of quinone-hydroquinone is enhanced by the help of the existence of this complex. This process is assisted by the presence of the stable Cu(III) state, which have been confirmed as [CuIII(tptm)Cl]PF6. The counter part of this process, oxygen evolution requires the four electron oxidation process. The model is now improved to polymerize the complex using Cu(I) and Fe(II) linkage. All these processes are unusual for these first transition metal ions. This is because the property of these ions is substantially modified by the presence of the novel tripodal carbanion. PS21.11 Sensitization of Light-Induced Charge Separation in Photosystem I by Synthetic Fluorescent Dyes A Nakamura (R&D Dep., Central Japan Railway), S Mizoguchi (IIS, University of Tokyo), Y Miyajima (IIS, University of Tokyo), Y Kato (IIS, University of Tokyo), T Watanabe (IIS, University of Tokyo) To drive the light-induced charge separation in photosystem (PS) I by

green light which cannot absorbed effectively by natural photosynthetic pigments, Rhodamine (Rh) derivatives were introduced covalently to the NH2 groups on the stromal and lumenal surfaces of PS I as the artificial light-harvesting antenna for green light. Because most of the antenna Chl a molecules clustered at just beneath the stromal and lumenal surfaces, we expected that RhB bounded to the surfaces can sensitize the antenna Chl a molecules within PS I and function as a mimic of soluble membrane extrinsic antenna complex such as phycobilisome. Whether the bounded Rh can function as the artificial antenna was examined by monitoring light-induced absorbance change at 700 nm (DA700) upon photooxidation of primary electron donor, P700, under green light which can excite principally Rh derivatives bound to PS I. When RhB derivatives were introduced covalently to PS I, the DA700 was enhanced 1.8-fold from that observed in unmodified PS I. This shows that Rh derivatives bound covalently to the PS I surfaces function as artificial light-harvesting antenna for green light to photooxidize P700. In addition to this, we synthesized polyacryrate-based amphiphilic polymer bearing Rh moiety as a model for membrane intrinsic antenna complex such as LHC in green plants. In this case, we expected that the hydrophobic parts of the polymer complexed with transmembrane regions of PS I. We also showed that the Rh-modified amphiphilic polymer can function as artificial light-harvesting antenna of PS I photochemistry for green light.

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PS22 - Perception of the Environment and Signalling PS22.1 Sensing and responding to high light stress

H2O2 signaling in Chlamydomonas reinhardtii requires chloroplast-mediated inactivation of H2O2 degrading enzymes

K Niyogi (University of California), X Li (University of California), Z Li (University of California), M Anwaruzzaman (University of California), H Ledford (University of California), B Chin (University of California), S Wakao (University of California), B Fischer (University of California)

A Krieger-Liszkay (CEA Saclay), S Lemaire (Université Paris-Sud), C Beck (Universität Freiburg), N Shao (Universität Freiburg)

Plants and algae need light for photosynthesis, but the intensity of sunlight can vary over several orders of magnitude and on timescales ranging from seconds to seasons. To optimize utilization of available light in different environments, photosynthetic organisms have evolved various ways of sensing and responding to changes in light intensity. In response to rapid fluctuations in light intensity, a nonphotochemical quenching mechanism that regulates photosynthetic light harvesting is induced by changes in thylakoid lumen pH. In plants, the PsbS protein associated with PS II appears to be involved in sensing lumen pH. A slower acclimation response to light intensity involves regulation of nuclear LHCB genes that encode PS II antenna proteins. Using Chlamydomonas reinhardtii as a model organism, we have examined the hypothesis that LHCB gene regulation in high light occurs in response to changes in plastoquinone redox state. We have also investigated the ability of Chlamydomonas to acclimate to specific forms of oxidative stress. High light stress induces acclimation to singlet oxygen stress, suggesting an overlap in high light and singlet oxygen response pathways.

PS22.2 Spatial dependence for H2O2-directed signalling in Arabidopsis leaves subject to high light. P Mullineaux (University of Essex) H2O2) generated from photosynthetic electron transport initiates intracellular and systemic signalling inducing protective and acclimatory mechanisms to environmental stress. H2O2 has been suggested to diffuse from the chloroplast to initiate this signalling. However, this model does not explain how H2O2 can diffuse to other subcellular compartments when the same stresses trigger an enhancement of antioxidant capacity in the cytosol. Therefore, we have proposed a hypothesis that argues for a spatial component for H2O2 signalling. In this hypothesis, a H2O2 signal is transduced in the chloroplast to a non-reactive oxygen species factor which exits the chloroplast, traverses the reducing environment of the cytosol to the plasma-membrane where a second extracellular production of H2O2 occurs. The increased antioxidant capacity of the cytosol confines H2O2 to the extracellular and chloroplast compartments. We are testing this hypothesis using high light – stressed Arabidopsis leaves which accumulate H2O2 in peri-veinal bundle sheath cells. These cells specifically express the APX2 gene which requires active photosynthetic electron transport, chloroplast-sourced H2O2 and a systemic induction of APX2 expression which requires extracellular H2O2. We propose that an abscisic acid (ABA) mediated signal exits the chloroplast stimulated by increased H2O2 production in the chloroplast. Results from transgenic lines and mutants altered in APX2 expression will be presented that supports the role of ABA as a signal from the chloroplast, supplies evidence for a spatial separation of increased antioxidant pools and H2O2.

PS22.3

Hydrogen peroxide is not only a harmful substance but also acts as a signaling molecule and influences the expression level of a significant number of genes. Transformants of Chlamydomonas reinhardtii carrying a nuclear-encoded reporter gene specifically activated by H2O2 (1) were investigated. The level of expression of the reporter gene depended not only on the H2O2 concentration but also on light. In the dark, the induction of the reporter gene by H2O2 was much lower than in the light. The expression level was correlated with a light-induced loss of H2O2 detoxifying enzyme activity. The inactivation of H2O2-consuming enzymes and the expression level of the reporter gene depended on photosynthetic electron transport since they were sensitive to DCMU, an inhibitor of photosystem II. Addition of aminotriazole, a catalase inhibitor, led to higher induction of the reporter gene by H2O2 in the dark. It is proposed that a controlled down-regulation of the activity of catalase and other detoxifying enzymes is necessary during photosynthetic electron transport to allow maintenance of a certain level of H2O2 required for signaling that enables the organism to acclimate to a change in environmental conditions. The role of thioredoxin and other disulfide bridge reducing compounds in this regulation mechanism will be discussed. (1) Shao, N., Krieger-Liszkay, A., Schroda, M., Beck, C.F. (2007) A reporter system for the individual detection of hydrogen peroxide and singlet oxygen: its use for the assay of reactive oxygen species produced in vivo. Plant J. (in press)

PS22.4 Action of reactive oxygen species in the photoinhibition of photosystem II Y Nishiyama (Ehime University), K Kojima (Ehime University), H Hayashi (Ehime University), S Allakhverdiev (Institute of Basic Biological Problems), N Murata (National Institute for Basic Biology) Photoinhibition of photosystem II (PSII) is due to the imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. Photodamage is initiated by the direct effects of light on the oxygen-evolving complex and, thus, photodamage to PSII is unavoidable. Studies of the effects of oxidative stress on photodamage and subsequent repair have revealed that reactive oxygen species (ROS) act primarily by inhibiting the repair of photodamaged PSII and not by damaging PSII directly. Thus, strong light has dual effects on PSII; it damages PSII directly and it inhibits the repair of PSII via production of ROS. Investigations of the ROS-induced inhibition of repair have demonstrated that ROS suppress the synthesis de novo of proteins that are required for the repair of PSII, such as the D1 protein. Moreover, analysis of polysomes has determined that a primary target for inhibition by ROS is the elongation step of translation. Investigations using a cyanobacterial translation system in vitro have revealed that elongation factor G might be the primary target, within the translational machinery, of inhibition by ROS. Here we present a new paradigm for the molecular action of ROS in photoinhibition.

PS22.5 Functional analysis of an

285 14th Photosynthesis Congress - PS07 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase, AtNUDX1, involved in repair of oxidative DNA damage in Arabidopsis thaliana T Ogawa (Department of Advanced Bioscience, Faculty of Agriculture, Kinki University), K Yoshimura (Department of Food and Nutritional Science, College of Bioscience and Biotechnology, Chubu University), S Shigeoka (Department of Advanced Bioscience, Faculty of Agriculture, Kinki University)

Synechocystis PCC6803 O Cheregi (Institute of Plant Biology, Biological Research Center, Szeged, Hungary ), P Kos (Institute of Plant Biology, Biological Research Center, Szeged, Hungary ), S Bottka (Institute of Plant Biology, Biological Research Center, Szeged, Hungary ), M Ashby (School of Biological and Chemical Sciences, Queen Mary, University of London, London, UK), I Vass (Institute of Plant Biology, Biological Research Center, Szeged, Hungary )

Cellular DNA, RNA, and their precursor nucleotides are at high risk of being oxidized by reactive oxygen species, resulting in the generation of oxidized forms such as 8-oxo-7,8-dihydro-2'-(deoxy)guanosine 5'-triphosphate [8-oxo-(d)GTP]. This oxidized base can pair with both adenine and cytosine, and thus would cause both replicational and translational errors. To prevent the mutagenic consequences of 8-oxo-G, organisms have developed cellular defense mechanisms by the sanitation of nucleotide pools and base excision repair pathway. Previously, we have reported that AtNUDX1 protein hydrolyzes 8-oxo-dGTP and completely suppresses increased frequency of spontaneous mutations in the E. coli mutT- strain (Ogawa et al. J. Biol. Chem. 2005, 280: 25277-25283). Here, we report the physiological function of AtNUDX1. AtNUDX1 had activity toward an oxidized ribonucleotide, 8-oxo-GTP, with high affinity (Km: 28.1 ?M). The phenotypic suppression of the lacZ amber mutation in an E. coli mutT- strain caused by the misincorporation of 8-oxo-GTP into the mRNA was significantly diminished by the expression of AtNUDX1. These findings suggest that AtNUDX1 prevents transcriptional errors in vivo. A confocal microscopic analysis using GFP fusion protein demonstrated that AtNUDX1 is distributed in the cytosol. The levels of 8-oxo-guanosine in genomic DNA were significantly increased in the KO-nudx1 plants compared with the wild-type plants under normal and oxidative stress (3 ?M paraquat) conditions. The results obtained here indicate that AtNUDX1 functions in cellular defense system against oxidative DNA and RNA damages through the sanitization of their precursor pools in the cytosol of Arabidopsis cells.

The mechanism by which UV-B radiation affects gene expression and also the way in which UV-B light is initially perceived is unknown. In cyanobacteria the acclimation responses are mediated by two component signaling systems comprising of a histidine kinase and a response regulator. To examine the potential role of the histidine kinase Hik33 in the UV-B response of the cyanobacterium Synechocystis 6803, we studied the UV-B induced expression of a series of eight genes, which are induced under variable stress conditions, and are known to be under the control of Hik33. UV-B induced expression of the hliA gene is enhanced about 2 fold in a mutant which lacks Hik33 gene as compared with the WT. Under high light (HL) conditions, the expression of hliA gene is also enhanced in the Hik33 mutant as compared with the WT. Our results point to a common signal transduction pathway for HL and UV-B light, in regulation of the hlia gene in Synechocystis 6803. The other seven genes examined in our experiments did not show significant differences of induction under UV-B in the WT and Hik33 deficient mutant, so, probably other histidine kinases control their expression. As a further step of an extensive investigation of the factors involved in UV-B signaling, we use antisense nucleotides specific for Rre26 and Rre31. This work was supported by the Hungarian Minister of Economy GVOP-3.1.1-2004-05-0096/3.0

PS22.6 The role of Heterotrimeric G-protein on chloroplast development

K Yoshida (Faculty of Agriculture, Utsunomiya University), D Yoshioka (Faculty of Agriculture, Utsunomiya University), K Inoue (Faculty of Agriculture, Utsunomiya University), S Takaichi (Department of Biology, Nippon Medical School), I Maeda (Faculty of Agriculture, Utsunomiya University)

Q Wei, W Wu, H Guangzhen, H Fenhong, J Huang (Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) The involvement of heterotrimeric guanine nucleotide-binding proteins (G-proteins) in chloroplast development has been demonstrated only by pharmaceutical data. In this work, using the variegation mutant thylakoid formation 1 (thf1), we present the genetic evidence that the mutant phenotype can be rescued either by expression of the constitutively active G protein alpha subunit (GPA1Q222L), or by activation of the endogenous GPA1, suggesting that the activated GPA1 have a role on chloroplast development in Arabidopsis. We further demonstrate that FtsH protease, composed of type A and type B subunits, is a critical downstream component in THF1 signaling pathway. Interestingly, overexpressing GPA1Q222L can also rescue the leaf-variegated phenotype of the ftsh2 mutant. Thus, we define a novel signaling pathway constituting of THF1, FtsH protease and GPA1 in regulation chloroplast development in Arabidopsis. Our work may be helpful for understanding the integrative signaling network that dynamically regulates chloroplast development in ever-changing environmental conditions.

PS22.7 Investigation of the role of histidine kinase Hik33 in UV-B induced stress responses in the cyanobacterium

PS22.8 Evaluation of colors in green mutants isolated from purple bacteria as a host for colorimetric whole-cell biosensors

The change in carotenoid-based bacterial color from yellow to red can be applied to whole-cell biosensors. An obvious change in color is however a critical factor for detecting target molecules by the naked eye in carotenoid-based biosensors. We generated several green mutants to emphasize the color change in such biosensors. The blue-green crtI-deleted mutant, Rhodopseudomonas palustris no.711, accumulated the colorless carotenoid precursor, phytoene. Green Rhodovulum sulfidophilum M31, isolated using chemical mutagenesis, accumulated neurosporene, a downstream product of phytoene. Another green mutant, Rhodobacter sphaeroides Ga, accumulated neurosporene and chloroxanthin, which are both downstream products of phytoene. All green mutants accumulated bacteriochlorophyll a. Photosynthetic membrane obtained from the green mutants all exhibited decreased absorption of wavelength range at 510-570 nm. Therefore, these indicate that the greenish bacterial colors were mainly caused by an existence of bacteriochlorophyll a and the changes in carotenoid composition in photosynthetic membrane. The colors of the green mutants and their wild-type strains were plotted in the CIE-L*a*b* color space, and the color difference (ΔE*ab) values between a green mutant and its wild-type were calculated. ΔE*ab values were higher in the green mutants than in Rdv. sulfidophilum CDM2, the yellowish host strain of reported biosensors. These data indicate that change in bacterial color from green to red is more distinguishable than that from yellow to red as a reporter signal of carotenoid-based whole-cell biosensors. Establishment of biosensors using the green

286 14th Photosynthesis Congress - PS07 mutants is in progress. This work was supported by New Energy and Industrial Technology Development Organization of Japan (05A22703a). PS22.9 Vitamin E: antioxidant properties in chloroplasts and functional interactions with carotenoids M Havaux (CEA/Cadarache, IBEB/SBVME, Laboratoire d'Ecophysiologie Moléculaire des Plantes, F-13108 Saint-Paul-lez-Durance, France), V Collin (CEA/Cadarache, IBEB/SBVME, Laboratoire d'Ecophysiologie Moléculaire des Plantes, F-13108 Saint-Paul-lez-Durance, France), P Rey (CEA/Cadarache, IBEB/SBVME, Laboratoire d'Ecophysiologie Moléculaire des Plantes, F-13108 Saint-Paul-lez-Durance, France), C Triantaphylidès (CEA/Cadarache, IBEB/SBVME, Laboratoire d'Ecophysiologie Moléculaire des Plantes, F-13108 The term ‘vitamin E’ describes the beneficial biological activity of a group of structurally related compounds, the tocochromanols, in animals and humans. Those compounds are composed of a chromanol head group and a prenyl side chain. Natural vitamin E includes four tocopherols and four tocotrienols, which are synthesized exclusively by oxygenic photosynthetic organisms. In leaves of vascular plants, α-tocopherol is the predominant form of vitamin E. A detailed analysis of tocochromanol distribution in chloroplasts isolated from young tobacco leaves showed that α-tocopherol is predominantly located in the thylakoid membranes. The protective role of vitamin E and the functional interactions between vitamin E and other plastid antioxidants (e.g. xanthophylls) were studied using Arabidopsis and tobacco mutant/transgenic plants that lack or over-accumulate vitamin E constituents and/or carotenoids. Mutants deficient in chlorophyll antennae were also used to investigate the influence of those proteins on the photoprotective activity of xanthophylls and vitamin E. This genetic approach was combined with new biochemical and biophysical methods that allow characterization, quantification and imaging of lipid peroxidation in vivo. Both tocopherols and tocotrienols were found to protect thylakoid membranes against photooxidative stress. We also observed that vitamin E and the xanthophyll zeaxanthin have overlapping functions, with lack of vitamin E being compensated by an increased level of zeaxanthin and vice versa. Lack of both compounds resulted in a very photosensitive phenotype. Moreover, vitamin E was found to be essential for tolerance of Arabidopsis towards oxidative stress induced by stress conditions different from high light, such as heavy metals. PS22.10 Inhibitions of allelopathy from Scenedesmus obliquus on photosynthesis and growth of Microcystis aeruginosa D SHI (Tianjin University of Science & Technology), X H Jia (Capital Normal University), R J Kang (Chinese Academy of Sciences), H M Li (Capital Normal University), Y Liu ( Tianjin University of Science Technology), Z Z An (Capital Normal University), D H Song (Tianjin University of Science Technology), G S Du (Capital Normal University) Microcystis aeruginosa has been overspreading in the world, as water eutrophication has been getting serious. The microcystin produced by the unicellular cyanobacterium may cause both human liver cancer and normal growth of aquatic organisms. Although physical and chemical techniques can control this “ecological cancer”, biological, particular plant and algal, controls have been considered effective. Among them microalgal allelopathy may be the most promising method. If the method is carried on the key is the selection of microalgal species. Fortunately, when M. aeruginosa was mix-cultivated with Scenedesmus obliquus together in BG11 medium, the growth of M. aeruginosa was inhibited obviously. The ratio of cell numbers between S. obliquus and M.

aeruginosa can be reduced to 1:100. Afterwords, M.aeruginosa was cultivated in the filtrated medium after cultivating S. obliquus and the cyanobacterial cells were also inhibited. Oxygen electrode measurement showed that the photosynthesis of M. aeruginosa cultivated in the filter medium was reduced. The allelopathic inhibition of S. obliquus on M. aeruginosa was able to be regulated by light, temperature, pH and mineral elements. The research of the inhibition mechanism indicated that the decrease of cell numbers dependent both on photosynthesis and cellular division. PS22.11 Chloroplastic NADPH photoperiod-dependent Arabidopsis

thioredoxin reductase mediates development of leaves in

A Lepistö (Department of Biology, University of Turku), S Kangasjärvi (Department of Biology, University of Turku), E Luomala (Agrifood Research Finland), K Hännikäinen (Department of Biology, University of Turku), G Brader (Department of Biological and Environmental Sciences, University of Helsinki), E Rintamäki (Department of Biology, University of Turku) A single copy gene for a chloroplastic NADPH thioredoxin reductase (NTRC) was recently found in the genome of Arabidopsis thaliana. To reveal the physiological significance of NTRC, we have characterized SALK T-DNA insertion mutant lines of the NTRC gene under various growth conditions. Homozygous ntrc plants showed retarded growth and a pale green phenotype when grown under short day conditions (8 h light/16 h dark). Accordingly, the number of chloroplasts per mesophyll cells and the total chlorophyll content per leaf area were significantly reduced in ntrc. Biochemical analysis revealed that the pale green phenotype of ntrc was not connected with deficiencies in the accumulation or function of the photosynthetic membrane protein complexes or with impaired carboxylation capacity of Rubisco. Nevertheless, low rate of photosynthetic CO2 fixation was observed in ntrc under growth conditions, probably due to doubled respiration rate and unbalanced transpiration of the leaves. Moreover, ntrc plants showed distinct developmental disorders when grown under short day conditions: mesophyll cells had irregular shape and their size was remarkably reduced, density of stomata had increased, and flowering and senescence were significantly delayed. Interestingly, the mutant phenotype was less distinct when plants were grown under long day conditions or under continuous light. The developmental deficiencies of ntrc were connected with hormonal imbalances: auxin content of ten-day-old ntrc plants was remarkably diminished as compared to wild type plants. Consistently, the phenotype of ntrc was partially recovered when grown on a medium supplemented with auxin.

PS22.12 LexA-regulated expression of the cyanobacterial RNA helicase, crhR L Patterson-Fortin (University of Alberta) Expression of the crhR RNA helicase gene in Synechocystis sp. strain PCC 6803 is regulated by the redox poise of the electron transport chain between carriers Qa of plastoquinone and Qo of cytochrome b6f. A change in electron flow through these carriers allows response to light-dark transitions via alteration of crhR transcript accumulation (1). The isolation and identification of Synechocystis LexA as the crhR regulatory protein provides initial clues to the mechanisms responsible for differential crhR transcript accumulation in response to the predominating redox/light environment. Competitive and non-competitive EMSA assays using recombinant LexA protein expressed in Escherichia coli indicate that LexA interacts with the crhR gene in a sequence specific manner. In vitro transcription and translation

287 14th Photosynthesis Congress - PS07 was used to further investigate the LexA-crhR relationship demonstrating that LexA negatively regulates crhR expression (2). In vivo analysis using a lexA partial mutant indicates LexA represses expression of crhR in the dark, under oxidizing conditions. RT-PCR analysis of dark-generated cDNAs showed that crhR transcripts accumulate at higher levels in the mutant than in the wildtype strain. LexA’s common association with DNA damage repair warranted analysis of lexA and recA levels under DNA damaging conditions. Conclusions drawn from transcript accumulation following ultraviolet irradiation and mitomycin-C treatment indicate that lexA and recA are not DNA damage inducible genes contrary to the established dogma in E. coli. Taken together, these results suggest a novel role for Synechocystis LexA in regulating expression of redox-regulated genes (2). Determination of the LexA binding site by DNA footprinting and the multimerization state of LexA has yielded additional insights into the mechanism by which LexA regulates crhR expression and its role in the cyanobacterial response to a changing light environment. (1) Kujat,S.L., and Owttrim,G.W. (2000) Plant Physiol., 124, 703-713. (2) Patterson-Fortin, L.M., Colvin, K.R., and Owttrim, G.W. (2006) Nucleic Acids Res., 34, 3446-3454. PS22.13 Light integral during the growth phase of the leaf as determinant for photosynthetic capacity? G Trouwborst (Wageningen University), W Van Ieperen (Wageningen University) In closed canopies, the photosynthetic capacity decreases from top to bottom. This decrease is interrelated with an exponential decrease in light intensity with increasing overlying LAI, a decrease in R/FR ratio and increasing leaf age. In many crop systems, increasing age and decreasing light intensity occur simultaneously for individual leaves: effects of aging and decreasing light intensity are inherently interacted. To separate these effects on the photosynthetic capacity of leaves, tomato plants were forced to grow horizontally, to guarantee always the same light intensity for all leaves on a plant. Experiments were done in a climate-controlled greenhouse compartment under natural light conditions in winter and spring. In winter no difference in Photosynthetic capacity was measured in leaves ranging from 20 to 70 days old. In spring, however, photosynthetic capacity decreased almost linearly with leaf age (20 -70 days old). In winter, the daily light integral was fairly stable while in spring the daily light integral increased almost linearly. Plotting the photosynthetic capacity against the light integral over three weeks during leaf growth resulted in a linear relation. This suggests that the light integral during leaf growth and expansion plays a role in the formation of the photosynthetic capacity. We suggest that in upright stands the decrease in photosynthetic capacity from top to bottom could also partly be explained by the effect of the natural light conditions during the growth phase of the leaves. Logically this effect should be mostly visible during increasing daily light conditions as it is in spring. PS22.14 Further characterization of cpPhi1, a recently identified redox component of PSII in higher plants. E Corteggiani Carpinelli (University of Padova), E Teardo (University of Padova), I Szabò (University of Padova), G Giacometti (University of Padova), E Bergantino (University of Padova) The cpPhi1protein was identified as a partner of interaction with the low molecular weight subunit of PSII PsbH. The latter is known to play an important role in the assembly of fully functional Photosystem II and in it’s repair in condition of high irradiance (Summer et al., 1997; Komenda, 2005; Iwai et al, 2006). The interaction of cpPhi1 with PsbH, suggested for the first time by a two hybrid test in yeast using the

N-terminus of PsbH as bait, was further confirmed by co-immunoprecipitation assays. Moreover the two proteins were found to be in close proximity using a cross linking reagent on solubilized thylakoid membranes prepared from Arabidopsis. We showed that cpPh1, isolated from higher plants of different species, exhibits a strong peroxidase activity and is a target for at least two post-translational modifications: phosphorilation and glutathyonilation. We also found evidences for a structural modification of the protein in response to different redox conditions, such as the subjectivity to be recognized by monoclonal antibodies. To further characterize the catalytic activity of the enzyme, we performed activity tests in the presence of different concentrations of reducing reagents and of KCN, in order to detect the eventual presence of an haem group in the active site. To give insights into the function of the protein we’ve start a fine characterization of the phenotype of cpPhi1 less mutants of Arabidopsis and we’ve set-up a partial purification of the protein to analyize by Mass Spectrometry the secondary modifications that the protein undergoes. PS22.15 Biophysical and Biochemical Characterization chloroplast-located Plant Glutamate Receptor

of

a

E Teardo (University of Padova), A Segalla (University of Padova), M Zanetti (University of Padova), M Zoratti (University of Padova), G Giacometti (University of Padova), I Szabò (University of Padova) Among the twenty genes encoding putative plant glutamate receptors in Arabidopsis thaliana, the putative product of the Atglr3.4 gene displays a targeting sequence for chloroplast location. Here we provide biochemical evidence for the presence of GLR3.4 protein in purified inner envelope membrane vesicles of chloroplasts. Furthermore, purified inner membrane vesicles harbour a channel displaying electrophysiological and pharmacological properties compatible with those predicted for plant glutamate receptors of the third subfamily. The physiological role of chloroplast-located plant glutamate receptor is indicated by the finding that various pharmacological inhibitors, known to act on these receptors, reduce photosynthetic efficiency in isolated, intact chloroplasts.

PS22.16 The acclimation of photosynthesis in cucumber leaves to different ratios of red and blue light S Hogewoning (Wageningen University), H Maljaars (Wageningen University), J Harbinson (Wageningen University) The scope of processes regulated by blue light, such as stomatal opening and chloroplast movement, has become clearer. However, little quantitative work has been done on the effect of blue light on leaf functioning. We investigated the effect of different ratios of red and blue light (from LEDs) on the photosynthetic characteristics of young cucumber leaves. The plants were grown hydroponically under 100 µmolm-2s-1 irradiance, of which 0, 7, 16, 31 and 49 % was blue. An increasing blue fraction (up to 31%) increased maximum photosynthesis up to four times that produced by red light alone. The photosynthesis rates at growth irradiance increased up to 16% blue (2-fold compared to red alone). The increased photosynthesis was accompanied by an increase in chlorophyll content per cm2, but not per mg dry weight. Thus the leaf weight per cm2 increased accordingly. The low photosynthesis rates at low blue light ratios were not, or at least not solely, due to lower internal CO2 concentrations. At light-limited irradiance levels photosynthesis was measured together with FPSII at 21% and 2% O2. The gross CO2 fixation was identically correlated with the product of FPSII and the absorbed irradiance for all blue light fractions at

288 14th Photosynthesis Congress - PS07 2 % O2. At 0% blue and 31% blue the irradiance/FPSII relationships were similar at both 2% and 21% O2 (370 ppm CO2), though between the light treatments the relationships differed greatly. Remarkably, at 7% blue FPSII declined considerably less with increasing irradiance at 2% O2, than it did at 21%. This phenomenon has not yet been explained. PS22.17 Identification of int N Tanabe (Kiki University), A Kimura (Kinki University), K Yoshimura (Chubu University), S Shigeoka (Kinki University) Alternative splicing of pre-mRNAs contributes greatly to the proteomic complexity and the increase in the coding potential of a genome. A regulatory mechanism for the alternative splicing of genes caused by environmental stress seems to exist in higher plants, since alternative splicing profiles are often affected by the stress conditions. Serine/arginine-rich (SR) proteins are involved in both the constitutive splicing and the alternative splicing in animals and plants. We demonstrated here the molecular characterization of a homologue of SR protein, atSR45a, in Arabidopsis plants. Six types of mRNA variants (atSR45a-1a~e and atSR45a-2) were generated by the alternative selection of transcriptional initiation sites and the alternative splicing of introns in atSR45a pre-mRNA. Both the atSR45a-1a and atSR45-2 mRNAs accumulated in response to high-light irradiation (400 µE m-2 s-1). Yeast two-hybrid system showed that atSR45a-1a and atSR45a-2 proteins, presumed mature forms, interact with U1-70K, which is the member of spliceosome assembly and is involved in the initial definition of 5' splice site. Furthermore, we demonstrated that interaction of both atSR45a-1a and atSR45-2 with U1-70K takes place through a specific association of their C-terminal SR-rich regions. They also interacted with SCL28, a SC35-like SR protein, through a their C-terminal SR-rich regions. By transient expression analysis using GFP fusion protein, the SR45a-1a and SR45a-2 proteins have been fond to be distributed in the nucleus. These results suggest that atSR45a serves as a component in the constitutive and/or alternative splicing. PS22.18 Characterization of singlet oxygen acclimation mutants in Chlamydomonas reinhardtii S Wakao (University of California Berkeley), B Chin (University of California Berkeley), H Ledford (University of California Berkeley), K Niyogi (University of California Berkeley) Singlet oxygen (1O2*) is a highly reactive type of oxygen species that is generated under excess light in the PSII reaction center. Using Rose Bengal as a photosensitizer, we have found that Chlamydomonas reinhardtii, a model photosynthetic eukaryote, exhibits a robust acclimation response to 1O2* stress. Acclimation was defined as the ability of a sublethal pretreatment with 1O2* to activate defense responses that subsequently enhance survival of 1O2* stress. To study the signaling pathway involved in acclimation to 1O2*, we have isolated two mutants, sos1 and sos2, that fail to acclimate to 1O2* stress. Both mutants and the wild type exhibit similar sensitivity to photosynthetic inhibitors such as methylviologen and metronidazole, which generate superoxide and then H2O2. sos1 is a recessive mutation caused by a plasmid that has inserted into a repetitive region without disrupting any annotated genes. A wild-type genomic fragment from the region partially rescues the phenotype, and transcripts in this region are being analyzed to identify the affected gene. sos2 is a semi-dominant mutation that has been mapped by positional cloning to a 45-kb region on chromosome VI. Two candidate genes within this region, one annotated

as a transcription factor and the other a hypothetical gene, showed aberrant mRNA levels when sos2 cells were treated with high light. Attempts to rescue sos2 by complementing with genomic fragments containing the candidate genes are in progress. PS22.19 Photoreaction of the cyanobacterial BLUF protein PixD studied by low temperature spectroscopy: Photoconversion without a Tyr8 residue F Yoshimasa (Nagoya University), Y Murai (Nagoya University), K Okajima (Osaka Prefecture University), M Ikeuchi (The University of Tokyo), S Itoh (Nagoya University) A new type of blue-light sensor, cyanobacterial BLUF domain protein PixD is known to regulate the phototaxis of Synechocystis sp. PCC6803 (1). The BLUF proteins show the 10 – 20 nm red shift of the absorption band of flavins upon the blue-light absorption. We investigated the photoconversion mechanism of TePixD protein (2, 3) of Thermosynechococcus elongatus BP-1 by the site-directed mutagenesis. The substitution of Tyr8 residue of TePixD, which has been postulated to be an electron donor to the flavin in the photo-induced charge separation mechanism, abolished the photoconversion activity at room temperature. We, however, found that the illumination at 80 K induced normal red-shifted forms in Y8F and Y8A mutant proteins. The red-shifted forms trapped were stable at 80 K, and decayed to the dark-adapted form by the dark annealing above 240 K. The accumulation rates of the red-shifted forms in Y8F and Y8A mutant proteins at 80 K were 43- and 137-times slower, and the maximum extents were about 1/2 and 1/4, respectively compared to those in the wild-type protein. The illumination at 150 K of the mutant proteins formed flavin anions without accumulation of the red-shifted form. It is concluded that the Tyr8 residue is not essential for the photoconversion and that the residue works to elevate the photoconversion efficiency and to stabilize the red-shifted product form. (1) Okajima et al., J. Biochem. (2005) 137, 741-750 (2) Fukushima et al. Biochemistry (2005) 44, 5149-5158 (3) Fukushima et al. Photochem. Photobiol. (2007) 83, 112-121 PS22.20 A glucose-6-phosphate/phosphate transported is essential for photoysnthetic acclimation in Aradidopsis thaliana K Athanasiou (University of Manchester), R Webster (University of Manchester), G Johnson (University of Manchester) We have been examining photosynthetic acclimation to light in Arabidopsis thaliana cv. Wassilewskija (Ws). Plants were grown at low light (LL) 100 µmol m-2 s-1 for 6 weeks and then transferred to high light (HL) 400 µmol m-2 s-1. First the acclimation profile of wt plant has been established by monitoring photosynthesis on an hourly base for 3 days and a daily basis for 9 days revealing two distinct acclimation patterns: transient changes in photosynthetic capacity occurred during the first two days followed by sustained changes observed from the third day. Affymetrix ATH1 GeneChips were used to reveal the global expression profile of the initiation of HL acclimated (after 4 hours in HL) comparing it with the HL acclimation two days later. While the expression of the majority of the photosynthetic genes did not change, significant changes were observed in the expression of genes involved in starch synthesis and degradation as well as the biosynthesis of trehalose-6-phosphate. Transcript that have been found to be highly induced (GPT2 a glucose-6-phosphate/phosphate translocator, BMY1 a beta-amylase) and highly repressed (TPS8 a trehalose-6-phosphate synthase) in both cases were monitored with real time RT-PCR for a period of 9 days revealing the expression pattern of these genes at different times of the day and for a period of 9 days. Finally the GPT2

