A36

Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system Short communication GYNHEUNG AN, BRIAN D. WATSON & CHIN C. CHIANG Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA Plant Physiol. 81:301 305, 1986 Abstract. Using a binary tumor-inducing (Ti) plasmid vector system, several plant species were

transformed with a kanamycin resistance marker (n~-om-ycinphosphotransferase gene). Four Nicotiana species, seven tomato cultivars, two potato cultivars, and Arabidopsis thaliana were transformed by the binary vector transformation method. In this method, various plant organ pieces were co-cultivated with Agrobacterium tumefaciens cells carrying the binary vector, pGA472, and a helper Ti plasmid. We have also demonstrated that a wild type Ti plasmid can be used as a helper to obtain a transformed plant.

Association of H+-translocating ATPase in the Golgi membrane system from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) MD. SHOWKAT ALI & TAKASHI AKAZAWA Research Institute for Biochemical Regulation, School of Agriculture, Nagoya University, Chikusa, Nagoya 464, Japan Plant Physiol. 81:222-227, 1986 Abstract. The Golgi complex and the disrupted vesicular membranes were prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using protoplasts as the starting material and employing linear sucrose density gradient centrifugation followed by osmolysis (Ali et al. [1985] Plant Cell Physiol 26:1119-1133). The isolated Golgi fraction was found to be enriched with marker enzyme activities and depleted of the activity of a typical mitochondrial marker enzyme, cytochrome c oxidase. Golgi complex, and vesicular membranes derived thereof were found to contain the specific ATPase (specific activity of about 0.5 to 0.7 micromoles per minute per milligram protein). Inhibitor studies suggested that the ATPase of Golgi was different from plasma membrane, tonoplast and mitochondrial ATPases as it was not inhibited by sodium vanadate, potassium nitrate, oligomycin and sodium azide. The sensitivity to N-ethylmaleimide further distinguished the Golgi ATPase from F 0 to FI ATPase of mitochondria. The internal acidification was measured by monitoring the difference in absorbance at 550 nanometers minus 600 nanometers using neutral red as a probe, The maximum rate detected with Golgi and disrupted membrane system was 0.49 and 0.61 optical density unit per minute per milligram protein, at pH 7.5, respectively, indicating that the proton pump activity was tightly associated with the Golgi membranes. In both cases, the acidification was inhibited 70 to 90% by various ionophores, indicating that the proton pump was electrogenic in nature. Both the Golgi ATPase activity and ATP-dependent acidification were profoundly inhibited by N,N'-dicyclohexylcarbodiimide, which also indicate that the two activities are

catalyzed by the same enzyme.

A37

Responses of cultured parsley cells to elicitors from phytopathogenic fungi Timing and dose dependency of elicitor-induced reactions ERICH KOMBRINK & KLAUS HAHLBROCK Max-Planck-lnstitut )~r Ziichtungsforschung, Abteilung Biochemie, 5000 K6ln 30, FRG Plant Physiol. 81:216-221, 1986 Abstract. Cultured parsley cells (Petroselinum crispum) responded to treatment with heat-released

soluble cell-wall fragments (elicitors) from several different phytopathogenic fungi by forming coumarin derivatives (phytoalexins). This response was preceded in all cases by large but transient increases in the activities of two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL). The activities of two hydrolytic enzymes, chitinase and 1,3-fl-glucanase, also increased strongly in elicitor-treated cells, whereas the activities of three enzymes participating in primary metabolism were affected differently by the elicitor treatment. Glucose-6-phosphate dehydrogenase increased, phosphofructokinase remained almost constant, and pyrophosphate:fructose-6-phosphate phosphotransferase declined sharply in activity. Different amounts of cell-wall preparations from various phytopathogenic fungi were required for maximum elicitor activity. While three oomycetes (Phytophthora spp.) yielded the most active elicitors studied (maximum coumarin accumulation at concentrations of about 10 microgram per milliliter), cell-wall preparations from an ascomycete and three deuteromycetes gave comparable results only at 10 to I00 times higher concentrations. Optimal induction of PAL, 4CL, and chitinase with Phytophthora elicitor required only about 1 microgram per milliliter, whereas 1,3-fl-glucanase induction showed a dose dependence similar to that observed for coumarins. The elicitor concentration had pronounced effects not only on the extent, but also on the timing of all induced reactions.

Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells GYNHEUNG

AN

Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA Plant Physiol. 81:86-91, 1986 Abstract. I have developed promoter expression binary vectors based on the tumor-inducing plasmid of Agrobacterium tumefaciens to facilitate elucidation of plant gene regulation. Promoter activity can be determined by inserting DNA fragments into the multiple cloning sites of the vectors forming transcriptional and/or translational fusions between the cat structural gene and an inserted promoter region. The activity of the nopaline synthase (nos) promoter was demonstrated with the vector. However, three animal promoters tested with this system showed no measurable activity in plant cells. Examination of 40 independently derived transformed tissues revealed a 200-fold difference in the nos promoter activity. Furthermore, there is no apparent correlation between the neomycin phosphotransferase and chloramphenicol acetyltransferase activities, although both genes are closely linked and undei" control of identical nos promoters. These results indicate that vast differences in promoter activity of transferred genes can occur within the same cell, as well as in independently derived cell lines.

