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6_2180.

CELL SPECIFIC EXPRESSION OF NADH-DEPENDENT GLUTAMATE SYNTHASE IN RICE PLANTS

Ishiyama K., Kojima S., Hayakawa T., Yamaya T.

Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi,
Aoba-ku, Sendai 981-8555, Japan

Keywords:

enzymes, gene expression, higher plants, N metabolism, transgenic plants

Following the supply of 1 mM NH₄⁺ for 24^h to the rice (Oryza sativa L. cv. Sasanishiki) seedlings, there were strong signals for the NADH-GOGAT protein in two cell layers of root surface, i.e. epidermis and exodermis (Planta, in press). In the developing leaves and grains, NADH-GOGAT protein localized in specific cell types of vascular tissues. To understand the cell-specific gene expression for NADH-GOGAT more precisely, we tested in situ detection of the mRNA. Also, an apparent promoter region for NADH-GOGAT gene was tested with Agrobacterium-mediated transgenic rice. The mRNA for NADH-GOGAT was expressed specifically in sclerenchyma cells, which locate between exodermis and cortex cells, and central cylinder, but not in epidermis or exodermis of the roots. When a chimeric gene construct having 3.7 kbp 5' upstream region of the NADH-GOGAT gene fused with GUS gene was introduced, signals for GUS activity in the R0 were detected specifically in the sclerenchyma cells and central cylinder. Thus, the 5' upstream region of the NADH-GOGAT gene possessed an information at least to determine the cell-specificity of the expression. The difference in the localization of NADH-GOGAT protein and NADH-GOGAT gene expression will be discussed.
This work was supported in part by JAPS-RFTF96L00604 and Grant-in-Aid for Scientific Reserach on Priority Area (No. 09274102).

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8_2840.

MECHANISM OF AUTO-INHIBITION OF NADP-MALATE DEHYDROGENASE BY ITS C-TERMINAL EXTENSION

Ruelland E., Decottignies P., Djukic N., Miginiac-Maslow M.

Institut de Biotechnologie des Plantes. ERS 569 CNRS Bat. 630 Université de Paris-Sud, 91405 Orsay Cedex. France

Keywords:

C metabolisms, enzymes, light activation

NADP-malate dehydrogenase is a chloroplastic enzyme activated in the light by the ferredoxin/thioredoxin system. The oxidized form of the enzyme possesses two disulfide bridges per subunit situated in sequence extensions. The C-terminal extension shields the access to the active site. In reducing conditions the N-terminal bridge is isomerized and the newly created disulfide bridge is then reduced. Consequently to this isomerization and reduction process the active site undergoes a conformational change yielding to an oxaloacetate binding site of high affinity. When the C-terminal bridge is reduced by thioredoxins, the C-terminal extension moves, thus unshielding the access to the active site. Using a site-directed mutagenesis approach, we identified the two most external C-terminal residues as the ones locking the C-terminal extension into the active site. The synergy between the reduction of the N-terminal bridge and the reduction of the C-terminal bridge was investigated. We therefore report in this poster the first molecular evidence that the total lack of activity of NADP-malate dehydrogenase under oxidative conditions might be in part due to an intrasteric inhibition.

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9_4139.

DAILY PATTERNS OF PHOTOSYNTHESIS IN TWO XEROPHYTE SHRUBS GROWING IN MEDITERRANEAN FIELD CONDITIONS

Munné-Bosch S., Nogués S., Alegre L.

Department of Plant Biology, University of Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain. S.N. present address: Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, Essex, UK.

Keywords:

adaptation, CO2 uptake, diurnal changes, drought stress, fluorescence

The effect of summer drought, dew and rainfall in early autumn on daily photosynthetic patterns and water relations were studied in two xerophyte shrubs: rosemary (Rosmarinus officinalis L.) and lavender (Lavandula stoechas L.) grown in Mediterranean field conditions. R. officinalis showed a water conservation strategy and remained under conditions of mild stress throughout the drought experiment, although soil water potential decreased to ca. -2.9 MPa. The maintenance of the maximum quantum yield of the photosystem II photochemistry (Fv/Fm=0.74) and the increase in the non-photochemical quenching of chlorophyll fluorescence (NPQ ca. 1 to ca. 3) demonstrated that in rosemary photoinhibition can be partly avoided by increases in the NPQ. In the same environmental growth conditions, L. stoechas was a water spender and it could suffered severe water stress (RWC lower than 30%). It showed slight photoinhibition, FV/Fm decreased from 0.76 to 0.72 and NPQ remained almost constant at ca. 1.6. Nevertheless, L. stoechas leaves can adsorbed water from the dew, which improved RWC and leaf water potential and slightly increased stomatal conductance, allowing the plant to cope with stress until the arrival of autumn rainfall.

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15_19962.

IDENTIFICATION OF METAL-BINDING LIGAND IN CATALYSIS BY MAIZE NADP MALIC ENZYME

RaoS.R., Kamath B.G., Bhagwat A.S.

Molecular Biology & Agriculture Division
Bhabha Atomic Research Centre
Trombay
Mumbai-400 085
India

Keywords:

C4, enzymes, active site, NADP-malic enzyme

Chemical modification by Woodward's reagent "K" (WRK), and metal (Fe²⁺-ascorbate) catalyzed oxidation system (MCO) were used to study the role of carboxylic groups as metal binding ligand in catalysis by maize NADP-Malic enzyme. WRK inactivation followed pseudo-first-order reaction kinetics. Since NADP⁺+Mg²⁺ offered almost complete protection against WRK inactivation, it was thought that the putative carboxylic acid residue might be involved in either NADP or Mg²⁺ binding. However, fluorescence enhancement studies showed loss of NADP binding on WRK modification. The second approach using MCO was more promising. The inactivation was almost complete and followed pseudo-first-order kinetics. Mg²⁺ offered considerable protection against inactivation. However, NADP and malate were ineffective, suggesting the involvement of the modified residue in metal binding. The inactivation results from cleavage between Asp³⁵² and Ilu³⁵³ of the enzyme subunit into two fragments of 32 kDa and 34 kDa. Possible role of Asp³⁵² in Me²⁺ binding will be discussed.

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18_20783.

THE ROLE OF THE C-TERMINAL EXTENSION OF SORGHUM NADP-MALATE DEHYDROGENASE IN THE INHIBITION OF ITS REDUCTIVE ACTIVATION BY NADP

Miginiac-Maslow M., Ruelland E., Schepens I., Issakidis-Bourguet E., Decottignies P.

Institut de Biotechnologie des Plantes, Bat. 630,
Université Paris-Sud, 91405 Orsay Cedex, France

Keywords:

C metabolisms, C4, dehydrogenases, enzymes, light activation, NADP

The chloroplastic NADP-dependent malate dehydrogenase is activated by light through the ferredoxin-thioredoxin electron transfer chain. The last step consists of a thiol-disulfide interchange with reduced thioredoxin yielding a reduced and active enzyme. This activation is prevented by incubation with the oxidized form of the cofactor. The suppression of the C-terminal disulfide bridge of the enzyme, either by reduction, or by substitution of the cysteines by site-directed mutagenesis renders the enzyme insensitive to the inhibitory effect of NADP and allows the activation to proceed. In the present study, the residues of the C-terminal extension responsible for this effect have been investigated by site-directed mutagenesis. The opening of the C-terminal disulfide bridge can be mimicked by the substitution of the penultimate glutamate by a glutamine, or by the deletion of the two most C-terminal residues. This result suggests an electrostatic interaction between the positively charged nicotinamide ring of the cofactor and the negatively charged C-terminal residues of the enzyme. The cofactor specificity of this effect is also investigated.

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29_23524.

ASSEMBLY OF FUNCTIONAL COMPONENTS OF CHLOROPLAST THYLAKOID MEMBRANES IN INTERMITTENT LIGHT

Sulemanov S.Yu., Guseinova I.M., Novruzova S.N., Aliev J.A.

Institute of Botany/Department of Molecular Genetic Bases of Production Processes
Patamdar Shosse, 40
370073 Baku
Azerbaijan

Keywords:

antennae, biogenesis, chloroplast, greening, higher plants, light regulation

Low-temperature fluorescence spectra (77K) of chloroplasts obtained during greening of wheat seedlings in intermittent light (LOO show peaks detected at 635, 687 (main) and 720nm. In plants transferred to continuous light (CL) the fluorescence spectrum are characterized with bands at 687, 696 and 740nm (main). In chloroplasts from LDC also appear a small peak of Chlb at 648nm, which after transfer to CL his intensity increases. Probably this corresponds to synthesis in these conditions of antenna systems LHCI and LHCII. At the same time a sharp rise takes place in the fluorescence intensity at 740nm due to restoration of the energy run off to the Chla form at 710-712nm from LHCI. It was suggested that the source of the longwave fluorescence band of the chloroplasts at 740nm is the Chla form at 710-712nm localized in a small protein.

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30_23341.

THE ROLE OF PHOTOSYNTHESIS OF VARIOUS ORGANS ON PROTEIN SYNTHESIS OF WHEAT GENOTYPE'S GRAIN UNDER WATER STRESS

Aliev J.A.

Institute of Botany/Department of Molecular Genetic Bases of Production Processes
Patamdar Shosse 40
370073 Baku
Azerbaijan

Keywords:

adaptation, biotic stress, C3, C4, drought stress, protein synthesis, whole plants

A protein synthesis in a grain of ear of wheat genotypes in crop on 30-45% is carried out at the expense of ear photosynthesis. Depending on value of ear photosynthesis and tolerance of plants under strong drought conditions genotypes accumulate 35-70% of proteins from their contents in plants at normal water supply. C4-cycle enzymes (PEP-carboxylase, NAD- and NADP-malate dehydrogenase, aspartate aminotransferase) in C3-plants play a certain role in their adaptation to stress. Values of ratio of true photosynthesis to photorespiration in leaves, ear elements of genotypes with various intensity of CO₃ assimilation are approximate (with increase of ratio at intensive genotypes). During ontogenesis there is a parallel increase of intensity of true photosynthesis and photorespiration. Attempts to low photorespiration with the purpose of increase of productivity are inexpedient.

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31_24403.

THE EFFECT OF WATER DEFICIT ON WHEAT THYLAKOID MEMBRANES

Ismailov M.A., Zulfugarov I.S., Aliev J.A.

Institute of Botany/Department of Molecular Genetic Bases of Production Processes
Patamdar Shosse 40
370073 Baku
Azerbaijan

Keywords:

ATP, biotic stress, drought stress, environmental stress, luminescence, membrane structure

In the present work we have used delayed luminescence (DL) to study the effect of drought on photosynthetic properties of wheat cultivars. Our data indicate the apparent decrease in photochemical efficiency under drought in all cultivars. On the other hand, we show that when plants leaves are subjected to water stress the slow DL kinetics are markedly affected. The changes in luminescence found in wheat are in good agreement with data found for other species, suggesting that drought stress primarily inhibited enzymatic sites consuming ATP and NADPH through alterations in the proportion of photochemical and energy dependent quenching. These results suggest that DL could be used for screening of wheat genotypes for drought tolerance.

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33_25091.

BIOSYNTHESIS OF ISOCYCLIC RING E OF BACTERIOCHLOROPHYLL A

Porra R.J., Urzinger M., Winkler W., Scheer H.

Botanisches Institut
Menzinger Str. 67
D-80638 München
Germany

Keywords:

Bchl, biosynthesis, mass spectrometry, purple bacteria, Isocyclic ring formation

When Mg-protoporphrin IX-monomethylester is converted to its 13¹-oxo derivative, cyclization occurs between C-13² and C-15 forming the isocylic ring E of chlorophylls. This 13¹-oxo form of the 13-propionate methylester sidechain arises by oxidation of the 13¹-hydroxy derivative which is probably formed either directly by an oxygenase attack at C-13¹ of the 13-propionate methylester or indirectly via conversion of the sidechain to a 13-acrylate methylester followed by hydration. Using ¹⁸O-labelling and mass spectrometry, we have shown that the 13¹-oxo group of BChl a in Rhodobacter sphaeroides, a photosynthetic bacterium forming pigments in the light at low O₂ tensions, is labelled by H₂¹⁸O by a hydratase mechanismSUP>O by a hydratase mechanism. In Roseobacter denitrificans, an obligate aerobe, the 13¹-oxo group is labelled by ¹⁸O₂ by an oxygenase as in higher plants and green algae. In Rhodovulum sulfidophilum which forms pigments both aerobically in the dark and anaerobically in the light, a hydratase is employed under anaerobic conditions but an oxygenase and hydratase co-operate when O₂ is present.

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34_24696.

CONTROL COEFFICIENT APPLICATION TO THE STUDY OF ELECTRON FLOW DISTRIBUTION BETWEEN LINEAR AND CYCLIC ELECTRON TRANSPORT AND THE CONDITIONS ESSENTIAL FOR APPEARENCE OF PHOTOSYNTHETIC OSCILLATIONS

Khuznetsova S.A., Kukushkin A.K.

Faculty of Physics, M.V. Lomonosov Moscow State University, Vorobjevy Gory, Moscow, 119899 Russia

Keywords:

ATP, Calvin-cycle, electron transport, modelling, NADPH

Control coefficients (C^y_p) of different parameters p with respect to all variables y are calculated in a theoretical model of photosynthesis for parameter sets giving oscillatory and nonoscillatory induction kinetics and differing in the constant of electron transport and ATP synthesis coupling, the coupling being smaller for oscillations. The comparison of rate constant C^y_p for oscillatory and nonoscillatory kinetics shows that: 1) in our model NADPH and DPGA are the most sensitive to parameter changes variables; 2) in oscillatory regime steady-state ATP concentration is strongly affected by the rates of ATP consuming reactions, ensuring a feed-back from the Calvin cycle to electron transport. The difference in cyclic and linear electron transport C^y_p with respect to NADP will be discussed.

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36_25358.

PATHWAYS OF ELECTRON TRANSPORT, REGULATION AND THE ROLE OF PLASTOQUINONE

Kukushkin A.K., Khuznetsova S.A.

Faculty of Physics, M.V. Lomonosov Moscow State University, Vorobjevy Gory, Moscow, 119899 Russia

Keywords:

alternative electron transport, electronic structure, modelling, photosystem 2, quinones

Using a theoretical model we have studied the effect of cyclic electron flow around the PS 2 on the dampening of photosynthetic oscillations. The base of this electron transport lies in the existence of plastoquinone at PS 2 donor side. As the rate of cyclic electron transport around the PS 2 increased, the oscillations dampened. By method of self-consistent field of antisymmetrical molecular orbital in p-electron approximation, we have studied the distribution of electron density in different redox states of plastoquinone (PQ). These distributions can explain the binding and dissociation of Q_B and reveal the place of quinone molecule association with the Fe²⁺ ion. The PQ dipole moment changes its direction after reduction, that may be important for LHC II regulation by kinase affected by the redox state of PQ. The regulation scheme of electron flow distribution between linear and cyclic electron transport based on the interaction of free and binded plastoquinone is discussed.

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39_27435.

MODULATION BY CALCIUM OF PEP CARBOXYLASE AND PEPC-PROTEIN KINASE FROM LEAVES OF AMARANTHUS HYPOCHONDRIACUS, A C4 PLANT

Raghavendra A.S., Parvathi K., Gayathri J.

Department of Plant Sciences, School of Life Sciences,
University of Hyderabad, Hyderabad 500046, INDIA

Keywords:

C4, calcium, light regulation, PEPC, phosphorylation/dephosphorylation

PEP carboxylase (PEPC) in leaves of C4 plants is activated in light due to phosphorylation of PEPC by a PEPC-protein kinase (PEPC-PK). Calcium (above 100 mM) inhibited the activity of PEPC, due to the competition with Mg²⁺. The inhibition by Ca²⁺ of PEPC was more at pH 7.8 than at pH 7.3. The phosphorylation of PEPC was stimulated by 10 mM Ca²⁺, while being inhibited by Ca²⁺ chelators, such as BAPTA and EGTA. The inhibition by BAPTA and EGTA was relieved by the addition of Ca²⁺ but not by the addition of Mg²⁺. Phosphorylation was restricted by Ca²⁺ or Ca²⁺ - calmodulin-dependent protein kinase inhibitors. These results suggest that phosphorylation of PEPC is mediated by Ca²⁺ dependent protein kinase in leaves of Amaranthus hypochondriacus and that a fine-tuning of Ca²⁺ in the cytosol is necessary for optimal activity of PEPC and PEPC-PK.

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47_30292.

A PARTIAL NUCLEOTIDE SEQUENSE OF 6.0 KB CHLOROPLAST DNA FRAGMENT OF CICER ARIETINUM

Ajalov V.A., Shahmuradov I.A., Suleymanova Z.J., Mamedov A.Ch., Aliev J.A.

Institute of Botany
Patamdar Shosse 40
370073 Baku
Azerbaijan

Keywords:

chloroplast, chloroplast development, chloroplast genes, evolution, recombinant DNA, sequence analysis

To study chloroplast genome organization the chloroplast genome library of chickpea (Cicer arietinum) has been created. Physical mapping and partial sequencing of the Bam HI fragment (6.0 kb) of the chloroplast DNA have been performed. The total length of sequenced DNA fragments is 2500 kb. To detect possible coding regions the sequenced DNA fragments of chickpea were compared with known complete chloroplast DNA sequences of rice, maize and tobacco from GenBank. The analyses revealed that the sequenced chickpea chloroplast DNA fragments contain the whole tRNAAsn(GUU) gene, 5'end of 4.5S rRNA, 3'ends of pet A and tobacco ORF 512 homologue, inner part of psa I. Pet A and 4.5S rRNA genes of chickpea have a higher homology with the corresponding tobacco genes (92% and 94%, respectively) than with the same genes from rice and maize (80% and 82%, 88% and 80%, respectively). Pet A and 4.5S rRNA genes of chickpea are located in the same genome region, while the corresponding genes of tobacco, rice and maize are located in separate areas (probably, as a result of genome rearrangement).

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49_30771.

INHIBITOR CONTROL OF TRANSCRIPTION IN WHEAT SEEDLINGS

Guseinova I.M., Suleimanov S.Yu., Mamedov A.Ch., Aliev J.A.

Institue of Botany/Department of Molecular Genetic Bases of Production Processes
Patamdar Shosse 40
370073 Baku
Azerbaijan

Keywords:

biosynthesis, chloroplast, higher plants, membrane protein, molecular biology, RNA

The investigation of transcription inhibitors effect - rifampicin and actynomicin D on the synthesisynomicin D on the synthesis of total RNA was carried out. In this purpose total RNA was isolated and was carried out electrophoretic and spectral analysis. It was found out that mRNA band intensity in agarose gel decreases in relation to the control. Obvious decrease of peak content at 260nm on absorption spectrum of pure preparation of total RNA also was observed. It was established that these inhibitors partially block mRNA synthesis and at the same time actynomicin D which block transcription process in nucleus as is suggested significantly effect. These data correspond to results obtained by us obviously on polypeptide composition of chloroplast thylakoid membrane which exposed to effect of transcription inhibitors. Principles of these inhibitors effect on transcription level in the cell are discussed.

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62_12172.

PHOTOSYNTHESIS AND A NEW BIOTECHNOLOGICAL APPROACH TO WHEAT CROP IMPROVEMENT AND ENVIRONMENTAL ADAPTATION

Kershanskaya O.I., Shpak E.V.

Institute of Plant Physiology, Genetics and Bioengineering, Ministry-Academy Sciences, 45 Timiryasev Str., Almaty, 480090, Kazakstan

Keywords:

crop improvement, photosynthetictests, productiveandadaptivewheatspecies

A real approach to plant production improvement is a regulation of the creation of physiology - genetic active photosynthetic apparatus by crop breeding. Increasing of photosynthetic apparatus activity can be created by a way of photosynthetic tests elaboration and application and its genetic determination in favorable and limiting environmental conditions. In these connection its necessary systematic study of photosynthetic indexes on different levels of photosynthetic apparatus organization, differentiation of selective collections by photosynthetic tests, revealing of forms - donors of high photosynthetic activity and creation of a model of optimal photosynthetic type wheat plant with high productivity and adaptability to environments. We have characterized 30 photosynthetic indices as tests on productivity and drought adaptability. Based on these indices with using of correlation, combine ability and polygene analysis we have determined four types of winter weat production processes in watered conditions of South - East of Kazakstan and have conducted search of wheat genotypes with high photosynthetic activity. The new lines of wheat - donors and the first productive sort of winter wheat with high photosynthetic activity were revealed and given to State examination.