289 14th Photosynthesis Congress - PS07 gene, identified as the most induce gene in microarrays, has been found to plays a significant role in HL acclimation – a T-DNA knockout mutant plant lacking this gene was seen to be deficient in acclimation. PS22.21 Stress response of multiple Hsp70 proteins from the cyanobacterium Synechocystis sp. PCC 6803 E Rupprecht (Department of Biochemistry and Molecular Biology, Center for Biochemistry and Molecular Cell Research, Faculty of Biology, Albert-Ludwigs-University Freiburg, Germany), E Fuhrmann (Department of Biochemistry and Molecular Biology, Center for Biochemistry and Molecular Cell Research, Faculty of Biology, Albert-Ludwigs-University Freiburg, Germany), D Schneider (Department of Biochemistry and Molecular Biology, Center for Biochemistry and Molecular Cell Research, Albert-Ludwigs-Univer Proteins of the 70 kDa heat shock protein family (Hsp70) are ubiquitously distributed in all kingdoms of life, and in eukaryotes members of this family are found in several organelles as well as in the cytosol. The main functions of the proteins are to ensure proper folding of polypeptide chains, to keep proteins in a translocation competent state after synthesis, and to prevent misfolding and aggregation of proteins e.g. caused by cellular stress. While in eukaryotes multiple Hsp70 proteins are expressed, which localize to different cellular compartments, in bacteria typically only one representative is present. Interestingly, cyanobacteria have a family of multiple Hsp70 (DnaK) proteins encoded in their genome. In the cyanobacterium Synechocystis sp. PCC 6803 at least three DnaK proteins are functionally expressed, two of which (DnaK2 and DnaK3) are essential for cell viability. From the three proteins, the DnaK2 protein shows the highest sequence homology to DnaK proteins from other bacteria and to Hsp70 proteins from chloroplasts, and therefore this protein has been suggested to have a privileged function. In our study, which was mainly based on the use of specific Synechocystis reporter strains and on Western blot analyses, we could show that mainly the expression of the DnaK2 protein altered significantly during cellular stresses. Based on our observations we propose different physiological functions of the multiple DnaK proteins within a cellular chaperone network in Synechocystis. Reference: Rupprecht, E. et al.; Three different DnaK proteins are functionally expressed in the cyanobacterium Synechocystis sp. PCC 6803; Microbiology, in press.

PS22.22 The role of GPT2 in photosynthesis R Webster (University of Manchester), K Athanasiou (University of Manchester), G Johnson (University of Manchester) The Arabidopsis thaliana genome contains 2 genes that encode glucose-6-phosphate/phosphate transporters presumed to be in the chloroplast envelope. One of these GPT1 has been found to be essential for pollen growth, however the role of the other, GPT2, is less well defined. It is known to be expressed in response to sucrose feeding and in senecsent leaves. We have observed that it is transiently massively up regulated when plants are transferred from low (100 µmol m-2 s-2) to high (400 µmol m-2 s-2) light. We are studying the role of GPT2 in photosynthesis, in particular using a T-DNA insertion mutant lacking this gene PS22.23 Group 2 sigma factors and stress responses in Synechocystis sp. PCC 6803

T Tyystjärvi (University of Turku), M Pollari (University of Turku), I Tuominen (University of Turku), L Gunnelius (University of Turku), E Aguire von Wobeser (University of Amsterdam), V Ruotsalainen (University of Turku), E Tyystjärvi (University of Turku), H Matthijs (University of Amsterdam), T Salminen (Åbo Academi University) The initiation of transcription, mediated by RNA polymerase holoenzyme, is the main determinant of gene regulation in eubacteria. Sigma factors are subunits of the RNA polymerase that recognise specific promoter elements. The cyanobacterium Synechocystis sp. PCC 6803 has nine sigma factors. The sigA gene encodes a primary sigma factor that is essential for cell viability. In order to study the roles of the primary-like sigma factors in Synechocystis , we have constructed single and double inactivation strains (DSigB, ?SigC, ?SigD, DSigE, DSigBC, DSigBD, DSigBE, DSigCD, DSigCE, DSigDE). All inactivation strains grew like the wild type under optimal growth conditions. Also the photosynthetic and PSII activities were similar in all strains. These results indicate that the primary-like sigma factors are not required for normal growth under optimal conditions although their transcripts were detected in the wild type strain. The expression of the primary and primary-like sigma factors is light dependent. Transcripts of all these genes diminish in the dark. When cells are shifted to light, the sigB gene is rapidly but transiently activated, whereas the expression of the sigA gene requires a newly synthesized protein factor and is only slowly activated to a steady-state level. The roles of different s factors in adaptation to bright light or to light-dark rhythm will be discussed. In addition, we have found that the SigB and SigC factors are important for the acclimation of Synechocystis cells to high temperature conditions, and regulate different acclimation routes. PS22.24 Characterization of a T-DNA inserted STN8 kinase mutant of Oryza sativa L. K Nath (Department of Biological sciences, Pusan National University), S Mishra (Department of Biological sciences, Pusan National University), Zulfugarov (Department of Biological sciences, Pusan National University), S Raffi (Department of Biological sciences, Pusan National University), G An (Department of Life Science, Pohang University of Science and Technology), C Lee (Department of Biological Sciences, Pusan National University) STN8 protein kinase is required for Photosystem II (PSII) core proteins phosphorylation. In this work, we used T-DNA inserted stn8 mutant line to characterize its physiological function. T-DNA insertion in the exon 2 of chromosome number 5 was confirmed by PCR using primer specific for genomic DNA and T-DNA border. To verify the lack of stn8 transcripts, RT-PCR analysis of mRNA was performed using specific primers for stn8 kinase gene. The transcript level was most abundant in leaves of wild type, but it was very low in roots. Inactivation of photochemical efficiency under highlight stress was severe in stn8 mutant than in wild type whereas recovery of PSII activity was faster in wild type. However, less difference in photochemical efficiency and its recovery was observed between WT and stn8 mutant after HL stress in the presence of lincomycin. Phosphorylated PSII core proteins was detected using phosphothreonine-specific antibody and it was found that with the lack of STN8 kinase, a marked decrease in the total amount of PSII core phosphoproteins in mutant plants, whereas LHCII phosphorylation was similar as in the wild type. More superoxide accumulation observed in mutant than in wild type leaves might be due to less NPQ in former. Taken together, these results suggested that STN8 kinase is required for PSII core phosphorylation and may be regulated by ROS accumulation thereby contributing to differential PSII inactivation and recovery in wild type and mutant plants. PS22.25

290 14th Photosynthesis Congress - PS07 The Mehler reaction as the link between environmental stresses and chloroplast redox signaling. I Strizh (Plant Physiology Department, Biological Faculty, Moscow State University, Russia) Oxygen photoreduction in chloroplasts in the Mehler reaction is amongst the well-known, but still confusing topics in photosynthesis. The history of this problem dates back to the 1951 when Alan Mehler had discovered hydrogen peroxide (H2O2) production in thylakoids under illumination. The most controversial topics were the intensity and the role of the Mehler reaction under environmental stresses. In the beginning of the 21st century the statement about the low rate of O2 uptake in chloroplasts was declared and the significance of this reaction was diminished. However, modern experimental technologies have rapidly extended our knowledge on H2O2-dependent signal transduction in plant. Current data on wide range of plant responses triggered by H2O2 accumulated in chloroplasts allow proposing that the Mehler reaction plays an important role in stress sensing and redox signaling. Although appreciable gaps are remaining in this area. Mechanisms that regulate H2O2 generation and accumulation in chloroplast are amongst the unresolved questions. It is supposed that potential ability to form reactive oxygen species in multiple sites of photosynthetic electron transport chain that was established from in vitro studies may be one of the crucial points for the in vivo regulation of the chloroplast signal transduction. The key intermediates and mechanisms of the signal transduction initiated from the Mehler reaction are not clear yet. To assemble the entire cell redox signaling network, we should use all benefits of classical biochemical and biophysical approaches with cooperation of the modern molecular biological and bioinformatics technologies. PS22.26 Comparative quantitative proteomics to investigate the remodelling of bioenergetic pathways under iron-deficiency in Chlamydomonas reinhardtii M Hippler (University of Münster), A Busch (University of Münster), J Allmer (University of Münster), E Ostendorf (University of Münster), M Zeller (ThermoFisher), H Kirchhoff (University of Münster), B Naumann (University of Münster) Remodelling of the photosynthetic apparatus is required to adapt to iron-deficiency in Chlamydomonas reinhardtii (1,2). To further investigate the impact of iron-deficiency on bioenergetic pathways, comparative proteomics was combined with spectroscopic as well as voltametric oxygen measurements to assess protein dynamics linked to functional properties of respiratory and photosynthetic machineries. Photo-heterotrophic and –autotrophic growth conditions were compared. Although photosynthetic electron transfer is largely compromised under iron-deficiency and the presence of acetate, our quantitative and spectroscopic data revealed that the functional antenna size of photosystem II (PSII) significantly increased. Concomitantly, stress related chloroplast polypeptides, like 2-cys peroxiredoxin and a stress-inducible light-harvesting protein, LhcSR3, as well as a novel light-harvesting protein and several proteins of unknown function were induced under iron-deprivation. Respiratory oxygen consumption did not decrease and accordingly, polypeptides of respiratory complexes, harbouring numerous iron-sulfur clusters, were only slightly diminished or even increased under low iron. Consequently, iron-deprivation induces a transition from photo-heterotrophic to primarily heterotrophic metabolism. In contrast, photosynthetic function was prioritized over respiratory function under photo-autotrophic and low iron conditions, indicating that a hierarchy for iron allocations within organelles of a single cell exists that is closely linked with the metabolic state of the cell.

BR>1. Moseley, J. L., Allinger, T., Herzog, S., Hoerth, P., Wehinger, E., Merchant, S., and Hippler, M. (2002) Embo J 21, 6709-6720 2. Naumann, B., Stauber, E. J., Busch, A., Sommer, F., and Hippler, M. (2005) J Biol Chem 280, 20431-20441 PS22.27 Membrane proteins from the cyanobacterium Synechocystis sp. PCC 6803 interacting with thioredoxin M Lindahl (Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, 41092 Sevilla, SPAIN), A Mata-Cabana (Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, 41092 Sevilla, SPAIN), F Florencio (Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, 41092 Sevilla, SPAIN) Cysteine dithiol/disulphide exchange forms the molecular basis for regulation of a wide variety of enzymatic activities and for transduction of cellular signals. Thus, the search for proteins with reactive, accessible cysteines is expected to contribute to the unravelling of new molecular mechanisms for enzyme regulation and signal transduction. Several methods have been designed for this purpose taking advantage of the interactions between thioredoxins and their protein substrates. Thioredoxins comprise a family of redox-active enzymes, which catalyse reduction of protein disulphides and sulphenic acids. Due to the inherent practical difficulties associated with studies of membrane proteins these have been largely overlooked in the many proteomic studies of thioredoxin-interacting proteins. We have developed a procedure to isolate membrane proteins interacting with thioredoxin by binding in situ to a monocysteinic His-tagged thioredoxin added directly to the intact membranes. Following fractionation and solubilisation of the membranes, thioredoxin target proteins were isolated by Ni-affinity chromatography and 2-DE SDS-PAGE under non-reducing/reducing conditions. Applying this method to total membranes, including thylakoid and plasma membranes, from the cyanobacterium Synechocystis sp. PCC 6803 we have identified 50 thioredoxin-interacting proteins. Among the newly identified thioredoxin targets are the ATP-binding subunits of several transporters and members of the AAA-family of ATPases. PS22.28 The Adaptation Mechanism to Copper Deficiency via microRNA in Arabidopsis H Yamasaki (Kyushu University), S E Abdel-Ghany (Colorado State University), C M Cohu (Colorado State University), Y Kobayashi (Kyushu University), M Pilon (Colorado State University), T Shikanai (Kyushu University) Copper is an essential micronutrient for all living organisms. However, excess copper is toxic because of a production of free radical via free copper. Therefore, intracellular levels of copper must be precisely regulated. In higher plants, major copper proteins are plastocyanin localizes to the thylacoid lumen of chloroplast, and copper/zinc superoxide dismutase localize to the cytoplasm and chloroplast stroma. Under copper limited conditions, expression of copper/zinc superoxide dismutase is down-regulated and its function is compensated by iron superoxide dismutase in chloroplasts. We present several evidences, indicating that one of microRNA, miR398, involves in this regulation in Arabidopsis thaliana, by directing the degradation of copper/zinc superoxide dismutase mRNAs when copper is limited. This regulation via miR398 takes place in response to changes in low copper levels less than 0.5 µM, indicating that miR398 is involved in a response to copper limitation. On the other hand, another major copper protein, plastocyanin which is involved in photosynthetic electron flow was not regulated via miR398. In low copper conditions, limited copper is transported to plastocyanin preferentially. We conclude that miR398 is a key factor in

291 14th Photosynthesis Congress - PS07 copper homeostasis in higher plants. PS22.29 Light acclimation of leaf chlorophyll content L Hallik (University of Tartu), O Kull (University of Tartu) Leaf chlorophyll as major photosynthetic pigment is easy to measure with both laboratory analysis and with remote sensing technology. However, use of this information is limited because total leaf chlorophyll content [Chls] is weakly related to its photosynthetic performance and predictors of [Chls] are not well understood. Acclimation of leaf [Chls] to increased irradiance consists of two opposing responses: an increase in the amount of photosynthetic apparatus to utilise extra energy, and a decrease in the amount of light harvesting complexes of the apparatus to avoid excess excitation of photosystems. We studied light acclimation of leaf chlorophyll [Chls] and nitrogen [N] contents and leaf dry mass per area (LMA) along a natural light gradient in the herbaceous and tree canopy. We found different acclimation patterns between the herbaceous and tree canopy. Light dependent regulation of the amount of leaf chlorophyll appears to consists of a series of morphological and physiological changes which operate in different time scales. Path analysis revealed threefold influence of light on leaf chlorophyll content. Increasing irradiance: (1) increases [Chls] through leaf morphogenesis and changes in LMA; (2) increases [Chls] through readjustment in the amount of photosynthetic apparatus as changes in [N] revealed; (3) decreases [Chls] to avoid excess excitation as revealed by decreased Chl/N ratio. Differences in time scales of these three processes together with differences in dynamics of leaf light environment between tree and herbaceous canopy are the probable reason of the found differences in acclimation patterns. PS22.30 Chlorophyll a Fluorescence emission and photosynthetic performances of green-algal lichens along a light regime gradient in the Mediterranean region M Piccotto (Department of Biology, University of Trieste - Italy), E Pittao (Department of Biology, University of Trieste - Italy), M Tretiach (Department of Biology, University of Trieste - Italy) The hypothesis that in lichens CO2 gas exchanges and chlorophyll a fluorescence (CaF) vary according to the light regimes of their original habitat, as observed in vascular plants, was tested analysing the photosynthetic performances of seven foliose phycolichens (Flavoparmelia caperata, Lobaria pulmonaria, Parmelina tiliacea, Parmotrema chinense, Xanthoparmelia tinctina, Xanthoria parietina, X. calcicola) collected from open, south-exposed rocks to densely shaded woods. In the laboratory the thalli were divided in two sub-samples. One subsample was used for building up a light response curve (LRC) at the specific optimum thallus water identified in a previous series of measurements. The other subsample was used to build up CaF emission curves at Iks and saturating PPFDs. Two fragments of c. 35 mg from each subsample were used to measure photosynthetic pigments in DMSO crude extracts. The results confirm that the chlorophyll content of lichens is highly correlated to Pg max. Surprisingly, however, the latter seems to be influenced by the nitrogen availability more than by the light regime of each lichen habitat. Some CaF parameters show not only clear differences between shade- and sun-exposed lichens, but also that the photosynthetic quantum conversion increases in good accordance with the ambient light regime of the species. Some unusual responses of CaF emission were observed: for instance, the low values of Fp compared

with Fm, even at the saturating PPFD inferred from LRC. They could be a matter of interest for biophysical studies on the photosynthetic mechanisms of these poikilohydric organisms. PS22.31 Photosynthesis and respiration regulation by the bacteriophytochrome RpBph1 in the bacterium Rhodopseudomonas palustris M Kojadinovic (CEA Cadarache, DSV/IBEB/LBC, UMR 6191 CNRS/CEA/Univ Aix-Marseille, St Paul-Lez-Durance, France), A Laugraud (Université Lyon 1, Pole Rhone-Alpin de Bioinformatique, UMR5558, Laboratoire de Biométrie et Biologie Evolutive, Villeurbanne, France), J Adriano (CEA Cadarache, DSV/IBEB/LBC, UMR 6191 CNRS/CEA/Univ Aix-Marseille, St Paul-Lez-Durance, France), P Bouyer (CEA Cadarache, DSV/IBEB/LBC, UMR 6191 CNRS/CEA/Univ Aix-Marseille, St Paul-Lez-Durance, France), J Fardoux (LSTM, IRD, CIR) The purple photosynthetic bacterium Rhodopseudomonas palustris is able to develop using photosynthesis or respiration. Out of the six bacteriophytochromes present in this species, one (RpBphP1) triggers the synthesis of the photosynthetic apparatus, counterbalancing the repressive effect of the transcription factor PpsR2. In the present work, we show that photosynthetic apparatus synthesis is coupled with a 40% to 50% respiratory activity limitation. Using bacteriophytochromes and PpsRs mutants, we demonstrate that both RpBphP1 and PpsR2 are involved in the regulation of the respiratory activity , RpBphP1 acting as a repressor and PpsR2 as a partial activator. Based on enzymatic and transcriptional studies, we demonstrate that this regulation does not occur on respiratory chain elements but rather on a central Krebs cycle enzymatic complex: the alpha-ketoglutarate dehydrogenase (KGD). Quantitative RT-PCR assays revealed that expression of sucA, encoding the E1 component of KGD and presenting a PpsR binding motif in its promoter region, is 28 % down regulated under far-red illumination versus dark conditions. This result was further confirmed by enzymatic assays, which showed a 20 to 30% decrease in KGD activity under far-red illumination. We propose that KGD down regulation provokes a decrease in reducing power available for the respiratory chain and therefore a respiratory activity limitation. Altogether our data indicate that, in the presence of light, R. palustris favors photosynthesis and limits respiration via the action of RpBphP1. This transcriptional regulation is complementary to the regulation occurring at the level of the bioenergetic chains which also favors photosynthetic activity versus respiration. PS22.32 A new photoactive protein acting as a sensor of light intensity: the Orange Carotenoid Protein (OCP) D Kirilovsky (CEA-CNRS), A Wilson (CEA-CNRS) Recently we demonstrated the existence of a blue-green light induced photoprotective mechanism for dissipating excess absorbed energy in cyanobacteria. A soluble carotenoid-binding protein (OCP), encoded by the slr1963 gene in Synechocystis PCC 6803, of previously unknown function, plays an essential role in this process. We proposed that blue light absorption by the carotenoid will induce changes in the carotenoid that will convert the dark OCP into an activated form able to absorb the energy coming from the phycobilisome and dissipate it as heat. To demonstrate this hypothesis, an active C-terminal His-tagged OCP was isolated and used for further studies. Blue-green light induces the transformation of the dark stable orange form to a red form. This conversion is reversible in darkness. The conversion is not stimulated by excitation of the red form. The kinetics of formation of the red OCP is light-intensity dependent. The kinetics of the recovery of the orange form in darkness is temperature dependent. While at 34°C the red form

292 14th Photosynthesis Congress - PS07 is unstable and thus high light intensities are needed to convert all the OCP to the red form, at 10°C, the red form is very stable and low light intensities are sufficient to convert all the orange OCP. Thus, the OCP seems to be a sensor of photoinhibitory conditions; the red OCP is accumulated under the conditions in which cyanobacteria need photoprotection, i.e. high light and light + cold. OCP is the first case of a photoactive protein with a carotenoid as the photoactive chromophore. PS22.33 Interactions between the photosystem II subunit PsbS and xanthophylls as studied in vivo G Bonente (University of Verona-Italy), S Caffarri (University Aix-Marseille II-France)), G Finazzi (IBPC Paris-France)), K Niyogi (california University Berkeley-USA)), R Bassi (University of Verona-Italy) The photosystem II subunit PsbS is essential for excess energy dissipation (qE), while both lutein and zeaxanthin are needed for its full activation. The interaction between PsbS and these xanthophylls has been studied in vivo, based on a model proposing two xanthophyll binding sites in PsbS, each activated by the protonation of a lumen-exposed glutamic acid residue binding DCCD (dicyclohexylcarbodiimide). First, we found that the single glutamate mutants differentially affect the reversible NPQ at low light. Both the amplitude and the kinetics of NPQ onset were markedly different in PsbS E122Q and PsbS E226Q, suggesting that their equivalence for NPQ establishment is not always conserved. To explore the possibility of selective xanthophyll binding by the two sites, PsbS point mutants on each of the two Glu residues PsbS E122Q and PsbS E226Q were crossed with the npq1 npq4 and lut2 npq4 mutants lacking respectively zeaxanthin and lutein. Double mutants PsbS(E122Q)npq1 and PsbS(E226Q)npq1 had no qE, while PsbS(E122Q)lut2, and PsbS(E226Q)lut2 showed a strong reduction in qE with respect to both lut2 and single glutamate mutants. These results are discussed in terms of supporting the “two xanthophyll binding sites” (Li et al. JBC ( 2004) 279, 22866) or an alternative “PsbS trigger” model in which PsbS could act as a pH sensor thus inducing conformational changes and quenching in one or more Lhcb proteins depending on their binding violaxanthin or zeaxanthin. PS22.34 Ozone effects on the metabolism and the antioxidant system in poplar leaves at different stages of development I Nogués (CNR), S Fares (CNR), F Loreto (CNR) During leaf development significant metabolic changes occur. Leaf development has been associated with a loss of photosynthetic activity, ad an increase in the oxidative damage which may be exacerbated by exposure to ozone. In this work poplar plants were subjected to a high but realistic ozone level (100 ppb) for ten days. Photosynthesis, stomatal conductance, methanol emission, photosynthetic metabolites (glucose, fructose) antioxidants (phenolics, ascorbic acid) and H2O2 and lipid peroxidation levels were measured in both, control and ozone-treated poplar leaves at two stages of development. Photosynthesis and stomatal conductance were not significantly affected by ozone in young and fully expanded leaves. However young control leaves showed a higher concentration of all studied metabolites and antioxidants than young ozone-treated leaves. Young ozone-treated leaves also showed a lower methanol emission than older leaves of the same plants, while methanol emission was highest in the youngest control leaves. This may indicate a faster development of ozone treated leaves or the interruption of leaf development due to ozone. In fully expanded leaves, ascorbic acid concentration was not affected by ozone, whereas phenolics content of treated leaves was higher than in control leaves. Thus we conclude that the ascorbate scavenging system is not a major defensive mechanism

against ozone, while induction of phenolics may play a significant role in protecting fully developed poplar leaves against ozone.

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PS23 - Global Climate Change - Terrestrial PS23.1 New Tracers of Global-Scale Photosynthesis during Climate Change D Yakir (Weizmann Institute of Science) Small imbalances between photosynthesis and respiration on global scale can significantly influence the atmospheric concentrations of CO2 and the rate of its increase due to fossil fuel burning. At present, photosynthesis by the land biosphere re-absorbs about 25% of the anthropogenic carbon emission, but this enhanced uptake is expected to reverse over time. The dynamics in global photosynthesis and respiration cannot be easily extrapolated from small-scale experiments and much effort is invested in developing means to assess photosynthesis from atmospheric measurements. Stable isotopes, 13C (in CO2) and 18O (in CO2 and O2), were shown to provide powerful natural tracers of photosynthesis. Here, I will briefly discuss two new tracers that were recently added to our arsenal: 17O and its anomalous ratios to its heavier companion, 18O; and COS (carbonyl sulfide) that, together with the uptake flux of CO2, diffuse into leaves and get hydrated with leaf water, into H2S and CO2. The 17O tracer is based on the idea that O2 acquires anomalous 17O/18O ratio in the stratosphere, but O2 cycling through photosynthesis and respiration brings this ratio back to “normal”. Variations in the 17O anomaly in tropospheric O2 can be interpreted as variations in the biological O2 cycle. The potential use of COS relies on the newly observed seasonal cycle in its atmospheric concentrations, which are ~6 times larger than those of CO2 as, roughly, would be expected if the COS is dominated by plant uptake, while CO2 reflects net exchange due to photosynthesis and respiration.

PS23.2 The Molecular Basis for Genotypic Variation in Soybean Photosynthetic Responses to Elevated Carbon Dioxide Concentration E Ainsworth (USDA ARS Photosynthesis Research Unit, University of Illinois), A Leakey (Department of Plant Biology, Institute for Genomic Biology), K Gillespie (Department of Plant Biology, Institute for Genomic Biology), F Xu (Institute for Genomic Biology), R Nelson (USDA ARS; University of Illinois), D Ort (USDA ARS; University of Illinois) A critical step in maximizing crop yield in a future of elevated atmospheric carbon dioxide concentration ([CO2]) is identifying genotypic variability in response to elevated [CO2] and understanding the molecular basis for the variation. We compared photosynthesis, leaf metabolites and global gene expression of three soybean cultivars grown at ambient [CO2] (380 ppm) and elevated [CO2] (550 ppm) at the Soybean Free Air Concentration Enrichment (SoyFACE) facility. The cultivars were selected because in previous years of the SoyFACE experiment Clark showed no yield enhancement at elevated [CO2] (+1%), Pioneer 93B15 showed moderate yield enhancement at elevated [CO2] (+13%) and Holt showed a large yield enhancement (+33%). While midday stimulation of photosynthesis did not differ substantially among cultivars, acclimation of photosynthesis measured as decreased maximum Rubisco activity, was only apparent in Pioneer 93B15 and Holt. All cultivars showed substantial increases in soluble sugars and starch at elevated [CO2]. However, there was significant variation among cultivars in investment of carbon in antioxidant metabolites and

total antioxidant capacity. The molecular basis for these changes in photosynthetic capacity and antioxidant metabolism are being investigated by expression profiling using Affymetrix gene chips. The aim of this research is to identify physiological, biochemical or molecular targets for improving the performance of soybean cultivars at elevated [CO2].

PS23.3 A new paradigm in plant photosynthesis: Direct and diffuse light are not equal C Brodersen (University of Vermont), T Vogelmann (University of Vermont), W Williams (St. Mary's College), H Gorton (St. Mary's College) Global-change scenarios suggest a trend of increasing diffuse light due to expected increases in cloud cover. Canopy-level estimates of plant community photosynthesis under diffuse light show increased productivity attributed to more uniform distribution of light within the forest canopy, yet the effect of the directional quality of light at the leaf level is unknown. Here we show that leaf-level photosynthesis in leaves grown in high light of both C3 and C4 plants is up to 10-15% higher under direct light compared to equivalent absorbed irradiances of diffuse light. Shade adapted leaves showed no preference for direct or diffuse light at all light intensities tested. The tissue depth at which maximum light absorption occurred within high light grown leaves was shifted up to 50 µm under direct vs. diffuse light, while shade adapted leaves showed no difference in light absorption profiles. Palisade cells may facilitate the utilization of direct light, and the thickness of that tissue may be responsible for the decreased rates of photosynthesis under diffuse light.