A38 Amino acid transport in suspension-cultured plant cells

111. Influence of pH buffers, calcium, and preincubation media on L-leucine uptake MARK A. SCHNEEGURT & CARL N. McDANIEL Department o f Biology, Rensselaer Polytechnic Institute, Troy, N Y 12180-3590, USA Plant Physiol. 81:36-40, 1986 Abstract. The rate at which L-leucine was transported into suspension-cultured Nicotiana tabacum cv Wisconsin 38 cells increased more than 2-fold over a period of hours when the cells were preincubated in a 1% sucrose solution. This increase in uptake rate was eliminated if certain tris buffers were included in the preincubation solution while other buffers had little effect. Calcium could reverse the effect of the inhibitory buffers only if the buffer and calcium were present together from the beginning of the preincubation period. It was the amine group of the inhibitory buffers which was responsible for the inhibition. Preincubation in a complete culture medium (EM Linsmaier, F Skoog 1965 Physiol Plant 18:100-127) led to minimal changes in L-leucine uptake rate over a 10 hour preincubation period indicating that the uptake rate was stabilized by this medium. The complete medium stabilized the L-leucine uptake rate as a result of its ionic composition and not because of its osmolarity. Most of the increased uptake rate observed after preincubation in a 1% sucrose solution could be inhibited by 2,4-dinitrophenol or carbonyl cyanide m-chlorophenyl hydrazone, or high concentrations of L-phenylalanine or L-leucine. Therefore much of the increase could be accounted for by an increase in active transport of L-leucine.

Amino acid transport in protoplasts isolated from soybean leaves CHARLES D. VERNOOY & W I L L Y L I N Central Research and Development Department, Experimental Station, E.I. du Pont de Nemours & Company, Wilmington, DE 19898, USA Plant Physiol. 81:8-11, 1986 Abstract. We isolated large quantities of mesophyll protoplasts from source and sink leaves of

soybean plants and examined them for amino acid uptake. Accumulation of amino acids in isolated protoplasts was linear for at least 40 minutes. Uptake kinetics revealed the presence of both saturable and linear components. Increasing external pH decreases the uptake. The uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazoneat 15 micromolar inhibited and fusicoccin at 10 micromolar stimulated amino acid uptake. Our data are consistent with a proton-cotransport mechanism for the uptake of L-glutamine and or-amino isobutyric acid into soybean mesophyll cells.

A39 Physiological changes in cultured sorghum cells in response to induced water stress II. Soluble carbohydrates and organic acids RONALD

J. N E W T O N ,

SHYAMALA

BHASKARAN,

JEFFREY

D.

PURYEAR & ROBERTA H. SMITH Department of Forest Science (R.J.N., J.D.P.) and Department of Soil and Crop Sciences (S.B., R.H.S.), Texas Agricultural Experiment Station, Texas A & M University, College Station, TX 77843, USA Plant Physiol. 81:62~629, 1986 Abstract. Eight cultivars Sorghum bicolor (L.) Moench were grown as callus cultures under induced,

prolonged water stress (8 weeks), with polyethylene glycol in the medium. Concentrations of soluble carbohydrates and organic acids in callus were measured at the end of the growth period to determine differences in response to prolonged water stress. Sucrose, glucose, fructose, and malate were the predominant solutes detected in all callus at all water potentials. All cultivars had high levels of solutes in the absence of water stress and low levels in the presence of prolonged water stress. However, at low water potentials, low levels of solutes were observed in drought-tolerant cultivar callus and high solute levels were observed in drought-susceptible cultivar callus. Estimated sucrose concentrations were significantly higher in water-stressed, susceptible cultivar callus. Large solute concentrations in susceptible cultivar callus were attributed to osmotic adjustment and/or reduced growth during water stress.

Rapid degradation of abnormal proteins in vacuoles from Acer pseudoplatanus L. cells HERVI~ CANUT,

GILBERT

ALIBERT,

ANTOINE

CARRASCO

&

ALAIN M. BOUDET Centre de Physiologie Vkg~tale de l'Universitk Paul Sabatier, 118 route de Narbonne, F-31062 Toulouse Cedex, France Plant Physiol. 81:460-463, 1986 Abstract. In Acer pseudoplatanus cells, the proteins synthesized in the presence of an amino acid

analog ([tac]p-fluorophenylalanine), were degraded more rapidly than normal ones ([~4C]phenylalanine as precursor). The degradation of an important part of these abnormal proteins occurred inside the vacuoles. The degradation process was not apparently associated to a specific proteolytic system but was related to a preferential transfer of these aberrant proteins from the cytoplasm to the vacuole.

A40

Hyoscyamine 6/I-Hydroxylase, a 2-Oxoglutarate-dependent Dioxygenase, in Alkaloid-producing root cultures TAKASHI

HASHIMOTO

& YASUYUKI

YAMADA

Research Centerfor Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Plant Physiol. 81:619~25, 1986 Abstract. Root cultures of various solanaceous plants grow well in vitro and produce large amounts of tropane alkaloids. Enzyme activity that converts hyoscyamine to 6fl-hydroxyhyoscyamine is present in cell-free extracts from cultured roots of Hyoscyamus niger L. The enzyme hyoscyamine 6/~-hydroxylase was purified 3.3-fold and characterized. The hydroxylation reaction has absolute requirements for hyoscyamine, 2-oxoglutarate, Fe2÷ ions and molecular oxygen, and ascorbate stimulates this reaction. Only the L-isomer of hyoscyamine serves as a substrate; D-hyoscyamine is nearly inactive. Comparisons were made with a number of root, shoot, and callus cultures of the Atropa, Datura, Duboisia, Hyoscyamus, and Nicotiana species for the presence of the hydroxylase activity. Decarboxylation of 2-oxoglutarate during the conversion reaction was studied using [l-l*C]-2-oxoglutarate. A 1:1 stoichiometry was shown between the hyoscyamine-dependentformation of CO 2 from 2-oxoglutarate and the hydroxylation of hyoscyamine. Therefore, the enzyme can be classified as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11 .-). Both the supply of hyoscyamine and the hydroxylase activity determine the amounts of 6fl-hydroxyhyoscyamine and scopolamine produced in alkaloid-producing cultures.