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65_20303.

CHLOROPLAST ATP SYNTHASE: IDENTIFICATION OF PROTEASE ACCESSIBLE SITES IN CF1

Malyan A.N., Vitseva O.I., Strotmann H.^*

Institute of Soil Science and Photosynthesis, Russian Academy of Sciences, Pushchino, Moscow Region 142292 Russia
^*Institute of Plant Biochemistry, Duesseldorf University, Duesseldorf, Germany

Keywords:

atomic model, ATP synthase, chloroplast, energy transduction, photophosphorylation, proteolysis

The structure of isolated and membrane-bound CF1 was studied by limited proteolysis, SDS-PAGE and sequence analysis. The N-terminal fragment of the a subunit was shown to have an exposed area including the bonds E17-G18, R21-E22-V23 and K24-V25. In the membrane-bound CF1 the bond b L14-E15 was identified. In the central domain area, the protease attacked bonds are (a) S86-S87, E125-S126, R127-L128, and (a) G102-G103. The protease accessibility of these bonds correlates with the surface location of similar bonds in mitochondrial F1 [Abrahams J.P. et al. (1994) Nature 370, 621-628]. In CFoCF1 the population of CF1 with a G160-R161 bond exposed to the outside decreases with membrane energyzation and increases with reduction of the g subunit S-S bond.

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66_20927.

TWO FORMS OF CARBONIC ANHYDRASE OF AMARANTH LEAVES

Guliev N.M., Babayev H.H., Bayramov Sh.M.

Institute of Botany
Patamdar Shosse 40
370073 Baku
Azerbaijan

Keywords:

C3, C4, carbonic anhydrase, whole plants

Carbonic anhydrase (carbonate hydro-lyase EC 4.2.1.1) catalysing reversible reaction of CO₂ hydration in C4-plant in comparison with C3-plant was investigated very poorly. During investigation carried out by us two forms of carbonic anhydrase of C4-plant amaranth were found. One form of enzyme is localised in soluble fraction of mesophyll cells, the other one - in chloroplasts of bundle sheath cells. Both forms of enzyme were obtained in highly-refined state. Carbonic anhydrase form localised in cytoplasm of mesophyll cells has molecular mass approximately 250 kDa, and enzyme form localised in chloroplasts of bundle sheath cells has molecular mass 150 kDa. Both forms of enzyme contain many SH-groups as well as zinc, which plays key role in appearance of catalytic activity. Investigation kinetics of CO₂ hydration reaction catalysed by both carbonic anhydrase forms showed that these forms of enzyme also differ from each other with its catalytic properties.

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67_21611.

COMPARATIVE ANALYSIS OF PROMOTER REGIONS OF RICE, TOBACCO AND MAIZE CHLOROPLAST GENES

Shahmuradov I.A., Mustafayev N.Sh., Zulfugarov I.S.

Institute of Botany/ Department Molecular Genetic Bases of Production Processes
Patamdar Shosse 40
370073 Baku
Azerbaijan

Keywords:

chloroplast, chloroplast genes, higher plants, nuclear genes, regulatory processes, computer analysis

To detect a possible common regulatory elements (besides of known canonical -35 and -10 boxes) of chloroplast genes transcription comparative computer analysis of promoter regions (-350 : +50) of all chloroplast genes from Oryza sativa, Nicotiana tabacum and Zea mays has been performed. DNA sequences of whole chloroplast genomes were obtained from GenBank. A search of putative RE was carried out by computer method "SITE" and FASTA program package. Although common RE for all analyzed genes was not found, areas of extensive and statistically nonrandom homology between chloroplast genes of various groups have been revealed. It is interesting that some of these common elements resemble the known regulatory elements in nuclear genes. Moreover, some of these elements have also been found in promoter regions some of chloroplast genes of other species. Data obtained suggest that at least some of analyzed genes contain additional RE exclusively related to chloroplast genes or resembling those in nuclear genes.

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140_5708.

A NOVEL PHOTOSYSTEM II-ASSOCIATED CARBONIC ANHYDRASE IN THE INORGANIC CARBON-CONCENTRATION PROCESS

Park Y.-I., Karlsson J., Rojdestvenski I., Pronina N., Klimov V., Öquist G. Samuelsson G.

Department of Plant Physiology
Umea University
Umea S90187
SWEDEN

Keywords:

carbonic anhydrase, CO2 concentrating mechanisms

Intracellular carbonic anhydrases (CA) in photosynthetic organisms are involved in the CO₂-concentrating mechanism (CCM), which helps overcome CO₂ limitation in the environment. In the green alga Chlamydomonas reinhardtii, this CCM is initiated and maintained by the pH-gradient created by photosystem (PS) II-mediated electron transport. We show here that photosynthesis is stimulated by the novel, intracellular a-CA bound to the chloroplast thylakoids. It is associated with PSII on the lumenal side of the chloroplast thylakoid membranes. This novel finding highlights that PSII is a feedforward regulator of CO₂ assimilation and a PSII-CA provides ample flux of CO₂ available for carboxylation.

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147_7768.

RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE LARGE SUBUNIT e N-METHYLTRANSFERASE AND SMALL SUBUNIT N-TERMINAL a N-METHYLTRANSFERASE ARE RELATED ENZYMES

Houtz R.L., Ying Z., Zheng Q., Royer M.

Department of Horticulture
N322D Agricultural Science Building - North
University of Kentucky
Lexington, Ky 40546-0091

Keywords:

processing enzymes, protein assembly, Rubisco, e N-methyltransferase, a N-methyltransferase

During investigations of the molecular basis for the absence of lysine-14 methylation in the LS of spinach Rubisco, two full-length cDNAs (S-40 and S-38) and a gene coding for a putative methyltransferase were isolated. S-38 and S-40 contain 60% and 62% identity with the amino acid sequences for pea and tobacco Rubisco LSMT respectively. However, the S-40 cDNA differs from S-38 by a 4-amino acid insert, a consequence of alternative 3' mRNA splicing and inclusion of 12 nucleotides from the 3' end of intron III. Engineering an identical insert into pea Rubisco LSMT resulted in complete loss of activity. Bacterially expressed forms of S-38 or S-40 did not exhibit methyltransferase activity towards the LS of spinach Rubisco. However, using spinach chloroplasts lysates it was discovered that both S-38 and S-40 catalyzed the incorporation of [H³-methyl] groups from S-adenosylmethionine into the N-terminal methionine residue of the SS of Rubisco. Thus, S-38 and S-40 represent the first available nucleotide and protein sequences for a eukaryotic protein N-terminal a amino methyltransferase. These results also suggest that protein a N-methyltransferases and protein e N-methyltransferases are related enzymes.

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151_8827.

STEPWISE ASSEMBLY OF ACTIVE a₁b₁-DIMERS AND a₃b₃-HEXAMERS FROM RECOMBINANT a AND b SUBUNITS OF THE RHODOSPIRILLUM RUBRUM ATP SYNTHASE

Du Z., Gromet-Elhanan Z.

Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

Keywords:

ATP synthase, chaperone proteins, HPLC, protein assembly, purple bacteria

The a subunit of the R. rubrum F₀F₁ ATP synthase (RrF₁a) was expressed in E. coli, and found to form inclusion bodies (Z. Du, Z. Gromet-Elhanan (1995) in: P. Mathis (Ed.), Photosynthesis: from Light to Biosphere, Vol. III, pp. 27-30, Kluwer Academic Publishers). We have established optimal conditions for refolding the expressed RrF₁a by following its capacity to stimulate, in a chaperonin-like manner (A. Avni, et. al., (1991) J. Biol. Chem. 266, 7317-7320), the restoration of ATP synthesis in b-less chromatophore-bound ATP synthase reconstituted with pure native RrF₁b. Size exclusion HPLC of the refolded RrF₁a revealed the appearance of a monomeric fraction well separated from all accompanying aggregated material. The isolated monomeric RrF₁a, as well as native or recombinant RrF₁b monomers show no ATPase activity. However, their incubation in presence of MgCl₂ and ATP results in formation of a₁b₁-heterodimers, that are active ATPases. Further studies have now demonstrated an additional step of assembly of the a₁b₁-dimers into the a₃b₃-hexamer, which forms the catalytic F₁ core complex, as illustrated in the bovine mitochondrial MF₁ crystal structure (J.P. Abrahams, et. al., (1994) Nature 370, 621-628).

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153_20696.

b SUBUNIT THR-159 AND GLU-184 OF THE RHODOSPIRILLUM RUBRUM ATP SYNTHASE ARE ESSENTIAL FOR ATP SYNTHESIS AND HYDROLYSIS

Nathanson L., Gromet-Elhanan Z.

Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, ISRAEL

Keywords:

ATPsynthase, calcium, magnesium, purplebacteria, site-directedmutagenesis

The crystal structure of bovine mitochondrial F₁-ATPase (Abrahams et al. (1994) Nature 370: 621-628) identified the MF₁b-T163 as a ligand to Mg²⁺ at the catalytic nucleotide binding sites, and MF₁b-E188 as appropriately positioned to activate a bound water molecule, promoting an inline nucleophilic attack on the nucleotide g-phosphate. We have mutated the equivalent T159 and E184 residues of the Rs. rubrum F₁b subunit and expressed them in soluble form in the system developed for wild type b (Nathanson L and Gromet-Elhanan Z (1998) J. Biol. Chem. in press). All mutated b subunits could bind, as the WT one, to the b-less chromatophore-bound ATP synthase. But the assembled E184 mutants did not restore any activity to the reconstituted enzyme complexes, indicating that this residue is absolutely essolutely essential for all catalytic activities of the ATP synthase. Among the T159 mutants only T159->S was active, restoring higher rates of Mg- and Mn-dependent ATP synthesis and hydrolysis than WT b, but even it could not restore any CaATPase activity. These findings indicate that b-S159 is a ligand for Mg²⁺, but not for Ca²⁺, suggesting that upon binding these cations F₁b catalytic sites acquire different conformational states. Our results provide a first plausible explanation for the absence of Ca-dependent energy-coupling in all ATP synthase complexes.

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160_25016.

LEAF CONDUCTANCE AND DARK RESPIRATION RATE IN TRANSGENIC TOBACCO PLANTS WHICH EXPRESS A scFv ANTIBODY AGAINST ABA

¹Peisker M., ²Artsaenko O., ³Fiedler U., ¹Wigger J., ⁴Phillips J., ¹Reinhardt I., ¹Conrad U.

¹Inst. für Pflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersleben, Germany;
²Univ. of Maryland, Chem. and Biochem. Eng., Baltimore, MD 21250, USA;
³MLU Halle-Wittenberg, Inst. für Biotechnologie, D-06120 Halle, Germany;
⁴QIAGEN GmbH, D-40724 Hilden, Germany

Keywords:

gas exchange, respiration, stomata, transgenic plants, abscisic acid

Ubiquitous expression of a single-chain variable fragment (scFv) antibody against ABA in leaves of Nicotiana tabacum results in highly increased transpiration rates and leaf conductances (g_L). In such leaves, g_L is nearly not influenced by CO₂ concentration and irradiance. Specific expression of the scFv antibody in mesophyll or guard cells does not give rise to altered stomatal characteristics. In our experiments, variations in g_L were regularly found to be significantly correlated with variations in dark respiration rate. This relationship is discussed with regard to the role of respiration in stomatal functioning.

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163_22891.

STRUCTURAL CHARACTERIZATION OF A MAJOR PHOTOSYNTHESIS GENE CLUSTER FROM HELIOBACILLUS MOBILIS

^*Xiong J., ⁺Inoue K., ^*Bauer C.E.

^*Department of Biology, Indiana University, Bloomington, IN 47405;
⁺Department of Biological Sciences, Kanagawa University, Hiratsuka, Kanagawa, 259-12, Japan

Keywords:

bacterial photosynthesis, bacterial reaction center, Bchl, genetic manipulation, heliobacteria

A 35.7 kb genomic DNA sequence of a major photosynthesis genes, has been obtained from the heliobacterium, Heliobacillus mobilis. This DNA fragment contains 28 open reading frames (ORFs), 19 of which are identified to be involved in photosynthesis. All the photosynthesis genes are tightly linked and transcribed unidirectionally. A unique four subunit gene organization for a cytochrome bc complex consisting of Rieske Fe-S protein, cytochrome b₆, subunit IV and a diheme cytochrome c was identified. Phylogenetic trees of various photosynthesis genes are constructed and analyzed, which show a consistent grouping of anoxygenic species distinct from that of oxygenic species. Our analysis also suggests that gene duplication events giving rise to the paralogous bchI and bchD genes might have occurred before the photosynthetic speciation, with the exception of purple bacteria. The evolutionary link of the heliobacterial reaction center core polypeptide PshA to both PSI type and PSII type reaction centers has been confirmed and analysed in relation to the origin of the photosystems and photosynthesis.

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167_23301.

DIRECT RESOLUTION OF SPECTRAL FINE-STRUCTURE AND ULTARFAST EXCITON DYNAMICS IN PLANT LHC II BY NON-LINEAR POLARIZATION SPECTROSCOPY IN THE FREQUENCY DOMAIN

Lokstein H., Schubert A., Leupold D., Voigt B.

Institut für Biologie, Humboldt-Universität zu Berlin, Unter den Linden 6, D-10099 Berlin, Germany
Max-Born-Institut, Rudower Chaussee 6, D-12474 Berlin, Germany

Keywords:

excited state dynamics, higher plants, laser spectroscopy, LHC II, light harvesting complexes

The structure of light-harvesting complex (LHC) II is known to 3.4Å resolution, but relations to function, i.e. reflection in optical spectra/exciton dynamics are not understood, yet. Non-linear Polarization Spectroscopy in the Frequency Domain (NLPF) yields ps/fs dynamics, spectral sub-structure and band-broadening modes at low (Schubert et al., BBA 1321 (1997) 195ff) as well as at room temperature (Lokstein et al., Biophys. J. 69 (1995) 1536ff). We report on NLPF experiments with LHC II in different states of aggregation (monomeric, trimeric, macro-aggregated). Though absorption spectra are rather similar, NLPF spectra reveal distinctive features. In particular, Chl a/b Q_y-band fine-structure is uniquely resolved at 77K, indicating intricate excitonic interactions among Chls. Implications for exciton dynamics and energy-transfer mechanisms are discussed.
(Supported by the DFG - grants Le 729/2-2 and Ho 1757/2-1.)

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170_8580.

PROPERTIES OF TIGHT NUCLEOTIDE BINDING SITE OF CHLOROPLAST COUPLING FACTOR CF₁ DEFICIENT IN THE d- AND e-SUBUNITS

Vitseva O.I., Malyan A.N.

Institute of Soil Science and Photosynthesis, RAS, Pushchino, Moscow Region, 142292, Russia

Keywords:

ATP, ATP synthase, chloroplast

The coupling factor CF₁ plays a key role in photosynthetic phosphorylation of ADP to ATP. In our work the role of three main CF₁ subunits (abg) in the nucleotide binding to CF₁ is considered. The effect of medium pH on the kinetics of the interaction of [¹⁴C]ATP and [¹⁴C]ADP with CF₁-de at substoichiometric nucleotide enzyme ratios was studied. Decreasing the pH from 8.0 to 6.0 results in a manyfold increase of the rate and extent of tight ATP binding, whereas ADP binding changes only slightly. The rate of hydrolysis of tightly bound ATP decreases; ATP dissociation becomes slower, while ADP dissociation slightly accelerates. Calculations show that acidification of the medium to pH 6.0 results in a 200-fold increase in tight nucleotide binding site affinity for ATP accompanied by a 3.5-fold decrease in its affinity for ADP. This suggests that the changes in site affinity and selectivity are controlled by protonation/deprotonation of specific acid-base groups located within the a-g subunits of CF₁; protonation of these grnation of these groups promotes stabilization of ATP binding while their deprotonation contributes to ADP binding.

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177_3159.

CALCIUM BINDING AT THE CFo H⁺ CHANNEL REGULATES THYLAKOID LUMEN ACIDIFICATION AND THROUGH THAT, CONTROLS VIOLAXANTHIN DEEPOXIDASE ACTIVITY AND PHOTOPROTECTION

Pan R.S., Dilley R.A.

Department of Biological Sciences
Purdue University
W. Lafayette, IN 47907 USA

Keywords:

calcium, energy dissipation, light stress, photoprotection, proton channel, xanthophyll cycle

The photoprotective xanthophyll cycle is triggered by lumen pH dropping near and below 6.0. Zeaxanthin formation is minimal under non-stressful conditions and it forms rapidly in over-energization conditions. This strong regulation correlates well with the occurrence of localized/delocalized proton gradient coupling, and the two coupling modes are, themselves, strongly regulated by Ca⁺⁺ binding to the CFo H⁺ channel. A logical hypothesis is that Ca⁺⁺ regulates the violaxanthin de-epoxidase activity and hence photoprotection by way of regulating the switching-over from localized [H⁺] gradient energy coupling to delocalized coupling. We tested this by measuring the viol. deepox. activity after various treatments that shift Ca⁺⁺ binding to the CFo. Displacing the Ca⁺⁺/CFo binding by (A) high KCl (B) EGTA or (C) excess light intensity (all assays done under coupling conditions), correlated with greatly increasing the viol. deepox. rate. In (A) and (B) adding Ca⁺⁺ reversed the effect and the rate of viol. deepox. activity was low. The results support the hypothesis that Ca⁺⁺/CFo H⁺ gating has a strong regulatory effect on the photoprotective mechanism in plants. This provides a biological rationale for why chloroplasts have evolved the switchable, dual energy coupling mechanism; i.e., both ATP formation and the V to Z activity sense the H⁺ gradient, and the buffering capacity of the sequestered domains provides the sensor that detects over-energization.

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181_1234.

ACTIVATING A-BRANCH ELECTRON TRANSFER IN BACTERIAL PHOTOSYNTHETIC REACTION CENTERS IMPOSES B-BRANCH CHARGE SEPARATION

Hartwich G., Mueller P., Richter M., Bieser G., Ogrodnik A., Scheer H., Michel-Beyerle M.-E.

Institut fuer Physikalische und Theoretische Chemie, TU Muenchen, D-85748 Garching and Botanisches Institut der LMU Muenchen, D-80638 Muenchen

Keywords:

bacterial reaction center, electron transfer, time-resolved spectroscopy, unidirectionality, modified reaction center

One of the fingerprint features of charge separation (CS) in bacterial photosynthetic reaction center (RC) is its exclusive unidirectionality along one of two branches of cofactors (active A-, inactive B-branch), which according to X-ray data are nearly symmetrical. In order to increase the probability of B-branch CS, electron transfer (ET) along the A-branch was significantly slowed down by selectively exchanging bacteriochlorophyll B_A against a structural analogue that in vitro is 130mV harder to reduce than B_A. Analysis of the spontaneous emission data of the primary donor's excited state (¹P^*) reveals a switching from a sequential CS route above 180K (¹P^* ® P⁺B_A^- ® P⁺H_A^-, H_A is the second electron acceptor at the A-Branch) to a superexchange route (¹P^* ® P⁺H_A^-) below 150K. The energy gap for ¹P^* ® P⁺B_A^- CS in the modified RC is DG₁=75 meV. A transient bleaching around 535 nm in addition to H_A bleaching at 546 nm can be attributed to the transient red uction of H_B indicative for ET along the unmodified B-branch. The quantum yield of P⁺H_B^- formation was determined and analysis of the data yield charge separation rates decreasing from ca. (700ps)^-1 at 290K to ca. (2ns)^-1 at 90K, revealing that CS along the unmodified B-branch is governed by a rather strong activation barrier for the sequential route (DG₁^B=90 meV for ¹P^* ® P⁺B_B^-).

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185_8456.

ROLE OF ANIONS IN THE REGULATION OF DISTRIBUTION OF ABSORBED EXCITATION ENERGY BETWEEN THE TWO PHOTOSYSTEMS IN SPINACH THYLAKOIDS

Jajoo A., Govindjee, Bharti S.