PS23.4 FLEX - FLuorescence EXplorer: a remote sensing approach to quantify spatio-temporal variations of photosynthetic efficiency from space U Rascher (Research Centre Jülich) Light use efficiency of photosynthesis dynamically adapts to environmental factors, which lead to complex spatio-temporal variations of photosynthesis on various scales from the leaf to the canopy level. Optical remote sensing techniques often failed to quantify photosynthetic light use efficiency within this fluctuating mosaic. In this presentation we summarize the results from several remote sensing projects for their potential to quantify light use efficiency from the level of single leaves to the canopy scale. The potential monitoring the fluorescence signal of chlorophyll by either active, laser induced or passive methods from a distance will be reviewed. Recently the FLuorescence EXplorer (FLEX) mission that proposed to launch a satellite for the global monitoring of steady-state chlorophyll fluorescence in terrestrial vegetation was selected for pre-phase A by European Space Agency (ESA). This method aims for mapping photosynthetic efficiency by quantifying steady state fluorescence in the so called Fraunhofer lines. The scientific background of this approach will be highlighted and first results from European campaigns will be presented: In two campaigns an air-borne sensor was used to measure steady state fluorescence and to relate this to photosynthetic status of agricultural systems in Spain and Southern France. Linking these results with ecosystem flux measurements and regional carbon modeling shows the

294 14th Photosynthesis Congress - PS07 way how direct quantification of photosynthesis may reduce uncertainties to predict plant mediated exchange processes. This presentation will be given on behalf of the Mission Assessment Group of ESA that is currently orchestrating the scientific activities around FLEX.

PS23.7 Relative importance of acclimation in respiration and photosynthesis in black spruce grown at high temperatures D Way (University of Toronto), R Sage (University of Toronto)

PS23.5 Warm simulated autumn growth conditions do not increase photosynthetic carbon gain but increase the dissipation of excess energy in Jack pine F Busch (University of Western Ontario), N Hüner (University of Western Ontario), I Ensminger (Max-Planck-Institut für Molekulare Pflanzenphysiologie) The seasonal development of trees of the boreal forest might be affected by climate change, as the trees will experience naturally decreasing daylength during autumn, while warmer air temperature will maintain photosynthesis and respiration. Working with controlled environments, we used a factorial design to assess the impact of photoperiod and temperature on the down-regulation of photosynthetic gas exchange and the mechanisms involved in the dissipation of energy in Jack pine (Pinus banksiana). Control treatments of plants grown under 16-h photoperiod and 22°C (representing warm summer conditions) and plants grown under 8-h/7°C (cool autumn) were compared to plants grown under warm autumn (8-h/22°C) and cool summer conditions (16-h/7°C). Assimilation and respiration rates under warm autumn conditions were only approximately half of those of the summer control and comparable to the cool autumn control. Different treatments appeared to govern different strategies for dissipating excess energy. Zeaxanthin did not appear to be a major contributor to safe dissipation of excess energy in the warm autumn treatment as it was the case in the other three treatments. Instead a strong alternative electron sink appeared to consume electrons by oxidizing the plastoquinone pool. We conclude that photosynthetic downregulation due to photoperiod appears to negate any potential for an increased carbon gain in connection with an extended growing season. Under warm autumn conditions, excess energy appears to be dissipated by a unique, as yet unidentified, mechanism which is not detected in pine seedlings grown under either normal warm summer or normal cold autumn conditions.

PS23.6 Effects of an extended drought period on grasslands at various altitudes in Switzerland U Feller (University of Bern), C Signarbieux (University of Bern) From recent investigations it can be concluded that extreme events (e.g heat waves, extreme drought periods) will become more relevant in Central Europe during the next decades (Schär et al., Nature 427, 332-336, 2004). The effects of an artificial drought period (installation of rain shelters for 10 weeks) on physiological traits of representative grassland species was investigated at two altitudes (400 and 1000 m a.s.l.) in Switzerland. The net assimilation rate (An) and stomatal conductance (gs) were affected in Phleum pratense and in other gramineae at both altitudes, while these effects were in dicots (Rumex obtusifolius, Trifolium repens) relevant only at the higher altitude. The decline of An was paralleled by a decrease of gs, but the intrinsic efficiency of photosystem II was not affected by the treatment. The still high An of Rumex in lowlands under drought may explain the dominance of this plant at the end of the drought period. Differences in the species composition and differences in reversible and irreversible damages caused by drought in the various species may contribute to the overall response of grasslands in a site-specific manner.

Black spruce (Picea mariana) is a dominant North American boreal species and its response to climate warming will determine the future of the boreal forest. We investigated how growth temperature affects photosynthesis, using CO2 response curves, temperature response curves, O2 sensitivity, and estimates of respiration for seedlings grown at cool (22/16°C) and warm (30/24°C) day/night temperatures. Below 30°C, warm-grown seedlings had lower net and gross CO2 assimilation rates than cool-grown seedlings, due to reduced Vcmax and Jmax. Above 30°C, warm-grown seedlings had slightly higher net CO2 assimilation rates than cool-grown trees; however, gross CO2 assimilation rates were identical because respiration acclimated in the warm-grown seedlings, resulting in lower dark and day respiration rates. Trees grown at high temperatures were significantly smaller and shorter than cool-grown seedlings, with lower net CO2 assimilation rates at their growth temperature. Higher growth temperatures will therefore likely have direct, negative impacts on the growth rate and carbon uptake of black spruce, and may reduce the carbon sink potential of the North American boreal forest. PS23.8 CO2 on growth and photosynthetic characteristics of chickpea M Singh (Indian Agricultural Research Institute, New Delhi, India), S Talwar (Indian Agricultural Research Institute, New Delhi, India), S Khetrapal (Indian Agricultural Research Institute, New Delhi, India), P Deshmukh (Indian Agricultural Research Institute, New Delhi, India) The concentration of CO2 in the atmospheric has increased almost 30% to a present level of about 379 µl l-1. Such an increase in CO2 may affect the growth and productivity of C3 crop species. There are no reports available on the response of chickpea (Cicer arietinum L.). to elevated CO2 and this investigation is the first attempt to study the effect of rising CO2 on growth and photosynthetic characteristics of this crop. The plants of chickpea cultivar Pusa 1108 were raised inside the open top chambers using earthen pots and exposed to ambient (370±20 µl l-1, CA) and elevated (550±50 µl l-1, CE) CO2 levels from germination till maturity of the crop. CE showed significant increase in shoot length, number of branches & leaves and leaf surface area per plant. Significant increase in dry weight of above and below ground plant parts was observed under CE. The concentration of starch, reducing and non-reducing sugars was more in plant grown under CE. Sucrose content was higher along with increased sucrose phosphate synthetase activity. Total carbon content increased and nitrogen content decreased, which resulted in higher C/N ratio. The soluble protein content decreased and the magnitude of reduction was almost similar at all the durations of CO2 exposure. There was no effect of CE on Rubisco activity or its activation state and on expression of rbc L but the expression of rbcS decreased marginally. The study concludes that rising CO2 in near future will be beneficial for chickpea crop.

PS23.9 Acclimation of photosynthesis in maize plants to high CO2 A Prins (University of Pretoria), S Driscoll (Rothamsted Research), J Muchwezi (University of Pretoria), P Verrier (Rothamsted Research), C Foyer (Rothamsted Research)

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While the dorso-ventral regulation of photosynthesis has been characterised with respect to CO2 availability and light orientation in dicotyledonous C3 leaves, little information is available on monocotyledonous C4 plants. Maize plants were therefore grown to maturity in near ambient CO2 (350 uL.L-1) and with CO2 enrichment (700 uL.L-1). Large decreases in leaf protein and chlorophyll were observed in plants grown at high CO2 for all leaves except the oldest and youngest two leaves on the stem. Similarly, photosynthesis was decreased by about 20% on a surface area basis in the young leaves of high CO2-grown plants and 17% in the senescent leaves. However, photosynthesis was greatly increased on a chlorophyll basis in high-CO2 grown plants and plant growth; development and biomass production were similar in both growth conditions. The senescence-dependent rise in leaf glucose and fructose was absent from leaves grown with CO2 enrichment. However, the abundance of invertase and sucrose synthase transcripts was similar in both growth conditions. While CO2 enrichment increased the pools of sucrose and starch in the youngest leaves, no significant effect of high CO2 on either carbohydrate was observed in the mature and senescent leaves. The abundance of sucrose phosphate synthase transcripts was similar in ambient and high-CO2 grown leaves and declined progressively with leaf age. Surprisingly, few transcripts associated with photosynthesis were modulated by growth with CO2 enrichment. However, the leaf epidermal structure was greatly modified, showing an increase in stomatal index and much larger cell sizes. Transpiration and stomatal conductance decreased per unit area in leaves grown with CO2 enrichment. However, the surface (adaxial/abaxial)-specific regulation of photosynthesis was largely unaffected by high CO2.

PS23.10 Gene Expression Profiling in Rice Grown under Elevated CO2 T Fukuda (Natl. Inst. Agrobiol. Sci.), H Sakai (Natl. Inst. Agro-Environ. Sci.), Y Taniguch (Natl. Inst. Agrobiol. Sci.), C Masumoto (Natl. Inst. Agrobiol. Sci.), W Cheng (Natl. Inst. Agro-Environ. Sci.), T Hasegawa (Natl. Inst. Agro-Environ. Sci.), M Miyao (Natl. Inst. Agrobiol. Sci.), H Fukayama (Kobe Univ.) The effects of long-term exposure to elevated CO2 (680 ppm versus ambient 380 ppm) on gene expression were studied in rice leaves grown with three different N conditions (0 g N, 1.0 g N, 2.0 g N) in a CO2 controlled greenhouse. Leaf soluble protein contents were lower in elevated CO2 than in ambient CO2 with all N conditions, showing a greater decline in lower N. Protein composition analysed by SDS-PAGE and 2-DE was not affected by elevated CO2. In contrast, gene expression was significantly affected by elevated CO2. Microarray analysis showed that expression of genes for enzymes involved in CO2 fixation (Rubisco, PGK, GAPDH and CA) was downregulated, whereas that of genes encoding enzymes for RuBP regeneration (SBPase, PtFBPase and PRK), starch synthesis (AGPase and SPS), and amino acid synthesis (GS2 and GOGAT) were upregulated in elevated CO2. These results suggest that a set of genes involved in a distinct metabolic pathway are co-regulated in elevated CO2. Cluster analysis of microarray data identified six transcription factors that likely participated in the responses to elevated CO2: Three genes encoding Synaptotagmin C, myb-like protein and retinaldehyde binding-like protein were downregulated, and that of the other three for proteins, each containing TPR-like, BTB/POZ and CCT domain, were upregulated by elevated CO2. Analyses of these transcription factor genes may provide a significant clue to understand the mechanisms for responses to elevated CO2.

PS23.11 Recovery of photosynthesis in the offspring of rice grown under free-air CO2 enrichment (FACE) G Chen (Institute of Plant Physiology and Ecology,Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences ) Rising CO2 concentration increases photosynthesis in C3 plants owing to the increase of its substrate concentration and inhibition of photorespiration. After long-term exposure to elevated CO2, however, the stimulatory effect decreases gradually so that photosynthesis is lower than that in plants grown in ambient air when measured at a same CO2 concentration. This phenomenon referred as down-regulation or acclimation of photosynthesis is often reported in CO2-enriched current generation plants. It has not been known whether the down-regulation is preserved or eliminated in the offspring from seeds of plants grown at elevated CO2. By free-air CO2 enrichment (FACE, 200µmol mol-1 above the ambient air CO2 concentration) experiments we found that leaf net photosynthetic rate (Pn) was significantly lower in CO2-enriched current regeneration rice plants (E0E) but not in the CO2-enriched rice offspring plants than that in rice plants grown in ambient air (E0A) when measured at a same CO2 concentration (580 µmol mol-1), indicating that the down-regulation of photosynthesis occurred in E0E but not in their successive generation (E1E-E5E). Interestingly, the similar differences in some physiological and biochemical indexes related to Pn were also observed between E1E and E0E. For example, the significant decreases in maximal carboxylation rate (Vcmax), maximal electron transport rate in vivo(Jmax), ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content, and sucrose-phosphate synthase (SPS) activity occurred in E0E but not in E1E and E2E. The finding may imply that plant photosynthesis can operate with an approximately constant capacity from generation to generation in increasing of atmospheric CO2 concentration, at least in rice-field ecosystem. PS23.12 The effects of elevated CO2 on tropical trees are related to succesional status and soil nutritional conditions C Martinez (Department of Biology, FFCLRP, University of Sao Paulo, Brazil.), E de Oliveira (Graduate Programme Comparative Biology, FFCLRP, USP), J Legracie Jr. (Graduate Programme Comparative Biology, FFCLRP, USP ), A Approbato (Graduate Programme Comparative Biology, FFCLRP, USP ) Predicting the effects of increasing atmospheric CO2 on plant species requires an understanding of the interactions between CO2 and other limiting environmental factors. Nutrient requirements for plant growth are expected to rise in response to the predicted changes in CO2 and temperature. The response of tropical tree species to elevated atmospheric concentrations of greenhouse gases has received little attention to date. We examined the effects of elevated CO2 concentrations and soil nutrient availability on gas exchange and growth of two pioneer (Croton urucurana and Cecropia pachystachia) and two late successional (Essenbeckia leiocarpa and Cariniana Legalis) tropical tree species. Plants were grown under open-top chambers at 380, 555 or 740 µmol mol-1 of atmospheric CO2. Pioneer plants grown under nutrient stress showed only half the photosynthetic stimulation (+22%) of plants with no stress treatment (+52%). Plant biomass responses to elevated CO2 were strongly affected by nutritional stress factor, but the magnitude of this response depended on functional group. The elevated CO2 significantly increased the total biomass, and stem, root and leaf

296 14th Photosynthesis Congress - PS07 biomasses only under the high nutrient, but not under low nutrient treatment. In late successional species, diurnal minimum Fv/Fm was significantly higher in the elevated CO2 treatments, decreasing the susceptibility of plants to suffer photoinhibition. These findings support the hypothesis that elevated CO2 would partially protect PSII from photoinhibition when growth under high nutrient level provides adequate sinks for extra photosynthate. Supported by FAPESP (Grant 2005/54804-7) PS23.13 Modification of photosynthesis temperature response by long-term growth in elevated CO2 and temperature in wheat field crops R Martinez-Carrasco (Institute for Natural Resources and agricultural Biology of Salamanca, CSIC), A Alonso (Institute for Natural Resources and agricultural Biology of Salamanca, CSIC), P Perez (Institute for Natural Resources and agricultural Biology of Salamanca, CSIC), R Morcuende (Institute for Natural Resources and agricultural Biology of Salamanca, CSIC) The temperature dependence of C3 photosynthesis is known to vary according to the growth environment. Atmospheric CO2 concentration and temperature are predicted to increase with climate change. To test whether long-term growth in elevated CO2 and temperature modifies photosynthesis temperature response, wheat (Triticum aestivum) was grown in ambient CO2 (370 µmol mol-1) and elevated CO2 (700 µmol mol-1) combined with ambient and 4 ºC warmer temperatures, using temperature gradient chambers in the field. Flag leaf photosynthesis was measured at temperatures ranging from 20 ºC to 35 ºC and 330 µmol mol-1 or lower CO2 concentrations between ear emergence and anthesis. The maximum rate of carboxylation was determined in vitro in the first year of the experiment and from the photosynthesis-intercellular CO2 response in the second year. Growth temperature had no effect on flag leaf photosynthesis in plants grown in ambient CO2, while it increased photosynthesis in elevated growth CO2. However, warmer growth temperatures did not modify the response of photosynthesis to measurement temperatures from 20 ºC to 35 ºC. The increase with temperature in photosynthesis was significantly higher in plants grown in elevated than ambient CO2. In association with this, growth in elevated CO2 increased the temperature response (activation energy) of the maximum rate of carboxylation. The results provide field evidence that growth under CO2 enrichment enhances the response of Rubisco activity to temperature in wheat.

conditions. The major objective of this study was to examine if the types of shoot development affect seasonal changes in the temperature response of photosynthesis. Three-year-old seedlings of Fagus crenata and Alnus hirsuta were grown in pots. The photosynthetic response curves to intercellular CO2 concentration were measured monthly at 4–5 temperatures ranging from 15 °C to 35 °C. In Fagus crenata, light-saturated rates of photosynthesis hit a peak after fully development and had a 50% decrease two months later. In Alnus hirsuta, however, light-saturated rates of photosynthesis maintained the same maximum values regardless of leaf flush time. The photosynthetic acclimation mechanism was further analysed by comparing the seasonal changes in leaf nitrogen concentration, leaf mass per leaf area, and temperature dependent changes in both the maximum rate of RuBP carboxylation and RuBP regeneration. PS23.15 Photosynthesis response of sorghum plants under changing environment E Aguirre (Paseo de la Universidad nº 7), A Muñoz-Rueda (Facultad de Ciencia y Tecnolgia), S Vicente (Paseo de la Universidad nº 7), A Mena-Petite (Facultad de Ciencia y Tecnologia), M Lacuesta (Paseo de la Universidad nº 7) Sweet sorghum is a multipurpose cereal of potential interest for several non-food uses. Although it is a C4 plant and uses nitrogen more efficiently than most C3 plants, changes in ambient conditions as change in soil water content, CO2 levels or nitrogen supply can alter the nitrogen demand and modify plant productivity. The aim of this work has been to evaluate the effect of several ambient conditions on photosynthesis and biomass production of the C4 plant Sorghum bicolor in order to estimate the response of this crop to the environmental conditions imposed by the Climate Change. Sorghum plants were grown during 60 days in a growth chamber and watered with three nitrate concentrations (5, 15 and 30 mM ). Plants were grown under different levels of CO2 (360 and 910ppm) and two irrigation treatments. The well watered plants (C) were watered during the 60 days period with the different nitrate solutions while the drought was imposed by withholding irrigation during the last 30 days. Results showed that photosynthesis rate in the third fully expanded leaf did not change significantly neither by nitrate concentration or drought conditions under ambient CO2. However, under elevated CO2 a decrease in photosynthesis rate could be observed with the highest nitrate concentration accompanied by a decrease in the maximum photosynthesis rate, light saturation point and biomass accumulation in high CO2. Key words: Sorghum, elevated CO2, nitrate, photosynthesis, biomass.

PS23.14 Seasonal changes in the temperature response of photosynthesis in leaves with different types of shoot development Q Han (Forestry and Forest Products Research Institute), Y Chiba (Forestry and Forest Products Reseach Institute) In evergreen species, light-saturated rates of photosynthesis and its optimum temperature range change within a growing season, in deciduous species, however, this photosynthetic acclimation is considered species specific. There are two widely different patterns of shoot growth in deciduous species. In one group of genera (determinant shoot development), such as Fagus, one single flush occurs at the early growing season and all leaves appear under the same air temperature conditions and they will experience different temperatures afterwards. In the second group of genera (indeterminant shoot growth), such as Alnus and Populus, leaf emergence and shoot elongation are prolonged, and both early and late leaves are produced under different temperature

This research was financially supported by grants of MEC BFU 2004/003503, UPV 00118.125-E15431/2003 and UPV 00118.125-E-14073/2001. Ernesto Aguirre Igartua is a recipient of a fellowship from the UPV/EHU.

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PS24 - Global Climate Change - Aquatic PS24.1 Can we predict how increased CO2 and temperature will affect oceanic photosynthesis in the future ocean? J LaRoche (IFM-GEOMAR Düsternbrooker Weg 20 Kiel 24105, Germany) The atmosphere and oceans are now being subjected to dramatic anthropogenic changes in concentrations of green-house gases, including CO2, which in turn lead to increase in global temperature. The present atmospheric CO2 concentrations and average global temperatures are now higher then they have been in the past 400 kyr and are expected to rise further. Because of the close exchange between the atmosphere and the ocean, the atmospheric forcing will have a large impact on the oceanic surface layer ecosystem, where marine photosynthesis occurs. This talk will concentrate on exploring the effect of increased CO2 and temperature on marine phytoplankton communities, which are composed of very diverse group of photosynthetic organisms. Temperature and CO2 are expected to have a direct effect on phytoplankton photosynthesis and growth but very little is known on the combined effect of these factors on the various groups of marine phytoplankton. The indirect effect of increasing temperature on ocean circulation and thermal stratification are perhaps even more important in determining light and nutrient availability to the diverse phytoplankton communities. Both the direct and indirect effect of temperature and CO2 changes will be discussed in the context of new genomic information available from diverse representative of marine phytoplankton.

PS24.2 Influence of nutrient supply and UV on photosynthesis and inorganic carbon concentrating mechanisms: relation to environmental change? J Raven (University of Dundee), J Beardall (Monash University), L Franklin (Smithsonian Environmental Research Centre), M Giordano (Universitá Politecnica delle Marche) About half of the anthropogenic emissions of CO2 over the last two centuries remain in the atmosphere, 20% is in terrestrial sinks and 30% is in the oceans, with a very significant fraction of this in the surface ocean. The oceans will continue to accumulate a fraction, although on physico-chemical grounds a decreasing fraction, of further anthropogenic CO2 emissions. Since external CO2 is the inorganic C species involved in controlling expression of inorganic carbon concentrating (CCMs) in the eukaryotic algae tested, the expression of CCMs is likely to decrease. This is predicted to change the resource cost of producing (energy, nitrogen, trace metals) and running (energy) the CCMs, and downstream effects on other photosynthetic components. There are also effects of the supply of PAR, nitrogen, phosphorus, iron and (?) zinc on the expression of the CCMs. Primary productivity can change with variations in UV fluxes, and UVB, at least in acute exposures, has less effect on CCMs than on downstream photosynthesis in some algae but the CCM is UVB-sensitive in at least one other. This range of interactions involving CCMs further complicate the prediction of effects of increasing sea surface CO2 on overall phytoplankton diversity and productivity, especially when possible changes in mixed layer depth are taken into account. The additional information needed to improve the predictive power of models of the effects on phytoplankton of these aspects of environmental changes will be outlined.

PS24.3 Repair rates explain differential susceptibility photoinactivation in prokaryotic picoplankters

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D Campbell (Mount Allison University), Z Finkel (Mount Allison University), C Six (Mount Allison University) Oceanic picophytoplankton include taxa with diverse photosynthetic antennae which have converged to grow as small coccoid cells. Understanding community structures, and modelling oceanic productivity, require knowledge of how these taxa exploit and respond to environmental variations. All oxygenic photobionts suffer photoinactivation of their PSII centres under illumination, and so for sustained growth, their capacity for PSII repair through protein turnover and synthesis must equal or exceed the rate of photoinactivation. We therefore studied short term photoacclimation in a panel of five representative strains of marine cyanobacteria, including offshore Prochlorooccus strains with Pcb integral membrane antennae, oceanic Synechococcus with phycoerythrin-rich phycobilisomes, and coastal Synechococcus with phycocyanin-rich phycobilisomes. All the species show similar quantum yields of ca. 1/1,800,000 photons for primary photoinactivation of PSII. The PSII antenna sizes, estimated using flash fluorometry, vary according to antenna type. More importantly, the maximum PSII repair rates vary at least 15 fold among strains, largely explaining the differences in their ability to exploit high or variable light. In species with slow repair and susceptibility to high light, the limiting step was on clearance of photoinactivated PsbA protein. We discuss these findings in the context of the interacting pressures of nutrient and light regimes on photoacclimation strategies in cells with differing antenna architectures.

PS24.4 Photosynthesis in the smallest free-living eukaryotic organism: light utilisation and capture in the picoeukaryote Ostreococcus P Cardol (IBPC), B Bailleul (IBPC)), E Derelle (Observatoire Océanologique de Banyuls)), F Rappaport (IBPC)), C Breyton (IBPC)), D Béal (IBPC)), H Moreau (Observatoire Océanologique de Banyuls)), G Finazzi (IBPC) CO2 assimilation by marine phytoplankton is a global scale process that maintains a homeostatic control on the planet’s climate by counteracting greenhouse effects. Although they represent a minor component of picoplankton in terms of cell number, picoeukaryotes are known to play a major role in primary productivity in oligotrophic areas, where they represent up to 80% of the autotrophic biomass. In this work, we investigate how the environment modifies the physiology of photosynthesis in picoeukaryotes by comparing two extreme ecotypes of the green alga Ostreococcus (Prasinophyceae order). In O. tauri, a surface/high light strain isolated in a coastal area, photosynthesis essentially reproduces that of land plants, with an equimolar PSI/PSII ratio, a highly efficient electron and proton transfer at high light, and a fast photoprotective response. Conversely, photosynthesis is drastically and constitutively different in the deep/low light oceanic strain RCC809 sp, owed likely to nutrient and light shortage. While antenna sizes of the two photosystems are increased, PSI content decreases by a factor of 3-4. Concomitantly we observe a modification in maximum photosynthesis and in the capacity to maintain a light driven transthylakoid proton gradient (?pH), whose size does not allow the onset of a fast response to fluctuating light intensity, but is still compatible with an efficient ATP cellular turnover

298 14th Photosynthesis Congress - PS07 By comparing the outcome of our study with previous reports in diatoms, we discuss possible strategies which allowed successful colonisation of the deep sea by photosynthetic eukaryotic taxa. PS24.5 Photoprotection capacities differ among plankton diatoms: Possible consequence on their spatial distribution related to fluctuations in the underwater light climate. J Lavaud (University of Konstanz) In order to cope with fluctuating light intensities, diatoms have developed various mechanisms for the regulation of photosynthesis and especially for the photoprotection of their photosynthetic apparatus. Among theses mechanisms, the dissipation of excess energy (or NPQ for ‘non-photochemical chlorophyll fluorescence quenching’) is considered as one of the most important mechanisms on a short time scale (seconds and minutes) during fast fluctuations of the light intensity. We recently showed a fundamental difference between diatom species from different marine habitats in their ability to cope with changes in irradiance (Lavaud, Strzepek and Kroth, Limnology and Oceanography, 52, in press). Estuarine species show a higher and more flexible capacity for photoprotection than oceanic and coastal species, and when exposed to excess light, the impairment of their photosynthetic capacity because of photoinhibition was reduced. This resulted in maintenance of growth in a fluctuating light regime, conferring the estuarine species an adaptive advantage. The ability of diatoms, and to a larger extent other phytoplankton (like the prochlorophytes), to occupy a wide range of ecological niches depends critically on their capacity to exploit the differences in underwater light climate. These results may explain how diatoms adapt to the challenge of maintaining optimal photosynthetic production in turbulent waters where the rate of light changes is high. PS24.6 Photoprotection capacities differ among plankton diatoms: Possible consequence on their spatial distribution related to fluctuations in the underwater light climate. J Lavaud (University of Konstanz) In order to cope with fluctuating light intensities, diatoms have developed various mechanisms for the regulation of photosynthesis and especially for the photoprotection of their photosynthetic apparatus. Among theses mechanisms, the dissipation of excess energy (or NPQ for ‘non-photochemical chlorophyll fluorescence quenching’) is considered as one of the most important mechanisms on a short time scale (seconds and minutes) during fast fluctuations of the light intensity. We recently showed a fundamental difference between diatom species from different marine habitats in their ability to cope with changes in irradiance (Lavaud, Strzepek and Kroth, Limnology and Oceanography, 52, in press). Estuarine species show a higher and more flexible capacity for photoprotection than oceanic and coastal species, and when exposed to excess light, the impairment of their photosynthetic capacity because of photoinhibition was reduced. This resulted in maintenance of growth in a fluctuating light regime, conferring the estuarine species an adaptive advantage. The ability of diatoms, and to a larger extent other phytoplankton (like the prochlorophytes), to occupy a wide range of ecological niches depends critically on their capacity to exploit the differences in underwater light climate. These results may explain how diatoms adapt to the challenge of maintaining optimal photosynthetic production in turbulent waters where the rate of light changes is high. PS24.7 What Limits CO2 Assimilation in Emiliania huxleyi? R Webster (University of Essex), R Geider (University of Essex), C

Raines (University of Essex) The oceans play a major role in the global carbon cycle, with approximately 40% of the Earth's photosynthesis each year occurring in aquatic marine environments. Fossil fuel consumption is leading to higher atmospheric CO2 concentration ([CO2]) and corresponding increases in [CO2] in seawater. This increase in CO2 may change the species composition of the phytoplankton community and the way in which organic carbon is cycled in the sea. Emiliania huxleyi is a key photosynthetic organism in the oceans, exhibiting extensive global blooms. Previous research has indicated that the photosynthesis rate of E. huxleyi is limited by inorganic carbon at current ambient atmospheric CO2 levels. Therefore this organism is likely to be a good indicator of the response of the ocean to rising [CO2]. This project determines the photosynthetic response of E. huxleyi to long-term changes in [CO2] and light, using continuous pH-stat cultures, under high (330 µmol m2 s-1) and low light (30 µmol m2 s-1) and high (750 µmol mol-1) and low (180 µmol mol-1) CO2. Photosynthetic light, and dissolved inorganic carbon, response were measured using a membrane inlet mass spectrometer. While the key photosynthetic protein RubisCO was characterised. The results show that there is a difference between the photosynthetic responses of E. huxleyi in response to light condition, but no long-term response to elevated [CO2] concentrations.

PS24.8 Nutrient limitation and acidification of glacial lake support the development of aerobic anoxygenic photosynthetic bacteria M Masin (Institute of Microbiology, Academy of Sciences of the Czech Republic) The industrial development resulted in acidification of many montaneous lakes. In this study we monitored the seasonal development of bacterial community in acidified freshwater, nutrient (especially phosphorus) limited glacial lake in Sumava Mountains (Czech Republic). Especially, we focused on aerobic anoxygenic photosynthetic bacteria (AAPs). AAPs are relatively newly identified bacterial group, which is studied in marine environments most frequently. However, the knowledge about these bacteria is very rare in fresh waters. We surveyed the Certovo Lake in the period of three years, and our monitoring brought very interesting findings. Infrared microscopy showed that AAPs form majority of bacterial biomass when peaked. Dominant AAPs were filamentous (bacteria with stalks); in stalks, there were concentrated photosynthetic structures. It seems that the nutrient concentration influences the length of these stalks. This massive development of filamentous bacteria was allowed probably thanks to the absence of crustacean zooplankton. This part of food net disappeared during the time of the lake acidification. On the contrary, AAPs were not present in significant amounts in eutrophic water bodies observed as a control objects. It seems that additional energy derived from photosynthesis is giving a crucial advantage to AAPs during the time of nutrient limitation. The finding that AAPs can be the major organisms of microbial loop is completely new. Therefore, these photosynthetic bacteria seem to be very important part of the microbial loop in oligotrophic and acidified environments, while they are negligible in environments with higher nutrient load. PS24.9 Synergistic effects of salinity and temperature in an extremophilic Antarctic alga (Chlamydomonas raudensis UWO 241). T Pocock (Department of Natural Sciences, Mid Sweden University, Sweden), A Vetterli (Department of Natural Sciences, Mid Sweden

299 14th Photosynthesis Congress - PS07 University), N Huner (Dept. of Biology, University of W. Ontario), S Falk (Department of Natural Sciences, Mid Sweden University, Sweden) We have studied the physiological stress responses of the Antarctic, extremophilic green alga Chlamydomonas raudensis UWO 241 (UWO) and its mesophilic counterpart C. raudensis SAG 49.72 (SAG) to changes in growth temperature and salinity. In contrast to SAG, UWO is a true psychrophile, it ceases to grow above 16 °C and dies at 20 °C and is halotolerant. Cells were grown at 20 µmol quanta m-2 s-1 at low and high temperature for each strain; 8 °C and 15 °C for UWO and 15 °C and 24 °C for SAG. The salinity varied from 2.0 µM NaCl up to the critical value of 1.3 M for UWO and 120 mM for SAG, respectively. We show that both strains increase their capacity for salt tolerance during growth at low temperatures. At low temperature growth was faster under high salinities for both strains. High salinity resulted in decreased FV/FM and increased 1-qp at both growth temperatures in UWO wheras this trend was only observed at low temperature in SAG. UWO, unlike SAG, showed no temperature effect on photosynthesis. The chl a/b-ratio was consistently around 2.0 for UWO, while this ratio was as high as 4.0 for SAG. The D1 protein in UWO was reduced by 50% and the PS II/PS I ratio by 60% during growth at high salinity while the reduction was only 10-20% for SAG. These results indicate that UWO is acclimating to high salinity by regulating its absorption cross section through changes in the number of PSU’s.