Proton fluxes as a response to external safinity in wild type and NaCl-adapted Nicotiana cell lines ABD-ELRAHEM REINHOLD

A. WATAD,

& HENRI

PIER-ANTONIO

PESCI, LEONORA

R. L E R N E R

Department of Botany, The Hebrew University of Jerusalem, 91904, lsrael Plant Physiol. 81:454~459, 1986 Abstract. Addition of 100 millimolar KC1, NaC1, or Na 2SO4 strongly promoted acidification of the medium by cells of Nicotiana tabacum/gossii in suspension culture. Acidification was greater in the case of NaCl-adapted than in that of wild type cells, and strikingly so in KCI medium when fusicoccin (FC) was present. Back-titration indicated that net proton secretion in KC1 medium was increased 4-fold by FC treatment in the case of adapted cells; but was not even doubled in wild type cells. Membrane potential was higher in NaCl-adapted cells. FC treatment hyperpolarized wild, but not NaCl-adapted cells, suggesting a higher degree of coupling between H + efflux and K + influx in adapted ceils; FC enhanced net K ÷ uptake in adapted but not in wild cells. Acidification by cells suspended in 10 millimolar KC1 was highly sensitive to vanadate, but that after addition of 100 millimolar KC1 or NaC1 was much less sensitive. Addition of 100 millimolar NaC1 to wild type cells already provided with 10 millimolar KCI briefly accelerated, then slowed down the rate of acidification. If the addition was made after acidification had already ceased, alkalization was observed, particularly in the presence of FC. The results are consistent with the operation of a N a + - H + antiporter.

A41 The effect o f salt concentration on auxin stability in culture media Short communication JAMES R. DUNLAP, STEPHEN KRESOVICH & ROBERT E. McGEE Subtropical Agricultural Research Laboratory, United States Department of Agriculture, Agricultural Research Service (J.R.D.) and Texas Agricultural Experiment Station (S.K., R.E.M.), Weslaco, TX 78596, USA Plant Physiol. 81:934-936, 1986 Abstract. The concentrations of indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA) and

2,4-dichlorophenoxyaceticacid (2,4-D) were followed for 35 days in cell-free liquid medium containing 100, 50, or 0% Murashige-Skoog (MS) salt base. Although the concentrations of NAA or 2,4-D remained constant the level of IAA decreased to only 11% of the original concentration after 35 days in the presence of 100% MS salt base. The observed rate of IAA degradation was accelerated by the presence of MS salts.

Auxin-controlled glycoprotein release into the medium of embryogenic carrot cells Short communication SHINOBU SATOH, HIROSHI KAMADA, HIROSHI HARADA & TADASHI FUJII Institute of Biological Sciences, University of Tsukuba, Sakura-mura Ibaraki, 305 Japan Plant Physiol. 81:931-933, 1986 Abstract. Glycoproteins released from carrot cells into culture media were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by staining with Coomassie brilliant blue or with the periodic-acid Schiff procedure. The appearance or disappearance of two glycoproteins of Mr 65,000 (GP65) and M r 57,000 (GP57) was closely related to the formation of somatic embroys. GP65 was released specifically from embryogenic cells cultured in a medium without 2,4-dichlorophenoxyacetic acid, in which they can form somatic embryos. GP57 was released from the same embryogenic cells, if they were cultured in a medium with 2,4dichlorophenoxyacetic acid, in which they cannot form somatic embryos. Nonembryogenic cells which cannot form somatic embryos, released only GP57.

A42

The induction of ethylene production from pear cell culture by cell wall fragments Short communication CINDY

B. T O N G ,

JOHN

M. LABAVITCH

& SHANG

FA YANG

Department of Vegetable Crops (C.B.T., S.F.Y.) and Department of Pomology (J.M.L.), University of California, Davis, CA 95616, USA Plant Physiol. 81:929-930, 1986 Abstract. Macerase, a pectinase-containing enzyme mixture, was used to digest cell walls isolated from cultured pear cells. Following digestion, the reaction mixture was boiled to inactivate enzymes. Addition of soluble aliquots of the mixture to suspension cultures of pear cells led to a rapid and transient production of ethylene by the cells.

Assay of photosynthetic oxygen evolution from single protoplasts L2 RLrDIGER

HAMPP,

WERNER

MEHRLE

& ULRICH

ZIMMERMANN

Biologie 1, Universitiit Tiibingen, D-7400 Tiibingen 1 (R.H.) and Institut J~r Biotechnologie, Universiti~t Wi~rzburg, D-8700 Wiirzburg ( W.M, U.Z.), FRG Plant Physiol. 81:854-858, 1986 Abstract. A semiquantitative assay for light-dependent 0 2 evolution by a single mesophyll protoplast is described. The assay indicator is the density of aerotactic bacteria (Pseudomonas aeruginosa, ATCC 10145; 'Engelmann experiment') attracted to the protoplast. Quantification is by dark field microphotometry. The sensitivity is about 50 femtomoles 02 per protoplast per minute. The results demonstrate the biphasic nature of 02 evolution of a single protoplast during photosynthetic induction. Computerized data acquisition yields traces which, until a steady state of photosynthetic 02 evolution is reached, are identical to ordinary 02 electrode traces.

A43

Possible involvement of calmodulin and the cytoskeleton in electrofusion of plant protoplasts Communication SHUNNOSUKE

ABE & JUNKO

TAKEDA

Institute of Agricultural Environment Control, College of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790, Japan (S.A.) and Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan (AT.) Plant Physiol. 81:1151-1155, 1986

Abstract. Calmodulin antagonists, trifluoperazine, chlorpromazine, calmidazolium, N-(6aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7), strongly inhibited the electrofusion of barley (Hordeum vulgare L. cv Moor) protoplasts with a marked increase of broken fusion products, after 60 minutes of incubation. W-5, a dechlorinated analog of W-7, was found less effective for the inhibition than W-7. Ethyleneglycol-bis(fl-aminoethylether)-N,N'-tetraacetic acid a Ca 2+ chelator, La 3+, a surface Ca :+ antagonist, and verapamil, a Ca 2+ channel blocker, also inhibited electrofusion. Dielectrophoresis was inhibited by La 3+. A microtubule inhibitor, vinblastine, inhibited electrofusion strongly while colchicine, slightly. A microfilament inhibitor, cytochalasin B, promoted fused cells to become spherical while phalloidin did not affect electrofusion.