School of Life Sciences
DA University
Vigyan Bhawan, Khandwa Road
Indore 452 001, MP, India

Keywords:

anion channels, bicarbonate, photosystem 1, photosystem 2

Role of cations in excitation energy distribution between the two photosystems of photosynthesis is well established. Evidence is provided for the first time, for an important role of anions in the regulation of distribution of absorbed light energy between the two photosystems, based on Chl a fluorescence yield, rates of electron transport in low light and 77K emission spectra : the Fv/Fm ratio (variable to maximul fluorescence) was decreased by inorganic anions even in the presence of DCMU, the PSII electron transport was decreased whereas PSI electron transport was increased and the F735/F685 ratio was increased. Such changes were observed with inorganic anions having different valencies (Cl^-, SO₄^2-, PO₄^3-). Higher the valency of inorganic anion, more was the energy transferred towards PSI. Change in the valency of the inorganic anions, thus, regulates distribution of absorbed light energy between the two photosystems. However, organic anions like acetate, succinate and citrate caused no significant changes in Fv/Fm ratio, and in rates of PSI and PSII electron transport, showing their ineffectiveness in regulating light energy distribution.

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188_11538.

EXPRESSION OF FATTY ACID DESATURASES IN MESOPHILIC AND THERMOPHILIC CYANOBACTERIA

Kiseleva L.L., Lyukevich A.A., Los D.A.

Institute of Plant Physiology
Botanicheskaya str., 35
127276 Moscow, Russia

Keywords:

acclimation, cyanobacteria, fatty-acid desaturation, gene expression, gene regulation

Cyanobacteria desaturate fatty acids which are bound to glycerol moiety using specific enzymes, desaturases. The mesophilic cyanobacterium Synechocystis sp. PCC 6803 has four fatty acid desaturases which introduce double b introduce double bonds at d 6, 9, 12, and w 3 positions of fatty acids. The expression of d 9 desaturase was found to be constitutive, whereas other desaturases are induced by low temperatures. The levels of the mRNA of d 6, 12 and w 3 desaturases increase 10-15-fold following the drop in temperature from 34 to 25^oC. When temperature increases back to 34^oC, the levels of the mRNA return to their original levels within 1 hour. The inducibility of the desaturases was also confirmed on the protein level. The thermophilic cyanobacterium Synechococcus vulcanus has only one desaturase that introduces the double bond at d 9 position. The desaturase was found to be low-temperature- and high light-inducible. A rapid increase in mRNA level was observed during the shift in temperature from 55 to 45^oC. In contrast to mesophilic cyanobacteria, the level mRNA of d 9 desaturase of S. vulcanus stayed at high level during the backward shift in temperature. The correlation between the photosynthetic activity and the level of the desaturase mRNA was observed in the thermophilic cyanobacterium indicating the energy-dependent mode of the induction of the desaturase gene.

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192_28105.

REACTION CENTRES FROM GREEN SULFUR BACTERIA

PermentierH.P., Francke C., Neerken S., Schmidt K.A., Franken E.M., Hager-Braun C.^*, Amesz J.

Department of Biophysics, University of Leiden, the Netherlands
^*Institute of Biochemistry, Odense University, Denmark

Keywords:

green sulfur bacteria, photochemistry, pigments, polypeptide composition, reaction centers

Reaction centre core (RCC) complexes of Prosthecochloris aestuarii and Chlorobium tepidum were purified by a simple and fast procedure. P. aestuarii preparations contained only PscA and PscB, and no bound Cyt (PscC) or FMO protein, whereas the C. tepidum RCC also contained Cyt. P. aestuarii RCC contains 16 BChls a, 4 Chls a 670 and presumably 1 chlorobactene and 1 rhodopin per RCC as determined by HPLC and photobleaching. The absorption spectrum showed 7 Bchl a bands at 783, 795, 800, 809, 819, 830 and 836 nm. A CD signal at 809 nm indicates strong interactions between at least some of the BChls a, while a signal at 837 nm might be due to the primary donor P840. A CD signal was also seen in the Chl a 670 band. Illumination caused an oxidation of P840. The low temperature difference spectrum showed a multitude of bands in the Qy region of both BChl a and Chl a 670, indicating electrochromism of the core pigments.
Supported by NWO and the EC (FMRX-CT96-0081).

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195_22798.

LIGHT-INDUCED CHANGES IN THE STRUCTURE AND FUNCTION OF THE THYLAKOID SYSTEM IN THE PRESENCE OF LIPOPHILIC AMINES

Opanasenko V., Agafonov A., Semenova G.

Institute of Soil Science and Photosynthesis RAS, Pushchino, 142 292, Russia

Keywords:

chloroplast transformation, coupling/uncoupling, electron microscopy, pH-gradients, photophosphorylation, cationtransport

Depending on structural peculiarities of amines presented in the reaction medium at 10-50 µM two major types of light-induced changes in thylakoids were revealed: 1) local fusions of non-appressed thylakoid membranes observed in the case of tertiary amines (TA) tetracaine or dibucaine; 2) heterogenic swelling of thylakoids induced by heterocyclic amines (HA⁺ like neutral red and 9-aminoacridine. The second one corresponds to the inhibition of ATP-synthesis and cation transport. The differences between TA/HA action may be accounted for the amine distribution in the thylakoid compartment. TA incorporate presumably in the lipid region of the inner membrane leaflet, HA cations localise in the domains formed by lumen-exposed proteins. These domains are supposed to keep networks of the cation transport pathways. Binding of HA+ to COO-groups of proteins breaks lateral and translumenal bonds that create the structure-function state of the thylakoid system.

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201_29411.

FROM CYTOCHROME C₆ TO PLASTOCYANIN: AN EVOLUTIONARY APPROACH

De la RosaM.A., Hervás M., Diaz-Quintana A., De la Cerda B., Molina-Heredia F.P., Balme A., Cavazza C., Navarro J.A.

Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla y CSIC, Américo Vespucio s/n, 41092-Sevilla, Spain

Keywords:

algae, cyanobacteria, cytochromes, electron transfer, laser spectroscopy, photosystem 1, plastocyanin

The transfer of electrons from the cytochrome b₆-f complex to photosystem I (PSI) is carried out by any of the two soluble metalloproteins cytochrome c₆ and plastocyanin. Comparative analyses in a wide variety of evolutionarily differentiated organisms have allowed us to suggest that cytochrome c₆ was discovered by the Nature before than plastocyanin. Evolution of photosynthetic organisms seems thus to have involved the substitution of cytochrome c₆ up to make the copper protein be the only electron carrier in higher plants. What is the reason for such replacement? In this lecture, the answer to this question will first be discussed on the basis of the relative bioavailability of copper and iron along the Earth life. An account of our most recent data on structural and functional features of these two metalloproteins will be presented. Particular attention will be paid to site-directed mutants of cyanobacterial cytochrome c₆ and plastocyanin.
Work supported by the Spanish Ministry of Education and Culture, the European Union, and the Andalusian Government.

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204_19319.

SITE-DIRECTED MUTANTS OF CYTOCHROME C₆ PROVIDE NEW INSIGHTS INTO THE INTERACTION BETWEEN PSI AND THE HEME PROTEIN

De la Cerda B., Molina-Heredia F.P., Hervás M., Navarro J.A., Díaz A., De la RosaM.A.

Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla y CSIC, Américo Vespucio s/n, 41092-Sevilla, Spain

Keywords:

algae, cyanobacteria, cytochromes, electron transfer, laser spectroscopy, photosystem 1, plastocyanin

Site-directed mutants of cytochrome c₆ (Cyt) are reported herein by the first time. The project is aimed at analyzing specific residues of Cyt that should play similar roles as others in plastocyanin. The cyanobacteria Synechocysteria Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7119 have been used as they exhibit different kinetic mechanisms of PSI reduction by Cyt: whereas the slightly acidic Synechocystis Cyt (pI, 5.6) fits a simple collisional model, the positively charged Anabaena protein (pI, 9.0) follows a complex mechanism with three different steps. The structure of Cyt from Monoraphidium braunii has been used as a template for modelling the wild-type structures of Synechocystis and Anabaena Cyt and further design of mutants. Changes have been made in the hydrophobic region surrounding the heme edge, as well as in the areas equivalent to the negative patches of eukaryotic Cyt. The set of mutants include F61A, R64D, D67R, D69R and S66D/S68D in Synechocystis, and V25E, V25A, K29H, K62E, R64E, K66E and D72K in Anabaena. Laser flash absorption spectroscopy analyses of PSI reduction by Cyt mutants have been performed.

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207_30293.

KINETIC MECHANISMS OF PSI REDUCTION BY PLASTOCYANIN AND CYTOCHROME C₆ IN THE ANCIENT CYANOBACTERIA PSEUDANABAENA SP. PCC 6903 AND PROCHLOROTHRIX HOLLANDICA

Navarro J.A.¹, Hervás M.¹, Babu C.R.², Molina-Heredia F.P.¹, Bullerjahn G.², De la RosaM.A.<sup>1

1</sup>Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla y CSIC, Spain
²Department of Biological Sciences, Bowling Green State University, Bowling Green, OH, USA

Keywords:

algae, cyanobacteria, cytochromes, electron transfer, laser spectroscopy, photosystem 1, plastocyanin

The cyanobacteria Pseudanabaena and Prochlorothrix diverged at the very beginning of the evolutionary tree of oxygen-evolving photosynthetic organisms. They both posses positively charged plastocyanin (Pc) and cytochrome c₆ (Cyt). The reaction mechanisms of PSI reduction by these metalloproteins have been analyzed in a comparative way by laser-flash absorption spectroscopy. In both organisms, Pc follows a two-step mechanism involving complex formation and electron transfer, but the complex is formed by means of attractive electrostatic interactions in Pseudanabaena whereas it is hydrophobic in Prochlorothrix. Cyt, in its turn, follows a three-step mechanism with rearrangement of redox partners within an intermediate electrostatic complex; the first, fast phase of electron transfer (t_1/2 ca. 9 µs) can represent as much as 80% of the total amplitude of the kinetic profile. These data reinforce our previous observations that PSI was first adapted, from an evolutionary point of view, to operate with positively charged Cyt rather than with Pc.

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213_32476.

¹⁵N MAS NMR SPECTROSCOPY ON PHOTOSYNTHETIC REACTION CENTRES

Soede-Huijbregts C., van Laren M., Boender G.J., Cappon H., Raap J., Lugtenburg J., Gast P., Hoff A.

Leiden Institute of Chemistry, Gorlaeus Laboratories, P.O.Box 9502, 2300 Leiden

Keywords:

bacterial reaction center, purple bacteria, solid state NMR, specifically 15N labelling

In the photosynthetic reaction centre of Rb. sphaeroides histidine residues are found in crucial positions. ¹⁵N-His enriched RCs can be analysed with spectroscopic techniques such as magic-angle-spinning solid-state ¹⁵N NMR spectroscopy (MAS-NMR) and ESR. The ¹⁵N NMR chemical shifts of the imidazole nitrogens will shift considerably when they are involved in metal-ion coordination and these interactions will therefore be easily observed in the solid-state MAS-NMR spectra of the ¹⁵N-His enriched RCs.
We developed a new route for the synthesis of specifically labelled L-histidine, which can be made in high enantiomeric purity, with labels at any position and combination of positions on a one to five gram scale. t-¹⁵N and p-¹⁵N-histidines were made in an overall yield of respectively 29% and 56% and were incorporated via growth on a synthetic medium containing the labelled histidine.
From ¹⁵N CP/MAS NMR spectroscopy the conclusion is that apparently both the pros and tele nitrogens carry atomic charges in the RC. Only a small fraction of the p-¹⁵N-His response can be associated with ¹⁵N|. Therefore the electronic structure of the reaction center may be much less straightforward than was assumed in model calculations for the ground state of the RC, where only neutral histidines were taken into account.

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214_32092.

STUDIES OF ELECTRON TRANSFER KINETICS IN PHOTOSYSTEM I AFTER INACTIVATION OF THE IRON-SULFUR CENTER FB: EVIDENCE FOR FB BEING THE DISTAL CLUSTER OF PHOTOSYSTEM I AND THE PARTNER OF FERREDOXIN.

Diaz-Quintana A., Leibl W., Sétif P.

CEA, Département de Biologie Cellulaire et Moléculaire, Section de Bioénergétique, CNRS, URA 2096, C.E. Saclay, 91191 Gif sur Yvette Cedex

Keywords:

electron transfer, ferredoxins, Fe-S centers, photosystem 1, spectroscopy

Reaction centers of photosystem I (PSI) contain three different [4Fe-4S] clusters FA, FB and FX. From the X-ray structure of PSI, it is known that the terminal electron acceptors (FA, FB) are distributed asymmetrically along the membrane normal, with one of them being reduced from FX and the other one reducing soluble ferredoxin (Fd). Kinetics of electron transfer have been measured in PSI from the cyanobacterium Synechocystis sp. PCC 6803 after inactivation of FB by treatment with HgCl₂. Photovoltage measurements indicate that, in the absence of FB, the t1/2 of FA reduction by FX is smaller than 150 ns. The first-order rate of ferredoxin reduction by FA-, within the PSI/Fd complex, has been calculated from measurements of P700⁺ decay. Compared to control PSI, this rate is several orders of magnitude smaller, resulting in inefficient ferredoxin reduction, in accordance with previous measurements. These results support FB as the diect partner of ferredoxin and as the more distal cluster of PSI with respect to the thylakoid membrane, in accordance with a linear electron transfer pathway (FX, FA, FB, Fd).

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218_4570.

ORIENTATION OF PIGMENTS IN THE PHOTOSYSTEM II CP47/D1/D2/CYT B₅₅₉ COMPLEX AND THE OXYGEN-EVOLVING CORE COMPLEX: A LINEAR DICHROISM STUDY

Hou J.M.^1,2, Dejonghe D.¹, Kuang T.-Y.², Breton J.<sup>1,2

1</sup>SBE/DBCM, Bat 532, CEA/Saclay, Gif-sur-Yvette Cedex 91191, France
²Lab of Photosynthesis, Institute of Botany of Botany, Chinese Academy of Sciences, Beijing 100093, P R China

Keywords:

linear dichroism, P680, photosystem 2, pigments, polarized spectroscopy, reaction centers, spectroscopy, orientation

Linear dichroism (LD) spectroscopy is one important technique in the study of the orientation and organization of pigments in the photosynthetic membrane complexes in vivo. In this work the orientation of the pigments in the isolated Photosystem II (PSII) CP47/D1/D2/Cyt b₅₅₉ complex and the oxygen-evolving core complex from spinach chloroplasts are analyzed and characterized by means of low temperature absorption and LD spectroscopies. Using the preparations containing different CP47 content in CP47/D1/D2/Cyt b₅₅₉ complex lead us to propose the orientation of chlorophylls, b-carotene, pheophytin with respect to the membrane plane. The absorption and LD spectra upon the photochemical oxidation of the PII oxygen-evolving core complex in the presence of silicomolybdate were obtained. The preliminary result of the bleaching at 681 nm is attributed to the LD signal of primary electron donor P680. The further study on the attribution of the LD signal is in progress using the PSII reaction center D1/D2/Cyt b₅₅₉ complex.
Supported by CEA, France and CAS, P R China.

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220_3291.

PHOTOSYNTHETIC PIGMENT CONTENT AND ESSENTIAL OIL YIELD OF OCIMUM BACILICUM L. DURING DIFFERENT STAGES OF GROWTH IN THE FIELD

Misra M.

Meena Misra, Aromatic & Medicinal Plants Division, Regional Research Laboratory (CSIR), Bhuabneswar-751013, India

Keywords:

biomass, productivity, chlorophyll, Ocimum, Sweet basil

Sweet basil (Ocimum basilicum L.) seeds were shown in rainy season and 2 week old seedlings were transplanted in the field. Plants were grown for one month for establishment and then sampling for analysis was done at weekly intervals. Plant height, number of branches and herbage yield increased till 70 days followed by maturation and senescence phase. Photosynthetic pigment content increased till 94 days of growth. Ocimum oil yield increased gradually till 79 days. Floral parts although produced good quality Ocimum oil with high fragrance, but its contribution to total oil yield is meagre. Foliage portions followed by the total herbage yielded the maximum amount of oil. Comparative studies showed that the oil yield of Ocimum oil from the foliage was 1.5 times more than the herbage in 57 day old plants and that was 10-20% more in mature plants. This study suggests that an increase in the Ocimum oil yield can be achieved by increasing the foliage yield of this crop.

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222_3627.

THERMOLUMINESCENCE PROPERTIES AND CHANGES IN D1 POLYPEPTIDE OF SPINACH CHLOROPLASTS DURING PHOTOINHIBITION OF SPINACH LEAF DISCS AT CHILLING, ROOM AND HIGH TEMPERATURES

Misra A.N.¹, Ramaswamy N.K.², Desai T.S.<sup>3

1</sup>Department of Botany, University of Pune, Pune-411007
²Biotechnology Division, and
³Molecular Biology Division, Bhava Atomic Research Station, Mumbai-400085, India

Keywords:

abiotic stress, chloroplast, D1 turnover, electron transport, light stress, photoinhibition, Qb, temperature stresses, thermoluminescence, spinach

Spinach leaf discs were treated with high light intensities at chilling (4^oC), room (25^oC) and high (40^oC) temperature. Photosynthesis as measured by oxygen evolution measurements using DCQ as electron acceptor was not affected at these temperatures within 90 minutes. However, high light treatment inhibited photosynthesis by 33% at 25^oC, 52% at 4^oC and 57% at 40^oC after 60 minutes of illumination. Thermoluminescence spectra of photoinhibited samples at room temperature showed a reduction in the TL intensity for peak V (C-band) and IV (B-band). Peak IV was more affected by photoinhibition compared to the peak V, indicating Q_B site is relatively more affected than Q_A. Chilling or high temperature accelerated these changes in TL intensity. The quantitative changes in peak IV of TL spectra were correlated to the changes in D1 protein content in the photoinhibited leaves and PS II activity at different temperatures. This study suggests that the relatively higher damage of PS II at 40^oC was due to an accelerated degradation of D1-polypeptide of the reaction center, which is depicted by the decreased thermoluminescence of peak IV (B-band).

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223_3406.

PHOTOSYNTHETIC PIGMENT-PROTEIN CONTENT, ELECTRON TRANSPORT ACTIVITY AND THERMO-LUMINESCENCE PROPERTIES OF CHLOROPLASTS ALONG THE DEVELOPMENTAL GRADIENT IN GREENING WHEAT (TRITICUM AESTIVUM L.) LEAVES

Misra A.N.^1,5, Dilnawaz F.¹, Mohapatra P.¹, Das M.¹, Sahu S.M.¹, Misra M.², Ramaswamy N.K.³, Desai T.S.<sup>4

1</sup>Department of Botany, Utkal University, Bhubaneswar-751004, India
²A & M Division, RRL, Bhubaneswar-751013, India
³Biotechnology Division, BARC, Mumbai-400085, India
⁴Molecular Biology Division, BARC, Mumbai-400085, India
⁵Department of Botany, University of Pune, Pune-411007, India.

Keywords:

biogenesis, chloroplast, chloroplast development, etioplasts, greening, photosystem 1, photosystem 2, Qa, Qb, Rubisco, thermoluminescence, thylakoid membranes, wheat

Wheat (Triticum aestivum L. cv. Sonalika) seedlings grown in darkness at 26 + 2^oC for 10 days on water soaked cotton. The etiolated seedlings were transferred to continuous illumination of 35 µmole^.m^-2.s^-1. The developmental gradient was studied along the leaf lamina. The development of chloroplasts was monitored through the accumulation of photosynthetic pigments chlorophyll and carotenoids, and the development of functional ability of thylakoid membrane bound photo system I and photo system II electron transport properties, and the stromal soluble Rubisco enzyme quantity and activity. The pigment accumulation pattern showed a variation in the time scale of greening of the leaf segments. There was a longer-lag phase of accumulation of photosynthetic pigments in the basal leaf segments compared to the apical segments. The appearance of photosynthetic ability of photosystem I, photosystem II and Rubisco activity was acropetal. This study showed a gradation of the developing chloroplasts along the leaf lamina in wheat seedlings. The development of thylakoid membrane components for light energy transduction and the stromal components for CO₂ fixation was in parallel sequence during chloroplast development in the greening leaves.

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224_4220.