300 14th Photosynthesis Congress - PS07

PS25 - Photosynthetic Mechanisms under Stress: regulation and improvement PS25.1 Photosynthesis under drought and salt stress - regulation mechanisms from the whole plant to cell M Chaves (Technical University of Lisbon; Instituto de Tecnologia Química e Biológica), J Flexas (Universitat de les Illes Balears), C Pinheiro (Instituto de Tecnologia Química e Biológica) Most climate change scenarios suggest that an increase in aridity will take place in many areas of the globe, placing huge constraints on plant growth and survival. Understanding how plants’ respond to drought and co-occurring stresses is of utmost importance to overcome these constraints and improve plant efficiency. Photosynthesis is one of the key processes to be affected by water deficits and salinity, either directly or via secondary effects, namely oxidative stress. Photosynthetic response to stress is complex because it involves the interplay of limitations taking place at different sites of the leaf/cell and at different time scales. Major advances took place in the knowledge of long- and short-distance signalling, which play a major role in the feedforward and the feedback control of photosynthesis response to drought and salt. It is acknowledged that signalling pathways leading to plants’ stress responses are interconnected at many levels. We will discuss specific and shared signalling responses as well as acclimation features that are species dependent and will help mitigate stress injuries.

PS25.2 Abiotic stresses: potential regulation of gene expression in photosynthetic cells by redox and energy state T Pfannschmidt, K Bräutigam, R Wagner, L Dietzel, Y Schröter, S Steiner, A Nykytenko (Friedrich-Schiller-University of Jena) Photosynthetic electron transport is performed by a chain of redox components that are electrochemically connected in series. Its efficiency depends on the balanced action of the photosystems and on the interaction with the dark reaction.. Plants are sessile and cannot escape from environmental conditions such as fluctuating illumination, limitation of CO2 fixation by low temperatures, low nutrient or water availability, or salinity, which disturb the homeostasis of the photosynthetic process. Photosynthetic organisms, therefore, developed various molecular acclimation mechanisms which maintain or restore photosynthetic efficiency under adverse conditions and counteract abiotic stresses. Recent studies indicate that redox signals from photosynthetic electron transport and reactive oxygen species (ROS) or ROS scavenging molecules such as glutathione, ascorbate or peroxiredoxins, play a central role in the regulation of acclimation and stress responses. We just begin to understand the underlying signalling network, but it is already obvious that gene regulation by redox signals is a major regulation process. Signalling cascades controlling the expression of chloroplast and nuclear genes have been identified and dissection of the different pathways is advancing. Because of the functional overlap with other plastid signals, photosynthetic redox signals can be defined as an additional class of retrograde signals. They represent a vital signal of mature chloroplasts which report their present functional state to the nucleus. By this means, the photosynthetic function of chloroplasts represents an important sensor that integrates various abiotic changes in the environment into corresponding molecular signals which, in turn, regulate cellular activities to counterbalance the environmental changes or stresses.

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PS25.3 High resolution monitoring of diurnal and sesaonal acclimation in the energy partitioning on photosystem II A Porcar-Castell (University of Helsinki), E Juurola (University of Helsinki), J Korhonen (University of Helsinki), P Hari (University of Helsinki), E Nikinmaa (University of Helsinki) The physiological acclimation of the light reactions of photosynthesis involves structural and biochemical adjustments in the thylakoid membrane that affect the energy partitioning in photosystem II (PSII). Acclimation occurs at time scales ranging from seconds and minutes (diurnal scale), to several days (seasonal scale). The energy partitioning in PSII can be monitored through chlorophyll fluorescence analysis combining dark-acclimation and saturating light techniques. Conventionally, estimation of the maximum quantum yield of photochemistry (?Pmax) through Fv/Fm requires the use of dark-acclimation clips that need to be placed directly on the leaf. Furthermore, the diurnal changes in the photochemical yield (?P) and yield of non-photochemical processes (?NP), as a response to diurnal changes in light and other environmental factors, can also be estimated by repeated saturating light pulses at different intervals. The main practical limitation to these techniques are the difficulty to follow the yields of these processes during prolonged periods and at short intervals. In addition, several fluorometers would be needed to simultaneously measure different leaves. We present here the results obtained with a new monitoring-PAM (Heinz Walz GmbH, Germany) intended to perform automatic long-term and high-resolution chlorophyll fluorescence measurements, in several leaves simultaneously. We monitored the diurnal and seasonal acclimation patterns inside a Scots pine canopy during spring recovery of photosynthesis. Light, temperature, Fs, Fm, ?P, and ?NP, were recorded at 5-10 minutes intervals (N=4), 24 hours, and 7 days. Results and potential of high-resolution monitoring of acclimation in PSII under natural conditions, as estimated through chlorophyll fluorescence analysis, are discussed.

PS25.4 Biotic damage to leaves universally down-regulates the transcription of photosynthesis genes E DeLucia (University of Illinois), D Bilgin (University of Illinois), J Zavala (University of Illinois), J Zhu (University of Illinois), S Clough (USDA ARS - University of Illinois), D Ort (USDA ARS - University of Illinois) In addition to producing a myriad of constitutive and induced defenses, a reduction in photosynthesis in remaining tissues may represent an indirect “cost” of herbivory and other forms of biotic damage. Using a comparative genomic approach we tested the hypothesis that biotic assault to leaf tissue causes a universal and balanced down-regulation of genes coding for photosynthetic proteins. Microarray data were obtained from public web sites or contributed by authors. The metadata included the transcription response to foliar damage inflicted by fungi, bacteria and virus, and by two insect feeding guilds, aphids and chewing insects. Fold-change data were subject to hierarchical clustering. With the exception of a putative chloroplast ferredoxin reductance, genes involved in light harvesting and photosynthetic electron transport were

301 14th Photosynthesis Congress - PS07 uniformly down-regulated following damage. In carbon metabolism, many of the genes coding for aldolase, ketolase and isomerase reactions were up-regulated, while those coding for Calvin cycle proteins were down-regulated. The response to physical wounding, powdery mildew, and aphid was mild; viral, bacterial and other fungal infections and chewing damage by caterpillars caused a relatively strong reduction in the expression of photosynthetic genes. The universal nature of the response suggests that the coordinated down regulation of photosynthesis following biotic assault may be adaptive, perhaps by redirecting resources to defense or by reducing the capacity for supra-optimal generation of reactive oxygen species PS25.5 Identification and characterisation of aquaporins in the grapevine, Vitis vinifera M Shelden (The Australian National University), B Kaiser (University of Adelaide), S Tyerman (University of Adelaide) Plant aquaporins belong to a large superfamily of proteins, the Major Intrinsic Proteins (MIPs). In many plant species the expression of aquaporin genes and their regulation has been linked to water stress, and recently it has been hypothesised that aquaporins may play a role in embolism repair. The formation of embolised vessels can lead to a reduction in hydraulic conductivity. This can result in stomatal closure, thus limiting transpiration and photosynthesis. The aim of this project was to identify and characterise aquaporin genes from grapevine, and ultimately assess the role of aquaporins in response to water-stress induced xylem embolism. Candidate genes were identified, by screening a Vitis vinifera cv. Cabernet Sauvignon cDNA library for aquaporin cDNAs encoding members of the Plasma membrane Intrinsic Protein (PIP) and Tonoplast Intrinsic Protein (TIP) subfamilies. The screen resulted in the identification of 11 full length and 2 partial length aquaporin cDNAs. Sequence analyses of these cDNAs reveal 5 are homologous to the PIP2 subfamily, six to PIP1 and two to the TIP subfamily. Functional expression in Xenopus oocytes showed PIP2 members have significantly higher water permeability compared to PIP1 aquaporins. VvPIP2;1 showed very high water permeability which was reduced by acidic cytosolic pH, as has been reported for other members of the PIP2 family. Transcript analysis (QPCR) of some of these AQP genes in petiole tissue, provides preliminary evidence that AQP expression may change in response to water stress in the water conducting pathway of grapevines. PS25.6 Investigation of the non-photochemical processes in photosynthetic bacteria and higher plants using interference of coherent radiation - a new approach K Rohacek (Biology Centre of AS CR, Institute of Plant Molecular Biology), M Kloz (Faculty of Biology, University of South Bohemia), D Bina (Biology Centre of AS CR, Institute of Plant Molecular Biology), F Batysta (Grammar School of J.V. Jirsik), F Vacha (Biology Centre of AS CR, Institute of Plant Molecular Biology) Photochemical processes (the charge separation in reaction centres followed by electron transport) and non-photochemical processes (heat dissipation and fluorescence) are the main mechanisms participating in a photosynthetic light energy conversion. They are accompanied with a pH-gradient formation, O2 production, CO2 fixation, heat propagation and many other processes, which result in dynamic changes in volume of chloroplasts or whole plant tissue. To register these changes, the photoacoustic techniques have been developed in 80's of the last century, which involve measurements of thermal volume expansion during photosynthesis in closed cells. In our contribution, we describe a new concept, in which the volume changes in chloroplasts,

photosynthetic bacteria, and whole plants are measured using interference of two coherent light beams produced by a HeNe laser (632.8 nm). The main advantage of this new interferometric method is the possibility to quantify the non-photochemical processes in photosynthetic samples measured in vivo using the parallel recording of interferograms and chlorophyll a fluorescence kinetics. In this way, the heat production in bacteria (Rhodobacter sphaeroides) treated with uncouplers (FCCP, gramicidine), as well as the O2 evolution, CO2 uptake and surface dilatations in leaves of higher plants (bean, hibiscus) stressed by high-light were studied. We found that the heat production was enhanced by approx. 30-40 % in stressed samples. Using interferometry, we have also observed pronounced dilatations in a leaf tissue of studied plants during photosynthesis. PS25.7 The photosynthetic response of a marine diatom to iron limitation M Lommer (IFM-GEOMAR, 24105 Kiel, Germany), S Proebst (IFM-GEOMAR, 24105 Kiel, Germany), A Allen (UMR8186, Dept of Biology, Ecole Normale Supérieure, 75230 Paris, France), U Maheswari (UMR8186, Dept of Biology, Ecole Normale Supérieure, 75230 Paris, France ), C Bowler (UMR8186, Dept of Biology, Ecole Normale Supérieure, 75230 Paris, France ), J LaRoche (IFM-GEOMAR, 24105 Kiel, Germany ) Much of contemporary ocean is Fe limited. Pennate diatoms are generally the most responsive to iron fertilization and seem best adapted to feast-and-famine regimes. Taking advantage of its recently completed genome sequence, we have combined transcriptomic and physiological approaches to explore the response of the photosynthetic pathway in the pennate diatom Phaeodactylum tricornutum grown under Fe limited vs Fe replete conditions. We find that photosynthesis as a Fe demanding process is strongly down regulated to cope with the reduced cellular iron quota. Electron micrographs confirm that the general reduction in cell size observed in Fe deficient conditions is accompanied by an almost complete reduction of the plastid thylakoid membrane system down to a core structure of just the pyrenoid surrounded by few membrane layers. Accordingly, CO2 fixation experiments using 14C-bicarbonate show a decrease of photosynthetic performance down to 1 % of the Fe replete level. Severe down-regulation of the genes encoding for plastid β-carbonic anhydrase (CA) and phosphoribulokinase (PRK), two enzymes supplying substrate for RuBisCO, and a decrease in expression of a HCO3- transporter provide a mechanistic explanation for the diminished flow of carbon through the Calvin cycle. Although chlorophyll levels decrease under Fe limitation, transcription profiles indicate a strong upregulation of genes involved in pigment biosynthesis pathways, likely indicating an enhanced de novo pigment biosynthesis required to produce specialized photoprotective pigment complexes. Overall, our results show that both the light and dark reactions of photosynthesis adjust to Fe-limited conditions. PS25.8 Manganese is a photoreceptor of photoinhibition E Tyystjärvi (University of Turku), M Hakala (University of Turku), P Sarvikas (University of Turku), T Tyystjärvi (University of Turku), M Keränen (University of Turku), E Pätsikkä (University of Turku), S Rantamäki (University of Turku), L Khriachtchev (University of Helsinki), J Guiamet (National University of La Plata) Photoinhibition of PSII can at least partly be explained by a mechanism triggered by the absorption of light by the manganese ions of the oxygen-evolving complex (OEC). After inhibition of OEC, the irreversible inhibition of PSII is completed by a chlorophyll-mediated

302 14th Photosynthesis Congress - PS07 reaction. The function of manganese as a photoreceptor of photoinhibition is supported by the finding that the action spectrum of photoinhibition resembles the absorption spectra of synthetic model complexes of OEC. We also show that OEC is not the only photosensitive manganese enzyme, but also manganese catalase, an enzyme containing an oxo-bridged dimanganese cluster, is sensitive to visible and ultraviolet light. Photoinhibition experiments in which we used short laser pulses as photoinhibitory illumination revealed that electron transfer via the primary donor P680 is insignificant for laser-induced photoinhibition, which also supports the role of Mn as a photoreceptor of photoinhibition. Laser pulse experiments suggest that the S1 state of OEC is the photosensitive state. Further evidence for the function of a two-photon reaction mechanism was obtained by studying mutant plants with impaired non-photochemical quenching of chlorophyll excitations. These experiments revealed that non-photochemical quenching protects PSII less than it would protect if chlorophyll was the only photoreceptor of photoinhibition under visible light. PS25.9 Reactive oxygen species accumulation in rice (Oryza sativa L.) mutant lacking PsbS protein of photosystem II differently regulates stress response genes I Zulfugarov (Department of Molecular Biology, Pusan National University), O Ham (Department of Molecular Biology, Pusan National University), S Mishra (Department of Molecular Biology, Pusan National University), P Gopalakrishnan Nair (Department of Molecular Biology, Pusan National University) Higher plants develop a variety of photoprotective mechanisms against photoinhibition or the light-dependent loss of photosynthetic efficiency. One of the mechanisms against photoinhibition is non-photochemical quenching, especially energy-dependent quenching (qE) of chlorophyll fluorescence. This component depends on three major parameters: the development of transthylakoid proton gradient (?pH), the amount of pigments involved in xanthophyll cycle, and the existence of a PsbS subunit in PSII. The OsPsbS-knockout mutant rice (Oryza sativa L.) plants completely lack qE. It has been shown that in the absence of qE plants possess extensive accumulation of the reactive oxygen species. We observed that in OsPsbS-knockout mutant rice plants the expression of the stress response genes are regulated differently. Our data shows that the expression pattern of the stress and defense response genes are regulated differently by different type of reactive oxygen species. The expression pattern of those genes related to antioxidant system with respect to the site of reactive oxygen species generation will be discussed. PS25.10 Photosynthetic Response to Environmental Stress in the Cyanobacterium Prochlorococcus: From Genome to Three-Dimensional Cellular Architecture C Ting (Williams College), C Hsieh (Wadsworth Center), S Sundararaman (Williams College), C Mannella (Wadsworth Center), M Marko (Wadsworth Center), E Haydu (Williams College), J McKenna (Williams College) Prochlorococcus is an abundant marine cyanobacterium that contributes nearly half of the net primary production in certain regions of the open ocean. With the availability of 11 complete Prochlorococcus genome sequences, we are addressing how differences at the genomic level translate into selective physiological advantages in the ability of strains to acclimate to environmental stress. The MED4 strain possesses one of the smallest genomes of any extant photosynthetic organism and belongs to a Prochlorococcus clade comprised of recently derived lineages. Our

comparisons of large-scale genome structure and composition, as well as specific photosynthesis and stress-related operons, indicate that major differences have evolved in this strain and its closest relatives, compared to strains belonging to more deeply branched clades. Through cryo-electron tomography, we have visualized the near-native structure of Prochlorococcus and have established that MED4 also possesses significant dissimilarities in its cellular architecture, notably in its cell wall and photosynthetic membranes1. These differences at the genetic and structural levels are expected to impact photosynthesis and stress response mechanisms. Data from our recent work on Prochlorococcus characterizing stress-induced alterations in photosynthetic capacity, cellular architecture, physiology, and protein expression support this and suggest that the MED4 strain is particularly sensitive to environmental stresses, such as temperature. The significance of our results will be discussed within the context of niche differentiation of Prochlorococcus ecotypes in the oceans. 1. Ting C.S. et al. (2007) Cryo-electron tomography reveals the comparative three-dimensional architecture of Prochlorococcus, a globally important marine cyanobacterium. Journal of Bacteriology (In Press) PS25.11 Physiological and photosynthetic toxicity of thallium in Synechocystis sp. PCC6803 M Aoki (Tokyo University of Pharmacy and Life Sciences), H Suematsu (Tokyo University of Pharmacy and Life Sciences), T Takahashi (Tokyo University of Pharmacy and Life Sciences), H Kumata (Tokyo University of Pharmacy and Life Sciences), K Fujiwara (Tokyo University of Pharmacy and Life Sciences) The physiological and photosynthetic toxicity mechanism of Tl(I) in a blue-green microalga, Synechocystis sp. PCC6803, was examined based on a series of batch culture experiments, determination of the pigments content and measurements of photosynthetic activities under the metal exposure conditions. Results showed that micro-moler level of Tl(I) drastically inhibit its growth, then 50% inhibitory concentration (IC50) was approx. 1.0 μM. An acclimating incubation with 0.5 μM Tl(I) for 72 hour bring no significant changes in IC50 of thallium for growth. Chlorophyll and phycobiliprotein content per cell density decreased by 70% and 85% during 72 hour incubation with 2.5 µM Tl(I), respectively. Results from pigments determination suggested that metabolic defect was rose by thallium exposure in Synechocystis sp. PCC6803. Then, to investigate the effect of thallium on energy generation process, acute dose-response of Tl(I) on photosynthetic O2 evolution activities were measured. No effect on net photosynthetic O2 evolution activity per chlorophyll basis was observed in 1.0 mM and below Tl(I) exposure, while 20 mM Tl(I) decrease the activity by 60%. Furthermore, 20 mM thallium did not affect 1,4 benzoquinone dependent PSII activity. These photosynthetic inhibition doses of thallium were approx. 2,000-fold higher than IC50 of growth. Thus, photosynthetic energy metabolism did not constitute a limiting factor of growth under the thallium exposure. These results confirmed that substance metabolic defect was the main process involved in thallium toxicity in Synechocystis sp. PCC6803. PS25.12 Cobalt effect on the bacteriochlorophyll biosynthesis pathway and magnesium metabolism in Rhodobacter sphaeroides strain R26.1 L Giotta (Dipartimento di Scienza dei Materiali, Università del Salento), F Italiano (Istituto per i Processi Chimico Fisici (CNR) - Sezione di Bari), F Pisani (Dipartimento di Biochimica e Biologia Molecolare Università degli studi di Bari), L Luigi R. Ceci (Istituto di Biomembrane e Bioenergetica (CNR) – Sezione di Bari), F De Leo (Istituto di

303 14th Photosynthesis Congress - PS07 Biomembrane e Bioenergetica (CNR) – Sezione di Bari), R Gallerani (Dipartimento di Biochimica e Biologia Molecolare - Università degli studi di Bar Rhodobacter sphaeroides is a purple, non-sulfur, facultative anaerobe, capable of anoxygenic photosynthesis, and is amongst the most metabolically diverse organisms known. In presence of high cobaltous ion concentration, the bacterium is still able to grow, although with a strong reduction in the photosynthetic apparatus biosynthesis. The decrease in Mg content found in cells treated with increasing cobalt concentrations suggests that the lower bacteriochlorophyll level is linked to the decreased magnesium availability. Functional experiments on enzymes involved in the tetrapyrrole biosynthetic pathway showed a tenfold decrease in the porphobilinogen deaminase specific enzymatic activity of water-soluble proteins extracted from cobalt exposed cells. This inhibition appears to be the result of two contributions: a) the direct effect of Co2+ on the enzyme, broadly described in literature, and b) a transcription or translation effect, strongly suggested by proteomic analysis. In order to study the influence of magnesium concentration on cobalt toxicity, growth experiments were performed with magnesium concentration increased to values higher than the physiological ones (up to 100 mM), for different cobalt concentrations. At higher cobalt concentrations the increase of magnesium concentration results in a significant increase in the growth rate and in cellular yield, reversing cobalt toxic effect. These results demonstrate the existence of an interrelation in the metabolism of magnesium and cobalt. It is likely that also a common transport system exists for magnesium and cobalt in R. sphaeroides, even though ultimate confirmation of this will require identification and mutagenesis of the genes involved. PS25.13 Oxygen evolution and chlorophyll fluorescence under extreme dehydration in the aquatic bryophyte Fontinalis antipyretica R da Cruz (Universidade de Lisboa), C Branquinho (Universidade de Lisboa), J Marques da Silva (Universidade de Lisboa) This work aims for a deeper insight into the physiological responses to extreme dehydration of the aquatic bryophyte Fontinalis antipyretica. Although studies have been done in terrestrial bryophytes, the underlying physiological mechanisms in aquatic ones have been overlooked. In intermittent Mediterranean streams, aquatic mosses can be submitted to total dryness, sometimes for 3 months, being exposed to high temperatures and low relative humidity (RH). Therefore, Fontinalis antipyretica specimens were subjected to different levels of RH (23%, 50% and 95%), for different periods of time (30 minutes up to 40 days). Simultaneous oxygen evolution and chlorophyll fluorescence measurements were made, before desiccation and during recovery through immersion in water. Loss of ions during rehydration was evaluated through conductivity measurements. After 30 minutes at 23% RH, oxygen production evidenced a great reduction together with a decrease in Fv/Fm, both indicative of the impact on the photosynthetic process. On the other hand specimens that were at 95% RH only showed a decrease on those parameters after 24 hours. Upon immersion in water, a burst of oxygen consumption was measured in the first 5-6 minutes especially on short term desiccation (30 minutes to 24 hours) at 23% and 50% RH. This was not a product of mitochondrial respiration, since it was insensible to mitochondrial respiratory inhibitors. It was also observed that samples exposed to lower RH presented an increase in oxygen production in the absence of light. Samples exposed to 23% and 50% RH after being immersed in water showed substantial loss of ions. PS25.14 Proteomic analysis of spring barley leaves and chloroplasts

treated with high temperature and irradiance M Datko (Slovak agricultural university Nitra), M Brestic (Slovak agricultural university Nitra), K Olsovska (Slovak agricultural university Nitra) Plant adaptations to environmental conditions such as high temperature and irradiance evokes protection responses. In our experiment the leaf and chloroplast proteins were analysed from spring barley plants by second-dimensional electrophoresis after their treatment with high temperature (40ºC) and high irradiance (above 1000 µmol.m-2.s-1). The 18 cm IPG strips at pH 4 – 7 were used to drive the first dimension electrophoresis. After that the second dimension was applied in 10% SDS polyacrylamid gel followed by the silver staining. After the quantitative and qualitative image analysis of samples the particular spots were selected and picked up from the gels and submited to MS analysis and N-terminal degradation. We found 16 spots in high temperature stressed plants and 4 spots in high irradiance treated plants which differed from control plants. We also found 69 quantitative differences in temperature treated plants compared to control plants indicating increased expresion of some proteins related to high temperature conditions. The presence of 14-3-3 protein was confirmed for the first time in the high temperature treated plants by second-dimensional electrophoresis. This protein may probably be envolved in regulation of heat shock transcription factors. PS25.15 Effect of Cobalt ions on the soluble proteome of Rhodobacter sphaeroides carotenoidless mutant. F Italiano (Dipartimento di Chimica - Università di Bari), F De Leo (Istituto di Biomembrane e Bioenergetica (CNR) – Sezione di Bari), F Pisani (Dipartimento di Biochimica e Biologia Molecolare - Università degli studi di Bari), L Ceci (Istituto di Biomembrane e Bioenergetica (CNR) – Sezione di Bari), R Gallerani (Dipartimento di Biochimica e Biologia Molecolare - Università degli studi di Bari), L Zolla (Università della Tuscia - Viterbo), S Rinalducci (Università della Tuscia - Viterbo), L Gio Rhodobacter sphaeroides is a well characterised photosynthetic facultative bacterium whose genome has been completely sequenced and is available to the scientific community(1). Interest in the characterization of its proteome is due to the observed capacity to grow in heavy metals polluted environments(2) and hence in its potential use in bio-remediation strategies. In particular, the strain R26.1 shows tolerance to Co2+ ions, up to 10 mM concentration. Interestingly tolerance to Co2+ is accompanied with a noteworthy detrimental effect on the photosynthetic apparatus of the microrganisms, and in details on the biosynthesis of photosynthetic pigments. Analysis of R. sphaeroides proteome from cells grown in control or Co2+ enriched media, was investigated by two dimensional electrophoresis. We have set up a 2DE PAGE protocol allowing to separate approximately 800 water-soluble proteins. 2DE map analysis identified 100 statistically different spots involved in cobalt response. Functional annotation could be made for 21 of them, suggesting the involvement of specific metabolic pathways in the adaptation of R. sphaeroides to high concentration of cobalt ions in growing media. (1) http://www.rhodobacter.org/ (2) Giotta L., et al. Chemosphere 62 (2006) 1490-1499.

PS25.16 Influence of elevated CO2 in plants exposed to short-term photoinhibitory light

304 14th Photosynthesis Congress - PS07 S Boisvert (Université du Québec à Trois-Rivières), S Govindachary (Université du Québec à Trois-Rivières), R Carpentier (Université du Québec à Trois-Rivières) Atmospheric CO2 is continued to increase from 370 µmol mol-1 and may attain about 500-900 µmol mol-1 by the end of this century. In plants grown with enriched CO2, photosynthetic rates are usually increased until they acclimatize to elevated CO2. Subsequently, the level of Rubisco declines. The response of field crops and other plants to abiotic stress like high or low light individually or in combination with low-to-high temperatures have been studied previously. However, the influence of elevated CO2 on interactions between plants and dynamic changes in the light environment remains to be clarified. In the present study, the effects of elevated CO2 in C3-plants exposed to high light intensity were examined. The quantum yield of photosystem II declined by ~35% in cucumber plant after exposure to high light under normal atmospheric CO2 concentration as deduced from chlorophyll fluorescence measurements. If CO2 surrounding the leaves was enhanced to ~ 710 µmol mol-1, around 45% of the total inhibition of the PSII electron transport found with normal CO2 level was restored. A similar response to CO2 enrichment was found in radish plant. Both plants markedly differed in their carboxylation efficiency and light saturated photosynthesis as detected from gas-exchange measurements. The magnitude of the fluorescence yield recovered in the dark within 30-60 s following exposure of plants to high light that correspond to the relaxation of the fast component of the non-photochemical quenching was not altered by elevated CO2.

PS25.17 Functional study of PSII and PSI energy use and dissipation mechanisms in chlorina mutants of barley in conditions of high light treatment M Brestic (Slovak Agricultural University Nitra), M Zivcak (Slovak Agricultural University Nitra), K Olsovska (Slovak Agricultural University Nitra), J Repkova (Slovak Agricultural University Nitra), M Datko (Slovak Agricultural University Nitra) Photosynthetic organisms dispose of mechanisms of their acclimation to high irradiance. The knowledge of how plants respond to high light and how they involve complementary regulation mechanisms help us to understand the limits in radiation use by plants. Changes of light intensity modulate plant photochemical and non-photochemical processes and their components. Although the net photosynthesis (ACO2) of barley plants from optimal conditions was similar in wild type and chlorophyll b-deficient chlorina f2 and 104 mutants, when exposed to high irradiance (1500 µmol.m-2.s-1) a considerable decrease of maximal photochemical PSII efficiency was measured in chlorina f2 as compared to chlorina 104 and wild type indicating a strong predisposition to photoinhibition. In the course of photosynthetic light curves the ETR of both mutants was higher in comparison to wild type, however its carboxylation efficiency (ACO2/ETR) was low. The ACO2/ETR in chlorina f2 was saturated at very low irradiances and its insufficient non-photochemical energy dissipation via NPQ reached the saturation at 400 µmol.m-2.s-1 of PPFD. The ETR in chlorina f2 was very high (160 µmol.m-2.s-1), however its cyclic electron transport was strenghtened. The increase of YI/YII in chlorina f2 was linear even above the saturated irradiance, while in wild type and chlorina 104 was steady-state. During the photoinhibition both mutants expressed a significant increase of non-photochemical energy quenching at PSI, however an increased sensitiveness of chlorina f2 may origin in a low transient quenching (qT). We stress that the absence of any regulation mechanism under stress may have a detrimental effect to photosynthesis.

PS25.18 Influence of salinity on the pigmentation, protein content and oxygen evolving activity in unicellular green alga Dunaliella salina J Aliyev (Botany), D Aliyeva (Botany), A Mammadov (Botany), I Azizov (Botany) Salt stress causes a decrease in the photosynthetic activity in higher plant and alga cells. The effect of different NaCl concentration on the O2 exchange and protein content in the green alga Dunaliella salina was investigated. It was established an optimum concentration of NaCl, at which an intensive biosynthesis of green pigments and functioning of photosynthetic apparatus were observed. Growth, expressed as number of cells ml-1, which was most rapid at 2M NaCl, was reduced at salinity extremes. Lower salt concentrations treatment of the cells had a lower effect on O2 produ?ing reactions, whereas high salinity (4M) markedly reduced oxygen evolution. We found that photosynthetic oxygen evolution activity were stimulated by 150% at 2M NaCl in comparision with the control (0.5M NaCl). The chlorophyll to carotenoid (Chl/Car) ratio was 11.6 : 1 under low salt and 0.8 : 1 under high salt concentrations. The divergent Chl/Car ratios were reflected in the coloration of the cells, which were green under low salt and yellow under high salt concentrations. High salt increases biosynthesis and turnover of proteins in Dunaliella salina. 9 novel polypeptides involved in the protective reactions and responsible for the normal cell metabolism were investigated. The intensity of some polypeptides of soluble proteins (mol. masses 150, 89, 77.5, 63, 46, 34.5, 28, 24 and 18) increased with increasing salt concentrations, whereas intensities of other polypeptide bands decreased. Cells were fractionated into nucleus, chloroplast, mitoxondrion and cytoplasm fractions. More abundant polypeptides were observed in chloroplast and cytoplasm fractions. 63, 46 and 28 kDa proteins were detected in all lines, but in different amounts. It is supposed that Dunaliella salina cells response to salinity of the medium by reconstruction of their photosynthetic metabolism. PS25.19 Photosyhthetic responses of Japanese lily to light conditions on the understory of a temperate deciduous forest M Kono (Kanagawa University) Light conditions on the understory of a temperate deciduous forest in Hiratuka Japan were determined and photosynthetic responses of Japanese lily, Erythronium japonicum Decne, were estimated. Photon flux densities (PFD) were determined every 1 second and their fluctuations were estimated by a Fourier analysis. From April to December, when canopy of the forest were covered by tree leaves, less than 1% of the PFD of the full sunlight at above the canopy were determined on the understory and the PFD fluctuated drastically within the short periods of time less than 5 seconds. From January to April, when most leaves in the canopy were fallen, ca. 40% of PFD were determined on the understory and fluctuated with cycles of 1-20 minutes. During 4 months in a year, strong PFD without drastic fluctuations were maintained. As E. japonicum living on the understory budded on middle March and fell their leaves on the early May, they were exposed only to the latter conditions. These two different light conditions were simulated by the light-emitting diode regulated by PC programs and E. japonicum were exposed. Its photosynthetic responses estimated by CO2 assimilation rates and chlorophyll fluorescence showed inefficient photosynthesis under low light with drastically fluctuations and E. japonicum only could respond to high light conditions in early spring on the understory of a temperate deciduous forest.