Effect of 2,4-Dichlorophenoxyacetic acid on the expression of embryogenic program in carrot Communication BORKIRD, J U N G H. C H O I & Z. R E N E E S U N G Department of Genetics, University of California, Berkeley, Berkeley, California 94720. USA CHUMPOL

Plant Physiol. 81: 1143 1146, 1986

Abstract. Embryogenesis in a wild carrot cell line, W001C, can start and progress up to the first morphogenetic stage (the globular-stage embryo) in 2,4-dichloropenoxyacetic acid (2,4-D). To clarify the quantitative effect of 2,4-D on this cell line, morphological and biochemical criteria have been used to monitor embryogenesis in the presence of increasing concentrations of 2,4-D. The biochemical criteria are the ability to inactivate cycloheximide and the expression of an embryogenic polypeptide E t . The results show that 2,4-D can affect embryogenesis in a quantitative manner but never fully suppresses embryogenesis unless it is coupled with high cell density.

A44

Potassium transport in suspension culture cells and protoplasts of carrot DANIEL R. BUSH & LOUIS JACOBSON Department of Plant and Soil Biology, University of California, Berkeley, CA 94720, USA Plant Physiol. 81:1022-1026, 1986 Abstract. The properties of potassium transport in carrot (Daucus carota L.) suspension culture cells and their isolated protoplasts were examined. Cells cultured in Murashige and Skoog (MS) medium (Plant Physiol 15:473-497) were potassium saturated and, consequently, they exhibited little net potassium accumulation. Cells that transport and accumulate potassium were derived from the MS-grown cells by culturing them in a potassium-free modified medium. The transport properties of the modified medium cells included: (a) smooth nonsaturating kinetics with 80% of the maximum rates occurring at 0. I millimolar KC1, (b) linear transport for at least 75 min, (c) alkaline pH optimum, (d) little accompanying anion uptake with increased malate concentrations balancing net increases in positive charge, and (3) little effect on transport by plasmolysis. Potassium transport activity appeared to be 50% lower in protoplasts isolated from the modified medium cells. Nevertheless, the protoplasts exhibited essentially the same kinetics, time course, pH response, and malate adjustment as the intact cells. We concluded from these results that the low potassium cells and their isolated protoplasts are ideally suited to investigating potassium transport at the cell level without the complications associated with multilayered and highly differentiated tissues.

A45

A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells STEVEN

C. H U B E R

& TAKASHI

AKAZAWA

Research Institute for Biochemical Regulation, School of Agriculture, Nagoya University, Chikusa, Nagoya 464, Japan Plant Physiol. 81:1008-1013, 1986

Abstract. Enzymes of sucrose degradation and glycolysis in cultured sycamore (Acerpseudoplatanus L.) cells were assayed and characterized in crude extracts and after partial purification, in an attempt to identify pathways for sucrose catabolism. Desalted cell extracts contained similar activities (20-40 nanomoles per milligram protein per minute) of sucrose synthase, neutral invertase, glucokinase, fructokinase, phosphofructokinase, and UDPglucose pyrophosphorylase (assayed with 2 micromolar pyrophosphate (PPi). PPi-linked phosphofructokinase activity was virtually dependent upon fructose 2,6-bisphosphate, and the maximum activity exceeded that of ATP-linked phosphofructokinase. Hexokinase activity, with glucose as substrate, was highly specific for ATP, whereas fructokinase activity was relatively nonspecific, At 1 millimolar nucleoside triphosphate, fructokinase activity decreased in the order: UTP > ATP > CTP > GTP. We propose two pathways for sucrose degradation. One involves invertase action, followed by classical glycolysis of hexose sugars, and the other is a novel pathway initiated by sucrose synthase. The Km for sucrose of sucrose synthase was severalfold lower than that of neutral invertase (15 versus 65 millimolar), which may determine carbon partitioning between the two pathways. The sucrose synthase pathway proposed involves cycling of uridylates and PPi. UDP glucose pyrophosphorylase, which is shown to be an effective 'PPi-scavenger,' would consume PPi and form UTP. The UTP could be then utilized in the UTP-linked fructokinase reaction, thereby forming UDP for sucrose synthase. The source of PPi is postulated to arise from the back reaction of PPi-linked phosphofructokinase. Sycamore cells contained a substantial endogenous pool of PPi (about 3 nanomoles per gram fresh weight, roughly the amount of ATP in these cells), and sufficient fructose 2,6-bisphosphate (0.09 nanomole per gram fresh weight) to activate the PPi-linked phosphofructokinase. Possible regulation and energetic differences between the sucrose synthase and invertase pathways are discussed.