SODIUM CHLORIDE SALT STRESS INDUCED CHANGES IN THYLAKOID PIGMENT-PROTEIN COMPLEXES AND PHOTOSYSTEM II ACTIVITY OF MUNG BEAN (VIGNA RADIATA L.) SEEDLINGS

Sahu S.M.¹, Misra A.N.^1,5, Misra M.², Ramaswamy N.K.³, Desai T.S.<sup>4

1</sup>Department of Botany, Utkal University, Bhubaneswar-751004, India
²A & M Division, RRL, Bhubaneswar-751013, India
³Biotechnology Division, BARC, Mumbai-400085, India
⁴Molecular Biology Division, BARC, Mumbai-400085, India
⁵Department of Botany, University of Pune, Pune-411007, India.

Keywords:

abiotic stress, chloroplast, electron transport, photosystem 2, pigment-protein complexes, Qa, salt stress, thylakoid membranes, mung bean, Indian mustard

Choroplasts are the first organelles to show visible symptoms of salt stress in plants. Plant responses to stress also varies with the time period of growth. Seedling stage is the most crucial for plant establishment and is also more prone to abiotic stress than the later stages of growth. The reaction of salt susceptible and salt resistant crops to external stress responses are reported to be different. So in the present study, mung bean (Vigna radiata L.) - a salt susceptible and Indian mustard (Brassica juncea Coss.) - a salt resistant crop was studied to find out the differences in stress responses of crops. Seedlings were grown in water soaked cotton under continuous illumination of 35 µmole m^-2 s^-1 at 26 + 1^oC. Salinity treatment of 0, 0.5 and 1.0% (w/v) was given to the seedlings at 6 day. Photosynthetic pigment content and PS II electron transport activity was reduced under salinity in both mung bean and Indian mustard. The pigment protein patterns of both the crops were similar. The thermoluminescence (TL) glow peaks suggested that the changes in the PS II activity is preferably due to an alteration in the quinone binding sites.

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225_31661.

THE EFFECT OF REDUCED PSI-G AND PSI-K PROTEIN LEVELS ON PHOTOSYNTHESIS IN BARLEY (HORDEUM VULGARE L.)

Gilpin M.J., Jensen A.M., Nielsen H.L., Scheller H.V., Müller B.L.

Dept. of Plant Biology, Royal Veterninary and Agricultural University, 40 Thorvaldsensvej, DK-1871, Frederiksberg C, Copenhagen, Denmark

Keywords:

abiotic stress, down regulation, gene expression, light harvesting complexes, mutational analysis, nucleargenes

The nuclear encoded proteins PSI-G and PSI-K are two of the 17 proteins which make up photosystem I of higher plants. Alignment of the protein sequences and phylogenetic analysis verified a high degree of homology between the two genes and it has been suggested the two genes evolved via gene duplication of a common ancestral gene. The functions of PSI-G and PSI-K have not yet been determined, although cross linking studies suggest a role in LHCI binding. We have adopted antisense/cosuppression strategies in barley, in an attempt to down regulate levels of PSI-G and PSI-K, and thus examine the effect of reduced PSI-G/PSI-K protein levels on photosynthesis. Immature barley embryos were transformed separately with PSI-G (sense) and PSI-K (antisense) constructs along with a selection plasmid (PDM803), via particle bombardment. Transformed plants were verified by PCR and mutants with reduced PSI-G or PSI-K protein levels identifeid by western blotting. In preliminay analyses, plants with reduced PSI-K were not visually affected and showed no change in low temperature fluorescence emmission. Second generation plants are currently being analysed and further characterisation of mutants will be presented.

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226_542.

SPECTRAL AND FUNCTIONAL CHL A/B ANTENNA HETEROGENEITY IN PROCHLOROTHRIX

Gulyaev B.A.¹, Tetenkin V.L.¹, Golitsin V.M.¹, BullerjahnG.S.<sup>2

1</sup>Dept. of Biophysics, Moscow State University, Moscow 119899
²Center for Photochemical Sciences, Bowling Green State Univ., Bowling Green OH, USA

Keywords:

antennae, fluorescence, prochlorophytes

P. hollandica is a divergent cyanobacterium containing a chlorophyll a/b light harvesting complex which is structurally and distinct from chloroplast LHC. We analyzed spectral parameters of P. hollandica cells grown under different conditions and chlorophyll-protein complexes. Absorption, fluorescence emission/excitation spectra were deconvoluted into components representing corrected sprectra of pigment protein complexes to get their relative concentration and quantum yield. Analysis of the data revealed two spectrally and functionally different pools of antenna complexes with a chl a/b ratio of ~5. One component (LH 687) energetically couples with PSI and yields the main 77K fluorescence band at 687 nm. Another component (LH 692) predominately couples with PS2 and exhibits a fluorescence peak at 692 nm. Our data show that LH 692 contains ~15% of whole chlorophyll in the membranes, PS1 core ~60% and the PS2 core ~10%. LH 687 includes 15% of chlorophyll but half of this complex is detached from PS1. This fraction of LH 687 may be involved as a mobile antenna.

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229_3313.

PHOTOTROPHIC PURPLE BACTERIA AS MODEL SYSTEMS IN THE RESEARCHES OF STRUCTURE AND FUNCTIONS OF PHYTOHORMONES

Serdyuk O.P., Smolygyna L.D.

Institute of Soil Science and Photosynthesis, RAS.
Pushchino, Moscow Region, 142 292 Russia

Keywords:

cytokinins, hormones, phylogeny, procaryote, purple bacteria

Two kinds of substances exibiting high cytokinin activity in bioassays - a purine cytokinin zeatinriboside (ZR) and new cytokinin-like substance 4-OH-phenethyl-alcohol (4-OH-PA) have been found in bacterium of a-subdivision Rs. rubrum. No any substances of purine line have been revealed neither in biomass, nor in the cultural medium of phototrophic purple bacterium C. minutissimum. 4-OH - benzoic acid and 4-OH-PA exibining cytokinin activity were observed in C. minutissimum growth medium. The distinctions on phytohormonal composition between these two families of purple phototrophic bacteria belonging to various evolutionary branches enable us to assume that cytokinins can serve as a taxon attribute in them. The cytokinins are able to regulate numerous processes in plants. The cytokinins excreted by non-phototrophic a-bacteria are used by them as signal substances in symbiostances in symbiotic plant-microoganism associations. Gen expression from bacteria to plant-host is observed under parasitic interaction between them. Many questions arose after discovery of cytokinins in phototrophic purple bacteria: a) what are the functions of intracellular and effluxed pytohormones in purple bacteria? b) do purple bacteria have a gen of biosynthesis of cytokinins or not? c) what is the mode of action of different kind of cytokinin in them? To answer all these questions joint efforts of various specialists are required.

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230_27743.

TEMPERATURE DEPENDENT HYSTERESIS WITH ACTINIC LIGHT INTENSITY OF THE PRIMARY DONOR OPTICAL ABSORPTION FOR REACTION CENTERS FROM RHODOBACTER SPAEROIDES

Gouchsha A.O.¹, Barabash Y.², Scott G.W.<sup>1

1</sup>Department of Chemistry, University of California, Riverside, Riverside, CA 92521
²Institute for Physics, Natl. Acad. Sci. Ukraine, 252650 MSP, Kyiv-22, Ukraine

Keywords:

bacterial reaction center, electron transfer, Qb, temperature dependence, nonlinear dynamics

Optical absorption hysteresis of bacterial reaction centers Rb. sphaeroides was studied at temperatures from -20C^o to +20^oC with actinic light intensity (I) variation. We measured the primary electron donor absorbance changes with variation of I from the dark to different I_max levels and back to the dark. For low levels of I_max (< 0.1 mW/cm²), we obtained optical absorption hysteresis loops, at T=+20^oC, that were similar to those reported in ref. [1]. Strikingly, at lower temperatures, there was a gradual decrease of the hysteresis gap, and it vanished for temperatures below -10^oC. For levels of I_max close to or greater than the saturation limit (I_max > 1 mW/cm²), the gap of the loop became much larger--more than 50% of the total bleaching amplitude. The gap, under saturation conditions, increased at lower temperatures. These experimental results are discussed within a non-linear dynamics formalism that takes into account that the detailed shape of the RCs effective potential and the control of this shape by the actinic light intensity.
[1] Goushcha A.O., Kharkyanen V.N. and Holzwarth A.R.: J. Phys. Chemistry, B 101 (1997) 259; Goushcha A.O., Dobrovolskii A.A., Kapustina M.T., Privalko A.V. and Kharkyanen V.N.: Phys. Lett. A 191 (1994) 393.

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235_3637.

THE INHIBITION OF PHOTOSYNTHESIS OF PEA AND CLOVER PROTOPLASTS BY HIGH CO₂ CONCENTRATION, AND PLASMALEMMA CARBONIC ANHYDRASE

Ignatova L.K., Romanova A.K., Moskvin O.V., Ivanov B.N.

Institute of Soil Science and Photosynthesis, RAS, Pushchino, 142292 Russia

Keywords:

carbonic anhydrase, CO2 uptake, elevated CO2 concentration, proton channel

An increase of CO₂ concentration in solution up to 500 µM resulted in 20 - 40% decline of light-induced oxygen evolution by pea protoplasts in pH range from 5.0 to 9.0. Such a decline occurred in clover protoplasts at fourfold CO₂ concentration. It was found that the above difference between pea and clover protoplasts came from 3 to 5 times higher buffer capacity of clover chloroplasts. Acetazolamide (AA) decreased the inhibitory effect of high CO₂ concentrations, and did not affect similar inhibition of oxygen evolution by formate. There was no influence of AA on oxygen evolution at optimal (100 µM) CO₂. Km(CO₂) of photosynthesis of pea protoplasts was 3.7 µM in the presence of AA, and 3.9 µM in its absence. Carbonic anhydrase (CA) activity of pea protoplasts covered 8%, and that of clover protoplasts covered 12% of total cell CA activity. Km(CO₂) of soluble CA was 20 mM, the Km(CO₂) of whole protoplasts CA activity (plasmalemma CA) was 104 mM.

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239_32682.

ROBUSTNESS AND TIME-SCALE HIERARCHY IN BIOLOGICAL SYSTEMS

Rojdestvenski I., Cottam M., Öquist G. Park Y.-I.

Department of Plant Physiology, Umea University, Umea 90187, Sweden, and
Department of Physics and Astronomy, University of Western Ontario, London N6A 3K7, Canada

Keywords:

acclimation, adaptation, C metabolisms, Calvin-cycle, electronic excitation transfer

We address the issue of robustness of biological systems with respect to microscopic parameters. We discuss the emerging of robustness as a consequence of time-scale hierarchy in biosystems, applying naive thermodynamic and dynamic assumptions. We discuss decoupling of physiological regulatory mechanisms via the time-scale hierarchy and illustrate our speculations using two model systems in photosynthetic apparatus of green plants as examples. The first model deals with the exciton propagation and trapping in photosystem II, the second being that of CO₂ assimilation in Chlamidomonas reinhardtii.

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243_5332.

PROTON SHIFTS FROM HIGH-FIELD 2-D AND 3-D HIGH-SPEED CP-MAS ¹³C-NMR DIPOLAR CORRELATION SPECTROSCOPY OF AGGREGATED BACTERIOCHLOROPHYLL C IN UNIFORMLY ¹³C-ENRICHED CHLOROSOMAL ANTENNAE OF CHLOROBIUM TEPIDUM

van Rossum B.-J.¹, Mulder F.M.¹, Boender G.J.¹, Balaban T.S.², Holzwarth A.R.², Schaffner K.², de Groot H.J.M.<sup>1

1</sup>Gorlaeus Laboratories, P.O.Box 9502 2300 RA Leiden, The Netherlands,
²MPI-Strahlenchemie Postfach 101365, D-45413 Mülheim a.d. Ruhr, Germany

Keywords:

antennae, chlorosomes, NMR

High-field (14.1 T) heteronuclear 2-D (¹H-¹³C) and 3-D (¹H-¹³C-¹³C) magic angle spinning NMR dipolar correlation spectroscopy was applied to determine solid state proton chemical shifts of the aggregated bacteriochlorophyll (BChl) c in intact uniformly ¹³C-enriched light harvesting chlorosomes of the green photosynthetic bacterium Chlorobium tepidum. From the heteronuclear correlation spectra, evidence is found for the existence of at least two well-defined interstack arrangements, since the 5-CH and the 7¹-CH₃ responses are divided over two sets of signals. For both fractions proton aggregation shifts are observed for the -CH(CH₃)-OH moiety and for protons in the vicinity of the rings A and C of the BChl c in the chlorosomes, relative to monomeric BChl c in solution. All methyl protons in this region (2¹ and 3²) and the 3¹-H are shifted upfield, which indicates ring-current efs ring-current effects from the next chlorophyll in the stack. This confirms the spatial stacking of the BChl deduced by Balaban et al. (1995), Biochemistry 34, 15259-15266, from ¹³C dipolar correlation spectroscopy.

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246_21721.

PHOTOSYNTHETIC RESPONSES TO DROUGHT OF TWO GRAPEVINE VARIETIES OF CONTRASTING ORIGIN

^1,2Schultz H.R.

¹UFR Viticulture, INRA/ENSA, 34060 Montpellier, France
²present address: Institut fuer Weinbau und Rebenzuechtung, Forschungsanstalt D-65366 Geisenheim, Germany

Keywords:

adaptation, CO2 uptake, genotypes, water stress, whole plants

A comparative study was conducted on adaptive responses to water deficit with 8-year old field-grown vines of the cultivars Grenache, of Mediterranean origin, and Syrah of mesic origin. Stomatal conductance (g), photosynthesis (A) and the stomatal sensitivity coefficient (k) (slope of the relationship between g and the 'Ball, Woodrow, Berry'-index ([A*(hs/Ca)] hs=rel. humidity, Ca=ambient CO₂-concentration) decreased more pronounced with increasing water deficit (pre-dawn water potential, Y_pd) for Grenache than Syrah. Chlorophyll fluorescence indicated down-regulation of photosystem II under high light and high temperature during the water stress for Grenache but to a lesser extend for Syrah. Osmotic adjustment did not occur. Differences in whole plant hydraulic conductance may be implicated in the differential responses of stomata. Model simulations of canopy A showed, that Syrah plants assimilaed almost 40% more than Grenache at the same Y_pd. Contrary to Syrah Grenache did not mature fruit, despite the minimum Y_pd being 0.55 MPa higher than for Syrah.

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248_1969.

THE EFFECTS OF INCREASED ATMOSPHERIC CO₂ ON GROWTH, CARBOHYDRATES AND PHOTOSYNTHESIS IN RADISH, RAPHANUS SATIVUS

Usuda H., Shimogawara K.

Lab. of Chem., Teikyo Univ., Hachioji, Japan

Keywords:

carbohydrate, CO2 uptake elevated CO2 concentration, gas exchange, rising CO2 concentration, source-sink

The effects of sink capacity on the regulation of the acclimation of photosynthetic capacity to elevated levels of carbon dioxide are important from a global perspective. We investigated the effects of elevated (750 ppm) and ambient (350 ppm) atmospheric CO₂ on growth, carbohydrate levels and photosynthesis in radish seedlings from 15 to 46d after planting. In radish, a major sink is the storage root and its thickening is initiated early. Elevated CO₂ increased the accumulation of dry matter by 111% but had no effect on the acclimation of the rate of photosynthesis or on the levels of carbohydrates in leaves at dawn. Elevated CO₂ increased the dry weight in storage roots by 105% 46d after planting. The sink capacity of the storage root seemed to be enhanced by elevated CO₂. This enhanced sink capacity seemed to be responsible for absorption of elevated levels of photosynthate and to result in the absence of any over-accumulation of carbohydrates in source leaves and the absence of negative acclimation of photosynthetic capacity at elevated level of CO₂. We will discuss about sink capacity.

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255_22740.

INFLUENCE OF CARBOHYDRATE CONTENTS ON SENESCENCE RELATED GENE EXPRESSION IN BARLEY LEAVES (HORDEUM VULGARE L.)

Haussühl K., van der Kooij T., Krupinska K.

Institute of Botany, University of Cologne, Germany

Keywords:

gene regulation, Rubisco, shading, source-sink

Flag leaves of cereals are known to be the main source for carbohydrates needed for grain filling (Stoy 1965, Physiol. Plant. Suppl. IV, 1-125). Therefore, the photosynthetic lifetime of these leaves is of high importance for grain yield. Senescence of barley flag leaves under field conditions has been characterized by various functional, biochemical and molecular parameters (Humbeck, Quast and Krupinska 1996, Plant Cell Environ. 19: 337-344). Barley flag- and penultimate leaves have been compared with respect to the expression of senescence-specific genes. Among those genes, the HvS40 gene (Becker and Apel 1993, Planta 189: 74-79) is predominantly active in senescing flag leaves, while e.g. the gene represented by cDNA HvSF27 is expressed in both types of leaves. In order to unravel the molecular mechanisms underlying differential expression of senescence related genes the contents of sucrose, fructose and glucose have been determined uring senescence in different types of leaves. The results suggest that an increase in sucrose content and in the ratio of sucrose to monosaccharides in flag leaves around the onset of senescence could be one of the factors regulating the expression of a subset of senescence related genes. An increase in sink strength of the reproductive organs by light deprivation of the ears does not affect the onset and time course of senescence of flag leaves, while ear removal accelerates this process.

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258_30802.

INTERFERENCE OF REDOX CHLOROPLASTS ENZYMES OF CINNAMIC DERIVATIVES

Céspedes A.C.L.¹, Salazar R.¹, Calderón J.S.¹, Perich F.², King-Díaz B.¹, Lotina-Hennsen B.<sup>1

1</sup>Instituto de Química, Facultad de Química, Universidad Nacional Autonoma de México, Ciudad Universitaria, C.P. 04510, D.F.México, México.
²Departamento de Ciencias Quimicas, Universidad de La Frontera, Temuco-Chile.

Keywords:

coupling/uncoupling, inhibitors, O2 evolution, photosystem 2, cinnamic acid

Methyl ester of 3,4-dihydroxy-trans-cinnamic acid isolated from Lolium perenne cv. Embassy and Nui have been found to act as an uncoupler and Hill reaction inhibitor in spinach chloroplasts. At 3 mM concentration, this compound inhibited ATP-synthesis (100%), basal (40%) and uncoupled (15%) electron transport from water to methylviologen. An enhanced phosphorylating electron flow from water to methylviologen was observed by using this methyl ester. By this compound electron transport in photosystem-I was enhanced, on the other hand it was inhibited 100% in photosystem-II from water to DCPIP, H₂O to SiMo and it was inhibited 50% from DPC to DCPIP. This suggests that the site of inhibition of methyl ester is located at the oxygen evolution level and the other target in the span of P₆₈₀ to Q_A redox enzymes. The presence of OH functional group at C-4 appeared to be important for interference sinerence since derivatives without it luck of any effect.

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260_18536.

PROTONATION PATTERNS OF THE PHOTOSYNTHETIC RC AND THE ENERGETICS OF THE DIFFERENT CHARGE AND PROTONATION STATES OF THE QUINONES

Rabenstein B., Ullmann G.M., Knapp E.W.

Institut fuer Kristallographie, FB-Chemie,
Freie Universitaet Berlin,
Takustrasse 6,
D-14195 Berlin, Germany

Keywords:

bacterial reaction center, electron transfer, electrostatic interaction, proton transfer, quinones

Electron-transfer (ET) reactions between the quinones in bacterial photosynthetic reaction centers (bRC) are coupled to proton uptake of titratable groups in the bRC and of Q_B. For the bRC from Rhodopseudomonas viridis the protonation patterns and energetics of the different states of quinone Q_A and Q_B were computed by solving Poisson-Boltzmann equation (PBE) of the heterogeneous dielectric with e=4 in protein and e=80 outside. Protonation patterns and energetics of the quinone states provide a picture fully consistent with known experimental results and allow to make predictions. In particular it turned out that the first ET from Q_A to Q_B is followed by a proton uptake at Q_B. The Q_BH decays again to Q_B(-) unless it is stabilized by a second electron. This explains why Q_BH can hardly be found experimentally. Alternatively the PBE was solved using energy minimized bRC structures for the different quinone states accounting for conformational flexibility. Since with this procedure nuclear polarization is explicitly considered, the value of the dielectric constant in the protein was reduced from 4 to 2. The data obtained from this computation are consistent with the former results. In the later case we observed the tendency that titratable groups prefer to adopt a fully protonated or deprotonated state. This is a consequence of the self-consistent energy minimization.