305 14th Photosynthesis Congress - PS07 PS25.20 Effect of action potential on photosynthesis and proton transport in the plant cell N Krupenina (Moscow State University), A Bulychev (Moscow State University) Generation of electrically-induced action potential (AP) in illuminated cells of Chara corallina was found to transiently suppress photosynthesis, disrupt circulating H+ fluxes across the plasmalemma, and give rise to non-photochemical quenching (NPQ) of chlorophyll fluorescence. The effect of AP on NPQ, most evident at high Fm’, was manifested as a strong long-lived drop of maximum fluorescence (Fm’). The sensitivity of NPQ to nigericin and a rapid release of AP-triggered NPQ in darkness indicate its relation to the thylakoid ∆pH. Simultaneous measurements of NPQ and external pH on small cell regions provide evidence that the thylakoid ∆pH in Chara is subject to spatial pattern coordinated with the external pH. The effect of AP on Fm’ disappeared if AP was triggered in darkness (even after dark periods ~10 s), indicating its dependence on photosynthetic electron transport. When electron transport (ET) was blocked with DCMU, AP had no effect on Fm’, despite high Fm’ level. When artificial cyclic ET around photosystem I (PSI) was supported by phenazine methosulfate in the presence of DCMU, the light-dependent NPQ was restored without restoration of AP-induced quenching. In the presence of methyl viologen, known to avert ET from ferredoxin, we observed strong irreversible Fm’ quenching after the first AP and no effect of AP on subsequent stimulations. The results suggest that the effect of AP depends on native linear electron flow, including ET on the acceptor side of PSI, while high Fm’ level, the capacity of generating ΔpH, and artificial ET are insufficient conditions. PS25.21 Water stress induces thermotolerance in bean plants photosynthetic apparatus J González-Cruz (Facultad de Ciencias Agronómicas. Universidad de Chile), C Pastenes (Facultad de Ciencias Agronómicas. Universidad de Chile) During their life time, higher plants are constantly exposed to environmental stresses against which they must develop protective mechanisms. It has been commonly observed that exposure to a particular stressing factor would induced protection against a different one, phenomenon known as crossed tolerance. Such is the case for water stress induced termotolerance of the photosynthetic apparatus in higher plants. We have found that the temperature threshold for non reversible damage in bean plants would be significantly increased when grown under drought conditions as observed by the temperature dependent minimal fluorescence (Fo) kinetics. Also, oxygen evolution under high CO2 air is negatively affected in water stressed plants up to a lesser extent compared to well watered plants. The basis for the observed termotolerance in bean plants grown under water stress conditions are discussed comparing their pigment content, PSII antenna size, xanthopyll content, lipids and fatty acids composition as well as leaf morphology compared to well watered plants. PS25.22 Adaptive variation of photosynthetic parameters in two subpopulations of Iris pumila L. on Deliblato Sands (Serbia)

from the same genotype. The initial plant (sun-exposed) has been spread by the rhyzomatheous growth in the direction of hawthorn shade where other plant established (semi-shaded). The separation of rhyzomes occurred 3-4 years ago. One group of plants (HI) grown in full sun exposure (PPFD at leaf level was in average 1018 µmolm-2s-1, mean leaf T was 28.1 oC) and second group of plants (LI) grown in semi-shade under hawthorn (PPFD at leaf level was in average 509 µmolm-2s-1, mean leaf T was 21.5 oC). All plants were exposed to middle drought (mean WD 25.8 %). Significant differences between HI and LI subpopulations were observed in growth characteristics: total biomass Tb, shoot:root ratio S/R and specific leaf area SLA were higher in LI plants, whereas leaf weight ratio LWR was higher in HI plants. At leaf level, significant differences were observed in net photosynthetic rate PN, efficiency of photosystem II Fv/Fm, transpiration rate E, stomatal conductance gS and total chlorophyll content ChT. In HI plants, PN peak appeared at about 10:00, and midday depression at 12:00. PN depression corresponded with diurnal stomatal conductance. Fv/Fm decreases were registered around 11:00, indicating the occurrence of photoinhibition. In LI plants, PN reached peak at about 11:45, whereas PN depression occurred at 13:00. Midday PN depression was mostly attributed to non-stomatal limitation. Midday PN depression was found to be associated with reversible inactivation of photosystem II. PS25.23 Photosynthetic and growth alternations in Iris pseudacorus L. along the soil moisture gradient Z Popovic (Insitute for Biological Research, Bulevar despota Stefana 142, 11060 Belgrade, Serbia), M Mijatovic (Insitute for Biological Research, Bulevar despota Stefana 142, 11060 Belgrade, Serbia) Ecophysiological responses of three clones of yellow iris exposed to different soil water regimes were compared. The first clone (CF) was exposed to continuous flooding in water depth of 70 cm, the second clone (PF) was exposed to shallow flooding (10 cm) and became periodically flooded when water level decreased (mid June). The third clone (NF) grown 2 m distant from the water and haven’t been exposed to flooding. In response to moisture decrease, plants reduced their expansion (height) and initiation of new leaves (number of leaves per plant). Thus, the highest biomass production, shoot: root ratio and number of leaves per plant were found in CF plants. Biomass production was slightly reduced, but plant height was significantly decreased in PF plants. The biomass production was mostly reduced in NF plants, in which the decrease in soil moisture was found to reduce both the leaf area and the leaf biomass per plant. Net photosynthesis PN, stomatal conductance gS, and transpiration E were significantly lower in NF plants. Photosynthesis was rather unaffected in PF plants until the decrease of water level led to the reduction of PN due to stomatal closure. Plant growth and photosynthesis were enhanced by continuous flooding and also favored by periodic flooding. In contrast, these parameters exhibited reduction in absence of flooding. The combined photosynthetic and growth responses of yellow iris are likely to contribute to the competitiveness of this species in flooded areas. However, the ability to alter these responses indicates that it could maintain under various soil moistures and colonize new habitats.

Z Popovic (Insitute for Biological Research, Bulevar despota Stefana 142, 11060 Belgrade, Serbia)

PS25.24 Gas Exchange Response of Sugar Beet (Beta vulgaris L.) Cultivars Grown under Salt Stress

Adaptive variations of photosynthetic parameters within the population of I. pumila were compared. Two analyzed groups of ramets originated

A Dadkhah (Ferdowsi University of Mashad), S Moghtader (Ferdowsi University of Mashad)

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Abstract This study investigated intra-specific variation in the growth parameters and photosynthetic rates of individual attached leaves of sugar beet (Beta vulgaris L.) plants during salinisation. Sugar beet plants grown in sand culture were gradually exposed to different levels of salinity (0, 50, 150 , 250 and 350 mM, NaCl + CaCl2 in 5:1 ratio). Although growth parameters such as leaf area and dry matter accumulation were stimulated or unaffected at low level of salinity (50 mM), higher salt concentrations significantly decreased all growth traits. At high level of salinity, cv P29 showed a significantly higher leaf area and total dry matter than others at 8 weeks of salt treatments. Net photosynthesis (ACO2) was plotted against computed leaf internal CO2 concentration (Ci), and the initial slope of this ACO2-Ci curve was used as a measure of photosynthetic ability. Leaves from plants exposed to 50 mM salinity showed little change in photosynthesis, whereas those treated to high level of salinity had up to 91.5 % inhibition, with increae in CO2 compensation point. Leaves appeared healthy and leaf chlorophyll content increased with increasing salinity. Although partial stomatal closure occurred with salinisation, reductions in photosynthesis were partly non-stomatal at high levels of salt treatment. Photosynthetic ability was inversly related to the concentration of either Na+ and Cl- in the leaf laminas sampled at the end of experimental period. Key Words: Gas Exchange, Photosynthesis, Growth, Sugar Beet, Salt Stress PS25.25 Photosystem II in the obliged halophyte Salicornia veneta C Pagliano (Università del Piemonte Orientale Amedeo Avogadro, Dipartimento di Scienze dell'Ambiente e della Vita), N La Rocca (Università di Padova, Dipartimento di Biologia), F Andreucci (Università del Piemonte Orientale Amedeo Avogadro, Dipartimento di Scienze dell'Ambiente e della Vita), G Forti (Università di Milano, Dipartimento di Biologia), N Rascio (Università di Padova, Dipartimento di Biologia) Halophytes are a heterogeneous group of plant species that can thrive on high salt concentrations, typical of extreme environments, such as salt marshes, salty inlands, coastal saline pans and swamps. Even though their physiology has been extensively investigated, few attention has been paid to their photosynthetic features. Searching for environmental conditions that could affect PSII, we studied the obliged halophyte Salicornia veneta, an annual species growing on salt marshes along the North Italic Adriatic Sea, where salinity may be extremely high and light intensity during the vegetative season can reach 1000-1500 µmol photons m-2 s-1. It was found that: 1) it contains a modified PSII, from which the PsbP and PsbQ proteins may be absent, indicating that under certain environmental conditions, their presence is not strictly necessary 2) in its natural environment, when light intensity is high some hundreds mmoles m-2 s-1, this plant is likely to spend most of the time in a highly quenched state, which could prevent from photoinhibition 3) in this plant the expression of psbP and psbQ genes is strongly down-regulated. In particular, when placed in saline solutions with decreasing ionic strength Salicornia veneta showed a modified expression of PsbP (not PsbQ). Anyway, even in plants exposed to distilled water the final level of this polypeptide was much lower than that observed in plants used as control, indicating that, eventually, only a fraction of centres contain a PsbP protein. PS25.26 Does Photorespiration increase under drought conditions? Insight from a study with C4 grasses.

E Carmo-Silva (Centro de Engenharia Biológica and Departamento de Biologia Vegetal, Faculdade de Ciências da Universidade de Lisboa; and Crop Performance and Improvement Division, Rothamsted Research), S Powers (Biomathematics and Bioinformatics Division, Rothamsted Research), A Keys (Crop Performance and Improvement Division, Rothamsted Research) The evolution of C4 photosynthesis resulted in increased CO2 concentrations at the Rubisco catalytic sites, decreasing the oxygenase activity of the enzyme and consequently the photorespiratory rate. Under drought conditions the intercellular CO2 concentration may decrease and photorespiration would then increase. With the global warming of the planet an understanding of plant responses to drought will become of increasing importance. C4 plants are better adapted to higher temperatures and drought conditions than C3 and may be more competitive in the changing environment. The C4 grasses Paspalum dilatatum Poiret (NADP-ME), Cynodon dactylon (L.) Pers (NAD-ME) and Zoysia japonica Steudel (PEPCK) were compared in their response to drought, imposed by water withdrawal. Despite the slight decrease of net CO2 assimilation observed at O2 concentrations greater than 10%, O2-response curves showed little evidence for photorespiratory gas-exchanges in these C4 grasses. In contrast, analysis of the amino acids content in leaves collected in the light and after a period of 30 seconds in darkness, provided evidence, particularly in the decrease of glycine content in the dark, that photorespiration was present and at different rates in the three species. Whilst the values of net CO2 assimilation were significantly decreased in dehydrated leaves of P. dilatatum, C. dactylon and Z. japonica compared to the control leaves of each species, the effect of dehydration on photorespiration was less clear. Results will be discussed in terms of the effects of drought stress on photosynthesis and photorespiration in C4 grasses with different decarboxylating mechanisms. PS25.27 LC/MS and Photoacoustic Analysis of Type-II Reaction Center Photoinhibition H Hou (UMass Dartmouth) The primary target of photodamage during the photoinhibition process in higher plants is located in the photosystem II. Using HPLC we observed the photodamage of pheophytin in the purified photosystem II reaction center complexes from spinach. Due to the significant similarities between the PSII and bacterial reaction centers in both structure and function, we investigate the photosensitivity of the bacterial photosynthetic reaction center. Our experimental data demonstrated: (1) The special pair P undergoes a two-step photodamage reaction, in which the first-step of photodamage is reversible; (2) One bacteriopheophytin molecule associated with photoactivity of the isolated RC from Rb. sphaeroides is damaged under strong light illumination; (3) The damaged bacteriopheophytin is located in the L subunit of the RC. Then we use LC/MS and pulsed, time-resolved spectroscopy to investigate the effects of strong light on the wild and mutant reaction centers PSII in vivo. The two mutants were pheophytin-D1 mutant, in which the pheophytin molecule in D1 protein were replaced by a chlorophyll, and pheophytin-D2 mutant, in which the pheophytin in D2 subunit was missing. LC/MS were also used to identify the possible products of photodamage process. In conclusion, our experimental data supports the idea that one of the two pheophytin or bacteriopheophytin molecules in photosynthetic centers is more susceptible to excess light. PS25.28 Physiological impacts of eastern dwarf mistletoe infection and developmental responses of host white spruce B Logan (Bowdoin College), J Reblin (Bowdoin College), R Butschek (Bowdoin College), C Hricko (Bowdoin College), A Hall (Bowdoin

307 14th Photosynthesis Congress - PS07 College), R Dunlavey (Bowdoin College), K Duran (Mesa State University), N Phillips (Boston University), D Tissue (University of Western Sydney) White spruce is acutely vulnerable to infection by eastern dwarf mistletoe, a native xylem- and phloem-tapping parasitic plant. White spruce stands along the Atlantic coast in Maine (USA) experience severe damage and increased mortality caused largely by eastern dwarf mistletoe infection. We quantified the effect of infection on host spruce needle morphology and anatomy as well as the effect of infection on stem hydraulic conductivity. Infection did not affect rates of host needle photosynthesis; however, infection led to a dramatic increase in host needle stomatal conductance early, but not later, in the growing season. One of the most readily visible effects of mistletoe infection of white spruce is a ~50% reduction in the size of host needles distal to infection. Since infection also reduces sapwood area-specific hydraulic conductivity by ~25%, the dramatic reduction in needle size may serve to re-establish the balance between the demand for water and the ability of infected branches to deliver water to distal foliage, compensating for reduced conductivity, greater host needle transpiration, and transpiration of the aerial shoots of the parasite. Consistent with these observations, branch xylem tension was unaffected by infection. Host developmental responses to infection (e.g., reductions in needle size) appear to preserve needle-and branch-scale function and longevity. Whole tree survival may be negatively impacted by these responses, as they prolong the survival of infected branches which may possess poor or negative carbon balance and also allow infected branches to serve as a source of parasite seed for further infection PS25.29 Modelling of the regulation of the photosynthetic processes and discussion of its possible meaning for photosynthesis T Karelina (Moskow State University), A Kukushkin (Moskow State University) Mathematical model of the photosynthesis developed in our group [1] allowed to describe many of the known experimental data on oscillations in photosynthesis without additional assumptions often used in other models [2]. The main goal of this work was to describe the experimental data on the non-linear answer of photosynthetic system to harmonically modulated irradiance [3]. The kinetic curves for CO2 assimilation rate, fluorescence and P700 absorption signal under such illumination would contain higher harmonics and have a rather complicated form. By our model we obtained the kinetic curves which were close to experimentally observed. We concluded, that the mechanisms leading to such a behavior are cyclic electron transfer around PS I and PQ redox-state depending energy distribution between photosystems. In the mathematical model without proposed feedback mechanisms stationary rate of CO2 assimilation increased with increasing intensity of the light absorbed by PS II until some value and then dropped abruptly to zero. If these feedback mechanisms were included in model, CO2 assimilation rate increased until saturation, then remained unchanged. We can make a conclusion that these mechanisms are necessary to keep photosynthetic apparatus from over-reduction, which could lead to blocking of the ATP synthesis and CO2 assimilation. These results may have significance for understanding regulatory mechanisms of photosynthesis. References [1] T. A. Karelina et al.// Biophysics, 2005, 50, 953-958. [2] A. Laisk, H. Eichelmann// Phil. Trans. R. Soc. London B. 1989, 323, 369-384. [3] L. Nedbal et al.// Biochim. Biophys. Acta. 2003, 1607, 5 - 17. +

PS25.30

First application of terephthalate as a fluorescent probe for hydroxyl radicals in thylakoid membranes I Šnyrychová (Department of Experimental Physics, Faculty of Science, Palacky University Olomouc, Czech Republic and Institute of Plant Biology, Biological Research Center, Szeged, Hungary), P Kós (Institute of Plant Biology, Biological Research Center, Szeged, Hungary), É Hideg (Institute of Plant Biology, Biological Research Center, Szeged, Hungary) Conversion of the non-fluorescent terephthalate into the highly fluorescent 2-hydroxyterephthalate by hydroxyl radicals has been recognized more than 40 years ago [1]. Even though terephthalate is well established as a specific, water-soluble fluorescent probe for hydroxyl radicals [2-3], its application in biology is very limited and the method has not been used for photosynthetic samples so far. We found that, despite some limitations (i.e. overlap of hydroxyterephthalate emission with chlorophyll absorption, quenching of hydroxyl radicals by sugars in buffers and by thylakoid membranes themselves), terephthalate can be used to study the stress-related production of hydroxyl radicals in thylakoids. As an example, we studied the origin of hydroxyl radicals produced in thylakoid membranes under UV-B irradiation [4]. Our data show that the presence of neither the directly detected hydroxyl radicals nor the other assumed reactive oxygen species was sufficient for structural degradation of D1 protein of Photosystem II reaction centres. In general, high selectivity of terephthalate hydroxylation towards hydroxyl radicals, relatively low toxicity, light-insensitivity and chemical stability of both probe and its hydroxylated form make this simple method highly promising for application in in vitro studies. [1] Armstrong et al. (1963) Can. J. Chem. 41: 1575 [2] Matthews (1980) Radiat. Res. 83: 27 [3] Fang et al. (1996) Ultrason. Sonochem. 3: 57 [4] Šnyrychová et al. (2007) submitted Acknowledgement: I.Š. was supported by a Visegrád Post-Graduate Scholarship S-023-2006 and by the grant MŠMT ČR MSM 6198959215. PS25.31 Higher plants photosynthetic antenna size is controlled by plastoquinone redox state at post-transcriptional rather than transcriptional level R Bassi (Università di Verona), S Frigerio (Università di Verona), T Morosinotto (Università di Padova), L Cattivelli (Centro sperimentare per la Cerealicoltura, Fiorenzuola d'arda (PC)), S Zorzan (Università di Verona), C Campoli (Centro Sperimentale per la cerealicoltura fiorenzuola d'arda (PC)), L Fantoni (Università di Modena), C Crosatti (Centro sperimentale per la Cerealicoltura Fiorenzuola d'arda (PC)), W Haehnel (University of Friburg) We have analyzed the effect of the plastoquinone redox state on the regulation of the light harvesting antenna size at transcriptional and proteomic level. This was approached by studying gene transcription and accumulation of lhc gene products in WT vs. the barley mutant viridis zb63, which is depleted in Photosystem I and has constitutively reduced plastoquinone. We show that the expression level of genes encoding antenna proteins is virtually unaffected in the mutant, with the exception of protease encoding genes; this stability of messenger level is not a peculiarity of antenna-encoding genes but it extends to all photosynthesis-related genes. In contrast, analysis of protein accumulation by 2D-PAGE shows, that the mutant undergoes strong reduction of its antenna size, with individual gene products having different levels of accumulation. We conclude that plastoquinone redox state plays an important role on the long-term regulation of chloroplast protein expression. However, this regulation is active at the post-transcriptional rather than transcriptional level. In addition, we show that epistatic regulation of translation is evident for PSI and PSII core subunits, while it is not effective for the polypeptides of Lhc antenna.

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PS25.32 Relatively high level of photosynthesis protection of rare coastal marsh plants in conditions of elevated soil salinity G Ievinsh (University of Latvia), U Andersone (University of Latvia), I Samsone (University of Latvia) Protection of photosynthesis is a crucial feature for coastal plants exhibiting periodical fluctuations in soil salinity. The aim of the present experiments was to investigate changes in photochemistry of photosystem II due to increased NaCl content by means of chlorophyll a fluorescence for two rare plant species from a coastal salt marsh of Baltic sea – Aster tripolium L. and Hydrocotyle vulgaris L. Plants were grown in controlled conditions with 0 - 400 mM NaCl (A. tripolium) or 0 - 100 mM NaCl (H. vulgaris). Both plant species had a certain degree of protection of photosynthetic apparatus against increased salinity by means of nonphotochemical quenching and/or ascorbate peroxidase activity. It can be concluded that A. tripolium is an obligate halophyte species exhibiting higher photosynthetic efficiency at 50 - 400 mM NaCl in comparison to control without added NaCl. Clonal plant H. vulgaris presumably exhibits adaptive morphological plasticity in conditions of moderately increased salinity (25 mM NaCl) instead of pronounced protection of photosystem II. PS25.33 The synthesis of thylakoid membrane proteins in wheat plants under salt stress S Suleymanov (Institute of Botany), I Huseynova (Institute of Botany) The response of wheat (Triticum durum L.) genotypes to salt stress was studied by growing the seedlings in the presence of 100 up to 250 mM NaCl. It was found out that lower salt concentrations treatment of plants had a little effect in the content and composition of thylakoid membrane. Some proteins presented in control samples either disappeared (48.5; 27.5, 25.5 ? 21.5 ?D?), or remained in slight amounts (b-subunit of CF1, and 34,5; 28.5; 24; 23; 19.5; 18; 17.5; 15 and 13 ?D? proteins) after being influenced by 150 mM NaCl. Upon increase of salt concentration up to 200 mM thylakoid membrane contained more plentiful contents of polypeptides (apoprotein CPI, a- and b-subunits of ?F1, 45.5 ?D? polypeptide of PS II) in comparison with the seedlings grown in 150 mM NaCl. The synthesis of 48.5 and 21.5 ?D? proteins was recovered. The noticeable increase was observed in the low-molecular polypeptides region of 19.5 -13 ?D?. Further increase of NaCl concentration up to 250 mM resulted in substantial increase of apoprotein ??I, CF1 proteins, and also 45.5, 34.5 and 28.5 kDa polypeptides. The new peak was detected in 51 ?D? region and synthesis of low-molecular proteins decreased noticeably. PS II activity in the treated preparations rose with increasing of sodium chloride concentration and reached to maximum at 200 mM, and then decreased sharply, whereas salinity reduced PS I activity insignificantly. The role of these proteins in protective mechanism against salt stress will be the focus of the future studies. PS25.34 Paraheliotropism in Robinia pseudoacacia plants: an efficient mean to cope with photoinhibition. C Arena (Dipartimento di Biologia Strutturale e Funzionale, Università di Napoli Federico II), L Vitale (Dipartimento di Biologia Strutturale e Funzionale, Università di Napoli Federico II), A Virzo De Santo (Dipartimento di Biologia Strutturale e Funzionale, Università di Napoli Federico II)

The exposure to high irradiance and temperature may result in photoinhibition. Paraheliotropism may represent an important strategy to decrease the potential for photodamages. The aim of this study was to assess the contribution of leaf movements to PSII photoprotection at high light and temperature in Robinia pseudoacacia. For this purpose, gas exchange and chlorophyll fluorescence measurements were performed at 10:00 and 12:00 am, and at 15:00 and 18:00 pm on attached blocked leaves (BL) and on control unblocked leaves (UL). At midday as well as at 15:00 and 18:00 pm, BL showed a decrease of net photosynthesis (An), stomatal conductance (gsH2O), quantum yield of electron transport (ΦPSII), percentage of inhibited photosynthesis by O2 (IPO), and PSII efficiency factor (F’q/F’v) as compared to values measured in the morning, whereas Ci/Ca ratio and NPQ increased significantly. Differently from BL, UL maintained the photosynthetic performance elevated throughout the day and higher compared to constrained leaves. At 18:00 pm, An, gsH2O, ΦPSII and F’q/F’v of BL showed a tendency to recovery compared to 15:00 pm even if the values remained lower than those measured at 10:00 am and in UL. Conversely to UL, no recovery was found in maximal PSII photochemical efficiency at the end of the study period. Data suggest that in R. pseudoacacia leaf movements represent an efficient and reversible strategy to overcome environmental stresses such as high light and temperature. Moreover paraheliotropism is able to protect photosystems avoiding photoinhibitory damage risks at no cost for plant in terms of An reduction. PS25.35 The conserved role of FtsH11 protease in protection of photosynthetic system from high temperature stress in higher plants J Chen (USDA-ARS, USA), Z Xin (USDA-ARS, USA), J Burke (USDA-ARS, USA) As sessile organisms, plants employ multiple mechanisms to cope with seasonal and daily temperature fluctuations associated with their habitats. To identify the various mechanisms of thermotolerance in plants, we isolated a series of thermosensitive mutants that are defective in growth and development at moderated high temperatures in Arabidopsis. One class of such mutants was shown by map-based cloning to have mutations on FtsH11 protease gene. The Arabidopsis genome contains 12 predicted FtsH genes, with all previously characterized FtsH proteases playing roles in the alleviation of light stress through degradation of unassembled thylakoid membrane proteins and photodamaged D1 protein. Under high light conditions at 21ºC, ftsh11 mutants were indistinguishable from wild type plants in photosynthesis capacity. However, under temperatures at 30ºC or above and normal light condition, ftsh11 mutants display a host of phenotypes typical of thermosensitivity: cotyledons of dark germinated mutant seeds failed to turn green after exposed to light, reduced photosynthetic capability, measured as chlorophyll content, chl a/b ratios, and PSII quantum yield in leaves, chlorosis, cessation of plant growth and development, and eventually death. Overexpression of wild-type FtsH11 from both Arabidopsis and pea was able to complement the thermosensitive phenotypes of ftsh11 mutants, suggesting a conserved role of FtsH11 in alleviation of damage caused by high temperature. Further analysis indicated that FtsH11 plays roles in the early stages of chloroplast biogenesis and the thermostability of photosynthetic systems. Our results suggest that FtsH11 plays essential roles in chloroplast biogenesis and in protection of photosystems from high temperature stress. PS25.36 Response of ferritin over-expressing tobacco plants to oxidative stress

309 14th Photosynthesis Congress - PS07 É Hideg (Institute of Plant Biology, Biological Research Center, Szeged, Hungary), K Török (Institute of Plant Biology, Biological Research Center, Szeged, Hungary), I Šnyrychová (Laboratory of Biophysics, Palacký University, Olomouc, Czech Republic), G Sándor (Institute of Plant Biology, Biological Research Center, Szeged, Hungary), E Szegedi (Research Institute for Viticulture and Enology, Kecskemét, Hungary), G V Horváth (Institute of Plant Biology, Biological Research Center, Szeged, Hungar In order to evaluate the role of ferritin as potential stress protector, stress-induced changes in photosynthetic activity and reactive oxygen production were compared in leaves of control and ferritin over-expressing tobacco plants under a variety of abiotic stress conditions, such as chemically induced oxidative stress, 290-320 nm ultraviolet radiation and photoinhibition by excess photosynthetically active radiation. Apart from standard biochemical assays, reactive oxygen production was measured with a newly developed imaging apparatus [1] capable of detecting changes in the fluorescence of the dansyl based ROS sensors 3-[N-(β-diethylaminoethyl)-N-dansyl]aminomethyl-2,2,5,5-tetramethyl2,5-dihydro-1H-pyrrole [2] and 3-(N-dansyl)aminomethyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole [3]. The effects on photosynthesis caused by the oxidative stress were assessed via chlorophyll fluorescence imaging and the saturation pulse method [4]. Our results show that the oxidative stress tolerance of transgenic, ferritin over-expressing tobacco was improved as compared to control plants. Acknowledgements: ROS sensors were synthesized at the Department of Organic and Medicinal Chemistry, University of Pécs, Hungary (please contact Dr Tamás Kálai for details at [email protected]). This work was supported by the Hungarian National Scientific Research Found (grant No. OTKA T049438) [1] Hideg É, Schreiber U. (2007) Photosynth Res, DOI: 10.1007/s11120-007-9146-4 [2] Kálai T, Hideg É, Vass I, Hideg K (1998) Free Rad Biol Med 24: 649-652 [3] Kálai T, Hankovszky OH, Hideg É, Jekő J, Hideg K (2002) ARKIVOC 2002 (iii): 112-120 [4] Schreiber U, Schliwa U, Bilger W (1986) Photosynth Res 10: 51-62 PS25.37 The thioredoxin-peroxiredoxin system in the cyanobacterium Synechocystis sp. PCC 6803 responds to changes in environmental conditions F Florencio (Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC), M Pérez-Pérez (Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC), A Mata-Cabana (Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC), A Sánchez-Riego (Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC), M Lindahl (Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC) The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains 5 genes encoding peroxiredoxins and 4 genes coding for thioredoxins. Phylogenetic analysis indicates that the former belong to the 2-Cys- (sll0755), Type II- (sll1621), 1-Cys- (slr1198) and PrxQ(sll0221and slr0242) families. Three of the thioredoxins fall into the established phylogenetic categories of prokaryotic m-type (sll0623), x-type (slr1139) and y-type (slr0233) thioredoxins. We have studied the expression of the different peroxiredoxin and thioredoxins under various environmental and nutritional stress conditions, such as high light intensity, oxidative stress, heat-shock and nitrogen starvation. Our results suggest that different environmental stimuli give rise to distinct response patterns. For example, under oxidative stress following addition of hydrogen peroxide to Synechocystis cultures the Type II

peroxiredoxin and PrxQ1 are strongly induced in contrast to the others, however only the y-type thioredoxin appears to respond to this stimulus. Furthermore, we have carried out an extensive enzymatic study of the activities of the different peroxiredoxins. This shows that all peroxiredoxins are able to receive reducing equivalent from each type of thioredoxin, but with different efficiencies. A detailed kinetic analysis of each peroxiredoxin with different electron donors and peroxide substrates will be presented. PS25.38 Effect of temperature and UV-B radiation on the expression of early light - induced proteins (ELIPs) in grapevine (Vitis vinifera L) M Pinto (Universidad de Chile), F Pinto (Universidad de Chile), M Berti (Universidad de Chile), A Riquelme (Universidad de Chile), W Sierralta (Universidad de Chile) Photoinhibition is an important stress in grapevine growing in desert areas where high light is normally accompanied by high temperature and high UV-B radiation. ELIPs are signaled as protective molecules in plants submitted to excess of light. These proteins have been essentially studied in annual plants like pea or barley, but much less in perennials like grapevine. In this work ELIPs expression was studied in mature and young leaves of grapevine. Plants were grown for three month under greenhouse conditions at 100 µmol PAR m-2 s-1 (12 h/day) and then exposed to different light intensities, temperatures and UV-B doses. Surprisingly no ELIPs induction was found in mature leaves (6 weeks-old). In young leaves (2 weeks old) the expression was notorious over 300 µmol PAR m-2 s-1 and over 15ºC. The maximum expression was found at 30ºC under high light intensity (1500 µmol PAR m-2 s-1). This result differs from these reported in annual plants, where ELIPs expression is normally increased by low temperatures (<10ºC). In time course experiments, ELIPs expression was notorious after 3 hours of exposure to high light and maximal after 4 hours. Using cut off filters in young leaves directly exposed to solar radiation it was found that UV-B radiation was more effective than UV-A to induce ELIPs expression. Fondecyt 1070788

PS25.39 Effect of water deficit on RUBISCO and carbonic anhydrase activities in different wheat genotypes N Guliyev (Institute of Botany), S Bayramov (Institute of Botany), H Babayev (Institute of Botany) Drought stress influences on all the metabolic pathways in plants and leads to the decrease of productivity. Photosynthetic assimilation of CO2 is more sensitive to water deficit among metabolic processes. Therefore, its investigation has a scientific and practical importance. An influence of drought stress on the dynamics of activities of some enzymes participating in CO2 initial fixation in bread and durum wheat genotypes differing in their drought tolerance and productivity was investigated under field conditions. An activity of the key enzyme of Calvin cycleribulose 1.5-bisphosphate carboxylase/oxygenase (RUBISCO) was primarily constant in plants with normal watering and subjected to drought. Carbonic anhydrase (CA) activity increased during the first period of drought in flag leaves of drought resistance genotypes, while it decreased at the last stage of vegetation in all genotypes. The total activities of RUBISCO and CA were lower in ear elements than in leaves and decreased less in flag leaves under water deficit. In ear elements, especially in maturation seeds CA activity was much less in comparison with flag leaves. It suggests that CA takes part only in CO2 photosynthetic assimilation. Mane case parallel changes of CA and

310 14th Photosynthesis Congress - PS07 RUBISCO activities in different wheat genotypes both subjected to drought stress and well watering plants indicate to the correlation between them. The results suggest that some genotypes obtain drought tolerance owing to CO2 concentrating mechanism.