A46 Light activation of pyruvate,Pi dikinase and N A D P - m a l a t e Dehydrogenase in mesophyll protoplasts of maize

Effect of DCMU, antimycin A, CCCP and phlorizin HITOSHI NAKAMOTO & GERALD E. EDWARDS Department of Botany, Washington State University, Pullman, WA 99164-4230, USA Plant Physiol: 82:312-315, 1986 Abstract. Pyruvate,Pi dikinase (PPDK, EC 2.7.9.1) and NADP-malate dehydrogenase (MDH, EC

1.1.1.82) were activated in the light and inactivated following a dark treatment in mesophyll protoplasts of maize. D C M U (up to 33 micromolar), an inhibitor of noncyclic electron transport, inhibited activation of MDH much more strongly than it did PPDK. Antimycin A (6.6-33 micromolar), an inhibitor of cyclic photophosphorylation, inhibited the activation of PPDK (up to 61%), but had little or no effect on activation of MDH. Carbonyl cyanide m-chlorophenylhydrazone (0.2-2 micromolar) and nigericin (0.4 micromolar), uncouplers of photophosphorylation, inhibited activation of PPDK while stimulating the activation of MDH. Phlorizin (0.33-1.7 millimolar), an inhibitor of the coupling factor for ATP synthesis, strongly inhibited activation of PPDK but only slightly effected light activation of MDH. These results suggest that noncyclic electron flow is required for activation of NADP-MDH and that photophosphorylation is required for activation, of PPDK.

Pea xyloglucan and cellulose

IV. Assembly of fl-glucans by pea protoplasts TAKAHISA HAYASHI, DANIEL R. POLONENKO, ANNE CAMIRAND & GORDON MACLACHLAN Department of Biology, McGill University, Montreal, Quebec, Canada H3A IB1 Plant Physiol. 82:301-306, 1986 Abstract. The synthesis and assembly of xyloglucan were examined during early stages of wall

regeneration by protoplasts isolated from growing regions of etiolated peas. During early stages of. cultivation, fluorescence microscopy showed that the protoplast surface bound Calcofluor and ammonium salt of 8-anilino-l-naphthalene sulfonic acid and, in time, it also bound fluorescent fucose-binding lectin. Based on chemical analysis, 1,3-fl-glucan was the main polysaccharide formed by protoplasts and xylogiucan and cellulose were minor wall components. Binding between cellulose and xyloglucan was not as strong as that in tissues of intact pea plants, i.e. mild alkali could dissolve most xyloglucan from the protoplast. However, the addition of exogenous pea xyloglucan into the culture medium stimulated the deposition of new polysaccharides into the protoplast wall and enhanced the close association of newly formed xyloghican with cellulose.

A47

Improved cytoplasmic delivery to plant protoplasts via pH-sensitive liposomes CHEN-YEN

WANG,

KAREN

W. HUGHES

& LEAF HUANG

Department of Biochemistry, University of Tennessee, Knoxville, Tennessee 37996-0840 ( C- Y. W., L.H.), and Department of Botany, University of Tennessee, Knoxville, Tennessee 37996-1100 (K.H.), USA Plant Physiol. 82:179-184, 1986 Abstract. We demonstrated that the liposomes composed of dioleolylphosphatidylethanolamine/ cholesterol/oleic acid (4:4: 2) dramatically release their contents at a pH of less than or equal to 6.0 and are capable of delivering their contents into the cytoplasm of higher plant protoplasts. This is shown by using a soluble fluorescent dye, calcein, as a liposome-entrapped marker. We found that calcein fluorescence was evenly distributed in the cytoplasm of wild carrot protoplasts after the incubation of protoplasts with liposomes in the presence of polyethylene glycol 6000. At 0.45 micro mole phospholipid per 6 x 105 protoplast, for example, the percentage of protoplasts which took up liposomes was 89% which was much higher than that achieved by conventional pH-insensitive liposomes. In this study, liposomes were prepared by a detergent dialysis method which avoided sonication and organic solvents. Thus macromolecules such as proteins and nucleic acids could be entrapped in the liposomes and delivered to the cytoplasm of the protoplasts.

C a 2+ -stimulated secretion of ~-Amylase during development in barley aleurone protoplasts

DOUGLAS SCOTT BUSH, MARIA-JESUS H U A N G & R U S S E L L L. J O N E S

CORNEJO,

CHUN-NONG

Department of Botany, University of California, Berkeley, CA 94720, USA Plant Physiol. 82:566-574, 1986 Abstract. The effects of gibberellic acid (GA 3) and Ca 2+ on the synthesis and secretion of or-amylase

from protoplasts of barley (Hordeum vulgate L. cv Himalaya) aleurone were studied. Protoplasts undergo dramatic morphological changes whether or not the incubation medium contains GA3, CaC12, or both. Incubation of protoplasts in medium containing both GA 3 and Ca 2+ , however, causes an increase in the or-amylase activity of both incubation medium and tissue extract relative to controls incubated in GA 3 or Ca 2+ alone. Isoelectric focusing shows that adding Ca 2+ to incubation media containing GA 3 increases the levels of ~t-amylase isozymes having high isoelectric points (pI). In the presence of GA 3 alone, only isozymes with low pIs accumulate. The increase in or-amylase activity in the incubation medium begins after 36 hours of incubation, and secretion is complete after about 72 hours. Protoplasts require continuous exposure to Ca 2+ to maintain elevated levels of or-amylase release. Immunoelectrophoresis shows that Ca 2+ stimulates the release of low-pI or-amylase isozymes by 3-fold and high-pI isozymes by 30-fold over controls incubated in GA 3alone. Immunochemical data also show that the half-maximum concentration for this response is between 5 and 10 millimolar CaC12. The response is not specific for Ca 2+ since Sr2+ can substitute, although less effectively than Ca 2+ . Pulse-labeling experiments show that or-amylase isozymes produced by aleurone protoplasts in response to GA 3 and Ca 2+ are newly synthesized. The effects of Ca 2+ on the process of enzyme synthesis and secretion is not mediated via an effect of this ion on or-amylase stability or on protoplast viability. We conclude that Ca 2+ directly affects the process of enzyme synthesis and transport. Experiments with protoplasts also argue against the direct involvement of the cell wall in Ca 2+-stimulated enzyme release.