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263_16351.

MODE OF RC OPERATION: A NEW INSIGHT INTO FAST/SLOW PROCESSES INTERPLAY

Christophorov L.N.

Bogoliubov Institute for Theoretical Physics NAS Ukraine, Kiev 252143 Ukraine

Keywords:

bacterial reaction center, charge separation, electron transfer, primary processes, electron-conformation interactions

Under sustained photo-induced electron flow the slow structural rearrangements of the RC protein within quinone acceptors binding pockets affect the electron transfer kinetics, so that the nonlinear feedback between the electron and structural subsystems leads to a threshold-like (with respect to the exciting light intensity) formation of nonequilibrium stable RC conformations with markedly distinct charge separation efficiency.
Coined in early 90's [1] and gaining first experimental evidence [2], this concept is developed at length, starting from a nonstandard stochastic theory of electron-conformation interactions in the RC up to qualitative and quantitative description of the recently observed intensity-dependent changes of stationary and kinetic characteristics of the Rb. sphaeroides RCs, employing no RC parameters except well-established [3].
1. L. Christophorov et al. J. Biol. Phys. 18, 191, 1992; BioSystems 5, 171, 1995; Phys. Lett. A205, 14, 1995
2. A. Goushcha et al. Phys. Lett. A191, 393, 1994; J. Phys. Chem. B101, 259 & 7612, 1997
3. L. Christophorov, A. Goushcha, A. Holzwarth, V. Kharkyanen. Phys. Rev. E (submitted)
Supported by the Ukrainian Fundam. Res. Found. (Grant 2.4/656) & Kiev SRC of Physics of the Alive.

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265_31353.

TEMPERATURE AND LIPID UNSATURATION EFFECTS ON PLASMA MEMBRANES AND THYLAKOID MEMBRANES OF SYNECHOCYSTIS SP. PCC 6803

^1,2Papageorgiou G.C., ^2,3Govindjee, ^2,3Govindjee R., ^2,4Mimuro M., ^1,4Stamatakis K., ¹Alygizaki-Zorba A., ²Murata N.

¹NRC Demokritos, Athens, Greece;
²National Institute of Basic Biology, Okazaki, Japan;
³University of Illinois Urbana-Champaign, IL, USA;
⁴Yamaguchi University, Yoshida, Japan

Keywords:

abiotic stress, acclimation, cyanobacteria, fluorescence, osmotic pressure, quenching

Polyunsaturated fatty acids exist in membrane lipids of wildtype (WT) Synechocystis cells but not in desA^-/desD^- mutant cells. Accordingly, membranes have different fluidities. We probed plasma membranes (PM) of intact cells by inducing chlorophyll a (Chl a) fluorescence changes with osmolality shifts and by measuring the electric conductivity of cell suspensions; and we probed thylakoid membrane (TM) by inducing light/dark acclimative changes of Chl a fluorescence. TM of mutant cells undergo reversible thermotropic transition at 20.5^oC. No such transition was detected in the TM of WT cells and the PM of both strains from 2 to 34^oC. Hyper-osmotic conditions suppress the Chl a fluorescence of photosystem II preferentially. Hyper-osmotic shocks caused a fast transient quenching only in WT cells and only at non-chilling temperatures. The transient quenching senses the higher fluidity of PM in WT cells.

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268_9620.

GAS EXCHANGE AND ENVIRONMENTAL PARAMETERS AT FBPase ANTISENSE POTATOES

Muschak M., Willmitzer L., Fisahn J.

Max-Planck-Institut f. Molekulare Pflanzenphysiologie
Karl-Liebknecht-Str. 25
D-14476 Golm
Germany

Keywords:

elevated CO2 concentration, gas exchange, stomata, transgenic plants, relative air humidity

Gas exchange measurements were performed to analyze the leaf conductances and assimilation rates of potato plants (Solanum tuberosum cv. Desireé) expressing an antisense construct against the chloroplastic fructose-1,6-bisphosphatase to increasing photon flux densities, different relative air humidities and elevated CO₂ concentrations. Assimilation rates, transpiration rates and leaf temperatures were observed during a stepwise increase of photon flux density. These experiments were carried out under atmospheric conditions and in air containing 500 ppm CO₂. In both gas atmospheres two levels of relative air humidity (60-70% and 70-80%) were applied in different sets of measurements. Intercellular CO₂ concentration, leaf conductance, air to leaf vapour pressure deficit, and instantaneous water use efficiency were determined. At 70-80% relative humidity the antisense plants revealed significantly higher leaf conductances as compared to wildtype plants while no difference emerged at 60-70%. Under a stepwise increase of photon flux density linear rflux density linear relationships between transpiration rate and air to leaf vapour pressure deficit or transpiration rate and leaf conductance were observed depending on the level of air humidity.The results are discussed with respect to stomatal control by environmental parameters and mesophyll photosynthesis.

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271_8201.

THE PROTEOLYTIC MACHINERY OF CHLOROPLASTS - HOMOLOGUES OF BACTERIAL PROTEASES

Adam Z.

Dept. of Agricultural Botany, The Hebrew University, Rehovot 76100, Israel

Keywords:

auxiliary enzymes, proteases, proteolysis, thylakoid membranes

Numerous proteolytic processes in chloroplasts have been documented throughout the years, yet the responsible proteases remained obscure. Recent attempts to identify chloroplast proteases have revealed a number of proteases, all homologues of known bacterial proteases. A homologue of the serine-type ATP-dependent Clp protease is found in the stroma, composed of a proteolytic subunit, ClpP, associated with an ATPase, either ClpC or ClpX, as a regulatory subunit. FtsH, an ATP-dependent metalloprotease, is an integral thylakoid membrane protein, with its catalytic and ATP-binding domains exposed to the stroma. Another serine protease, DegP, is independent of ATP and is found tightly bound to the lumenal side of thylakoid membranes. Three of these proteins, ClpC, ClpX and DegP contain PDZ-like domains in their C-termini. These domains are implicated in protein-protein interactions in other systems, and may be important for substrate recognition and/or binding. The possible physiological substrates of these proteases will be discussed.

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272_14564.

USING SYNTHETIC MODEL SYSTEMS TO UNDERSTAND CHARGE SEPARATION AND SPIN DYNAMICS IN PHOTOSYNTHETIC REACTION CENTERS

Wasielewski M.R.

Chemistry Division, Argonne National Laboratory, Argonne, Illinois 60439 USA and Department of Chemistry, Northwestern University, Evanston, Illinois 60208 USA

Keywords:

electron transfer, EPR, femtosecond spectroscopy, model systems, primary events, reaction centers

Biomimetic studies of photosynthetic charge separation utilize supermolecules that are designed to mimic several key properties of the reaction center protein, e.g. 1) high quantum yield charge separation initiated from a photoexcited singlet state, 2) multi-step electron transfer to increase the lifetime of the charge separated radical pair product, 3) fast rates of charge separation and charge recombination, 4) temperature independent electron transfer rates, and 5) non-Boltzmann spin population of the radical pair product states. Most reaction center models fulfill only a subset of these criteria. We will report on photosynthetic model systems that closely mimic the five features listed above with particular emphasis on understanding how molecular structure influences the electron transfer and spin dynamics of radical pair formation as well as the unique spin-polarized triplet state previously found only in photosynthetic reaction centers.
Supported by the US DOE.

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276_15513.

EFFECT OF NITROGEN AVAILABILITY ON SULFATE ASSIMILATION IN ARABIDOPSIS THALIANA

Koprivova A., Brunold C., Kopriva S.

Institute of Plant Physiology, Altenbergrain 21, 3013 Bern

Keywords:

enzymes, gene expression, molecular biology, N metabolism, S metabolism

Assimilatory sulfate reduction is a pathway used by procaryots, fungi, and photosynthetic organisms to convert inorganic sulfate to sulfide. Both assimilatory sulfate and nitrate reduction are necessary for the synthesis of amino acids and regulatory interactions between these pathways are well documented. Using A. thaliana we analysed the effect of nitrogen defficiency and of the addition of different nitrogen sources on extractable activity, and enzyme and mRNA accumulation of APS reductase (APR) and O-acetylserine thiolyase (OAS-TL). After 72h without nitrogen the APR activity decreased to 60% and 30% in shoots and roots, respectively. Addition of nitrate restored the APR activity in both shoots and roots, whereas NH₄⁺ and Gln/Asn increased the APR activity in roots only. N-deprivation caused an transient increase of accumulation of mRNA coding for APR1, APR2, sulfite reductase, and cytosolic OAS-TL, followed by a substantial decrease. Western analysis revealed that the decrease and restoration of APR activity correlated with the APR protein amounts. Altogether, our results are consistent with the hypothesis according to which N-defficiency represses the expression of genes coding for key enzymes of sulfate reduction leading to a decrease in flux through the sulfate assimilation pathway.

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284_17308.

MOLECULAR CLONING AND CHARACTERIZATION OF CYTOSOLIC ISOFORM OF RIBULOSE-5-PHOSPHATE 3-EPIMERASE FROM RICE

Kopriva S., Koprivova A., Suss K.-H.

Institute of Plant Physiology
Altenbergrain 21
3013 Bern
Switzerland

Keywords:

C metabolisms, cytosolic enzymes, molecular biology, phylogeny

The oxidative pentose phosphate pathway (OPPP) supplies NADPH for various biosynthetic pathways and its metabolic intermediates can be utilized for the biosynthesis of nucleic acids, amino acids, fatty acids, terpenes, and phenolic compounds. Its localisation in plants is a matter of a great controversy. Originally, this pathway was supposed to be located in the cytosol; nowadays, the most accepted view is that there is a complete pathway in chloroplasts and an incomplete one in the cytosol, lacking the regenerative enzymes. We have characterized a cytosolic isoform of ribulose 5-phosphate 3-epimerase (RPE) from rice. The 24 kDa protein is 40% identical with RPE from potato chloroplasts and 52% with the cytosolic enzyme from yeast and human. Overexpression of the recombinant protein in E. coli lead to an increase of epimerase activity in bacterial extracts. Both Northern and Western analyses of rice revealed predominant location of cytosolic RPE mRNA and protein in roots. Taken together, these results demonstrate that the plant cells have the potential for a complete cytosolic oxidative pentose phosphate cycle.

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286_18617.

THE INFLUENCE OF DCCD ON KINETICS AND pH-DEPENDENCE OF ZEAXANTHIN FORMATION

Jahns P., Heyde S.

University of Düsseldorf, Plant Biochemistry, D-40225 Düsseldorf, Germany

Keywords:

carotenoids, energotenoids, energy dissipation, light harvesting complexes, pH-regulation, xanthophyll cycle

The influence of N,N-Dicyclohexylcarbodiimide (DCCD) on the kinetics and the pH dependence of the de-epoxidation reactions of the xanthophyll cycle has been investigated in isolated pea thylakoids. Zeaxanthin (Zeax) formation was found to be slowed down in presence of DCCD. The second step (antheraxanthin to Zeax) of the reaction sequence was more affected than the violaxanthin to antheraxanthin conversion. Furthermore, the decrease of de-epoxidase activity at increasing pH was found to be shifted by about 0.3 pH units to more alkaline pH values in presence of DCCD. This was paralleled by a less pronounced cooperativity for the pH-dependence of de-epoxidation. Comparative studies with antenna depleted thylakoids from intermittent light grown plants and analyses of the xanthophyll conversion in different antenna subcomplexes support the view that binding of DCCD to antenna proteins is most probably responsible for the retarded kinetics but DCCD-binding to the violaxanthin de-epoxidase for the altered pH-dependence.

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290_19823.

CRITICAL ROLE OF TURGOR FOR THE PROLIFERATION OF SYNECHOCOCCUS SP. PCC 7942 AT UNFAVORABLE SALINITIES

LadasN.P., Stamatakis K., Papageorgiou G.C.

NRC Demokritos, Institute of Biology, Athens 15310, Greece

Keywords:

abiotic stress, cyanobacteria, fluorescence, osmoregulators, salt stress, salt tolerance

Sucrose accumulation enables the fresh-water cyanobacterium Synechococcus sp. PCC 7942 to tolerate salinities up to 0.4M NaCl. The genetic transformation to a strain (PAMCOD) capable of constitutive accumulation of 60-80 mM glycine betaine shifts the upper limit of salt tolerance to approx. 0.5M NaCl. We measured turgor threshold fluorimetrically [Papageorgiou GC and Alygizaki-Zorba A., Biochim. Biophys. Acta 1335 (1997) 1-4] and sucrose accumulation during culturing these strains in the presence of 0.4M NaCl. In wild-type (WT) cells, sucrose and turgor threshold reached maximal values (9 mol ^. mol Chl^-1 and 0.70 Osm^.kg^-1, respectively) after 25h and declined thereafter. WT cells did not proliferate. Transformed cells began to proliferate rapidly after 75h when cytoplasmic sucrose reached 20 mol ^. mol Chl^-1 and turgor threshold 0.85 Osm^.kg^-1. Our results emphasize the critical role of turgor for cell proliferation at unfavorable salinities. Transformed cells proliferated because of enhanced sucrose synthesis in the presence of glycine betaine.

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292_25725.

COUPLING OF PHYCOBILISOMES TO REACTION CENTRES IN CYANOBACTERIA

Mullineaux C.W., Ashby M.K., Emlyn-Jones D.

Department of Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, UK

Keywords:

cyanobacteria, light acclimation, light harvesting complexes, phycobiliproteins, thylakoid membranes

A combination of confocal microscopy, spectroscopy and mutagenesis has been used to study the way in which phycobilisomes are structurally and functionally coupled to reaction centres. The results suggest a “dynamic equilibrium” model for the organisation of phycobilisomes and reaction centres in the thylakoid membranes of cyanobacteria. Phycobilisomes are weakly coupled to reaction centres: they frequently detach and diffuse before coupling to another reaction centre. Phycobilisomes can be functionally coupled to PSII or PSI: the relative coupling to the two photosystems depends on the binding constants and the PSII/PSI ratio. Phycobilisome-reaction centre coupling is a major site of regulation of the photosynthetic apparatus. We have used mutagenesis to identify factors involved in both long and short-term regulation of light-harvesting in cyanobacteria. We propose that the regulatory mechanisms work by altering the relative binding constants of phycobilisomes for PSII and PSI.

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293_31287.

CHLOROPHYLL a FLUORESCENCE SENSES WATER AND NaCl FLUXES ACROSS PLASMA MEMBRANES OF CYANOBACTERIUM SYNECHOCOCCUS SP. PCC 7942

Stamatakis K., Ladas N., Alygizaki-Zorba A., Papageorgiou G.C.

NRC Demokritos, Institute of Biology, Athens, Greece 153 10

Keywords:

abiotic stress, cation channels, cyanobacteria, fluorescence, salt stress

Chlorophyll a (Chl a) fluorescence senses osmotic volume changes of cyanobacterial cells (Papageorgiou G.C. and Alygizaki-Zorba A., Biochim Biophys Acta 1335 [1977] 1-4; Papageorgiou G.C., Alygizaki-Zorba A., Ladas N. and Murata N. Physiol Plantarum [1998] in press). With this property we tracked cell volume changes in suspensions of DCMU-treated (20 mM) and dark-acclimated Synechococcus sp. PCC 7942 after NaCl concentration jumps. After NaCl jump fluorescence first declined (t_1/2 ~10 ms) because of cytoplasmic water efflux, and then rose (t_1/2 ~200 ms) to a higher level because of NaCl and water influx. Fluorescence rise was more extensive in starved cells than in normal cells indicating reduced active Na⁺ ion expulsion. Because of NaCl uptake turgor threshold increased from 0.195 Osm^.kg^-1 (cells in BG11 medium) to 0.460 Osm^.kg^-1 (cells in BG11 plus 1M NaCl). Microhematocrit cell packing proved that NaCl does not induce cells contraction; sorbitol does. Chl a fluorescence is a dynamic sensor of water and solute fluxes across plasma membranes of cyanobacteria.

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302_6347.

ISOLATION AND CHARACTERISATION OF ACCLIMATION-DEFECTIVE MUTANTS OF ARABIDOPSIS THALIANA

Walters R.G., Shephard F., Horton P.

Robert Hill Institute, Dept of Molecular Biology and Biotechnology,
University of Sheffield, Sheffield S10 2TN, UK

Keywords:

acclimation, fluorescence imaging, light regulation, mutants, chloroplast composition

Many higher plants are able to modify the composition of the photosynthetic apparatus in response to changes in the environment. Such acclimation enables them to optimise the use of scarce resources and to avoid damage due to excess absorption of incident light. Wild type plants show changes in response to varying growth light within 2-3 days of a change in growth conditions; these are readily monitored via the maximum photosynthetic rate (P_max) and the Chl a/b ratio.
We have used imaging of chlorophyll fluorescence to screen the T-DNA-tagged "Feldmann" lines of Arabidopsis thaliana for mutants which are defective in acclimation. Several lines with widely differi with widely differing phenotypes have been identified: Two mutants retain the ability to acclimate to growth irradiance, but have reduced P_max compared to the wild type (Chl a/b is unchanged), whether grown in low or high light; two further mutants are also capable of acclimation, but respond to a change in growth conditions more slowly than the wild-type; and a fifth mutant grows normally under constant growth conditions, but is unable to respond to an increase in irradiance.
Current work aims to identify the mutated genes, and to complete the physiological characterisation of the defects in acclimation. Combining these approaches promises to advance our understanding of the molecular mechanisms involved in the regulation of acclimation.

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303_7559.

THE AFFINITY OF BINDING OF DIFFERENT XANTHOPHYLLS TO LHCII COMPLEXES

RubanA.V., Lee P., Young A.J., Horton P.

Robert Hill Institute,University of Sheffield, Sheffield, S10 2TN, UK (AVR, PL & PH). School of Biological and Earth Sciences, Liverpool John Moores University, Liverpool, L3 1AF UK (AJY).

Keywords:

LHC II, light stress, non-photochemical quenching, pigment binding, xanthophyll cycle, carotenoidbinding

Using nondenaturing IEF and sucrose gradient centrifugation combined with different types of detergent and enzymatic treatments we have estimated the relative affinity of binding of a number of xanthophylls to different LHCII. Monomers and trimers of LHCIIb, and 2 minor LHC’s, Cp26 and Cp29 were isolated either from dark-adapted or light-treated leaves to compare binding affinities of violaxanthin and zeaxanthin. It was found that violaxanthin is very weakly bound to LHCIIb trimers compared to lutein. For LHCIIb trimer the relative binding strength was neoxanthin > lutein > zeaxanthin > violaxanthin whereas for monomer it was lutein > neoxanthin > violaxanthin > zeaxanthin. Binding strength of all pigments to the minor antenna was lower than to LHCIIb, and the relative affinity of different xanthophylls was similar to that for the LHCIIb monomer. Efficiency of violaxanthin deepoxidation was shown to be highest in those complexes, where xanthophyll cycle carotenois have lowest affinity of binding (LHCIIb and Cp26). It is suggested that violaxanthin available for de-epoxidation is mostly loosely bound/periferal to LHC.

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304_14628.

INTERRELATED TRANSFORMATION OF CHLOROPLAST ULTRASTRUCTURE AND MORPHOGENESIS OF PLANTS CAUSED BY UV-A RADIATION

Selga M., Selga T.

Institute of Biology, University of Latvia 3 Miera St., Salaspils LV-2169, LATVIA

Keywords:

chloroplast, chloroplast differentiation, chloroplast transformation, light stress, regulatory processes

Tomato (Solanum lycopersicum L.) a/o plants were cultivated in controlled conditions and treated with UV-A radiation (l_max = 365 nm). Fission of chloroplasts immediately after UV-A treatment and during next 10-20 days in mature mesophyll cells was induced. Chloroplasts contained rarefied stroma areas filled with fibrillar and granular structures (nucleoids). Successive stages of chloroplast changes was stated: 1) twine of thylakoids in concentric coils; 2) joining of tips of thylakoids to the chloroplast envelope; 3) moving of thylakoids in the form of "U" or trefoil figure; 4) association between the nucleus and the chloroplast, containing duple/triplex membrane system; 5) formation of crosswalls in the chloroplast; 6) separation of 2-3 daughter chloroplasts. This reflect: photoinhibition and efflux of protons from thylakoids to periplastida space; change, locomotion and activation of chloroplast nucleoids; exchange of substances between chloroplasts and nucleus what stimulate transcription and replication; intensified biogenesis of thylakoids, fast change of epigenesis to accelerated growth, blossoming, production and senescence of plants induced by UV-A radiation.