PS25.40 Heat Stress Induces an Aggregation of the Light-harvesting Complex of Photosystem II in Spinach Plants C Lu (Institute of Botany, Chinese Academy of Sciences) Whole spinach plants were subjected to heat stress (25-50oC) in the dark for 30 min. At temperatures higher than 35oC, CO2 assimilation rate decreased significantly. The maximal efficiency of PSII photochemistry remained unchanged until 45oC and decreased only slightly at 50oC. Non-photochemical quenching increased significantly either in the absence or presence of dithiothreitol. There was an appearance of the characteristic band at around 698 nm in 77K fluorescence emission spectra of leaves. Native green-gel of thylakoid membranes isolated immediately from heat-stressed leaves showed that much pigment-protein complexes remained aggregated in the stacking gel. The analyses of SDS-PAGE and immunoblotting demonstrated that the aggregates were composed of the main light-harvesting complex of PSII (LHCIIb). To characterize the aggregates, isolated PSII core complexes were incubated at 25-50oC in the dark for 10 min. At temperatures over 35oC, much pigment-protein complexes remained aggregated in the stacking gel of native green-gel and immunoblotting analyses showed that the aggregates were composed of LHCIIb. In addition, isolated LHCII was also incubated at 25-50oC in the dark for 10 min. Much LHCII remained aggregated in the stacking gel of native green-gel at temperatures over 35o. Massive aggregation of LHCII was clearly observed by using microscope images, which was companied by a significant increase in fluorescence quenching. There was a linear relationship between the formation of LHCII aggregates and non-photochemical quenching in vivo. The results in this study suggest that LHCII aggregates may represent a protective mechanism to dissipate excess excitation energy in heat-stressed plants. PS25.41 Genome-wide analysis of gene functions by chlorophyll fluorescence K Sonoike (University of Tokyo), H Ozaki (University of Tokyo) Since chlorophyll fluorescence reflects the redox state of photosynthetic electron transport chain, monitoring of chlorophyll fluorescence was successfully applied for the screening of photosynthesis-related genes. In the case of cyanobacteria, prokaryotic organisms, photosynthetic and other metabolic pathways are not separated in organelles. Therefore, we assume that monitoring chlorophyll fluorescence in cyanobacteria has potentiality to detect the effect of the wide range of gene-disruption. We created the mutant library of Synechocystis sp. PCC 6803, which covers 15% of genes in the genome. Chlorophyll fluorescence measurements of these mutants revealed that disruption of about half of the genes (most of them are not directly involved in photosynthesis) could affect the chlorophyll fluorescence indicating the cross-talk between photosynthesis and very wide range of other cellular activities in cyanobacteria. We further constructed the database of the fluorescence kinetics, and develope the method to quantify the change of “shape” of the fluorencence kinetics to rank the similarity between any two mutants. When we searched in the database for those that showed similar fluorescence kinetics with that of sll1961 mutant, which has defects in the regulation of photosystem stoichiometry under high light condition, eight mutants showed different photosystem stoichiometry under high light condition out of fifteen mutants ranked high in the list. This method

is not based on the theoretical relationship between gene fucntion and fluorescence kinetics, but is based on simple “similarity” of fluorescence kinetics. Thus, we hope the method could be applid to any other physiological function other than the regulation of photosystem stoichiometry. PS25.42 Influence of salt stress and copper toxicity on photosynthetic parameters and antioxidative defense components in leaves of a succulent CAM plant L Bartha ("Babes-Bolyai" University, Dept. Biology), L Fodorpataki ("Babes-Bolyai" University, Dept. Biology) Increased concentrations of salt and of heavy metals affect more and more terrestrial habitats and represent stress factors for many plants, inducing physiological responses reflected in acclimation processes. Because development of tolerance includes energy-consuming restoration of ion homeostasis and turgor, accumulation of compatible solutes, synthesis of stress proteins and detoxification of reactive oxygen species, it adversely affects the photosynthetic metabolism and its energetic budget. The aim of the present study is to reveal the influence of high concentrations of sodium chloride (150 mM) and of copper sulfate (50 micromoles), separately and simultaneously, on the quantum yield efficiency of PSII and on other functional parameters of photochemical processes revealed by induced chlorophyll fluorescence, as well as on the effectiveness of the antioxidative protective system of plant cells under conditions of enhanced formation of reactive oxygen species. Photosynthetic parameters are investigated in the leaves of the succulent CAM plant Kalanchoe daigremontiana, suitable for restoration of vegetation on degraded soils. Salt and copper reaching the leaves induce changes in the potential and effective quantum yield efficiency, as well as in the non-photochemical quenching of chlorophyll fluorescence and in the composition of light-harvesting complexes. Stress tolerance of the antioxidative machinery of leaves is discussed as reflected by changes in ascorbate peroxidase and superoxide dismutase activities, and in the ratio between the reduced ascorbate and the oxidized dehydroascorbate levels. Differences and common features between salt stress and copper toxicity, as well as their interactions are discussed. PS25.43 Differential sensitivity of the photosynthetic apparatus of a freshwater green alga and of duckweed exposed to salinity and heavy metal stress L Fodorpataki ("Babes-Bolyai" University, Dept. Biology), L Bartha ("Babes-Bolyai" University, Dept. Biology) In terrestrial plants salt stress highly overlaps with drought, but the latter does not exist in aquatic environments, so aquatic plants did not develop protective strategies against water deficit. This makes freshwater plants more susceptible to increasing saliniti, which may occur e.g. upon intense evaporation of water from lakes, in relation with climate warming. Water pollution with heavy metals interferes with mechanisms of salt tolerance and cross-tolerance may occur upon combined stress conditions. Salt-stressed plants exhibit a decrease in photosynthetic efficiency, but it is not clear how this actually occurs. The present study investigates the changes induced by salt and copper stress in photosynthetic parameters of a cosmopolitan freshwater green alga (Scenedesmus opoliensis) and of duckweed (Lemna minor), both originating from the same kind of habitatand both being highly suitable for bioindication of water quality. Growth rate and net photosynthetic production were established in cultures grown for 15 days under constant photon flux density (90 micromole m-2s-1), oxygen evolution, photosynthetic pigment content and parameters of conventional and

311 14th Photosynthesis Congress - PS07 modulated chlorophyll fluorescence were determined on the 5th day of treatment. Duckweed is much more sensitive to high concentration of NaCl than the freshwater alga, this being well reflected by decrease in the quantum yield efficiency of PSII (both Fv/Fm and Φ), and by disorganization of light-harvesting complexes. The green alga is less tolerant to copper than the duckweed, stress effects being reflected by depletion of oxygen and biomass production. Cross tolerance between salt and copper stress is more pronounced in the alga. PS25.44 Loss of Photosystem II during Dark-Induced Leaf Senescence in a Stay-Green Rice Variety, SNU-SG1 and a Mutant, sgr M Oh (National Institute of Crop Science, Rural Development Administration), T Park (National Institute of Crop Science, Rural Development Administration), W Yang (National Institute of Crop Science, Rural Development Administration), K Kwak (National Institute of Crop Science, Rural Development Administration), J Shin (National Institute of Crop Science, Rural Development Administration) During leaf senescence, the most characteristic visible change is leaf yellowing due to the preferential breakdown of chlorophyll with concomitant chloroplast degradation. In this study, we examined the characteristics for the stay-greenness during dark-induced senescence (DIS) in two stay-green varieties, SNU-SG1 and sgr. During DIS, chlorophyll loss was delayed in SNU-SG1 and sgr compared with that in wild type (WT), but the photochemical efficiency of PSII (Fv/Fm) was not. The content of the functional PSII during DIS, estimated as (1/Fo 1/Fm) was high in WT, but low in SNU-SG1 and sgr. In both varieties, Fo parameter increased during DIS, indicating the detachment of LHCII in PSII. In western blot analysis, the D1 and LHCII of WT were detected even when leaves turned yellowed. However, D1 protein in sgr completely disappeared after 2 d without significant decrease in LHCII. These results suggest that the delay in chlorophyll degradation in both SNU-SG1 variety and sgr mutant was non-functional and largely attributed to the high stability of LHCII with an early degradation of D1 protein as a key protein of PSII. PS25.45 Improved photoautotrophic survival of Chlamydomonas mutants under high light and oxidative stress B Forster (The Australian National University), B Osmond (The Australian National University), B Pogson (The Australian National University) Survival and growth of photosynthetic organisms under strong light and the associated oxidative stress require effective photoprotection and repair mechanisms to ensure maintenance of photosynthetic activity, particularly of the readily photoinactivated photosystem II. We isolated very high light resistant (VHLR), nuclear mutants from the green alga Chlamydomonas reinhardtii that sustain fast growth under excess light levels normally lethal to the wild type. At 30% of full sunlight, these mutants are phenotypically similar to wild type in growth, structure and function of photosystems as well as in zeaxanthin-mediated photoprotection. However, VHLR mutants show constitutive and induced adjustments, potentially necessary for survival under extreme light stress, such as faster D1 protein synthesis for photosystem II repair, increased tolerance to reactive oxygen species (ROS) and lower ROS accumulation. Acclimation to excess light is associated with stable downregulation of the photosynthetic apparatus and altered electron flow through the photosystems in these mutants. On the whole cell level, comparative proteomics of wild type and VHLR mutants identified differentially expressed proteins in various biological functions. Our investigations strongly indicate that enhanced high light resistance is

associated with increased tolerance to ROS that has resulted from coordinated changes in several photoprotective and photoacclimation processes. This suggests VHLR mutants affect “regulatory master switches” which determine the balance between the induction of photoprotective mechanisms and photosynthetic efficiency. PS25.46 Proteomic analyses of the effects of drought stress on the expression of thylakoid membrane proteins of rice (Oryza sativa. L) J Liang (College of Bioscience and Biotechnology,Yangzhou University, China), Y fan (Yangzhou University) The thylakoid membrane system is the site of undertaking photosynthetic electron transport which converts light energy into chemical energy, and contains four abundant multi-subunit protein complexes (Photosystem I, II, the ATP synthase, and the cytochrome b6f complex). In addition, the thylakoid membrane might also contain many other proteins that are involved in the coordination of these complexes and protection against abiotic stresses. We constructed high-resolution two dimensional electrophoresis maps of the thylakoid membrane proteins purified from intact rice chloroplasts and analyzed the effects of soil drying treatment on the expression of thylakoid membrane proteins. At least 300–350 different thylakoid membrane proteins were detected. The expression levels of at least twenty proteins were affected by drought treatment, of which three proteins disappeared, three proteins were newly synthesized, seven proteins up-regulated (three times higher) and seven proteins down-regulated (three times lower) when exposed to soil drought. Mass spectrometry analysis showed that twelve proteins had a clear function or functional domain, whereas other eight proteins were hypothetical proteins. However, blast results indicated that the eight hypothetical proteins had predicated roles in PSI-mediated processes. Combined the results of 2-DE - MALDI-TOF/MS and BLAST with the physiological data , we concluded that drought-induced inhibition of leaf photosynthesis were largely through the effects on the expression of thylakoid membrane proteins, which was involved mainly in photosystem I-mediated photochemical processes. PS25.47 Evaluation of acid stress tolerance of mutants of Synechocystis sp. PCC 6803 lacking signal transduction related gene, sigB, H Ohta (Dept. of Biol. Fac of Sci. Tokyo Univ. of Sci.), Y Shibata (Tokyo Univ. of Sci.), Y Haseyama (Tokyo Univ. of Sci.), Y Yoshino (Tokyo Univ. of Sci.), T Suzuki (Tokyo Univ. of Sci.), A Moriyama (Tokyo Univ. of Sci.), I Enami (Tokyo Univ. of Sci.), M Ikeuchi (University of Tokyo) DNA micro array analysis has been identified the genes upregulated by acid stress in cyanobacteria Synechocystis sp. PCC 6803[Photosynthesis Research 84, 225-230, 2005]. Among these genes, we focused on sigB, sigD and patA which are involving signal transduction and investigated the impact of these genes null mutation on the survival of Synechocystis cells at low pH medium. The survivals of sigB and sigD mutants at pH 6.0 were lower than the survival of wild-type cells. Quantitative real time RT-PCR revealed that sigD expression was 33% in the DsigB mutant relative to wild type cells. These results suggest that SigB and SigD are required for acid tolerance and SigB is located in upstream of SigD in signal transduction pathway of acid stress response in the Synechocystis sp. PCC 6803. PS25.48 Maize (Zea maize L.) performance under drought: Decreased photosynthetic area vs. decreased efficiency of PSII

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P Ehsanzadeh (Isfahan University of Technology-Iran) Crop production in arid and semiarid conditions is faced with water limitation. Corn production in Iran accounts for a considerable amount of the annual irrigation water use of this drought-prone country. Thus a field experiment was conducted to investigate the impact of irrigation regimes on growth, chlorophyll fluorescence and grain yield of maize in 2006 at the Research Farm, College of Agriculture, Isfahan University of Technology, using a 3-replicate split-plot RCBD. Four irrigation regimes (I1, I2, I3 and I4, representing no drought, slight, moderate and severe drought, respectively) were the main plots and four corn hybrids (SC 704, SC700, SC500 and TC647) served as subplots. Chlorophyll fluorescence parameters such as F0, Fm and Fv/Fm in addition to LAI and grain yield were studied. While hybrids differed in their F0 at leaf10 stage, they differed with regard to Fm at tasseling stage. No significant difference was found between irrigation regimes in terms of chlorophyll fluorescence parameters, though LAI decreased with drought. Grain yield differed among hybrids and irrigation regimes, with the I4 producing the lowest grain yield, compared to other irrigation regimes. SC700 outperformed other hybrids in terms of grain yield. It seems that, at least with the present study, a decreased photosynthetic area leaves a more pronounced impact on plant productivity, compared to a decreased efficiency of PSII under drought conditions. Key words: Maize, chlorophyll fluorescence, grain yield.

PS25.49 Light Induced Energy Dissipation in Iron-Starved Cyanobacteria : Roles of OCP and IsiA Proteins A WILSON (CEA CNRS), C Boulay (CEA CNRS), A Wilde (Humboldt University), C Kerfeld (University of California), D Kirilovsky (CEA CNRS) In response to iron deficiency, cyanobacteria synthesize the Iron-stress-induced chlorophyll-binding protein, IsiA. This protein protects cyanobacterial cells against iron stress. It has been proposed that the protective role of IsiA is related to a blue light-induced non-photochemical fluorescence quenching (NPQ) mechanism. In iron-replete cyanobacteria cell cultures, strong blue light is known to induce a mechanism dissipating excess absorbed energy in the phycobilisome, the extramembranal antenna of cyanobacteria. In this photoprotective mechanism the soluble Orange-Carotenoid Protein (OCP) plays an essential role. Here, we demonstrate that in iron-starved cells, blue light is unable to quench fluorescence in the absence of the phycobilisomes or the OCP. In contrast, the absence of IsiA does not affect the induction of fluorescence quenching or its recovery. We conclude that in cyanobacteria grown under iron starvation conditions, the blue light-induced non-photochemical quenching involves the phycobilisome-OCP related energy dissipation mechanism and not IsiA. IsiA, however, does seem to protect the cells from the oxidative stress generated by iron starvation, initially by increasing the size of the Photosystem I antenna. Subsequently, the IsiA converts the excess energy absorbed by the phycobilisomes into heat through a mechanism different from the dynamic and reversible light-induced NPQ processes. PS25.50 Effect Of NaCl And Exogenously Supplied ABA On Xanthophyll Cycle Pigments And Energy Dissipation In Rice Plants Under High Light Stress P Sharma (Goa University), J Vaz (Goa University)

Since violaxanthin (V) serves as a common precursor for abscisic acid (ABA), a plant hormone, involved in stress adaptation and zeaxanthin (Z) a carotenoid, which is involved in energy dissipation, therefore, plants supplied with exogenous ABA should show a better protection under high light treatment, since V pool shall be available for the formation of Z, while in plants subjected to salt stress the increase in ABA levels, presumably from V, may limit the V pool for Z formation, undermining energy dissipation function. In the present study rice (Oryza sativa L. cv. Jyothi) plants grown in the shade were fed with ABA (10 mM) through roots or subjected to salt stress (100 mM) to investigate their influence on the light-dependent Z formation and energy dissipation. Plants (both +ABA/–ABA and +NaCl/-NaCl) were then treated with high light by exposing them to direct sunlight (1600-2000 µmol m-2s-1 PFD). Leaf samples were collected for chlorophyll fluorescence measurements, pigment and ABA analysis at different intervals during the exposure. Since a common pool of V acts as a precursor to ABA as well as Z formation: V would convert either to ABA or to Z or both depending on stress level. In our study plants subjected to salinity stress showed an increase in the endogenous ABA level while Z was absent. Fv/Fm and qN remained more or less same in treated and untreated plants. Neoxanthin (N) content were much higher in salt grown plants but declined initially after onset of sun light treatment. However, N content increased after longer duration of the exposure. When plants were grown with exogenously supplied ABA and subsequently exposed to sunlight most of the V was made available for conversion to Z and this was correlated with higher level of qN and better photoprotection. Data with respect to these experiments will be presented in the congress. PS25.51 Evaluation of early vigour and photosynthesis of industrial chicory in relation to temperature S Devacht (Institute for agricultural and fisheries research (ILVO)), P Lootens (Institute for agricultural and fisheries research (ILVO)), L Carlier (Institute for agricultural and fisheries research (ILVO)), J Baert (Institute for agricultural and fisheries research (ILVO)), J Van Waes (Institute for agricultural and fisheries research (ILVO)), E Van Bockstaele (Institute for agricultural and fisheries research (ILVO)) The aim of this study is to evaluate the effect of cold stress on the early vigour and the photosynthesis efficiency for industrial chicory, Chicorium intybus L. From a preliminary experiment, with 17 industrial chicory cultivars/lines, eight are chosen for their contrasting early vigour at 10°C. The selected ones are thorough evaluated for cold stress by growing them in growth chambers at 16°C (reference), 8°C (intermediate) and 4°C (stress). First, the germination duration of the cultivars/lines at 20°C is determined. There is a variation from 1 to 3 days. Based on this information the plants were sown at 20°C at different days to exclude the germination phase in the further experiments. For the cold stress experiments all plants are evaluated at the same stage (seedlings with two cotyledons, germinated at 20°C) when the appropriate growth temperature, 16°C, 8°C or 4°C, is applied. The following analyses are performed to study the effect of cold stress: (i) growth analysis, (ii) photosynthesis and chlorophyll fluorescence measurements for the evaluation of the photosynthesis process and (iii) evaluation of the pigment concentration. The evaluation of the performed analyses through statistical and correlation analysis offers a first glance on the discriminating power of the parameters. These analyses showed significant differences in discriminating power for the different cultivars and growth temperatures for the following parameters: the non-photochemical quenching, the yield of the photochemical and non-photochemical processes and the photosynthetic quantum efficiency. These parameters could in the future be used for the physiological screening method.

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PS25.52 LuminoTox: Enlarging toxicity response by using different photosynthetic systems

PS25.54 Chloroplast proteomics and fluorescence visualise systemic viral infection in plants

N Boucher (Lab_Bell), F Bellemare (Lab_Bell), L Lorrain (Lab_Bell), M Rouette (Lab_Bell)

M Barón ( Department of Biochemistry, Cell and Molecular Biology of Plants. Estación Experimental del Zaidín. CSIC. C/ Profesor Albareda, nº 1. C.P. 18008. Granada. Spain ), M Pineda (Department of Biochemistry, Cell and Molecular Biology of Plants. Estación Experimental del Zaidín. CSIC. C/ Profesor Albareda, nº 1. C.P. 18008. Granada. Spain), C Sajnani (Department of Biochemistry, Cell and Molecular Biology of Plants. Estación Experimental del Zaidín. CSIC. C/ Profesor Albareda, nº 1. C.P. 180

A biosensor based on the LuminoTox Analyzer has been developed by Lab_Bell and successfully incorporated into field assessment for the detection of toxic molecules in water. The photosynthetic electron transport chain is the target of several toxic molecules found in water, affecting the growth of phytoplankton and aquatic plants. This characteristic makes the photosynthetic process very effective in the detection of water pollutants. A hand-hold fluorescence apparatus, included in the LuminoTox test, was designed to measure photosystem II photochemical yield. The presence of toxic agents altering the photosynthetic electron transport chain is measured by the decrease in the photochemical yield. As a means of further exploring the scope of the LuminoTox application and of selecting a biomaterial to complement the instrument design, three photosynthetic systems were exposed to several chemical substances (herbicides, metals and organic solvents): photosynthetic enzyme complexes (PECs), isolated from spinach leaves and stabilized to improve their use at 22oC, one freshwater green algae specie: Chlorella vulgaris, one marine green algae, Dunaliella tertiolecta. Different experimental conditions (incubation time, illumination conditions, chlorophyll concentration and temperature) were tested to allow toxicity detection. The three systems were compared for their degree of sensitivity to the substance of interest and their capability to impact the photosynthetic yield upon quickly and with reproducibility. The difference in the experimental protocol used for persistent organic pollutants and metal cations permitted a selective detection when Chlorella vulgaris is employed as biomaterial. PS25.53 Plants without Significant Superoxide Dismutase Activity in the Chloroplast and Cytosol: Reconsideration of the Biological Role of SOD in Photosynthesis. M Pilon (Colorado State University), S Abdel-Ghany (Colorado State University), C Cohu (Colorado State University), K Gogolin (Colorado State University) The regulation and function of SOD enzymes has been studied mainly in the context of oxidative stress. We found that the availability of the Cu cofactor and not stress, is the main regulatory signal for Cu/ZnSOD and FeSOD expression in plants. When Cu is plentiful, Cu/ZnSODs are expressed and FeSOD is switched off. When Cu is limiting, plants use FeSOD and turn off Cu/ZnSOD to save Cu for use by Plastocyanin. The down-regulation of Cu/ZnSOD expression, involves a microRNA, miR398 (see abstract by Yamasaki et al.). The copper cofactor is delivered to Cu/ZnSODs by a Cu chaperone for SOD (CCS), which has isoforms both in the cytosol and plastids, but which is encoded by a single gene in Arabidopsis. A CCS-KO line, when grown on Cu-sufficient media or soil, has no measurable Cu/ZnSOD or FeSOD activity. Unexpectedly, these plants, which are without SOD activity in the cytosol or the plastids and that only have the constitutive MnSOD in the mitochondria, are phenotypically very similar to WT plants, even when stressed, as judged from growth and chlorophyll fluorescence analyses. We note that our observations of a mild phenotype contrast with a report in the literature on the function of stromal Cu/ZnSOD. However, our observations are further supported by the phenotypes of plants that have disrupted regulation of miRNA398 and by the phenotypes of Cu-transporter mutants. We propose that the function of SOD enzymes should be reconsidered: perhaps Cu/ZnSODs are more important for metal homeostasis as compared to oxidative stress responses.

imaging

to

Multicolour fluorescence (MCF) induced by UV light is a sensitive and specific tool to provide information about the primary and secondary metabolism of the plants by obtaining signals of the chlorophyll fluorescence and blue-green fluorescence, respectively. We have followed the systemic infection of Nicotiana benthamiana plants with the Pepper mild mottle virus (PMMoV) by means of a MCF set up. Blue-green fluorescence (BGF) increases mainly in the abaxial leaf side during pathogenesis and the corresponding images showed a clear vein-associated pattern in both old and young leaves of the infected plants. Chlorogenic acid was demonstrated to be the main contributor to the BGF emission in N. benthamiana plants. In addition, we have analysed the chloroplast proteome of Nicotiana benthamiana by using 2-D electrophoresis and mass spectrometry followed by database searching. To improve the resolution of the 2D gels, we have made separate maps for the low and the high pH range. At least 200 protein spots were detected and 72 identified, being some of them isoforms of different multiprotein families. In addition, changes in this chloroplast proteome induced by the PMMoV infection were investigated after 14 days post-inoculation. Viral infection induced the down-regulation of several photosynthetic proteins. L. Chaerle*, M. Pineda*, D. Van Der Straeten, M. Barón. Plant Cell Physiol. 2006. 47: 1323-1336. (*co-first authors) M. L. Pérez-Bueno, M. Ciscato, M. vandeVen, I. García-Luque, R. Valcke, M. Barón. 2006. Photosynth Res. 90: 11-123. C. Sajnani, J. L. Zurita, M. Roncel, J.M. Ortega, M. Barón, J.M. Ducruet. 2007. New Phytol. doi:10.1111/j.1469-8137.2007.02074.X

PS25.55 Plastochromanol, a ‘new’ lipophilic antioxidant in Arabidopsis leaves, is synthesized by tocopherol cyclase. The effect of high-light on the level of prenyllipid antioxidants R Szymanska (Jagiellonian University Cracow Poland), J Kruk (Jagiellonian University Cracow Poland) Plastochromanol, a γ-tocotrienol homologue with a longer side-chain, has been known for years as the component of linseed oil and a minor component of leaves of some plants, however its biosynthesis and function remains unclear. In the present study, we have identified plastochromanol in Arabidopsis leaves and this lipophilic antioxidant was shown to be synthesized by tocopherol cyclase, the key enzyme in tocopherols biosynthesis pathway, as revealed the analysis of vte1 Arabidopsis tocopherol cyclase mutant. Dissection of γ-tocopherol methyltransferase activity (vte4 mutant) did not affect plastochromanol synthesis. The analysis of age-dependent plastochromanol content in the wild-type showed that its level increases tenfold during development, reaching about 50% of total tocochromanols in three-months old plants. Tocopherols content increased 3-4 times during that time. Plastochromanol was detected also in seeds at 10% of all tocochromanols. Arabidopsis wild-type plants grown under high-light conditions for 4-5

314 14th Photosynthesis Congress - PS07 weeks showed increased level of α-tocopherol, especially in older leaves, while the higher content of plastochromanol was found in young leaves. For the vte4 mutant, high-light stimulated γ-tocopherol synthesis in both type of leaves, while plastochromanol level was increased only in young leaves but to a higher extent than in the wild-type. However, the most affected among prenyllipids was the content of plastoquinone. Its content, especially of the reduced form, increased over tenfold under high-light conditions as compared to low-light grown plants. The obtained results were discussed in terms of the antioxidant function of lipophilic antioxidants during high-light stress and the molecular mechanism of action of these compounds. PS25.56 Acclimation of Norway spruce photosynthetic apparatus to combined exposure to higher irradiance and temperature M Štroch (Ostrava University), J Podolinská (Ostrava University), D Vrábl (University of South Bohemia), J Kalina (Ostrava University), V Špunda (Ostrava University) The diurnal dynamics of photosynthetic activity, utilization of absorbed light energy within photosystem II (PSII) and xanthophyll cycle activity was studied in 4-year old seedlings of Norway spruce (Picea abies [L.] Karst.) under laboratory conditions. Plants were initially acclimated to low irradiance together with moderate air temperature (maximum PPFD at midday approx. 300 μmol m-2 s-1, maximum temperature 25 °C). Then, the irradiance was increased (maximum PPFD approx. 1000 μmol m-2 s-1), while the daily course of temperature remained unchanged. Finally, only the temperature over the entire day was increased (midday maximum 35 °C). The duration of all three acclimation regimes was 13 days. Acclimation to both elevated irradiance and elevated temperature resulted in a strong reduction of the net CO2 assimilation rate, particularly during noon hours. This reduction was associated with decreased stomatal conductance. PSII photoinhibition was observed only after the first day of exposure to increased irradiance. Acclimation to elevated temperature led even to increase of the maximal PSII photochemical efficiency (FV/FM). We conclude that under increased acclimation irradiance the enhanced PSII electron transport rate, increased efficiency of thermal energy dissipation and reduction of light-harvesting complexes contributed to the efficient photoprotection of PSII. Under combined treatment of elevated irradiance and elevated temperature the increase of photorespiration was the dominating protective process leading to the optimal function of PSII. Enhanced photorespiration resulted in the lower demand for xanthophyll cycle-dependent thermal dissipation that was associated with decreased pool of xanthophyll cycle pigments and decreased degree of violaxanthin de-epoxidation. PS25.57 Differences between rice and wheat in the temperature responses of photosynthesis and plant growth T Nagai (Tohoku University), A Makino (Tohoku University), T Mae (Tohoku University) In this study, we examined the effects of growth temperature on photosynthesis and plant growth in rice (Oryza sativa L. cv. Notohikari) and wheat (Triticum aestivum L. cv. Ias). Plants were grown hydroponically under the day/night temperature regime of 19/16°C, 25/19°C and 30/24°C.The optimal temperatures of the light-saturated rate of photosynthesis at Ci = 28 Pa (A) were 35 in rice and 25 to 30°C in wheat, respectively, and A below 25°C was always higher in wheat than in rice. These temperature responses for A wasn’t affected by growth temperature, although the photosynthetic acclimation to low temperature was observed for only the rice plant grown at 19/16° C. The

maximal plant biomass was found to be at 30/24°C in rice and at 25/19°C in wheat. Thus, rice is more adaptive to higher temperature than wheat in both photosynthesis and plant growth. However, when the temperature responses of the net assimilation rate (NAR) at the level of the whole plant were estimated, these were largely different from those of the rate of photosynthesis per unit of leaf area in both species. Therefore, the difference in the temperature responses of plant growth between rice and wheat couldn't be explained only by a difference in the temperature responses of photosynthesis. PS25.58 Suppression of non-photochemical Arabidopsis exposed to ionizing radiation

quenching

in

Y Moon (ARTI), J Kim (ARTI), J Kim (ARTI), M Lee (ARTI), B Chung (ARTI) Non-photochemical quenching (NPQ) of chlorophyll fluorescence has been known to be involved in protection of photosystems against photoinhibition through the dissipation of excess light absorbed by photosynthetic pigments. In the present study, we aimed to elucidate effect of ionizing radiation on NPQ by comparing alterations in the development and release of NPQ after gamma-irradiation between the wild-type and the npq1-2 mutant of Arabidopsis. The npq1-2 mutant cannot develop a normal NPQ under excess light, since it is defective in de-epoxidase activity for conversion of violaxanthin to zeaxanthin. Gamma-irradiation with a dose of 200 Gy inhibited the development of NPQ in both the wild-type and mutant but more noticeably in the latter. In detail, the initial rise and plateau level of NPQ were inhibited time-dependently after gamma-irradiation. In contrast, the release of NPQ was nearly unaffected in both the wild-type and mutant. Moreover, the photochemical efficiency of photosystem II (PSII), Fv/Fm, was almost the same in both the wild-type and mutant throughout the post-irradiation period of 5 d. HPLC analysis of photosynthetic pigments revealed that the inhibition of the NPQ development could be correlated with the decreasing levels in xanthophyll pigments after gamma-irradiation. The obtained results will be also discussed with those from photoinhibition induced by non-ionizing radiations such as visible light and UV-B.