A48 NADH nitrate reductase and NAD(P)H nitrate reductase in genetic variants and regenerating callus of maize GEORGE SORGER, DINSDALE O. GOODEN, ELIZABETH D. EARLE & JOANNE McKINNON Department of Biology, McMaster University, Hamilton, Ontario, L8S 4K1, Canada (G.S., D.O.G., J.M.) ; and Department of Plant Breeding and Biometry, Cornell University, Ithaca, N Y 14853 (E,D.E.), USA Plant Physiol. 82:473-478, 1986 Abstract. Different organs of maize seedlings are known to contain different complements of NADH

and NAD(P)H nitrate reductase (NR) activity. The study of the genetic programming that gives rise to such differences can be initiated by looking for genetic variants exhibiting different patterns of distribution of the above enzymes. We demonstrate in this work that scutella of very young maize seedlings contain NADH NR almost exclusively and that this activity is gradually replaced, as the seedling ages, with NAD(P)H NR. Leaves in the seedlings contain exclusively the NADH NR activity. A genetic variant is described that contains much reduced levels of NAD(P)H NR activity but not of NADH NR activity in the scutellum. This same variant exhibits a relatively low level of NAD(P)H NR but normal NADH NR activity in seedling root tips. These observations suggest that the genetic program used to specify the scutellar complement of NR activity shares some common components with the genetic program used to determine the young root tip complement of NR activities. Parts of regenerating callus at different stages of differentiation were examined to determine when the differences in NR complement begin to appear. The same pattern o f N A D H NR and NAD(P)H NR activities was found in unorganized as well as in organized callus, in recognizable root-like and even in green shoot-like material, both activities being present in all these tissues. An examination of the NR complement in different organs of a number of siblings originating from a cross involving transposon Mu-containing parents and having different levels of leaf NADH NR activity shows that the leaf NADH NR activity content and the scutellum NAD(P)H NR activity content are relatively independent of each other, indicating that the genetic programs specifying the NR content of these organs are not tightly coupled, if at all.

A49 Polyamine metabolism and osmotic stress

H. Improvement of oat protoplasts by an inhibitor of arginine decarboxylase ANTONIO FERNANDEZ TIBURCIO, RAVINDAR KAUR-SAWHNEY & ARTHUR W. GALSTON Department of Biology, Yale University, New Haven, CT 06511, USA Plant Physiol. 82:375-378, 1986 Abstract. We have attempted to improve the viability of cereal mesophyll protoplasts by pretreat-

ment of leaves with DL-ct-difluoromethylarginine (DFMA), a specific 'suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.

Polyamine metabolism and osmotic stress

I. Relation to protoplast viability ANTONIO FERNANDEZ TIBURCIO, MARIA ANTONIA MASDI~U, FRANt~OISE M. DUMORTIER & ARTHUR W. GALSTON Department of Biology, Yale University, New Haven, CT 06511, USA Plant Physiol. 82:369-374, 1986 Abstract. Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a

rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine, and spermine (HE Flores, A W Galston 1984 Plant Physiol 75:102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, and Vigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.

A50

Carbon assimilation in carrot cells in liquid culture JAN KANABUS, RAY A. BRESSAN & NICHOLAS C. CARPITA Department of Botany and Plant Pathology (J.K., N.C.C.) and Department of Horticulture (R.A.B.), Purdue University, West Lafayette, IN 47907, USA Plant Physiol. 82:363-368, 1986 Abstract. Assimilation of carbohydrates by carrot (Daucus carota L. cv Danvers) cells in liquid culture was studied to delineate the major metabolic pathways used in transformation of external carbohydrates to UDP-glucose. The cells grown on either sucrose or glucose for several years proved equally capable of utilizing each of these sugars. Sucrose was rapidly hydrolyzed extracellularly to glucose and fructose, and glucose was preferentially taken up. Uptake of fructose was slower and delayed until glucose was nearly depleted from the medium. Concentrations of cellular sugars, mainly glucose and sucrose, increased during late logarithmic phase of growth and decreased during the plateau phase. Continuous labeling of the cells with D-[t4C]glucose resulted in rapid accumulation of radioactivity in glucose-6-phosphate and UDP-glucose. Because there was virtually no uptake of sucrose, UDP-glucose was likely derived from glucose-1-phosphate in a reaction catalyzed by UDP-glucose pyrophosphorylase and not directly from sucrose. Concentrations of major nucleotides and nucleotide sugars were maximal during the early logarithmic phase of growth and decreased several-fold in the stationary phase. A modified 'energy charge' for adenylates calculated with the omission of AMP decreased steadily from 0.9 to 0.8 during the course of culture cycle. An analogous uracil nucleotide ratio was considerably lower (0.85) during early culture, decreased to about 0.7 for the entire logarithmic phase, and returned to initial values as cells entered stationary phase. The uracil nucleotide ratio may provide a useful index to assess the coupling between the energy available in phosphoanhydride bond in adenine nucleotides and the demand for sugar for polysaccharide synthesis through uridine diphosphate-sugar pools.

Effect of 5-Fluorouracil on growth and morphogenesis of tissue cultures of Nicotiana sylvestris FRANCESCO MARIA RESTIVO & FRANCESCA TASSI Institute of Genetics, University of Parma, 43100 Parma, Italy Plant Cell Physiol. 27(5):785-790, 1986 Abstract. Tissue cultures of Nicotiana sylvestris treated with increasing concentration of 5fluorouracil (5-FU) showed a differential effect of the drug on growth and morphogenesis. In the range of concentrations tested, 5-FU did not inhibit increases in fresh weight, but it inhibited the production of shoots. The effect of 5-FU on shoot production was reversible and depended on the time of adding 5-FU to the tissue culture. Uracil and thymine did not counteract the effect of 5-FU on morphogenesis, whereas uracil partially reduced the inhibition of growth under some culture conditions.