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312_4258.

THE MUTATION D1-D61N IN PHOTOSYSTEM II OF SYNECHOCYSTIS SP. PCC 6803 RETARDS S-STATE TRANSITIONS WITHOUT AFFECTING ELECTRON TRANSFER FROM Y_Z TO P₆₈₀⁺

Hundelt M.¹, Hays A.M.², Debus R.J.², Junge W.<sup>1

1</sup>Universitaet Osnabrueck, FB Biologie/Chemie, Abt. Biophysik, Barbarastr. 11, 49076 Osnabrück
²Riverside CA, USA

Keywords:

cyanobacteria, electron transfer, O2 evolution, photosystem 2

The influence of single-site mutations at Asp61 of D1 of photosystem II (PSII) was studied with cells and oxygen-evolving core particles (1) from Synechocystis. The fast relaxation of the S-states in these particles (2) allowed repetitive experiments on S-state transitions. The half-rise times of S₁®S₂ and S₂®S₃ were doubled for D61N compared to WT. The flash-induced release of oxygen from cells was slowed tenfold. The rate of P₆₈₀⁺-reduction was similar in D61N and WT, i.e., the mutation slowed S-state transitions without affecting Y_Z and P₆₈₀. These results are indicative of a selective effect of the charge in position D1-61 on the Mn-cluster.
(1) Kirilovsky, D.L., Boussac, A., Van Mieghem, F.J.E., Ducruet, J.M., Setif, P., Yu, J., Vermaas, W.F.J., and Rutherford, A.W. (1992). Biochemistry 31, 2099-2107.
(2) Haumann, M., Hundelt, M., Jahns, P., Chroni, S., Bögershausen, O., Ghanotakis, D., and Junge, W. (1997). FEBS Lett. 410, 243-248.

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313_4687.

GENERATION OF C. REINHARDTII MUTANTS THAT PHOTOPRODUCE HYDROGEN FROM WATER IN THE PRESENCE OF OXYGEN

Ghirardi M.L., Flynn T., Iyers A., Forestier M., Melis A., Danielson P., Seibert M.

National Renewable Energy Laboratory, Golden, CO 80401; Dept. Plant and Microbial Biology, University of California, Berkeley CA 94710; and Dept. of Biological Sciences, University of Denver, Denver CO 80208.

Keywords:

mutants

The photoproduction of hydrogen by anaerobically-induced algae through the reversible hydrogenase pathway is inhibited by very low levels of oxygen. We have developed two selection procedures and a chemochromic screen to rapidly identify and isolate desirable mutants with increased rates of hydrogen evolution and higher tolerance to oxygen. Concomitantly, efforts are being made to clone the C. reinhardtii reversible hydrogydrogenase gene by (1) PCR techniques using degenerate primers, and (2) probing a subtraction expression library from induced minus non-induced cells with scpecific polyclonal antibodies generated against a synthetic oligopeptide containing the published N-terminal sequence of the purified hydrogenase.

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315_23969.

PHOTOSYNTHETIC CHARACTERISTICS OF CAM SUCCULENTS WITH HIGH PRODUCTIVITY

Nobel P.S.

University of California, Los Angeles
Los Angeles, California 90095-1606

Keywords:

CAM, CO2 uptake, environmental stress, productivity, whole plants

Responses of net CO₂ uptake over 24-h periods with respect to day/night air temperatures, total daily photosynthetic photon flux (PPF), and soil water status under controlled conditions in environmental chambers have been determined for about 10 species exhibiting Crassulacean Acid Metabolism (CAM). This has enabled predictions of net CO₂ uptake under field conditions and also has led to design of plant spacing that maximizes annual net CO₂ uptake per unit ground area based on the PPF responses. Certain CAM plants (two Opuntia species and two Agave species) had measured dry weight productivities averaging 43 metric tons per hectare per year, which exceeds that of C₃ agronomic species and C₃ trees and is only slightly less than the most highly productive C₄ species. The high productivity of these CAM species can be rationalized based on daily net CO₂ exchange and cellular photosynthetic properties. However, highly productive cacti are not tolerant of low temperature, which has major implications for their cultivation in regions of the United States and other countries where air temperatures annually fall below about 5^oC.

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316_5718.

PHOSPHORIBULOKINASE: 3-DIMENSIONAL STRUCTURE AND CATALYTIC MECHANISM

Miziorko H.M., Runquist J.A., Harrison D.H.T.

Biochemistry Department
Medical College of Wisconsin
Milwaukee, WI 53226
U.S.A.

Keywords:

C metabolisms, Calvin-cycle, enzymes, sugar phosphates, X-ray diffraction, phosphoribulokinase

The structure of octameric R. sphaeroides phosphoribulokinase (PRK) has been determined to 2.5Å resolution. PRK folds into a 7-member mixed b sheet surrounded by a helices; a similar fold characterizes the nucleotide monophosphate (NMP) kinases. Sequence identity between eukaryotic and prokaryotic PRKs, as well as buried surface area, suggests the identity of the functional dimer conserved throughout evolution. This dimer assignment is compatible with thioredoxin-mediated regulation of eukaryotic PRK activity. While PRK crystals do not contain bound substrate, identification of Walker A and B motifs allows assignment of the ATP binding site. Observation of a NMP kinase fold and mutagenesis results suggest the roles of several active site amino acids. These include the general base that deprotonates Ru5P's C1 hydroxyl as well as the residues that bind Ru5P. (USDA-NRICRGP Photosynthesis [HMM]; Herman Frasch Fdn. [DHTH])

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319_2116.

RECOVERY OF THE PHOTOSYNTHETIC APPARATUS FROM PHOTOINHIBITION DURING DARK INCUBATION OF THE GREEN ALGA DUNALIELLA SALINA

Melis A., Polle J.E.W.

University of California, Berkeley
Dept. of Plant and Microbial Biology
411 Koshland Hall
Berkeley, CA 94720-3102
USA

Keywords:

algae, light harvesting complexes, photoinhibition, photosystem 1, photosystem 2

The light-independent recovery from photoinhibition was monitored upon a transition of HL-grown Dunaliella salina to darkness. Upon dark incubation, the Chl a /Chl b ratio of the cells decreased promptly with a half-time of 2h from about 12:1 to about 5.5:1. The amount of photodamaged PSII, measured from the relative amount of a 160 kD protein complex containing the inactive D1-protein, decreased during dark incubation after an initial lag period. Concomitantly, the amount of functional PSII, measured from the 32 kD form of D1, remained constant. The results suggest that, upon dark incubation, photodamaged D1 is removed by degradation from the thylakoid membrane. During dark incubation the level of LHCII proteins increased in the cell accompanied by alterations in the composition of the LHCII proteins. In addition, the number of Photosystem I centers increased in the dark. The results show that recovery from photoinhibition upon a HL®Dark transition, in general, resembles that of a HL®LL transition. We conclude that several processes involved in the recovery of D. salina from photoinhibition are light-independent.
The work was supported by a grant from the USDA-NRICGP.

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320_23364.

SITE DIRECTED AND SUPPRESSOR MUTATION OF THE LIGANDS OF Fx IN PHOTOSYSTEM I OF SYNECHOCYSTIS SP. PCC 6803

¹Zeng M.T., ²Sagi I., ³Evans M.C.W., ¹Carmeli C.

¹Department of Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel;
²Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel
³Department of Biology, University College, London WC1E 6BT London, U.K.

Keywords:

cyanobacteria, electron transfer, Fe-S centers, mutants, photosystem 1

Intrasubunit interactions in the environment of the iron-sulfur cluster Fx in PSI of Synechocystis sp. PCC 6803 were studied by site directed mutation and suppressor mutation. Aspartate 566 adjacent to one of the cysteine ligands of Fx Cys565 was replaced by glutamate. The resulting D566E mutant in PsaB of the heterodimer prevented functional assembly of PSI in the thylakoids of the cyanobacterium. A stable functional PSI containing all three iron-sulfur centers Fx and FA/B was assembled in the suppressor mutant D566E/L416P. A modified yet stable PSI was also formed in the double mutation D566E/C565S. The results were interpreted to suggest that a stable Fx was required for assembly of PSI. The stability of Fx is supported by a salt bridge formed between Asp566 which is adjacent to Cys565 which is one of the cysteine ligands of the iron-sulfur cluster and Lys413 located on interhelical loops between helixs VIII-XI and VI-VII, respectively.

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321_7098.

PHOSPHOGLYCOLATE AS A POSSIBLE SIGNAL FOR CYANOBACTERIAL ACCLIMATION TO LOW AMBIENT CO₂

ShochatG>

Shochat S., Zer H., Rosenbaum A., Kaplan A.

The Avron-Evenari Minerva Center, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel

Keywords:

acclimation, CO2 concentrating mechanisms, cyanobacteria, phosphorylation/dephosphorylation, photorespiration

The signal involved in the acclimation of cyanobacteria to changing CO₂ concentration and the signal transduction path are not yet recognized. Acclimation requires light and is affected by the CO₂/O₂ concentration ratio. Thus, it was suggested that phosphoglycolate which accumulates following exposure to low CO₂ might serve as the internal signal. Over expression of cbbZ (from Ralstonia eutropha, kindly provided by Dr. B. Bowien) was used to raise the level of phosphoglycolate phosphatase (PGPase) in Synechococcus sp. PCC 7942. The mutant grew like the wild type under high- and low CO₂. PGPase activity in the wild type increased following exposure to low CO₂ and subsequently declined to the level observed under high CO₂. PGPase activity in the mutant was higher then in the wild type and was unaffected by exposure to low CO₂. The patterns of polypeptide phosphorylation and their rate of synthesis showed several changes in wild type cells following transfer from high- to low CO₂ but not in the mutant. When transferred from high- to low CO₂ concentration the apparent photosynthetic affinity for external inorganic carbon (representing the extent of acclimation to low CO₂) increased much slower in the mutant. These data indicated that phosphoglycolate level may serve as an internal signal for acclimation to changing CO₂ level.

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322_7284.

SELF-AGGREGATES OF SYNTHETIC ZINC 20-SUBSTITUTED CHLORINS AS MODELS FOR CHLOROSOMAL PHOTOSYNTHETIC ANTENNAE

Tamiaki H., Tsudzuki S., Nagata Y., Kureishi Y.

Department of Bioscience and Biotechnology, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan

Keywords:

assembly, Bchl, chlorosomes, light harvesting complexes, model systems

Zinc complexes of 20-substituted 3¹-hydroxy-13¹-oxo-chlorins were prepared for a model as bacteriochlorophyll-c possessing a methyl group at the 20-position, which is major light-harvesting pigment of photosynthetic green bacteria.
In a polar organic solvent such as tetrahydrofuran (THF), all the synthetic zinc chlorins gave sharp and small CD peaks, characteristic of monomeric species. The monomeric Q_y peaks were red-shifted in the order of the bulkiness of the 20-substituents (H < F < Cl < Br < Me < CF₃).
In a non-polar organic solvent (e.g., 1% THF-hexane) or an aqueous solution of a detergent (e.g., a-lecithin), the synthetic compounds aggregated to give oligomers with more broad and red-shifted peaks than the corresponding monomer. The red-shifted visible and large S-shaped CD spectra in the aggregate solutions were dependent upon the 20-substituents, although the FT-IR spectra were almost identical. Therefore, the local structure of the self-aggregates were the same (Zn^...3¹-OH^...O=C-13¹ and p-p interaction) but the supramolecular structures were controlled by the 20-substituents.

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331_8924.

DOMAIN-SPECIFIC RANDOM MUTAGENESIS IN LIGHT HARVESTING CHLOROPHYLL a/b PROTEIN (LHCII)

Heinemann B., Paulsen H.

Institute for General Botany
Müllerweg 6, D-55099 Mainz, Germany

Keywords:

amino acid sequences, complex formation, fluorescence, genetic manipulation, LHC II

The crystal structure of the major light-harvesting chlorophyll a/b complex (LHCII) suggests functional contributions of amino acids in chlorophyll binding or complex stabilisation. Most of these amino acids are located in the a-helical membrane-spanning protein domains, whereas much less is known about the functional significance of amino acids in other domains, such as the hydrophilic loops. We devised an experimental procedure to identify those amino acids in specific protein domains that are involved directly or indirectly in pigment binding or complex stabilisation. This in-vitro procedure follows an evolutionary concept in that it includes random mutagenesis of individual protein domains and subsequent selection of functionally altered mutants. Domain-directed mutagenesis is performed by mutagenic PCR amplification of a light harvesting protein gene in a bacterial expression vector creating a library of proteins each carrying unique mutations in the investigated domain. Mutants are then grown in individual wells in microtiterplates. As all following steps, overexpression of light harvesting protein, pigment binding and spectroscopic assay for stabile pigment-protein complex formation, are performed in one-vessel reactions on the same microtiter plate, several hundred mutants can be screened with reasonable effort. From clones where light harvesting protein is unable to bind pigment, DNA is isolated and sequenced in order to establish which aminoacid has been changed.

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335_3348.

REGULATION OF AMMONIUM ASSIMILATION IN CYANOBACTERIA

Florencio F.J., Garcia-Dominguez M., Martin-Figueroa E., Crespo-Gonzalez J.L., Navarro F., Muro-Pastor M.I., Reyes J.C.

Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC. CICIC. AVDA Américo Vespucio S/N, 41092-Sevilla, Spain

Keywords:

cyanobacteria, light regulation, metabolic processes, N metabolism, regulatory processes

Cyanobacteria are photosynthetic prokaryotes that carry out oxygenic photosynthesis like plants. Most cyanobacteria can use nitrate or ammonium ions as nitrogen source, and some strains are also able to fix dinitrogen. Although the existence of NAD- and NADP-dependent glutamate dehydrogenases has been reported in cyanobacteria, ammonium is mainly incorporated into carbon skeletons through the GS-GOGAT pathway Glutamine synthetase (GS) catalyzes the ATP-dependent synthesis of glutamine from ammonium and glutamate. Glutamine is not only utilized for protein synthesis but also for the synthesis of a number of nitrogen-containing metabolites such as purines, pyrimidines and amino sugars. Because of the importance of glutamine in nitrogen metabolism, it is not surprising that both the catalytic activity and the synthesis of GS are finely regulated in many organisms. We have studied the GS_GOGAT pathway, mainly in two cyanobacteria, Synechocystis 6803 and Anabaena 7120. The first one contains two different GS, GSI and GSIII, and two GOGATs, one depending on ferredoxin and another one on NADH, as electron donor, while Anabaena 7120 has only a GS, and one Fd-GOGAT. The effect that different environmental conditions such as the nitrogen source, carbon and nitrogen availability and li availability and light-dark transitions will be analize respect to the genes and the proteins involved in ammonium assimilation.
This work has been financed by grant PB94-1444 from DGICYT and by the Junta de Andalucia.

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338_28266.

NEW STRATEGIES TO EXPLORE THE INTRASUBUNIT INTERACTIONS IN THE ENVIROMENT OF THE IRON-SULFUR CLUSTER Fx IN PSI FROM SYNECHOCYSTIS

Gong X.M., Carmeli C.

Department of Biochemistry, Tel-Aviv University, Tel-Aviv, 69978, Israel

Keywords:

cyanobacteria, electron transfer, Fe-S centers, mutants, photosystem 1

The iron-sulfur cluster Fx is bound to the heterodimeric PsaA and PsaB subunits, the PSI reaction center complex, by four conserved cysteines in both of the heterodimer to form a rare interpolypeptide [4Fe-4S] cluster and has an unusually electronegative redox potential. The site directed single and double mutations of the amino acid residues adjacent to the ligands and of the ligands of the iron in PsaB subunit were used to modify PSI. Induction of suppressor mutation by PCR random mutagenesis or chemical reagents yielded valuable information on the intrasubunit interactions of the amino acids in the environment of Fx in PSI from Synechocystis sp. PCC 6803. Fx which functions as intermediate in the electron-transfer chain was also found to play a central role in the proper assembly of PSI. Analysis of the function suppressor mutations unveiled conformational changes resulting from the intrasubunit interactions. PsaC minus mutants lacking Fa and Fb were selected to facilitate the measurement of the structure of Fx by x-ray absorption (EXAFS).

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341_29425.

THERMOLUMINESCENCE ANALYSIS OF NOVEL D1 PROTEIN MUTANTS OF SYNECHOCYSTIS 6803

Keränen M.¹, Mulo P.¹, Aro E.-M.¹, Govindjee², Tyystjärvi E.<sup>1

1</sup>Dept. of Biology, Univ. Turku, FIN-20014 Turku, Finland;
²Dept. of Biophysics and Computational Biology, Univ. Urbana at Urbana-Champaign, IL 61801-3707, USA

Keywords:

cyanobacteria, D1 protein, mutants, photosystem 2, thermoluminescence

The relationship between the electron transfer capacity of PSII and thermoluminescence glow curves was studied in three Synechocystis 6803 mutants. The mutants have deletions in the stromal loop between the fourth and fifth membrane spanning helix of the D1 protein: the putative cleavage region of D1 was deleted from CD (del[G240-V249]), the PEST-like sequence from PD (del[R225-F239]), and both from PCD. PD grows autotrophically but CD and PCD do not. The position of the Q band measured in the presence of DCMU was the same in all three mutants and in the AR control strain. In AR, the B band was at about 15^oC higher than the Q band, but the mutants had the peak of the B band at the same temperature as the Q band. This suggests a destabilization of Q_B^- in all three mutants. The difference between the autotrophic PD and non-autotrophic CD and PCD is apparently in the rate of quinone/hydroquinone exchange at the Q_B site.

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342_29748.

INORGANIC ANIONS INDUCE STATE CHANGES IN SPINACH THYLAKOID MEMBRANES

Jajoo A., Bharti S., Govindjee

School of Life Sciences,
Vigyan Bhavan, Khandwa Road
Devi Ahilya University
Indore - 452001 (M.P.)
INDIA

Keywords:

anions, energy dissipation, photosystem 1, photosystem 2, thylakoid membranes, state change

Role of cations in excitation energy distribution between the two photosystems of photosynthesis is well established. This work provides evidence, for the first time, for an important role of anions in the regulation of distribution of absorbed light energy between the two photosystems. Inorganic anions (Cl^-, SO₄^2-, PO₄^3-) caused redistribution of energy more in favour of Photosystem I, by causing state changes, as judged from measurements of Chlorophyll a fluorescence transients, rates of electron transport in low light and 77K emission spectra: the Fv/Fm ratio (variable to maximal fluorescence) was decreased by inorganic anions even in the presence of DCMU, the PS II electron transport was decreased whereas PS I electron transport was increased and the F735(77KemissionfromPSI) / F685(77KemissionfromPSII) ratio was increased. However organic anions like acetate, succinate and citrate caused no significant changes in Fv/Fm ratio and in rates of PSI and PSII electron transport showing their ineffectiveness in regulating light energy distribution.

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347_30109.

SELF-ASSEMBLY OF SYNTHETIC ZINC CHLORINS IN AQUEOUS MICROHETEROGENEOUS MEDIA: STRUCTURAL AND FUNCTIONAL MODELS FOR CHLOROSOMES

Miyatake T.¹, Tamiaki H.¹, Holzwarth A.R.², Schaffner K.<sup>2

1</sup>Department of Bioscience and Biotechnology, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
²Max-Planck-Institut für Strahlenchemie, Postfach 101365, D-45413 Mźlheim an der Ruhr, Germany

Keywords:

aggregates, antennae, chlorosomes, light harvesting complexes, model systems

Self-assembled aggregates of a synthetic zinc chlorin in an aqueous suspension with either a-lecithin or Triton X-100 exhibit unique structural and functional properties. Absorption, circular dichroism, fluorescence and resonance Raman spectra indicate that the supramolecular structure is very similar to that of the bacteriochlorophyll c aggregates in non-polar organic solvents and in chlorosomes. The nature of the aggregates is controlled by structure and/or concentration of the added surfactants. Furthermore, in the presence of a small amount of metal-free bacteriochlorin, energy transfer from the aggregated zinc chlorin to the bacteriochlorin occurs in the aqueous micellar suspension. This result shows that bacteriochlorin and aggregates of zinc chlorin in an aqueous microheterogeneous medium adequately mimic the structure and function of natural chlorosomes.