PS25.59 Differential radiation sensitivities of Arabidopsis seedlings at various developmental stages J Kim (ARTI), Y Moon (ARTI), S Wi (ARTI), J Kim (ARTI), M Lee (ARTI), B Chung (ARTI) Radiation sensitivity of Arabidopsis seedlings was investigated at different developmental stages. Seedlings were exposed to γ-rays of 200 Gy at one vegetative and three reproductive stages such as 23, 30, 36, and 43 d after sowing the seeds (DAS). The growth inhibition of inflorescence stems was dependent on the stage at which the seedlings were irradiated with γ-rays. The stem growth was almost suspended in the 23-DAS seedlings during the post-irradiation period of 16 d but nearly normal in the 43-DAS ones. However, the seedlings irradiated at certain reproductive stages, 30 and 36 DAS, showed a marked delay in leaf senescence in spite of inhibited growth of inflorescence stems and abnormalities in leaf morphology. The delayed senescence in the irradiated leaves of the 30- and 36-DAS seedlings was correlated with the increased levels of chlorophylls, carotenoids, and anthocyanins. Moreover, these leaves were functionally delayed in terms of the photosynthetic activities, e.g., Fv/Fm and ETRmax. Microscopic analyses revealed that the increased numbers of chloroplasts and the integrity of thylakoid membranes contributed to the enhanced photosynthetic activities in the irradiated leaves of the 30- and 36-DAS seedlings at 16 d after the γ-irradiation, which corresponds to 46 and 52

315 14th Photosynthesis Congress - PS07 DAS, respectively. The obtained results demonstrate that ionizing radiation could induce an enhancement of photosynthetic activity through various phenotypic and physiological changes in photosynthetic apparatuses with a great dependency on specific reproductive stages.

BR> PS25.62 Plastoquinol as a singlet oxygen scavenger in photosystem II J Kruk (Jagiellonian University, Krakow, Poland), A Trebst (Bochum University, Germany)

PS25.60 Reasons for increased photosynthesis of Arabidopsis plants after gamma-irradiation at the reproductive stage J Kim (ARTI), Y Moon (ARTI), J Kim (ARTI), S Lee (ARTI), J Lee (ARTI), B Chung (ARTI) Our previous work to investigate differential radiation sensitivities of Arabidopsis plants at various developmental stages suggested that ionizing radiation could induce an enhancement of photosynthetic activity through various phenotypic and physiological changes in photosynthetic apparatuses with a great dependency on specific reproductive stages. As a further study of this work, we attempted to reveal reasons for increased photosynthesis and delayed leaf senescence in Arabidopsis plants irradiated with gamma-rays of 200 Gy at 33 d after sowing the seeds (DAS). Chlorophyll fluorescence and transmission electron microscopy (TEM) analyses indicated that the leaf senescence in the irradiated plants was actually delayed both in the functional and structural meanings. It was also shown by immunoblot analysis that core proteins of photosystems, e.g. D1 and PsaA/B, were not in progress of degradation at the later reproductive stage of 16 d after the irradiation (DAI). Moreover, quantitative real-time RT-PCR analysis showed that transcriptions of psbA, psaB, and rbcL were significantly higher in the irradiated leaves at 16 DAI than in the control ones. The obtained results suggest that increased photosynthesis of Arabidopsis plants after gamma-irradiation at the reproductive stage could be correlated with a substantial and concerted increase of gene expressions involving in both light-driven photosynthetic electron transport and stromal enzyme reactions. PS25.61 Two types of binding sites for chloride exists in PSII-enriched membranes of spinach under temperature stress A Jajoo (Devi Ahilya University), A Tiwari (Devi Ahilya University), S Bharti (Devi Ahilya University) Effects of heat were studied in photosystem II (PSII)-enriched membranes isolated from spinach in relation to Cl¯-induced activation of PSII complex. Cl¯-sufficient membranes and low-Cl¯ membranes were used. The presence of Cl¯ in the reaction medium did accelerate oxygen evolution, which remained unaffected by heat treatment up to 40ºC in PSII membranes. Heat resistance of Cl¯-induced activation of oxygen evolution was found to be independent of the presence of ‘bound Cl¯’ in the preparations. However, the functional stability of the PSII complex during heat treatment showed a marked dependence on the presence of bound Cl¯ in PSII. Electron paramagnetic resonance study of manganese (Mn) release per reaction center YD+ showed that there was little loss of Mn2+ up to 42ºC in our preparations, although the PSII activity was significantly lowered. These observations imply that the site of action of Cl¯ causing direct activation of oxygen evolution was different from the site of primary heat damage. A differential response of chloride binding sites to heat stress was observed. The high-affinity (tightly bound, slow exchanging) site of chloride is affected earlier (37ºC) while low-affinity (loosely bound, fast exchanging) site gets affected at higher temperatures (40ºC).

We have found that in control Chlamydomonas reinhardtii cells, under high-light stress, the level of reduced plastoquinone considerably increases while in the presence of pyrazolate, an inhibitor of plastoquinone and tocopherol biosynthesis, the content of reduced plastoquinone quickly decreases, similarly to α-tocopherol. Photodegradation of both prenyllipids was partially reversed by diphenylamine, an singlet oxygen scavenger. It was concluded that under high-light stress plastoquinol, as well as α-tocopherol is degraded as a result of a scavenging reaction of singlet oxygen generated in photosystem II. The lack of photodegradation of α-tocopherol and of the reduced plastoquinone in the absence of the inhibitor is due to a fast turnover of both prenyllipids, i.e., their degradation is compensated by the fast biosynthesis. We have also found that the level of α-tocopherol quinone, an oxidation product of α-tocopherol, increases as the α-tocopherol is consumed. The same correlation was also observed for γ-tocopherol and its quinone form. Moreover, in the presence of pyrazolate under low light growth conditions, the synthesis of plastoquinone-C, an hydroxylated plastoquinone derivative, was stimulated in contrast to plastoquinone, indicating for the first time a functional role for plastoquinone-C. It is also suggesting that the two plastoquinones may have different biosynthetic pathways in Chlamydomonas reinhardtii. PS25.63 Does elevated CO2 mitigate the salt effect on photosynthesis in barley cultivars? U Pérez-López (University of Basque Country), A Robredo (University of Basque Country), M Lacuesta (University of Basque Country), A Mena-Petite (University of Basque Country), A Muñoz-Rueda (University of Basque Country) Stomatal conductance and photosynthesis are very responsive to the osmotic stress caused by salinity, a major abiotic stress in agriculture worldwide. In parallel, global atmospheric carbon dioxide concentration is rising and could reach 700ppm by the end of the 21st century. CO2 enrichment also changes the behaviour of the stomas and assimilation constitutive levels. The impact of these environmental future conditions on photosynthetic response was studied in two barley cultivars, submitted to 4 salinity treatments (0, 80, 160 and 240mM) and 2 CO2 levels (350 and 700ppm) for 14 days. Under ambient CO2, salinity caused a greater decrease of CHL-a than CHL-b. At 240mM NaCl, the assimilation of CO2 decreased by 40% and 30% in Alpha and Iranis, respectively. However, when plants were submitted to elevated CO2, the reduction was less pronounced. Under the most extreme salt treatment, stomatal conductance decreased approximately a 75% in both cvs. and at both CO2 treatments. Since transpiration rate decreased more than assimilation did, increased instantaneous water use efficiency (ITE) was observed in both cvs. and for both CO2 concentrations. Under elevated CO2, photosynthesis acclimation was noticed. Nevertheless, under salinity the acclimation was reduced. This fact could suggest that plants under stressful conditions would need the consumption of extra carbohydrates, increasing sink demand, reducing thus, the acclimation. The lower decreases of pigment content and assimilation rates and the

316 14th Photosynthesis Congress - PS07 higher rates of ITE observed in plants grown under salinity and elevated CO2 would indicate a better photosynthetic capacity than their counterparts at ambient CO2.

PS25.64 Effect of Trifluoroacetic acid, a degradation product of fluorinated hydrocarbons, on photosynthesis of C3 and C4 crop plants G Krüger (North-West University), M Smit (North-West University), J Pienaar (North-West University), R Van Heerden (North-West University), L Weissflog (Helmholz Centre for Environmental Research), R Strasser (University of Geneva) TFA is a pollutant generated in the atmosphere by the degradation of hydrofluorocarbons (HFCs), as well as other anthropogenic sources such as teflon-cookware. The aim of this study was to quantify the physiological and biochemical basis of the inhibition of photosynthesis by trifluoroacetic acid (TFA) in Phaseolus vulgaris and Zea mays, representing crop plants with C3 and C4 photosynthetic pathways respectively. Photosynthetic gas exchange and fast phase fluorescence kinetics were measured in parallel over a 12 day period in plants cultivated in a hydroponic system under strictly controlled conditions. The TFA concentrations used ranged from 0.625 to 160 mg Na-TFA dm-3 corresponding to some levels found in the environment. Although initial stimulation of some photosynthetic parameters was observed at low concentrations, marked inhibition occurred at higher concentrations. The inhibitory effect was reflected by chlorophyll fluorescence and photosynthetic gas exchange derived parameters. Analysis of photosynthetic gas exchange revealed that besides constraints on mesophyll processes such as Rubisco activity and RuBP regeneration, stomatal limitation increased with TFA concentration. Although the mechanism of inhibition was similar in both species, the C4 plant were affected more severely. In depth analysis of the fast phase fluorescence transients indicated TFA-induced uncoupling of the OEC and inhibition of electron transport beyond QA including constraints on FNR-function.

PS25.65 Changes in O-J-I-P fluorescence rise kinetics during dark chilling provide insight into genotype-specific effects on photosynthesis and N2 fixation in soybean R Van Heerden (North-West University), A Strauss (North-West University), G Krüger (North-West University), R Strasser (University of Geneva) Dark chilling affects growth and yield of warm-climate crops such as soybean [Glycine max (L.) Merr.]. Several studies have investigated chilling stress effects on photosynthesis and other aspects of metabolism, but none have compared effects of whole plant chilling (shoots and roots) with that of aboveground chilling in legumes. This is important since low root temperatures might induce additional constraints, such as inhibition of N2 fixation, thereby aggravating chilling stress symptoms. Effects of dark chilling on photosystem II, shoot growth, leaf ureide content and photosynthetic capacity were studied in two soybean genotypes, Highveld Top (chilling tolerant) and PAN809 (chilling sensitive), in experiments comparing effects of whole plant chilling (WPC) with that of shoot chilling (SC). Both treatments inhibited shoot growth in PAN809, but not Highveld Top. Also, WPC in PAN809

caused a decrease in leaf ureide content followed by severe chlorosis and alterations in O-J-I-P fluorescence rise kinetics, distinct from SC. A noteworthy difference was the appearance of a ΔK-peak in the O-J-I-P fluorescence rise in response to WPC. These genotypic and treatment differences were reflected in the degree of inhibition of CO2 assimilation rates. The appearance of a ΔK-peak, coupled with growth inhibition, reduced ureide content, chlorosis and lower CO2 assimilation rates, provide mechanistic information about how WPC might have aggravated chilling stress symptoms in PAN809. We introduce a model explaining how chilling soil temperatures might trigger N-limitation in sensitive genotypes and how characteristic changes in O-J-I-P fluorescence rise kinetics are linked to changes in carbon and nitrogen metabolism.

PS25.66 Superoxide production cytochrome b559

by

photosystem

II:

role

of

C Gross (University of Freiburg), N Bondarava (University of Freiburg), A Krieger-Liszkay (University of Freiburg; CEA Saclay) At high light intensities the photosynthetic electron transport chain is saturated, leading to the production of reactive oxygen species. In addition to the Mehler reaction in photosystem I, electrons can be transferred from photosystem II to oxygen, resulting in superoxide formation. Putative electron donors are pheophytin, QA, single reduced QB or semiquinone localized in the membrane. Cytochrome b559, which is closely attached to the PSII reaction center, has also been proposed to be involved in superoxide formation. Cytochrome b559 can be oxidized or reduced in the light and its midpoint potential can vary between 0-400 mV depending on pH, the integrity of the reaction center and other factors. From a thermodynamical point of view it seems possible that the low potential form (0-80 mV) can transfer electrons to oxygen. Further we have studied a tobacco mutant with a single amino acid exchange in the ß-subunit (PsbF) of cyt b559 [1]. The mutant is able to grow photoautotrophically but is retarded and suffers from photoinhibition. Based on these characteristics we conclude that the electron transfer pathway from cyt b559 to oxygen is no longer functional, therefore no superoxide is produced by PSII. The lack of superoxide formation seems to be responsible for the increased susceptibility of PSII to high light intensities. Thus, these data support a protective role for cytochrome b559. [1] N. Bondarava, L. De Pascalis, S. Al-Babili, C. Goussias, J. Golecki, P. Beyer, R. Bock, A. Krieger-Liszkay, 2003, J Biol Chem 278, 13554-13560. PS25.67 Evidences for interaction of PsbS with photosynthetic complexes in maize thylakoids G Giacometti (University of Padova), E Teardo (University of Padova), P Polverino de Laureto (University of Padova), A Segalla (University of Padova), T Morosinotto (University of Padova), F Dalla Vecchia (University of Padova), I Szabò (University of Padova) The PsbS subunit of Photosystem II (PSII) has received much attention in the past few years, given its crucial role in photoprotection of higher plants. The exact location of this small subunit in thylakoids is also debated. Possible interaction partners of PsbS have been identified by immunoaffinity and immunoprecipitation, performed with mildly solubilized whole thylakoid membrane. Components of Photosystem II and I, of the cytochrome b6f complex as well as of ATP synthase can be co-immunoprecipitated by using highly specific anti-PsbS antibodies. Our results suggest multiple location of PsbS in the thylakoid membrane

317 14th Photosynthesis Congress - PS07 and point to an unexpected lateral mobility of this PSII subunit. As revealed by immunogold labelling with antibody against PsbS, the protein is associated either with granal membranes or prevalently with stroma lamellae in low or high-intensity light-treated intact leaves, respectively. This finding is consistent with the capability of PsbS to interact with complexes located in stroma lamellae. Interactions are being further confirmed by cross-linking experiments under various physiological conditions. PS25.68 The rate of photosynthesis remains relatively high at moderately high temperatures in Arabidopsis thaliana rca mutant expressing thermostable chimeric Rubisco Activase. A Portis Jr (United States Department of Agriculture, Agricultural Research Service), A Kumar (United States Department of Agriculture, Agricultural Research Service), C Li (Institute of Human Virology) The rate of photosynthesis declines at moderately high temperatures (30-42oC) in temperate plants like Arabidopsis. The decline is due to deactivation of Rubisco which in turn is due to a reduced ability of activase to activate Rubisco (Crafts-Brandner and Salvucci, PNAS 97:13430-13435, 2000). We created a more thermostable activase for Arabidopsis by replacing the Rubisco recognition domain in the more thermostable tobacco activase with that from Arabidopsis. In vitro studies confirmed that the chimeric activase is more thermostable that the native Arabidopsis activase. We than transformed the Arabidopsis rca mutant and selected lines with high expression of this chimeric activase. The initial studies indicate that the rate of photosynthesis as measured by gas exchange is higher than the wild type in the transgenic lines at both 30 and 38oC. The recovery of photosynthesis is also better in the transgenic lines when the plants are returned to lower temperature (23oC).

PS25.69 Effects of hypergravity on the chlorophyll content and growth of root and shoot during development in rice plants P Vidyasagar (University of Pune), S jagtap (University of Pune), A Nirhali (University of Pune), S Bhaskaran (University of Pune), V Hase (University of Pune) Gravitational force on the earth is a pervasive environmental parameter that affects directly or indirectly, virtually all life on the planet. Establishing the role of gravity in regulating the basic life processes in plants requires information about how these processes are affected under micro and hypergravity conditions. Earlier studies on hypergravity effects showed modification in the metabolism of cell wall components, promotion of metaxylem development and decrease in extensibility of secondary cell walls in Arabidopsis thaliana. In the present study, the effects of hypergravity on the growth and development of rice seeds have been studied. Rice seeds (PRH-10 obtained from National Seeds Corporation, Govt. of India) were suspended in water in a test tube and were exposed to hypergravity ranging from 500-3000g for 10 minutes. Seeds for each value of g were grown on 0.8% agar under ambient conditions and light intensity of 1250 Lux for 16 hours per day. Seeds unexposed to hypergravity grown under the same conditions acted as control. Length of roots and shoots were measured. Chlorophyll was extracted on the 5th day and absorption and fluorescence spectra were recorded in both control and hypergravity samples. The cross sections of the roots were obtained and studied under the microscope. The results obtained showed that the chlorophyll content was less in the samples exposed to hypergravity. The roots showed changes in the diameter of cells at the core. These and similar results will be discussed.

PS25.70 Overexpression of Protochlorophyllide oxidoreductase C Results in Tolerance to Singlet oxygen-mrdiated Oxidative Stress in Plants B Tripathy (Jawaharlal Nehru University), G Pattanayak (Jawaharlal Nehru University) Arabidopsis thaliana protochlorophyllide oxidoreductase C gene (porC) that codes for the mature part of PORC protein having 335 amino acids was overexpressed in Arabidopsis by Agrobacterium mediated transformation. Transgenic plants accumulated higher chlorophyll than their wild types. Overexpression of porC resulted in efficient metabolisim of Chl biosynthetic intermediates to Chl and consequent reduced accumulation of Chl biosynthetic intermediate i.e., Pchlide. When WT plants were exposed to light stress (330 μmoles photons m-2 s-1) they turned purple due to increased accumulation of anthocyanin. Under identical conditions PORCx plants were green and accumulated small amounts of anthocyanins. PORCx plants in light stress accumulated smaller amounts of Pchlide leading to reduced accumulation of singlet oxygen and minimal expression of CHS gene. Singlet oxygen caused substantial damage to the photosynthetic apparatus leading to loss of Fv/Fm, electron transport rate, quantum yield of PSII and photochemical quenching in WT plants. Light stress did not affect the photosynthetic function of PORCx plants. The 5-aminolevulinic acid (ALA), a precursor of chlorophylls, when sprayed at sunset induced plants to accumulate excess protochlorophyllide (Pchlide). In the morning when the Sunlight fell on the plants, most of Pchlide due to limitation of POR were not photo-transformed to Chlide and acted as the photo-sensitizer. Non-photo-transformed Pchlide generated singlet oxygen that killed the WT plants. When ALA was sprayed on WT and transgenic plants the latter very efficiently converted the photosensitizer Pchlide to Chlide due to over-expressed PORC. Therefore, the transgenic plants had only marginal damage due to the herbicide ALA. PS25.71 A rapid method for determining the fraction of functional Photosystem II in whole leaf tissue after photoinhibition W Chow (Australian National University), P Losciale (University of Bologna), R Oguchi (University of Tokyo), A Hope (Flinders University), L Corelli-Grappadelli (University of Bologna), L Hendrickson (Australian National University) Assaying the number of functional Photosystem II (PSII) complexes by the oxygen yield from leaf tissue per saturating, single-turnover flash (assuming that each functional PSII evolves one oxygen molecule after four flashes) is one of the most direct methods but time-consuming. The ratio of variable to maximum chlorophyll fluorescence (Fv/Fm) in leaves can be correlated with the oxygen yield per flash, and is rapid and non-intrusive, but suffers from being only representative of chloroplasts near the leaf surface; consequently, the exact correlation depends on the internal leaf structure and on which leaf surface is being measured. Our results show that the average Fv/Fm of the adaxial and abaxial surfaces has a reasonable linear correlation with the oxygen yield per flash after varied extents of photoinactivation of PSII. However, we obtained a much better linear correlation between (a) the integrated, transient electron flow to P700+ (S) after superimposing a single-turnover flash on steady background far-red light and (b) the relative oxygen yield per flash. Leaves of C3 and C4 plants, woody and herbaceous species, wild type and a chlorophyll b-less mutant, and monocot and dicot plants gave a single straight line, which seems to be a universal relation for predicting the relative oxygen yield per flash. Measurement of S is non-intrusive, rapid and applicable to attached leaves; it may even be applicable in the field in future.

318 14th Photosynthesis Congress - PS07 transport was inhibited by chromium. PS25.72 Effects of nitrogen and/or sulphur deprivation on the regulation of photosynthesis in barley seedlings L Vitale (Dipartimento di Biologia Strutturale e Funzionale, Universita' di Napoli Federico II), S Carfagna (Dipartimento delle Scienze Biologiche, Universita' di Napoli Federico II), S Esposito (Dipartimento di Biologia Strutturale e Funzionale, Universita' di Napoli Federico II), C Arena (Dipartimento di Biologia Strutturale e Funzionale, Universita' di Napoli Federico II) The present study investigates the effects of nitrogen and/or sulphur deprivation on photosynthetic apparatus in barley seedlings grown in hydroponics culture. For this reason four different growth conditions were induced at 100 PFD and 25°C: Control, -N, -S, -N-S and simultaneous measurements of gas exchange and chlorophyll a fluorescence were performed as well as total protein content, glutathione and cysteine levels in leaves were determined. No difference was observed in AN, qP and ETR/AN among -N, -S and control (C) plants, differently from NPQ that increased significantly in –S compared to C and –N. In -N-S plants a reduction of AN and qP as well as a significant increase of NPQ and ETR/AN were found compared to other plant groups; moreover high levels of glutathione and cysteine were detected. Among the different growth conditions,–N and –N-S plants, exhibited the lowest total protein content. Nevertheless, despite the strong AN decline in –N–S condition, the maximum photochemical PSII efficiency (Fv/Fm) resulted the same for all plant groups. Data indicate that the simultaneous starvation of N and S leads to an alteration of plant metabolism that affects photosynthesis. However, since no difference in maximum photochemical efficiency was found, it may be hypothesized that in –N–S seedlings processes other than photosynthesis and thermal dissipation are effective in photoprotection at low light too. In -N-S plants, the highest glutathione content could represent a mean to face oxidative stress triggered within photosynthetic membranes by carbon fixation decline. PS25.73 Alteration of energy dissipation by chromium effect in xanthophylls deficient mutant of Chlamydomonas reinhardtii F Perreault (University of Quebec in Montreal, Department of Chemistry, TOXEN), N Ait Ali (University of Quebec in Montreal, Department of Chemistry, TOXEN), C Saison (University of Quebec in Montreal, Department of Chemistry, TOXEN), P Juneau (University of Quebec in Montreal, Department of Biology, TOXEN), R Popovic (University of Quebec in Montreal, Department of Chemistry, TOXEN) Energy dissipation via Photosystem II and I activity was investigated when xanthophylls deficient mutant of Chlamydomonas reinhardtii was exposed to chromium effect. To determine simultaneous chromium and light intensity effect on PSII and PSI functional properties, algal mutant lacking xanthophylls cycle was exposed for 24h to different chromium concentration. Methodological approach was based on Dual-PAM-100 fluorimeter, western blot analysis and determination of free radicals formation. For energy dissipation via Photosystem II and I we assumed to be an addition the operational quantum yield + regulated non-photochemical quenching + non-regulated non-photochemical quenching = 1. For xanthophyll deficient mutant npq1, non-regulated non-photochemical energy dissipation was the highest when electron transport was completely inhibited by chromium while regulated non-photochemical quenching was the highest when chromium inhibition of PSII-PSI was partial. Strong photo inhibitory effect and formation of free radicals were observed when algal mutant was exposed to chromium effect and high light intensity. In this report, we discussed possible model explaining xanthophylls role when PSII-PSI electron

PS25.74 Drought Resistance Ranking of four Wheat Cultivars Probed by the fast Fluorescence Rise OJIP I Sabour (université libre de Bruxelles), M Eyletters (Université Libre de Bruxelles), A Oukarroum (University of Geneva), R Strasser (University of Geneva) The progression of desertification and global warming forces the farmers to select new cultivars, which are optimally adapted to the local climate. Usually there are many new lines of Wheat available, but their performance as a function of the changing climate is mostly unknown. Standard testing needs a lot of time and labor. Therefore only a few lines can be really tested. An alternative is to use biophysical essays which are fast and easy to do on many plants as seedlings with many repetitions permitting to do statistical treatments. These measurements however are indirect and the results and conclusions have to be considered as good guesses which increase the chance to select the optimal cultivars for field trials. The fast fluorescence transient of seedlings allows the determination of a so called Drought Factor Index DFI. This makes it possible to test and rank many cultivars for drought resistance. We investigated four local lines of Wheat from Morocco, two hard and two soft grains. The fluorescence data confirmed the classification done empirically by the local farmers. Data of two levels of drought stress and for two durations will be shown and discussed. The determination of specific and phenomenological fluxes and quantum yields and the determination of sensible inhibition sites using the JIP-test equations allow making some propositions about the acting reaction mechanism.

PS25.75 Suppression of non-photochemical quenching in Arabidopsis exposed to ionizing radiation J Kim (ARTI) Non-photochemical quenching (NPQ) of chlorophyll fluorescence has been known to be involved in protection of photosystems against photoinhibition through the dissipation of excess light absorbed by photosynthetic pigments. In the present study, we aimed to elucidate effect of ionizing radiation on NPQ by comparing alterations in the development and release of NPQ after gamma-irradiation between the wild-type and the npq1-2 mutant of Arabidopsis. The npq1-2 mutant cannot develop a normal NPQ under excess light, since it is defective in de-epoxidase activity for conversion of violaxanthin to zeaxanthin. Gamma-irradiation with a dose of 200 Gy inhibited the development of NPQ in both the wild-type and mutant but more noticeably in the latter. In detail, the initial rise and plateau level of NPQ were inhibited time-dependently after gamma-irradiation. In contrast, the release of NPQ was nearly unaffected in both the wild-type and mutant. Moreover, the photochemical efficiency of photosystem II (PSII), Fv/Fm, was almost the same in both the wild-type and mutant throughout the post-irradiation period of 5 d. HPLC analysis of photosynthetic pigments revealed that the inhibition of the NPQ development could be correlated with the decreasing levels in xanthophyll pigments after gamma-irradiation. The obtained results will be also discussed with those from photoinhibition induced by non-ionizing radiations such as visible light and UV-B. PS25.76 Remote monitoring of cold and light stress induced effects on photosynthesis using laser induced fluorescence transient (LIFT) technique

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R Pieruschka (Carnegie Institution of Washington), D Klimov (Monterey Bay Aquarium Research Institute), U Rasher (Forschungszentrum Jülich), Z Kolber (Monterey Bay Aquarium Research Institute ), J Berry (Carnegie Institution of Washington) The interaction of plants with their environment is a very dynamic and variable system. Studying the underlying processes is important for understanding and modeling plant stress response to changing environmental conditions. Stress impact on photosynthesis includes damage and repair mechanisms acting simultaneously and leaves at different locations within a canopy experience different stress levels. Thus, continuous and spatially distributed monitoring is necessary to assess the dynamic response of plants. Yet, limited access to many canopies and scale of observation with portable instrumentation make it difficult to examine stress responses at canopy or ecosystem scale. We report here on the application of a recently developed technique, Laser Induced Fluorescence Transient (LIFT), for continuous remote measurement of photosynthetic efficiency of selected leaves at a distance of up to 50 m. The LIFT approach was tested in a field experiment monitoring the combined effect of low temperatures and high light intensity on four different plant species during the early winter in California. We observed a reduction in maximum and effective quantum yield, in quantum efficiency of electron transport and, absorption cross section of photosystem II as the temperatures fell. These measures with the LIFT correlated well with (more limited) sampling by pulse amplitude modulated (PAM) fluoromentry and gas exchange. The ability to make continuous, automatic and remote measurements of photosynthetic efficiency of leaves with the LIFT provides a new approach for studying the heterogeneity of stress effects and for integrating these effects from the leaf to the canopy scale. PS25.77 Action Spectrum of Photoinhibition In Vivo Measured from the Wild Type and npq1-2 and npq4-1 Mutants of Arabidopsis thaliana P Sarvikas (University of Turku), E Tyystjärvi (University of Turku), M Hakala (University of Turku), E Pätsikkä (University of Turku), T Tyystjärvi (University of Turku) Photoinhibition is light-induced inactivation of Photosystem II (PSII). We measured the action spectrum of photoinhibition from leaves of wild type Arabidopsis thaliana L. and from the npq1-2 and npq4-1 mutants defective in nonphotochemical quenching (NPQ) that converts chlorophyll excitations to heat. The in vivo action spectrum of photoinhibition was found to closely resemble earlier in vitro action spectra, and comparison with the potential photoreceptors of photoinhibition suggests that both manganese ions of the oxygen-evolving complex and chlorophylls of PSII antenna function as photoreceptors of photoinhibition. In accordance with the function of two types of photoreceptors in photoinhibition, NPQ was found to offer only partial protection (20-25 %) against visible-light-induced photoinhibition. The low protective efficiency of NPQ suggests that the chlorophyll antenna of PSII is not the principal photoreceptor of photoinhibition. Comparison of the action spectrum of photoinhibition with the emission spectrum of sunlight shows that under natural conditions the UV part of sunlight can be responsible for major part of photoinhibition. PS25.78 Elevated zeaxanthin bound to oligomeric LHCII enhances the resistance of Arabidopsis to photo-oxidative stress by a lipid-protective, anti-oxidant mechanism M Johnson (Department of Molecular Biology & Biotechnology, University of Sheffield), M Havaux (CEA-Cadarache, France), C

Triantaphylides (CEA-Cadarache, France), B Ksas (CEA-Cadarache, France), A Pascal (CEA-Saclay, France), B Robert (CEA-Saclay, France), P Davison (Department of Molecular Biology and Biotechnology, University of Sheffield), A Ruban (School of Biological & Chemical Sciences, Queen Mary College, London), P Horton (Department of Molecular Biology and Biotechnology, University of The xanthophyll cycle has a major role in protecting plants from photo-oxidative stress, although the mechanism of its action is unclear. Here, we have investigated Arabidopsis plants overexpressing a gene encoding β-carotene hydroxylase, containing nearly three-times the amount of xanthophyll cycle carotenoids present in the wild-type. In high light at low temperature wild-type plants exhibited symptoms of severe oxidative stress - lipid peroxidation, chlorophyll bleaching and photoinhibition. In transformed plants, which accumulate over twice as much zeaxanthin as the wild-type, these symptoms were significantly ameliorated. The capacity of non-photochemical quenching is not significantly different in transformed plants compared to wild-type and therefore an enhancement of this process cannot be the cause of the stress tolerant phenotype. Rather, it is concluded that it results from the anti-oxidant effect of zeaxanthin. 80-90% of violaxanthin and zeaxanthin in wild-type and transformed plants was localized to an oligomeric LHCII fraction prepared from thylakoid membranes. The binding of these pigments in intact membranes was confirmed by resonance Raman spectroscopy. Based on the structural model of LHCII, we suggest that the protein/lipid interface is the active site for the anti-oxidant activity of zeaxanthin, which mediates stress tolerance by the protection of bound lipids.