A51

Effects of Glyphosate on the shikimate pathway and regulation of Phenylalanine Ammonia-lyase in Cryptomeria and Perilla cell suspension cultures NARIYUKI I S H I K U R A l, S U S U M U TAKESHIMA I & SEIJI MITSUI 2

TERAMOTO

l, Y A S U N O B U

i Department of Biology, Faculty of Science, Kumamoto University, Kurokami, Kumamoto 860, Japan; eBiological Laboratory, College of Medical Science, Kumamoto University, Kuhonji, Kumamoto 862, Japan Plant Cell Physiol. 27(4):677-684, 1986 Abstract. Treatment of Cryptomeria and Perilla cell suspension cultures with glyphosate resulted in a marked suppression of the formation of flavans and caffeic acid derivatives, respectively, while it caused only a slight decline in the cell growth. In contrast with 3-deoxy-o-arabino-heptulosonate (DAHP) synthase-Mn isozyme, DAHP synthase-Co isozyme from Cryptomeria and Perillacells was much more sensitive to inhibition by glyphosate. The addition of 1 to 2 mld glyphosate caused an accumulation of shikimate and quinate and a reduction of L-phenylalanine in both cell cultures. The inhibition of phenylalanine ammonia-lyase (PAL) activity by glyphosate was reversed by exogenously supplied L-phenylalanine to near the control level. Cycloheximide and actinomycin D nullified the recovery by exogenous L-phenylalanine on PAL activity. L-Phenylalanine itself promoted PAL activity to some extent. No recovery of PAL activity in L-~-aminooxy-/~-phenylpropionate (L-AOPP)-treated cell cultures could be observed by the addition of L-phenylalanine. Therefore, L-AOPP seems to inhibit the formation of PAL, though it has been considered a competitive inhibitor.

Glucosylation of salicyl alcohol by Gardenia jasminoides cell cultures HAJIME HIROMU

MIZUKAMI,

TOSHIMITSU

TERAO,

AKEMI

AMANO

&

OHASHI

Faculty of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi, Nagasaki 852, Japan Plant Cell Physiol. 27(4):645~50, 1986 Abstract. Cultured ceils of Gardenia jasminoides produced both salicin and isosalicin from exogenously supplied salicyl alcohol. The glucosylation activity of the cells was highest in the exponential phase of growth and ca. 70% of the added substrate was converted to the glucosides within 4 days. The rate of glucosylation was also dependent on the medium composition such as auxin and sucrose concentrations. The ratio of salicin to isosalicin formed from salicyl alcohol was influenced by the growth stage of the cultured cells. Salicin was converted to isosalicin when exogenously added to the culture.

A52

Introduction of functional RNA into plant protoplasts by electroporation KAZUYA

OKADA

~, T o s h i y u k i N a g a t a 2 & I t a r u T a k e b e ~

tDepartment of Biology, Faculty of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464, Japan; 2Department of Cell Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji-cho, Okazaki 444, Japan Plant Cell Physiol. 27(4):619~26, 1986 Abstract. A simple apparatus was constructed for producing electric discharge of varying intensities

between two electrodes in a spectrophotometer cuvette. This apparatus was used to study the conditions for efficient introduction of functional RNA into plant protoplasts using RNAs of tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). Electroporation under optimal conditions resulted in the production of TMV and CMV in 80% of the protoplasts from tobacco cell line BY2. Infection by TMV-RNA occurred in 60% of Vinea rosea suspension culture protoplasts and in 40% of tobacco mesophyll protoplasts. Electroporation in the presence of TMV and CMV particles also resulted in infection, suggesting that pores larger than 30 nm are formed. The advantages and uses of the electrical method for introduction of functional RNA are discussed.

Growth and aspartate kinase activity in wheat cell suspension culture: effects of lysine analogs and aspartate-derived amino acids YASUYUKI Y A M A D A I, R O S A R I N K U M P A I S A L l, T A K A S H I HASHIMOTO l, Y U K I H I R O SUGIMOTO 2 & AKINORI SUZUKI 2

1Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan; 2Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Tokyo 113, Japan Plant Cell Physiol. 27(4):607q517, 1986 Abstract. The effects of lysine analogs and aspartate-derived amino acids on the growth of wheat

cell suspension culture were studied. S-(2-Aminoethyl)-L-cysteine (AEC). 6-hydroxylysine (DHL) and trans-lysene caused complete growth inhibition at 1.0 raM. The growth inhibition of lysine analogs were, in the order of decreasing effectiveness; AEC /> DHL, trans-lysene > oxalysine, homolysine and lysyne, cis-Lysene and methyllysine were not inhibitory even at concentrations of 10mM, Lysine effectively relieved growth inhibition induced by the lysine analogs. Lysine plus threonine showed concerted inhibition, which was relieved by the addition of methionine. Activity of aspartate kinase extracted from wheat cell suspension culture was strongly inhibited by L-lysine; 0.75 to 1 m~a of lysine was required for half-maximal inhibition. Threonine and methionine, individually or in combination with lysine, showed no inhibitory effect on the enzyme activity. S-Adenosylmethionine, when added with lysine in equimolar concentrations, enhanced the feedback inhibition by lysine, lowering the concentration of lysine for half-maximal inhibition to 0.13 raM. The aspartate kinase isolated from the cells cultured in the presence of 5 mM lysine did not differ in regulatory properties from the enzyme from the cells cultured without lysine. AEC at 5 mM inhibited the enzyme activity by 50%. Other lysine analogs were not inhibitory to the enzyme activity even at 10 mM. Growth inhibition of wheat suspension culture by aspartate-derived amino acids and lysine analogs were discussed in relation to their inhibitory effects on aspartate kinase activity.