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348_13336.

THE ANALYSIS OF THE PHOTOSYNTHETIC APPARATUS OF FRESHWATER PHYTOPLANKTON: PROBLEMS - PROGRESS - PERSPECTIVES

Wilhelm C.

University of Leipzig, Institute of Botany - Plant Physiology -, Johannisallee 21, D-4103 Leipzig, Germany.

Keywords:

adaptation, algae, ecology,tion, algae, ecology, fluorescence

Freshwater habitats have to cope with dynamic changes in nutrient availability (especially P and C), light and temperature, which leads to fast changes in the biomass, the population structure and in the productivity. Eutrophication endagers water quality and makes sanitation measures necessary. The different strategies to reduce the risk of phytoplankton blooms genereally introduce or strengthen limiting factors leading to physiological adaptations which can counteract the sanitation measure. The paper shows how the physiologicial state of the phototrophs can be monitored. Obviously, a concerted action by pigment-fingerprinting of carotenoids, chlorophylls and chlorophyll degradation products together with PAM fluorometry can be used as a powerful tool to control the efficiency of sanitation measures. The data also demonstrate that under field conditions physiological states can be observed which had been never analysed so far.

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350_18938.

EFFECTS OF MECHANICAL WOUNDING, CURRENT APPLICATION AND HEAT TREATMENT ON CHLOROPHYLL FLUORESCENCE AND PIGMENT COMPOSITION OF TOMATO PLANTS

Herde O., Pena-Cortes H., Willmitzer L., Fisahn J.

Max-Planck-Institut f. Molekulare Pflanzenphysiologie
Karl-Liebknecht-Str. 25
14476 Golm
Germany

Keywords:

abiotic stress, Chl fluorescence induction, pigments, xanthophyll cycle

Abiotic stress conditions that lower a plant's photosynthetic rate, such as light stress, water stress, nutrient stress, or temperature stress, increase the degree to which absorbed light can be excessive, increasing the need for energy dissipation. A wide variety of environmental stresses has now been shown to induce increases in the levels of xanthophyll cycle-dependent energy dissipation.
Chlorophyll fluorescence quenching parameters and pigment content of wild-type tomato plants were analyzed upon mechanical wounding, current application and heat treatment. Upon all stimuli the non-photochemical quenching raised significantly in local and systemic tissue. Vice versa, the photochemical quenching decreased. Total chlorophyll content remained almost unchanged, zeaxanthin content increased 5-6 fold, antheraxanthin content increased also, but to a minor extend, violaxanthin and neoxanthin on the other hand decreased significantly. These data indicate a tight parallelism between the processes induced by light-stress and those induced by mechanical wounding, current application and heat treatment on the level of photosynthesis and pigment composition.

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351_19297.

LIGHT INTENSITY ACCLIMATION OF THE PHOTOSYNTHETIC APPARATUS IN CHLOROBIUM TEPIDUM

Vannini C., Granata M., Balsemin M.T., Gerola P.D., Vianelli A.

Dept. of Structural and Functional Biology, University of Milano, via Ravasi 2, 21100 Varese, Italy.

Keywords:

bacterial photosynthesis, green sulfur bacteria, light acclimation, pigments, regulatory processes

We have studied the process of light acclimation of green sulfur bacterial photosynthetic apparatus in Chlorobium tepidum grown in batch cultures at white light intensities ranging from 0.3 to 250 µE m^-2s^-1. We observed a generalized decrease in the amount of pigments by increasing the light intensity. These changes especially occur in the range between a few and about 60 µE m^-2s^-1. The latter value corresponds to the onset of the saturation of the growth rate as a function of light intensity, while the former corresponds to very low values of the growth rate. Pigment analyses of chlorosomes do not show appreciable variations in the pigment ratios. On the contrary, from absorption spectra of cytoplasmic membranes, an enrichment in carotenoids is seen at the highest light intensity.

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353_8144.

RESONANCE RAMAN SPECTROSCOPY OF THE LHCB4 PROTEIN (CP29): A STRUCTURAL INVESTIGATION OF THE PIGMENT BINDING SITES

Pascal A.A.¹, Sandonŕ D.², Croce R.², Bassi R.², Robert B.<sup>1

1</sup>SBPM / DBCM, CE-Saclay, 91191 Gif-sur-Yvette, Cedex, France.
²Facoltŕ di Scienze, Biotecnologie Vegetali, Universitŕ di Verona, Italia.

Keywords:

antennae, electronic excitation transfer, light harvesting complexes, pigment binding, Raman spectroscopy

The minor photosystem II antenna protein CP29 can be reconstituted from recombinant Lhcb4 polypeptide and isolated pigments (Giuffra et al. (1996), Eur. J. Biochem. 238 112-120). We are using resonance Raman spectroscopy to investigate pigment-protein interactions in wild-type CP29 and mutants missing each of seven of the eight chlorophyll-binding sites. The binding-site properties at each of these seven positions (and, by extension, the eighth position) are considered in relation to the atomic model of the major photosystem II light-harvesting protein, LHCIIb (Kühlbrandt et al. (1994), Nature 367 614-621). In addition the observed flexibility of some of the sites (chlorophyll a or b), and the nature of xanthophyll-binding, are discussed.

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355_9327.

PHOTOSYNTHESIS AND ANTIOXIDANT METABOLISM IN MAIZE LEAVES SUBJECTED TO LOW TEMPERATURES

Foyer C.H.¹, Harbinson J.², Kingston-Smith A.H.<sup>1

1</sup>Institute of Grassland and Environmental Research, Department of Environmental Biology, Plas Goggerddan, Aberystwyth, WALES - UK, SY23 3EB.
²Agrotechnological Research Institute, Postbus 17, 6700 AA Wageningen, The Netherlands.

Keywords:

abiotic stress, active oxygen, chilling, fluorescence, Mehler reaction, photochemistry

When maize plants are grown at suboptimal temmperatures (below 20^oC), growth and photosynthesis are inhibited. Losses in total chlorophyll and maximal extractable Rubisco activity occur, but the relationships between the photosystems and between electron transport and CO₂ fixation are only slightly affected by growth temperature such that the integration and regulation of photosynthesis are largely unchanged. Differential compartmentation of antioxidants between the bundle sheath and mesophyll cells requires that oxidised ascorbate and glutathione are transported from the bundle sheath to the mesophyll for re-reduction. Exposure to low temperatures or to the pro-oxidant herbicide methylviologen causes preferential oxidative damage to bundle sheath proteins suggesting that this compartment is difficient in antioxidant defences in these stress conditions.

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356_28394.

CYCLODEXTRINS AS A TOOL FOR STUDYING THE ROLE OF GLYCEROLIPIDS IN SPINACH THYLAKOID MEMBRANES

¹Duchene S., ¹Siegenthaler P.A., ²Srivastava A., ²Strasser R.

¹Laboratoire de Physiologie végétale, Université de Neuchâtel, Rue Emile-Argand 13, CH-2007 Neuchâtel, Switzerland
²Laboratoire de Bioénergétique, Université de Genève, Ch. des Embrouchis 10, CH-1254 Jussy-Lullier, Switzerland

Keywords:

chloroplast, electron transport, fluorescence, glycerolipids, membrane structure, cyclodextrins

Cyclodextrins (CD) molecules are cyclic oligosaccharides consisting of 6, 7 or 8 glucopyranose units linked by alpha (1-4) bonds. They adopt a torus shape and are able to bind lipids within their hydrophobic cavity to form water soluble guest-CD inclusion complexes. We have used a-permethylated CD to investigate the extent of lipid depletion in thylakoid membranes (TM) and the concomitant effect on the uncoupled electron flow in photosystem II and photosystem I. The effect of lipid depletion was also studied on the low temperature fluorescence spectra, the ratio F695/F740 and the fluorescence transients OJIP. In conclusion, the CD-induced removal of lipids from TM results in important alterations of their functions. The results will be discussed in terms of the role of each lipid class in the photosynthetic functions of thylakoid membranes.
Supported by the Swiss National Science Fundation to PAS Nr. 31.432.97.95.

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359_3051.

ACCLIMATION OF LOLIUM TEMULENTUM TO GROWTH AT ELEVATED CO₂

Lewis C.E.¹, Causton D.R.², Foyer C.H.<sup>1

1</sup>Institute of Grassland and Environmental Research, Department of Environmental Biology, Plas Goggerddan, Aberystwyth, WALES - UK, SY23 3EB.
²Institute of Biological Sciences, University of Wales, Aberystwyth, WALES - UK, SY23 3DD.

Keywords:

acclimation, alternative electron transport, carbohydrate, elevated CO2 concentration, Rubisco, Lolium temulentum

The fructan former, Lolium temulentum, was grown in air and at elevated CO₂ (700 µmol mol^-1), and at high (500 µmol m^-2 s^-1, HL) and low (150 µmol m^-2 s^-1, LL). Photosynthesis and respiration were stimulated by growth at high CO₂. Maximal Rubisco activities were unaltered by CO₂ concentration, but a marked decrease in activation state occurred. Biomass accumulation, foliar carbohydrate composition and content were similar in plants grown either in air or elevated CO₂. Foliar C:N ratios either decreased or were comparable with plants grown in air. Since changes in Rubisco activation state did not offset the increases in light-saturated photosynthesis at elevated CO₂, and biomass was largely unchanged, we conclude that partitioning of carbon between biomass accumulation and respiration is modified in favour of the latter such that elevated respiration rates offset increased carbon gain at elevated CO₂ concentrations.

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360_19018.

2-CYS PEROXIREDOXIN IS PART OF THE ANTIOXIDANT NETWORK OF CHLOROPLASTS

Baier M., Dietz K.-J.

Universität Bielefeld, Lehrstuhl für Stoffwechselphysiologie und Biochemie der Pflanzen, Universitätsstraße 25, 33615 Bielefeld

Keywords:

antioxidants, chloroplast, oxidative stress, peroxidases, photoinhibition, unsaturated fatty acids

Oxygenic metabolism in chloroplasts may lead to peroxidation of a wide range of biomolecules which in turn damage other cellular constituents. Until recently, detoxification of organic peroxides was an unsolved question. The 2-Cys peroxiredoxin (2-CP) is the first reductase of organic peroxides identified in the chloroplast stroma. It is homologous to animal, yeast and bacterial proteins. Its chloroplast localisation indicates a novel physiological function in plants. Transgenic Arabidopsis with reduced level of 2-CP showed growth retardation and reduced photosynthetic activity under certain conditions. Our results indicate that the 2-CP is of central importance in the antioxidant network of chloroplasts and protects the photosynthetic apparatus from being damaged by organic peroxides.

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361_19717.

THE ROLE OF NIFS IN THE BIOGENESIS OF FeS PROTEINS IN CYANOBACTERIA

Jaschkowitz K., Seidler A.

Department of Plant Biochemistry, Ruhr-University Bochum, Germany

Keywords:

assembly, biogenesis, biosynthesis, cyanobacteria, Fe-S centers

In Azotobacter vinelandii and Escherichia coli NifS has been shown to be involved in the incorporation of FeS centers by mobilizing sulfur from cysteine (1). A similar but not identical activity has been purified from the cyanobacterium Synechocystis PCC 6714. This enzyme, termed cysteine desulfurylase or C-DES, is mobilising sulfur from cysteine to form holo-ferredoxin (2). However, unlike NifS which binds the mobilised sulfur transiently to a cysteine side chain, this enzyme does not bind the sulfur but might form free cysteine persulfide instead. In the genome sequence of Synechocystis PCC 6803 there are three orfs with homology to nifS genes. We have made attempts to inactivate the three genes and to study the phenotype of the mutants. However, the mutants have not segregated yet, indicating that all three nifS-like genes might be essential under chemoheterotrophic growth conditions. In order to study the function of the three gene products we have overexpressed them in E. coli. The results of the functional studies of one of them, the gene product of orf slr0387, will be presented.
1) Zheng, L., White, R. H., Cash, V.L. and Dean, D.R. (1994) Biochemistry 33, 4714-4720; Zheng, L. and Dean, D.R. (1995) J. Biol. Chem. 269, 18723-18726; Flint, D.H. (1996) J. Biol. Chem. 271, 1608-1674
2) Leibrecht, I. and Kessler, D. (1997) J. Biol. Chem. 272, 10442-10447

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363_19863.

PHOTOSYNTHETIC OXYGEN EXCHANGE IN TWO WHEAT VARIETIES OF DIFFERENT DROUGHT RESISTANCE UNDER WATER STRESS

Lysenko G.G., Strizh I.G., Zhigalova T.V.

Department of Plant Physiology, Faculty of Biology, Moscow State University, 119899, Moscow, Vorobjovy Gory, Russia

Keywords:

alternative electron transport, Chl fluorescence induction, dessiccation, Mehler reaction, photoinhibition

Desiccation suppresses CO₂-fixati CO₂-fixation whereas photosynthetic electron transport from water to PS1 (ETC) is more resistant and possibly by this conditions oxygen may accept electrons in ETC. We investigated electron transport and Chl fluorescence induction in leaves of two wheat var. with different drought resistance. Under water stress the rate of fluorescence quenching decreased in drought-sensitive (DS) var. and increased in drought-resistant (DR) var. Simultaneously electron transport from PS2 to PS1 decreased only in DS var. Water stress increased oxygen reduction in PS1 of the DS var. whereas in the DR plants oxygen reduction increased in PS2. One can suggest that the electron flow to oxygen in PS2 prevents photoinhibition of DR var. Experiments with isolated PS2 membrane fragments supported it. It was showed that photoinhibition equally inhibited oxygen release and the oxygen reduction in PS2.

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369_29133.

GLUCOSE INHIBITS CHLOROPHYLL A AND PHYCOCYANOBILIN BIOSYNTHESIS IN UNICELLULAR RED ALGA GALDIERIA AT COPROPORPHYRINOGEN III FORMATION STAGE

Stadnichuk I.N., Rakhimberdieva M.G., Karapetyan N.V., Bolychevtseva Y.V., Yurina N.P., Selyakh I.O.^*

A.N. Bakh Institute of Biochemistry, Russian Academy of Sciences, 117071 Moscow, Russia;
^*M.V. Lomonosov Moscow State University, 119899 Moscow, Russia

Keywords:

glucose regulation, light regulation, microalgae, phycobiliproteins, porphyrins, Chl biosynthesis

The addition of 1% D-glucose to the culture of the unicellular red alga Galdieria partita, capable of heterotrophic growth, caused an increase in the cell content of chlorophyll a and of phycobiliproteins. At the same time, switching G. partita from autotrophic growth to heterotrophic nutrition led to the excretion of coproporphyrinogen III into the growth medium. Thus, the presence of D-glucose caused a reduction of pigment content in the cell, due to the inhibition of chlorophyll a and phycocyanobilin biosynthesis at the stage of the transformation of their universal precursor, coproporphyrinogen III, to protoporphyrinogen IX. In the mixotrophic culture of G. partita light induces both the biosynthesis of photosynthetic pigments and the excretion of coproporphyrinogen III. It is supposed that light affects an earlier stage in the biosynthetic pathway of chlorophyll a and phycocyanobilin than does D-glucose.

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370_5359.

THE D1-CP43 AGGREGATES IN PHOTOSYSTEM II FORMED BY THE ACCEPTOR-SIDE PHOTOINHIBITION ARE THE SUBSTRATE FOR AN ATP-DEPENDENT STROMA PROTEASE

Nakatani E., Yamamoto Y.

Department of Biology, Faculty of Science,
Okayama University,�@Okayama 700-8530, Japan

Keywords:

D1 protein, D1 turnover, degradation pathways, photoinhibition, photosystem 2

Under light stress conditions, the reaction center-binding protein D1 in photosystem II turns over rapidly. The protease responsible for the degradation of the D1 protein and the form of D1 as the substrate for the enzyme are not known exactly. We report here that the aggregates between D1 and CP43, which are formed under strong illumination of spinach photosystem II membranes under the aerobic conditions, are efficiently degraded by an enzyme located in the stroma. The D1-CP43 aggregates are produced both by the donor-side and the acceptor-side photoinhibition, and the products of the acceptor-side photoinhibition that are formed by the action of active oxygen molecules are recognized as the substrate by the stroma enzyme. Undamaged D1 proteins and the D1 aggregates produced by the donor-side photoinhibition by the endogenous cation radicals, namely P680⁺ and Tyr Z⁺, are not susceptible to the stroma protease. The protease is serine-type and the protease activity is dependent on ATP.

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373_5998.

DEVELOPMENT OF THE PHOTOSYNTHETIC APPARATUS IN AUSTRALIAN RAINFOREST LEAVES

Robinson S.A., Russell A.W.

Plant Molecular Ecophysiology Research Group, University of Wollongong, Northfields Avenue, NSW 2522, Australia

Keywords:

fluorescence, ontogenesis, photoinhibition, photoprotection, pigments

Leaves of rainforest species often show asynchronous development of the various components of the photosynthetic apparatus, thus increasing the susceptibility of young leaves to photoinhibition. We are investigating, in several Australian rainforest species, the regulation of this vital process and the importance of photoprotection and photoinhibition to leaf development and seedling establishment.
Gap-specialist and shade tolerant species have been established in sun and shade conditions. These species show a range of leaf longevity, cyanic pigmentation and rates of greening. Changes in chlorophyll fluorescence parameters, leaf pigments and the concentration of key photosynthetic proteins have been determined during leaf development for all species.
The results show differences in photosynthetic development between species and provide insights into the relationship between light environment and developmental patterns.

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375_15248.

LIGHT-DEPENDENT MASSIVE CYCLING OF INORGANIC CARBON IN CYANOBACTERIA

Tchernov D., Keren N., Luz B., Ronen-Tarazi M., Kaplan A.

The Avron-Evenari Minerva Center, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel

Keywords:

acclimation, CO2 concentrating mechanisms, CO2 uptake, cyanobacteria, light acclimation

Massive CO₂ uptake was observed in Synechococcus sp. PCC 7942. Consequently, the concentration of dissolved CO₂ was considerably lower than expected at equilibrium. The resulting "standing CO₂ gradient" was observed in wild type cells; in cells treated with iodoacetamide which inhibited photosynthetic CO₂ fixation; and in high-CO₂-requiring mutants that did not exhibit net photosynthesis under low CO₂ concentration. The extent of massive C_i cycling, due to CO₂ uptake and HCO₃^- efflux, increased with light intensity and with the degree to which the light harvesting complexes transferred energy to the reaction center. The efficiency of energy transfer was affected by the light intensity and duration, and by the availability of C_i. Simultaneous measurements of gas exchange and fluorescence parameters by the membrane inlet mass spectrometer and PAM thus enable analysis of the interrelations between C_i fluxes and photosynthetic electron transfer.

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378_14116.

TEMPERATURE ACCLIMATION OF THE PHOTOSYTNHETIC APPARATUS: BALANCING REGENERATION AND CARBOXYLATION OF RIBULOSE BISPHOSPHATE

Hikosaka K., Murakami A., Hirose T.

Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai 980-8578, Japan

Keywords:

acclimation, adaptation, gas exchange, modelling, nutrients, Rubisco, temperature acclimation, temperature dependence, temperature stresses, nitrogen use

Temperature dependence of photosynthesis is known to change even in the same individual subjected to changing temperature regimes. In many species, the optimal of photosynthesis is low when the plant is grown low temperatures. Hikosaka (1997, Ann Bot 80: 721-730) theoretically predicted that change in N partitioning among photosynthetic components can alter the temperature dependence of photosynthesis. Following this model, we investigated a temperate evergreen tree, Quercus myrsinaefolia. The optimal temperature that maximises the photosynthetic rate at normal CO₂ was found to be 25 and 32^oC for leaves grown at 15 and 30^oC, respectively. We focused on two processes, carboxylation and regeneration of RuBP with gas exchange method. Temperature dependence of both two processes varied depending on growth temperature. Using a biochemical model, we estimated the potential rate of the two processes at various temperatures under normal CO₂. In 15^oC-grown leaves, the photosynthetic rate was limited solely by RuBP carboxylation under any temperature, while in 30^oC-grown leaves, the photosynthetic rate was limited by RuBP regeneration below 22^oC, but limited by RuBP carboxylation above 22^oC. It was concluded that the 1) changes in temperature dependence of carboxylation and regeneration of RuBP and 2) changes in the balance of these two processes altered temperature dependence of the photosynthetic rate.