PS25.79 Response to ozone of beech (Fagus sylvatica L.) seedlings under competition, in an open-top chamber experiment. A chlorophyll fluorescence analysis F Bussotti (University of Florence (IT)), C Cascio (University of Florence (IT)), R Strasser (University of Geneva (CH)) This study was carried out within the open-top chamber (OTC) research facility of the Swiss Federal Institute WSL within the Lattecaldo forest nursery in Southern Switzerland. The setting of the experimental site an the ground level of ozone were described in previous papers (VanderHeyden et al., 2001). The experiment performed in the year 2004 consisted in small populations of beech (Fagus sylvatica L.) seedling (2-years old) growing in pure culture and under the competition of viburnum (Viburnum lantana L.) seedlings, in sub-plots across 4 non filtered (NF, i.e. treated with ambient air) and 4 charcoal-filtered (CF, i.e. treated with air with 50% of the ambient air ozone). Chlorophyll a fluorescence in the PS II was measured once a month from June to September 2004 with a direct fluorimeter HandyPea (Hansatech, UK). The rising fluorescence transients were analysed by means of the JIP-test (Strasser et al., 2000). The PS II efficiency tended to decrease over the time in the NF chamber respect to the CF ones, on the beech plants growing without competition, but a slight increase was evidenced the first months. On the other hands, the presence of Vibrnum lantana (that grows faster than beech) seemed to protect the photosynthesis machinery of the beech plants growing under competition. These results are discussed in relation to the importance of local conditions to protect the renewal of the forest against ozone. References: Strasser A. et al., 2000. In: Yunus M, Pathre U, Mohanty P (eds). Probing photosynthesis: mechanisms, regulation and adaptation. London, UK.: Taylor & Francis, pp. 445-483. VanderHeyden D.J. et al., 2001. Env.Poll. 111: 321-331. PS25.80 Water use and water use efficiency in two durum wheat

320 14th Photosynthesis Congress - PS07 cultivars contrasting for yield capacity F Rizza (C.R.A. Istituto Sperimentale per la Cerealicoltura, Fiorenzuola d'Arda, Italy), L Matteu (C.R.A. Istituto Sperimentale per la Cerealicoltura, Foggia, Italy), J Ghashghaie (Laboratoire d’Ecologie, Systématique et Evolution (ESE), Université de Paris-Sud ,France), A Mastrangelo (C.R.A. Istituto Sperimentale per la Cerealicoltura, Foggia, Italy), A Stanca (C.R.A. Istituto Sperimentale per la Cerealicoltura, Fiorenzuola d'Arda, Italy), F Badeck (Potsdam Institute for Climate Impact Research The aim of this work was to analyse the response to water stress in two durum wheat cultivars (“Ofanto” and “Cappelli”) employed as parents of a segregating population, being contrasting for their yield performance. In a growth chamber experiment plants were grown at early stage under dryand irrigated conditions. Gas exchange and mass accumulation were measured throughout the experiment. “Ofanto” consumed more water and used soil water resources more rapidly on incipient drought. Consistently “Ofanto” showed a higher stomatal conductance, resulting in a lower water use efficency (WUE), compared to “Cappelli”.The different behaviour of the two genotypes was consistent with the results of field experiments conducted at Foggia (Southern Italy) in rainfed conditions and with supplementary irrigation. Leaf temperature measured at anthesis on flag leaf using an infrared thermometer showed a trend for lower leaf temperatures in “Ofanto”. Carbon discrimination (D), analysed in the grains harvested from the field trials as a measure of the integrated WUE during the kernel development (Farquahar and Richards, 1984 Aust. J. Plant Physiol. 11: 539-552, 1984), was significantly higher in “Ofanto” than in “Cappelli”, in both rainfed and irrigated trials. The overall results agree in suggesting a constitutive difference in stomatal responses in “Ofanto” and “Cappelli”, with consequences for water use efficiency. These traits together with other physiological and yield associated traits will be studied in the segregating population to verify their association and their contribution to yield stability in presence of water stress.

PS25.81 Analysis of the variability of photosynthesis parameters in tree seedlings under ozone and water stress conditions. C Cascio (University of Florence, IT), F Bussotti (University of Florence, IT), R Strasser (University of Geneva, IT) The analysis of the variability of the photosynthesis properties and responses (within and among individuals), is important in assessing the impact of stress factors in plant population and communities, as well as in supporting remote sensing techniques in stress assessment. Photosynthesis parameters, namely the Performance Index (PIABS), assessed by direct fluorescence techniques, were taken over several measurement campaigns in open-top chamber experiments at Lattecaldo (South Switzerland) and Curno (North Italy), from 2001 to 2006. These experiments consisted in treatments of seedling belonging to different tree species, with non filtered (NF, i.e. treated with ambient air) and charcoal-filtered (CF, i.e. treated with air with 50% of the ambient air ozone) air, and were aimed to detect the impact of ground levels of ozone and their interaction with water shortage conditions. The variability of PIABS was tested by mean of the coefficient of variation (CV = st./dev/Mean expressed as percent). CV changes over time, and it is higher on the begin of the season and lower in the central months of the summer. CV was usually higher in stressful conditions (NF chambers vs. the CF ones; D vs. W conditions) and is negatively correlated with PIABS. This finding indicates that moderate stress factors act selectively on different individuals, and differential responses are enhanced. The negative effects of ozone and drought were evidenced only on few members of the population.

PS25.82 Phenotypic response of PSII photochemistry to low temperature: comparison between winter and spring barley (Hordeum vulgare) genotypes F Rizza (C.R.A. Istituto Sperimentale per la Cerealicoltura, Fiorenzuola d'Arda, Italy), D Pagani (C.R.A. Istituto Sperimentale per la Cerealicoltura, Fiorenzuola d'Arda, Italy), F Badeck (Potsdam Institute for Climate Impact Research (PIK), Potsdam, Germany), A Soltész (Agricultural Research Institute of the Hungarian Academy of Sciences (MTA), Martonvásár, Hungary) A key element of plant adaptation to cold is represented by the capacity for morpho-physiological adjustments that allow herbaceous plants to retain growth and photosynthetic capacity at low temperature. Short-term and long-term adjustments in photosynthesis contribute to re-establish the balance between photophysical and photochemical processes transforming light and energy used by metabolic sinks (Ensminger et al., Phys.Plant. 126:28-44, 2006). The genotypic variability for traits related to these mechanisms is little explored in cereals. We show a first characterization of nine barley cultivars of different origin and winter hardiness. Plants were subjected to hardening temperatures for 0 to 11 weeks. Adjustments in photosynthetic characteristics were monitored in the last developed leaf by chlorophyll fluorescence analysis and correlated to growth. Results show genetic variability in development and photosynthesis-related traits as a result of both constitutive and cold-induced mechanisms. In all tested genotypes Fv/Fm decreased in a reversible way after exposure to hardening. Cold-induced changes in the actual quantum yield of the PSII photochemisrty (FPSII) were more pronounced in winter than in spring genotypes showing a significant decrease during the first period at 3°C, a subsequent return to the initial state, (at about 25 days) and a final increase. This higher PSII quantum efficiency reflects an improved capacity for photosynthetic electron transport, that may be ascribed to a more efficient sink activity. PS25.83 The Nostoc sp. PCC7120 CP43s Y Gerchman (Haifa University in Oranim) Nostoc sp. PCC 7120 (a.k.a. Anabaena PCC 7120) is a filamentous, heterocystous cyanobacteria. Its full genome was published in 2001, and revealed, among others, five psbA genes, coding for three versions of D1 - the main photosystem II protein, and five psbC genes coding for five different versions of CP43 like proteins. The CP43 protein is believed to be involved in light harvesting and transfer to the PSII reaction center. A shorter version, IsiA, has been found to express under iron stress and create a ‘light harvesting complex like’ structure around PSI. Of the five different predicted 7120 CP43s one is highly homologue to the Synechocystis sp. PCC 6803 CP43 while another to the Synechocystis IsiA. The other three are of unknown function and intermediate sequence homology, missing the CP43s 'E loop', suggesting that like IsiA they probably associate with Photosystem I. We are currently looking into the role of the different CP43s in the life of Nostoc 7120 PS25.84 Chlorophyll fluorescence and photosystem II activity of tomato leaves as affected by Prohexadione-calcium and irradiance A Giannakoula, I Ilias (Aristotle University of Thessaloniki) Abstract: The objective of this research was to investigate the effects of growth retardant Prohexadione-Calcium on the growth and physiological

321 14th Photosynthesis Congress - PS07 parameters of tomatoes (Lycopersicon esculentum Mill., cv Karla and cv. Hari Moran) grown under a glass greenhouse environments. Main stem length of tomatoes plants decreased in a quadratic pattern as the concentration of Prohexadione-Ca increased. In tomatoes cv. ‘Carla’ and cv. ‘Hari Moran’, 100 mgl-1 resulted in 14.5 and 12.1% shorter plants respectively. Higher concentrations (200 and 300 mgl-1) resulted in even shorter plants than control plants. The application of 300 mgl-1 of Prohexadione-Calcium resulted in diminution of the leaf chlorophyll concentration, and also the values of chlorophyll fluorescence of the leaves were significantly lower compared to control. In both cultivars Prohexadione-Calcium at 300 mgl-1 significantly affected chlorophyll fluorescence parameters (Fo, Fm, Fv, Fv/Fm, Fv/Fo). Electron transport rate (ETR), real photochemical efficiency of PSII ( FPSII ), photochemical quenching (qP) and non-photochemical quenching (qN), were recorded under different irradiances (66, 96, 136, 226, 336, 536, 811, 1211, 1911 and 3111µmol m s, I66 to I3111, respectively) with PAM-2000 fluorometer. Key Words. Lycopersicon, Prohexadione-calcium, chlorophyll fluorescent, Growth inhibition growth retardant . photosystem2, photoinhibition PS25.85 Altered gene expression in cold adapted and cold sensitive algae can be studied using microarrays R Hutchison (Richard Stockton College), T Nguyen (Richard Stockton College), A Yekkirala (North Dakota State University), M Anderson (North Dakota State University)

of both known and unknown genes that have been annotated. We are developing a resource for such functional genomic analysis of photosynthesis using insertional mutagenesis of the nuclear genome. We aim to generate a total of 80,000 insertional mutants in Chlamydomonas and are subjecting these to a number of phenotypic screens. To date, 45,000 insertional mutants have been generated and screened to identify ~2500 mutants that are pigment deficient, light sensitive, non-photosynthetic, or hypersensitive to reactive oxygen species. As flanking sequence from each mutant exhibiting a phenotype becomes available, it is marked on a track named ChlreInsMut in version 3.0 of the JGI genome browser. Individual mutants are currently available to researchers as mated zygospore stocks and cell streaks. The work presented at this meeting will detail how researchers can find flanking sequence data and information on the phenotype of each mutant. Two mutants representing insertions in one known and one unknown gene will be described in detail in order to highlight the different uses of the population. This work is supported by the National Science Foundation (grant no. MCB-0235878). PS25.87 The Concentration of the Target State of Laser-Pulse-Induced Photoinhibition Increases in the Dark M Hakala (University of Turku), M Keränen (University of Turku), T Tyystjärvi (University of Turku), L Khriachtchev (University of Helsinki), E Tyystjärvi (University of Turku) We measured photoinhibition of Photosystem II (PSII) in thylakoids illuminated with saturating laser pulses. Photoinhibitory efficiency was found to increase in proportion to the square of the pulse energy. This result cannot be explained by assuming that photoinhibition is caused by singlet oxygen produced in the charge recombination reactions. We also treated thylakoids with pulse doublets in which the first pulse oxidized the primary donor P680 and the second pulse hit PSII before reoxidation

Microarrays for the model organism Chlamydomonas reinhardtii have been developed and can be used to study the effects of stress adaptation at the gene expression level. In this poster, a cDNA microarray from Chlamydomonas reinhardtii was cross-hybridized with a cold-adapted chlamydomonad, “Chlamydomonas” cf. altera. in order to understand how these organism might differ in their response to low temperatures. For the microarray experiments, algal cultures were grown alternatively at 20˚C (normal temperature) and 4˚C (cold stress temperature). The first treatment comprised a 1 day chilling (4˚C) period and the second treatment, a 7 day chilling (4˚C) period. C. reinhardtii was grown at 20˚C and used as the control for both treatments. We found that all the expressed spots showed hybridization of both the probes in all treatments and the number of good spots averaged more than 92% for all the experiments. Quantitative PCR analyses performed on selected genes from both algae were consistent with the microarray results. Overall, those genes associated with metabolic processes (such as photosynthesis and respiration) were up-regulated in the cold-adapted algae after 24 hours of 4oC exposure, and returned to normal levels after 7 days of cold temperature exposure. The pattern for the cold-sensitive Chlamydomonas reinhardtii was different, up-regulating the same metabolic genes only after 7 days. Our work shows that cross-species hybridization of Chlamydomonas reinhardtii cDNA microarrays with other related chlamydomonads is possible.

of P680+. The second pulse was found to be equally photoinhibitory as the first pulse, suggesting that photoinhibition does not depend on electron transfer via P680. UV pulses were more photoinhibitory than visible-light pulses. As manganese absorbs UV light better than visible light, we suggest that the Mn ions of the oxygen-evolving complex act as photoreceptors of laser-pulse-induced photoinhibition, similarly as they function under continuous light. Pulses fired with long pulse-to-pulse intervals are more photoinhibitory than pulses fired with short intervals. This finding and the pulse intensity dependence of the photoinhibitory efficiency indicates that the target state of laser-pulse photoinhibition increases with the recombination reactions that increase the concentration of the S1 state during the pulse-to-pulse interval. A similar pulse interval dependency of photoinhibitory efficiency was seen also under UV pulses, suggesting that the target of laser-pulse photoinhibition is the same whether UV or visible pulses are used. We suggest a model that covers photoinhibition under laser pulse illumination, under continuous light and under the combination of these two.

PS25.86 Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii

PS25.88 Gaba and hormone responses of sesame plant (Sesamum indicum L.) under different abiotic stresses

R Dent (University of California), M Kobayashi (University of California), K Niyogi (University of California)

M Bor (Ege University), F Özdemir (Ege University), I Türkan (Ege University)

As the sequencing and annotation of the full genome of the eukaryotic green alga Chlamydomonas reinhardtii nears completion, researchers are looking to new horizons that have been made available by this milestone. One such uncharted territory is the functional characterization

Non protein amino acid 4 amino butyrate gamma aminobutyrate (GABA) is found in a large range of organisms including bacteria, yeasts, plants and animals. GABA has relations with regulation of cytoplasmic pH, Ca+2 signalling and defense system against stressors

322 14th Photosynthesis Congress - PS07 including heat shock, wounding, anoxia and water stress in plants. Although the enzymatic and regulatory steps for GABA synthesis are well known in plants, the implication of GABA accumulation in response to different stressors and plant hormones remains unclear. We aimed to study the relation between GABA, auxins, gibberellins, abscisic acid and cytokinins in Sesame plant under PEG mediated drought, salt stress, high temperature and heavy metal stresses. Plant growth responses, photosynthesis, GABA and hormone level changes will be investigated and discussed under different stress conditions. PS25.89 Effect of herbicides (diuron and oxadiazon) on photosynthetic energy dissipation processes of different species of cyanobacteria C Deblois (University of Quebec in Montreal), B Qiu (Central China Normal University), P Juneau (University of Quebec in Montreal) Blooms of cyanobacteria are a major concern for freshwater ecosystems because of the cyanotoxin production and economic impact of these blooms. Despite a better understanding of the role of environmental factors such as nutrient and light availability on cyanobacteria proliferation, little is known about the physiology of cyanobacteria and their photosynthetic activity. Understanding photosynthesis of these organisms can be a major step in the comprehension of toxic cyanobacterial blooms. By using Pulse Amplitude Modulated fluorometry (PAM), rapid rise fluorescence (PEA) and Fluorescence Induction and Relaxation system (FIRe), we have investigated the energy dissipation processes in species from three genera of cyanobacteria (Synechococcus sp., Synechocystis sp. and Microcystis aeruginosa) when exposed to two herbicides having different modes of action (diuron and oxadiazon). For all studied species, the maximal photosystem II quantum yield (FM) was not affected by diuron, but the operational photosystem II quantum yield (F'M) was decreased by 50% at 48, 5.0 and 2.7 nM diuron for M. aeruginosa, Synechocystis sp. and Synechoccocus sp., respectively. In presence of 2.89 µM oxadiazon (maximum solubility in water), the only affected specie was Synechoccocus sp. (10-15 % decrease for F'M). These data showed that cyanobacteria have different sensitivity to herbicide effect. Our results concerning higher tolerance of M. aeruginosa may explain why it is one of the most frequently reported species in cyanobacterial bloom events. PS25.90 Effect of light intensity on energy dissipation processes in photosynthesis of the freshwater diatom Cyclotella sp. G Vernouillet (University of Quebec in Montreal), C Deblois (University of Quebec in Montreal), P Juneau (University of Quebec in Montreal) Phytoplankton has developed strategies, at the photosynthetic apparatus level, to cope with fluctuating light intensities found in their environment. These strategies will be reflected in energy dissipation via photochemical and non-photochemical pathways. Nevertheless, even in presence of protection mechanisms excessive light can produce modification of the photosynthetic apparatus. By using Pulse-Amplitude-Modulated fluorometry and rapid rise fluorescence (PEA) we investigated the effect of high light intensity on a diatom, Cyclotella sp.. Dark-adapted Cyclotella sp. previously exposed to high light intensity (1000 µE m-2 s-1) demonstrated a strong diminution of fluorescence levels FO and FM compared to algae exposed to 75 µE m-2 s-1. The use of NH4Cl, as an uncoupler which eliminates the pH gradient in thylakoid membranes, restored the FM level, therefore suggesting the presence of non-photochemical quenching. The mechanism, by which this non-photochemical quenching was built, was further studied by

using different inhibitors of the photosynthetic and respiratory systems permitting to discuss about a possible model of energy dissipation processes triggered under strong light intensity. PS25.91 Time scale of the appearance of Cd photosynthetically competent poplar leaves

effects

on

F Láng (Eötvös University), P Szegi (Eötvös University), B Basa (Eötvös University), Á Solti (Eötvös University), L Gáspár (Eötvös University), L Tamás (Eötvös University), I Mészáros (Eötvös University), É Sárvári (Eötvös University) Cd exposure causes diverse effects on plant growth and metabolism including photosynthesis. However, direct effects have not been revealed yet. Trying to resolve the problem we followed the appearance of Cd effects on photosynthetic performance of the third leaf of hydroponically cultured poplar (Populus glauca var. Kopeczkii) plants during a two-week treatment with 10 µM Cd(NO3)2. Growth and Chl concentration of leaves were affected only slightly. Decrease of Chl a/b ratio started at the 3rd day. The accumulation of LHCII and PSI were slightly diminished. Quantitative real-time RT-PCR studies referred to regulation of Lhca1-3,5 genes and their Lil1 (ELIP) relative at transcriptional level. While the transcript level of Lhca1-3 genes decreased gradually, a transient increase in those of Lhca5 and Lil1 were detected at the beginning of Cd treatment. The amount of PSII core was reduced more strongly. However, the actual quantum efficiency of PSII did not change in spite of an early (after 3 days) decrease in CO2 fixation, and reduced stomatal conductance. Ascorbate peroxidase (APX) activity and malondialdehyde content showed a later decrease and increase, respectively. In conclusion, decreased CO2 fixation/stomatal conductance and thylakoid reorganization were the first detectable symptoms in the case of photosynthetically competent leaves. This work was supported by grant T-043646 (OTKA). PS25.92 Role of plastoquinone redox state in plant responce under high temperature N Pshybytko (National Academy of Sciences of Belarus), J Kruk (Jagiellonian University), L Kabashnikova (National Academy of Sciences of Belarus), K Strzalka (Jagiellonian University) The effect of high temperature treatment (40ºC, 3h, illumination at 100 µmol m-2s-1) on the photosynthetic electron flow in chloroplast of different age barley seedlings was investigated. The thermoinduced partial inhibition of electron flow on PSII acceptor side in 4-days-old leaves was shown by measurements of oxygen evolution using of benzoquinone or potassium ferricyanide as electron acceptors and following QA¯ reoxidation kinetics in absence and presence exogenous electron acceptors DCBQ and DMBQ. Using HPLC analysis, the increase in oxidation of photoactive plastoquinone pool in young leaves under heating was shown. In old 11-days-old leaves the heat treatment limited as photosynthetic electron flow as oxygen evolution. The same effects of heat shock on oxygen evolution in presence of benzoquinone and ferricyanide demonstrated inhibition of electron flow on donor side of PSII only. However, rise in part of non-active PSII was observed. Addition of exogenous acceptors DCBQ and DMBQ and donor DPC show that the thermoinduced decrease of electron transport rate on acceptor side was caused by structural damage of PSII. The decrease in size of photoactive PQ-pool and its oxidized and reduced parts in old leaves under heat treatment was shown. It is proposed that thermoinduced change of redox state of PQ-pool and redistribution of plastoquinone molecules between photoactive and non-photoactive pools are the mechanisms regulating response of the photosynthetic apparatus on stress reactions.

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1 - Photosynthesis and Education 1.1 How and why do we need to teach the evolution of photosynthesis? R Blankenship (Washington University), A Haffa (Monterrey Bay Aquarium Research Institute) The teaching of evolution in schools has long been a controversial societal issue, especially in secondary school education in the USA. In recent years, repeated attempts to rewrite science standards and modify textbooks to downplay evolution or present alternatives have been made. The most visible of these recent efforts has been spearheaded by the Intelligent Design (ID) movement. ID proposes that certain biological systems are “irreducibly complex”, in that they are so complicated that it is impossible that they arose via the gradual accumulation of mutations and therefore had to have been created by an “intelligent designer”. Photosynthesis is a process often portrayed as irreducibly complex in the ID literature and thus it becomes a central issue in this larger societal debate. It is important that scientists clearly articulate the existing evidence relating to the origin and evolution of photosynthesis and communicate this information to the community at large in a way that is both accessible and scientifically valid.

1.2 Effective teaching methods for Systems and Molecular Biology J Pritchard (University of Birmingham) The burgeoning DNA sequence information on the internet can be daunting to students, yet aspects of it must be taught at A level and in University Biology courses. To stimulate ideas for teaching in this area I have put together a web site in which various online tools for manipulating and comparing gene sequences. The site uses salt tolerance in Arabidopsis as its starting point but can be tailored towards systems related to photosynthesis with appropriate modification of the text and the starting gene sequences. The aim of the exercise is to use online data banks to examine the function and relationship between gene sequences, in particular in the model plant Arabidopsis. It addresses questions how you can predict gene function from the DNA sequence through to different ways of thinking about the relationship between genes in evolution. Specifically in the exercise students will: 1. Find out how they can use DNA sequence to determine function of a gene 2. Use online resources to find out the function of a DNA gene sequence 3. Find genes closely related to the example given 4. Determined which chromosomes the genes are on 5. Thought about the evolutionary processes that these data illustrate. 6. Compared gene sequences The site will be presented and the problems and advantages of this approach will be discussed.

1.3 Bioenergetics – have we forgotten how and why to teach it? E Evans (Liverpool John Moores University) Bioenergetics makes the world go round! The transduction of energy by biological systems is core to the existence of the Biosphere, and therefore core to all teaching of any Bioscience. The challenge in

teaching Bioenergetics is to contextualise the content for the student class, whether medical students, ecologists or biochemists. The popular scientific press carries many stories which are underpinned by, for example, the activity of methylotrophs or faulty replication in mitochondria. The use of such material in aiding understanding and maintaining interest in Bioenergetics in student groups will be discussed.

1.4 How and why we need to include the past discoveries, and much more, in teaching photosynthesis in the future? Govindjee (University of Illinois at Urbana-Champaign) ‘Life is Bottled Sunshine’ is a four word summary of the significance of photosynthesis for life on Earth. We all know the importance of the audio visual tools for communicating scientific principles to the students. During my 40 years of teaching BS to PhD level students, I have found that they have learned faster and with ease when they were encouraged to imagine themselves either (a) as molecules participating in exciton transfer from antenna to reaction center molecules, or as electrons/ protons moving through the Z-scheme2; or (b) as scientists of the past describing their own results. Further, the inclusion of photographs and personal aspects of the discoverers fascinates students as they absorb the scientific concepts. In addition, simple movies such as that showing Photosystem II, and rotating ATP Synthase imprints the dynamic nature of the processes in their minds. An integrated approach is needed. I plan to show selected examples of the tools I have used. They include the use of the internet (‘Photosynthesis and the Web, 2008’, by Orr and Govindjee, in ‘Photosynthesis Research’) and of stories and photographs (Discoveries in Photosynthesis, edited by Govindjee et al., 2006). I plan to ask: Can the history of photosynthesis research help us correct student’s misconceptions? In my experience, a historical perspective inspires students to learn and encourages a desire to solve problems facing the world. [2See, for fun, the Z-Scheme movie by Ohio State Football team: http://www.youtube.com/watch?v=XsZlPeT3D10&eurl=].I thank Howard Gest, Robert Blankenship and David Walker for their help.

1.5 A New Leaf in Time D Walker (University of Sheffield) Over the last decade the availability and increasing speed of Broadband has made it commonplace to turn to the Internet for information. Digital books with both narrative and hyperlinks offer a means of structuring this retrieval process and even leavening the most complex science with cartoons and sound for those who might not otherwise readily turn to a more formal style of presentation. An example of this approach will be illustrated.

1.6 Student-led investigative laboratories designed to examine the acclimation of photosynthesis and energy dissipation B Logan (Bowdoin College), J Reblin (Bowdoin College) The photosynthetic apparatus acclimates to the growth environment to

324 14th Photosynthesis Congress - PS07 optimize carbon assimilation. However, most plants absorb more light than they can process via the Calvin cycle (i.e. excess light). Since excess light can cause cellular damage, plants have evolved a mechanism referred to as energy dissipation, which safely converts absorbed light energy to heat. Over multiple time-scales (minutes to seasons) plants adjust their carotenoid and chlorophyll composition in response to levels of excess light absorption. Here we describe student-led investigative laboratories designed to examine the acclimation of both photosynthesis and energy dissipation to the growth light environment. In preliminary laboratories we introduce students to the measurement of photosynthetic oxygen evolution and leaf pigment analysis (via HPLC), while in lecture we discuss the physiological ecology of photosynthesis and energy dissipation. In small groups, students then propose research questions, which they examine over the next several weeks and present as journal-style manuscripts. These laboratories enhance student’s understanding of the effect of environmental stress on plant bioenergetics. Furthermore, they foster a greater appreciation for research and enhance engagement because students are exploring their own experimental questions. (Course materials and example manuscripts will be available on CD.) 1.7 Teaching Biophysics of Photosynthesis and Ecology at Faculty of Physics of Lomonosov Moscow State University A Kukushkin (Moscow State University, Faculty of Physics) Our students receive these lectures during their fifth year at University. They have studied applied and theoretical physics, pure and computing mathematics, chemical and quantum chemistry, biology, radio physics and spectroscopy. The basic themes of the course are the following. What is Biosphere? Ecosystems. The structure of Biosphere. Energy flows in Biosphere. Productivity of Biosphere. Destructive factors of Biosphere. Anthropogenic influence. Demographic explosion. Fundamentals of Photosynthesis. Photosynthetic pigments. Photosynthetic units. Light-harvesting antennae. Reaction centers. Migration of energy. Electron carriers. Substances accumulated energy in primary processes of photosynthesis: NADPH, ATP; their electronic structure. The dark biochemistry: Calvin-Benson cycle, starch and sucrose synthesis. C3 and C4 photosynthesis. CAM metabolism. The fundamentals of evaluation of plant state and photosynthetic efficiency. Fluorescence, carbon dioxide fixation, oxygen evolving, electron transport, delayed luminescence, thermoluminescence and the action potential in plants. The photosynthetic regulation under changing of external conditions. The ways of regulation. The experimental studies of regulation. The damped oscillations of different components of photosynthetic systems in higher plants. The theoretical models of regulation in photosynthesis. The ecological problems and photosynthesis. The greenhouse effect. Photosynthesis and atmosphere. The influence of temperature on photosynthesis. The possible ways of solution of urgent ecological problems. The renewable resources of energy. References: G.E.Edwards, D.A.Walker. C3, C4, Mechanism, and Cellular and Environmental Regulation of Photosynthesis. Blackwell Scientific Publications Ltd, Oxford, 1983. D.A. Walker. Energy, Plants and Man. Oxygraphic Limited, Brighton, 1992. A.K. Kukushkin,, A.N. Tikhonov, Lectures on biophysics of plants photosynthesis. MGU Press, Moscow, p.1-320, 1988 (in Russian)