A53

Cultivation of rice protoplasts and their transformation mediated by Agrobacterium spheroplasts AKIKO BABA, SEIICHIRO HASEZAWA & KUNIHIKO SYONO Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153, Japan Plant Cell Physiol. 27(3):463-471, 1986 Abstract. Improvement of the cultivation of rice (Oryza sativa L.) protoplasts isolated from

suspension cultures led to their division at a frequency of 5 to 10%. Rapidly growing colonies were obtained on a hormone-free medium when Agrobacterium tumefaciens spheroplasts were introduced into the protoplasts by polyethylene glycol treatment. Opines corresponding to the strains of A. tumefaciens used for the spheroplast treatments were detected in some of these colonies at a frequency of about 10 -4. Using radioactive precursors, [t4C]-ct-ketoglutaric acid and [3H]-arginine, activities of nopaline synthase, a marker enzyme of nopaline-type crown gall were also detected in some of these clones. These results show that the rice cells were transformed by Ti plasmid introduced by the spheroplast method.

Induction and differentiation of callus from embryos of Cocos nucifera L. by IAA-conjugates NEERA BHALLA-SARIN, SUMAN BAGGA, SUDHIR K. SOPORY & SIPRA GUHA-MUKHERJEE Plant Research Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India Plant Cell Reports 5:322-324, 1986 Abstract. Calli from young embryos of Cocos nucifera L. were induced on B5 medium supplemented

with IAA-conjugates (IAA-asp or IAA-ala) at a concentration of 2.0mg/1 and callusing was increased by about 10% if both IAA-conjugates, IAA-asp and IAA-ala were added together. Differentiation of shoots and roots was achieved by transferring calli to B5 medium supplemented with either IAA-asp (2.0mg/1) + Kn(2.0mg/1) or NAA (2.0mg/1). Complete plantlets were obtained on B5 medium supplemented with NAA (0.5mg/1) + BAP (2.0mg/l) + PVP (1.0g/I).

A54

Rapeseed embryo development in culture on high osmoticum is similar to that in seeds RUTH R. FINKELSTEIN & MARTHA L. C R O U C H Biology Department, Indiana University, Bloomington, Indiana 47405, USA Plant Physiol. 81:907-912, 1986 Abstract. The development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without high osmoticum (0.48 and 0.69 molar sorbitol) was compared with normal development in situ to investigate the role of a drying environment in embryo maturation. Sensitivity to osmoticum was assayed in terms of its ability to mimic normal development, i.e. to both suppress germination and maintain 12 S storage protein (cruciferin) synthesis at levels comparable to those seen in the developing seed. The osmotic conditions used block germination of pre-desiccation stage embryos but were not sufficient to prevent desiccation stage embryos from taking up water and germinating. At all stages tested, the osmotically treated embryos had approximately normal levels of cruciferin mRNA. Measurements of endogenous abscisic acid (ABA) levels by radioimmunoassay indicated that the osmotic effects on germination and gene expression were not mediated by elevated embryonic ABA. Comparison of the kinetics of osmotic and ABA effects on gene expression showed that the osmotic effect is more rapid. These results are consistent with the hypothesis that ABA acts by inhibiting water uptake, which mechanically prevents germination and affects gene expression in some unknown manner.

Effect of phytochrome on the uptake of gibberellin by protoplasts of Nicotiana glutinosa L. L. D A L E K E P P L E R 1 & D A N M E R T Z 2 Division of Biological Sciences, University of Missouri, CO 65211, USA Plant Cell Physiol. 27(5):861-865, 1986 Abstract. The effect of red light on gibberellin uptake by Nicotiana glutinosa L. protoplasts .was

determined. Five minutes of red light caused over a 50% inhibition of GA3 uptake within 2 minutes. Five minutes of far red light completely reversed the red light effect. The antagonistic effect of far red light indicates that gibberellin uptake is under phytochrome control. The rapid inhibition of gibberellin uptake indicates that phytochrome regulates the permeability properties of the plasma membrane as an initial response and not the intracellular binding of gibberellin.

A55

lnterspecific hybridization of Phaseolus vulgaris L. and Phaseolus angustissimus A. Gray using in vitro embryo culture TELEMACHOS BELIVANIS ~ & CLAIRE DOR]~2 1Fonctionnaire du Minist~re de l'Agriculture de Grbce, en stage ?t la Station de G~n~tique et d'Am~lioration des Flames, INRA, F-78000 Versailles, France; 21NRA, Station de G~n~tique et d'Am~lioration des Plantes, route de Saint-Cyr, F-78000 Versailles, France Plant Cell Reports 5:329-331, 1986 Abstract. The introgression of new desirable characters is very necessary in common bean (Phaseolus

vulgaris L.). Interspecific hybridization of P. vulgaris L. with P. angustissimus A. Gray, a wild species with narrow leaves, could be successfully achieved for the first time using embryo culture. Sixteen to twenty-three-day old embryos could grow to plantlets when they were cultivated on modified Monnier's medium. The leaf shape of the hybrid plants obtained showed intermediary traits between the two parents. A chromosome stock doubling could be achieved for one embryo.

Production of Nicotiana tabacum x Nicotiana acuminata hybrid by ovule culture SUMIO IWAI, CHIE KISHI, KAZUO NAKATA & NOBUMARO KAWASHIMA Central Research Institute, Japan Tobacco Inc., 6-2 Umegaoka, Mldori-ku, Yokohama, Kanagawa 227, Japan Plant Cell Reports 5:403-404, 1986 Abstract. Using a conventional sexual crossing technique, Nicotiana tabacum x N. acuminata was not produced. After the fertilized ovules were cultured for 20 days in a liquid Nitsch H medium, germination was observed. The roots grew rapidly but leaves did not. However, plantlets were produced in an H medium containing Benzyladenine or Kinetin(0.014). 1 mg/1). The plantlets grew and flowered in a greenhouse. The chromosome number of the hybrid was 36 and its morphological characteristics were intermediate between those of parental species.