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379_7696.

STRUCTURAL CHARACTERIZATION OF MEGACOMPLEXES OF PHOTOSYSTEM II AND LHCII OF GRANA MEMBRANES

Dekker J.P., Van Roon H., BoekemaE.J.^*

Dept. of Physics and Astronomy, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands
^*Dept. of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

Keywords:

electron microscopy, LHC II, light harvesting complexes, photosystem 2, supramolecular structures

Spinach PSII membranes were incompletely solubilized by a mild detergent, partially purified by gel filtration chromatography and structurally analyzed by electron microscopy, multivariate statistical analysis and classification procedures. Two new types of dimeric supercomplexes of PSII and LHCII were resolved, one with CP24 and trimeric LHCII in a second binding position, the other with trimeric LHCII in an additional third binding position. Two types of tetrameric megacomplexes of PSII and LHCII were also detected in substantial amounts. The results suggest that CP26 and CP24 play important roles in the association of the dimeric complexes and that most excitation energy flows via the minor LHC proteins to the PSII core complex.

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380_26343.

REFINED ANALYSIS OF SUPERCOMPLEXES OF PHOTOSYSTEM II AND LHCII OF GRANA MEMBRANES

BoekemaE.J., Van Roon H.^*, Dekker J.P.^*

Dept. of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
^*Dept. of Physics and Astronomy, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands

Keywords:

electron microscopy, LHC II, OEC proteins, photosystem 2, ultrastructure

Spinach PSII membranes were incompletely solubilized by a mild detergent, partially purified by gel filtration chromatography and structurally analyzed by electron microscopy using negatively stained specimens. Large numbers of supercomplexes of PSII and LHCII were analyzed by multivariate statistical analysis and classification procedures. The results suggest the way of orientation of trimeric LHCII in the first and second binding positions, and identify the positions at which the largest variation occur in the electron density. We propose a model in which CP43 and CP47 are localized at opposite sides of the D1/D2 heterodimer and show that the densities attributed to CP47, CP43 and the extrinsic 33 kDa OEC subunit show the smallest amount of variation.

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388_27879.

MOLECULAR REQUIREMENTS OF CHLOROPHYLLS FOR THE ANTENNA FUNCTION OF THEIR SELF-ASSEMBLED FORMS

Oba T., Tamiaki H.

Department of Bioscience and Biotechnology, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan

Keywords:

aggregates, assembly, chlorosomes, model systems

We synthesized Zn-chlorophylls (Zn-Chls) and examined their aggregation behaviors in relation to the supramolecular structure and the function of the Chl self-aggregate in chlorosome. (1) A Zn-Chl having an OH group at C7¹ yielded an aggregate Qy band sharper and less red-shifted than the chlorosome-like one of Zn-3¹-OH-Chl (natural type), despite using the same molecular linkages (OH ^... Zn and OH ^... O=C). It is concluded that not only the molecular linkages but the linear location of OH, C=O, and Mg in the molecule is crucial for the formation of the aggregates by Chl self-organization [1]. (2) The C7¹-substituents could 'tune' the antenna function of the aggregates quite differently from the way the C8-substituents do, as demonstrated by Zn-3¹-OH-Chls having bulky substituents (OMe, OCOMe, and OCO^tBu) at C7¹ which did not affect the mode of the molecular linkage. (3) We are also studying a possibility that the chlorosomal self-aggregate is an associate of Chl dimers by use of a novel synthetic Zn-Chl dimer.
[1] T. Oba, H. Tamiaki, Photochem. Photobiol., 67, 295-303 (1998).

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389_28415.

OPTIMISATION OF LEAF PHOTOSYNTHESIS UNDER LOW IRRADIANCE

Cailly A.-L., Genty B.

Laboratoire d'Ecophysiologie végétale, CNRS, URA 2154, bât.362, Université Paris XI-Orsay, 91405 Orsay cedex, France.

Keywords:

light distribution, O2 evolution, quantum yield, sun and shade leaves

The efficiency of leaf photosynthesis under low irradiance was investigated in Lamium galeobdolon Cr. (a non obligate shade plant) grown under sun and shade light regimes (spectral quality and irradiance) and in a wild type and a mutant, chlorina f2, of barley lacking the peripheral LHCIIb. Leaf O₂ evolution waf O₂ evolution was measured under saturating CO₂ either under growth light regimes or under monochromatic light (590 nm, 650 nm and 680 nm). The quantum yield of O₂ evolution, estimated on the basis of the absorbed light, was found to be high in all materials, (i.e. 0.103 at 590 nm for wild type of barley) and weakly dependent on wavelength and on simulated light regime. This result implies that whatever light acclimation was, leaves are capable of achieving a consistently balanced distribution of excitation energy between PSI and PSII. The significance of changes in stoechiometries of PSI:PSII reaction centres is discussed in this context.

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390_30637.

PHOTOHYDROGEN PRODUCTION - HYDROGENASES IN THE GREEN ALGA SCENEDESMUS OBLIQUUS

Wuenschiers R., Schulz R.

FB Biology/Botany, Philipps-University, Karl-von-Frisch-Str., D-35032 Marburg, Germany, wuenschi@mailer.uni-marburg.de

Keywords:

bioreactors, green algae, hydrogen production, hydrogenase, thioredoxins

After adaptation to anaerobic conditions the unicellular green alga Scenedesmus obliquus shows the ability to produce molecular hydrogen in the light. Responsible for this reaction are enzymes namend hydrogenases. In case of photohydrogen production they receive electrons via ferredoxin. Thus the electrons are derived from the photosynthetic splitting of water. The hydrogenase under investigation seems to be regulated by thioredoxin and the partial pressure of oxygen.
Besides biochemical and physiological data on the hydrogenases of Scenedesmus obliquus the possibilities to use green algae for the photobiological production of hydrogen gas as energy source will be presented.

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392_7100.

SELF-AGGREGATES OF SYNTHETIC BACTERIOCHLOROPHYLL-F DERIVATIVES

Tamiaki H., Kubo M.

Department of Bioscience and Biotechnology, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan

Keywords:

aggregates, Bchl, chlorosomes, model systems, pigments

We synthesized zinc complexes of methyl bacteriopheophorbide-f and its 3¹-demethyl homologue as the model compound of bacteriochlorophyll-f, which possesses a formyl group at the 7-position. The synthetic model compounds were characterized mass and ¹H-NMR spectroscopies. The separation of 3¹-epimers of methyl bacteriopheophorbide-f was done by high-performance liquid chromatography. Visible, fluorescence and CD spectra showed that the model compounds self-aggregates to form oligomers in nonpolar solvents. The optical spectra of the present (7-CHO) and bacteriochlorophyll-d (7-CH₃) aggregates indicated that the 7-substituents did not disturb the formation of the self-aggregates to afford a similar supramolecular assembly.

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393_7370.

RELEVANCE OF THE NAD(P)-REDUCING HYDROGENASE OF SYNECHOCYSTIS 6803 TO PHOTOSYNTHESIS

Appel J., Schulz R.

FB Biology-Botany, Philipps-University, Karl-von-Frisch-Str., D-35032 Marburg, Germany, appel@mailer.uni-marburg.de

Keywords:

cyanobacteria, electron transport, hydrogenase, NADH-dehydrogenase

Because of high sequence homologies of three ORFs of the operon of the NAD(P)-reducing hydrogenase of Synechocystis to subunits of the complex I (ndh) it was suggested that this hydrogenase is a part of this large membrane complex. Physiological investigations of a set of hydrogenase (hox) as well as ndh-mutants are presented and are supporting this hypothesis. The complex I of cyanobacteria is acting at the interface of the photosynthetic and respiratory electron transport in the thylakoid membrane. A hydrogenase linked to it might be a controlling device for a proper redox poising of the photosynthetic membrane. This fine tuning of the electron transport may be important since an autotrophic culture of Synechocystis permanently shows a low but detectable hydrogenase activity and planktic cyanobacteria are living under rapidly changing light conditions.

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398_25570.

CO₂ DIFFUSION IN LEAVES

Genty B., Meyer S., Piel C., Badeck F., Liozon R.

Laboratoire d'Ecologie Végétale, CNRS, URA 2154, Université Paris Sud, Bât. 362, 91405 Orsay, France

Keywords:

fluorescence imaging, gas exchange, stomata, whole leaf, gas diffusion

CO₂ diffusion into the leaf to Rubisco depends on stomatal conductance and on the path CO₂ follows inside the leaf mesophyll from the substomatal cavities to the catalytic sites of Rubisco. Recent methodological advances have permitted to show that in tree leaves a large drop in the CO₂ partial pressure may occur along this path. Two components determined the internal conductance in the mesophyll, i) the gaseous diffusion through intercellular air spaces, ii) the diffusion from the cell wall through the liquid phase to the carboxylation site. The question arises as to what extent gaseous versus liquid phase diffusion contributes in determining internal conductance. To estimate gaseous versus liquid phase conductance, in this study, we introduce an approach combining chlorophyll fluorescence imaging and measurements of leaf gas exchange in He atmosphere in which the diffusivity of CO₂ is largely enhanced. We show that the limitation of diffusion through intercellular air spaces accounts for the larger part of the limitation to CO₂ transfer in the mesophyll in leaves of ligneous plants. Significance of this specificity of leaves of ligneous plants and its relationship with anatomy, stomatal diffusion and distribution of photosynthetic activity is discussed.

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399_26653.

DIFFUSIVE AND NON-DIFFUSIVE LIMITATIONS OF HETEROGENEOUS PHOTOSYNTHESIS OVER ROSA LEAF

Meyer S., Genty B.

Laboratoire d'Ecophysiologie Végétale, CNRS, URA 2154, bât. 362, Université Paris Sud, 91405 Orsay, France.

Keywords:

fluorescence imaging, gas exchange, stomata, whole leaf, photosynthetic heterogeneity

The contribution of stomatal conductance and photosynthetic metabolism in determining heterogeneous photosynthesis was investigated during ABA-feeding or rapid dehydration on detached leaves of Rosa rubiginosa L. Spatial distribution of photosynthesis was mapped by computing quantum yield of photosystem II photochemistry from leafphotochemistry from leaf chlorophyll fluorescence images. Both ABA-feeding and dehydration treatments induced heterogeneous decrease in photosynthetic activity over the leaf area mainly by inducing a patchy closure of stomata. A heterogeneous non-diffusive limitation was also identified by a limited capacity of photosynthetic electron flow during transient saturating CO₂ supply. The spatial patterns of diffusive and non-diffusive limitations of photosynthesis were correlated; the sites with the lowest stomatal conductance showed the lowest capacity for electron transport. This correlation, which was also found for control leaves held near the compensation point, is discussed.

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400_27628.

PROTON UPTAKE AND ELECTRON TRANSFER IN REACTION CENTERS FROM RB. SPHAEROIDES MODIFIED IN THE PROXIMITY OF A WATER CHAIN

Tandori J.^1,2, Sebban P.¹, Michel H.³, Baciou L.<sup>1

1</sup>CGM, CNRS, Gif/Yvette, France,
²Dept. of Biophysics, JATE University, Szeged, Hungary,
³MPIBP, Frankfurt, Germany

Keywords:

bacterial reaction center, electron transfer, mutants, proton transfer, purple bacteria

In order to investigate the role of the water chains in the reaction center from Rb. sphaeroides, proline L209, which is adjacent to one of the water chain, was mutated to residues F, Y, W, E, T and Q. We have analyzed the effects of the mutations on the second electron transfer and the coupled proton uptake on the second flash. In all of the aromatic mutants, both processes slowed down compared to the wild type. This is not observed in the other studied mutants. The temperature dependence of these rates have been also studied. The same activation energies (DH°) are found for the proton and electron transfer processes in the wild type and in most of the mutants. However, DH° for the proton uptake is significantly higher in PL209W and smaller in PL209E compared to the DH° for the second electron transfer. We shall discuss how the proton transfer pathways may be affected differently by the large aromatic residue (W) and by the charged sde chain (E).

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404_2431.

ELUCIDATING FUNCTIONAL ASPECTS OF LHC PHOSPHORYLATION BY DNA INSERTIONAL MUTAGENESIS IN THE GREEN ALGA CHLAMYDOMONAS REINHARDTII

Kruse O.¹, Mullineaux C.W.², NixonP.J.³, Schmid G.H.<sup>1

1</sup>Biologie/Zellphysiol.,Universität Bielefeld,33501 Bielefeld, Germany
²Department of Biology,University College London, U.K.
³Department of Biochemistry, Imperial College London, U.K.

Keywords:

LHC II, molecular biology, mutational analysis, phosphorylation/dephosphorylation, protein kinase

A nuclear transformation mutant (stm1) of Chlamydomonas reinhardtii lacking the ability of state1:state2 transition and showing a drastic decrease in LHCII peptide phosphorylation was constructed by insertional mutagenesis of the ARG7 gene using the method of Debuchy et al., EMBO 8 (10) 2803, 1989. The identification of stm1 from the generated library has been achieved by various screening methods including the estimation of state1:state2 transition by fluorescence imaging and the rate of 'in vivo' LHCII peptide ³²P phosphorylation by phosphoimages. Stm1 grows photoautotrophically at standard light conditions indicating that the random integration of the ARG7 gene did not result in a damage of the photosynthetic electron chain and its related protein complexes. Providing that genetic experiments confirm a close linkage of the mutation to the inserted gene, the results indicate that stm1 is a suitable candidate for investigations concerning the identification of a gene product which is strongly related to the process of the reversible LHCII phosphorylation or its regulation, namely the LHC kinase or a possible redox sensor.

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406_3256.

EVIDENCE FOR THE EXISTENCE OF A PSEUDOGENE FOR THE LARGE SUBUNIT OF RUBISCO WITHIN THE CHLOROPLAST DNA OF NORWAY SPRUCE (PICEA ABIES)

Sutter A., Philipps A., Ochs G., Wild A.

Institute for General Botany
Johannes Gutenberg University
D-55099 Mainz
Germany

Keywords:

chloroplast genes, molecular biology, Rubisco, sequence analysis, gymnosperms

Although the structure and organisation of chloroplast genome has been extensively studied in angiosperms, only limited sequence analysis has been carried out on chloroplast DNA (ctDNA) from gymnosperms. Among them the mapping and sequencing of the entire chloroplast genome from black pine (Pinus thunbergii) has revealed some unusual features compared with ctDNA structures in the majority of flowering plant species. To confirm these unusual structures or to find new deviations, we have cloned and sequenced approximately 30% of the chloroplast genome from Norway spruce. The cloned fragments contain genes for PSI- or PSII-polypeptides, electron transport components, ATP synthase polypeptides, ribosomal RNAs and proteins, several tRNA species, and numerous 'open reading frames' (ORFs). Surprisingly, the rbcL gene which codes for the large subunit of Rubisco seems to be duplicated in the ctDNA of Picea abies. But one rbcL gene copy lacks the entire 5' region including the start codon ATG (81 bp) and is therefore unable to function (rbcL pseudogene).

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416_17153.

EPR STUDY OF MERCURY ACTION ON THE PHOTOSYNTHETIC APPARATUS OF SPINACH CHLOROPLASTS

Sersen F., Kralova K., Bumbalova A.

Comenius University
Faculty of Natural Sciences
Institute of Chemistry
Mlynska dolina CH2
842 15 Bratislava
Slovakia

Keywords:

EPR, fluorescence, heavy metals, photosystem 1, photosystem 2

The effect of mercury on the photosynthetic apparatus of spinach chloroplasts for a wide concentration range of HgCl₂ (molar ratio chlorophyll:HgCl₂ = 1:1 up to 1:10) has been studied by EPR spectroscopy. Direct evidence has been presented that at higher concentrations HgCl₂ interacts with the intermediates Z⁺/D⁺, i.e. radicals of Tyr_Z and Tyr_D located in D₁ and D₂ proteins (for Chl:HgCl₂ £ 1:2.5) and releases Mn²⁺ ions from the manganese cluster situated in the water splitting complex (for Chl:HgCl₂ £ 1:10). In this work it has been shown tas been shown the first time by EPR spectroscopy that HgCl₂ oxidizes the core of PS 1 (P700) in the dark if the molar ratio Chl:HgCl₂ £ 1:1. This effect of HgCl₂ on PS 1, as well as the inhibition of the direct, cyclic and non-cyclic reduction of P700⁺ by HgCl₂, have been confirmed also in the subchloroplast particles of PS 1. The irreversible accumulation of mercury in chloroplasts as well as the formation of organometallic complexes of mercury with amino acids contained in chloroplast proteins have been confirmed.

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426_30378.

Mg²⁺-INDUCED LHC II AGGREGATION KINETICS AND ITS MIGRATION IN THYLAKOID MEMBRANE

LinS.-Q., Wen X.-G., Kuang T.-Y.

Photosynthesis Lab., Institute of Botany, Chinese Academy of Sciences, 100093 Beijing, China

Keywords:

fluorescence, LHC II, magnesium, Sub-band

The Mg²⁺-induced chl a fluorescence enhancement of thylakoid membrane at room temperature was demonstrated to represent the migration of the LHC II-containing particles from stroma to grana regions (Briantais, 1986). Two different types of the LHC II-containing particles were resolved in the time course of the Mg²⁺-induced chl a fluorescence enhancement, e.g. the migration process and their different features in the activation energy, etc. were also compared (Lin et al., 1992, 1995).
The Mg²⁺-induced changes of the 77K chl a fluorescence spectrum of wheat thylakoid membrane were further analyzed by the Gaussian sub-band deconvolution. It consisted of F681 (LHC II trimer), F699 (LHC II aggregate), F685 and F695 (PS II), F729 and F740 (PS I) as well as F738 (vibration band), but no F712 (vibration band) at all. Among them, F681 (LHC II trimer) decreased by 34%, F699 (LHC II aggregate) increased by 79% and the F699/F681 ratio was enhanced by 1.72 times. Furthermore, from the analysis of the kinetics for the sub-band F699/F681 ratio, it could also be fitted by two exponential components. The time constant was 5.63±1.92sec and 88.64±5.53sec, resp.
It was suggested that the two types of the LHC II-containing particles were aggregated at different time constant as indicated above while they were migrating at different speed in the thylakoid membrane. The implication of the aggregation process for the two types of the LHC II-containing particles was also discussed.

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431_30729.

TRANSCRIPTIONAL REGULATION OF PLASTIDIAL AND MICROSOMAL w-3 FATTY ACID DESATURASE GENES IN ARABIDOPSIS THALIANA

Matsuda O., Kusumi J., Iba K.

Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581 Japan

Keywords:

fatty-acid desaturation, gene expression

Trienoic fatty acid (TFA) is a major constituent in membrane lipids of higher plants. This fatty acid is assumed to take an important role in plant adaptation to environmental stresses. The w-3 fatty acid desaturases catalyze the final step of biosynthetic pathway of TFA and are closely related to the control of their relative composition in the membrane lipids. In Arabidopsis, three loci for the w-3 desaturase have been characterized and the corresponding genes (FAD3/7/8) were isolated. We analysed in detail the expression of these genes. We will discuss effects of aging, application of plant hormones, active oxigen species (antioxidants), and temperature changes on the transcriptional regulation of these genes.

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432_3523.

WHICH MECHANISM OF EXCITATION TRANSFER WORKS IN ANTENNA SYSTEMS OF PHOTOSYNTHETIC BACTERIA AMONG FORSTER, SUPEREXCHANGE, AND EXCITON?

Kakitani T., Kimura A.

Department of Physics, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan

Keywords:

antennae, energy transduction, excited state dynamics, light harvesting complexes

We developed a new theory of excitation transfer applicable to intermediate couplings between donor and acceptor. We found that a new mechanism exists between the Forster mechanism (weak coupling limit)and the exciton mechanism (strong coupling limit). The new mechanism is termed as superexchange in analogy with the superexchange mechanism of the electron transfer.
Applying this theory to the antenna systems of photosynthetic bacteria, we could assign that the excitation transfer of B800-B800 is Forster type but mostly using vibrationally unrelaxed state in the initial photo-excited state, the excitation transfer of B800-B850 is mostly usual Forster type, and the excitation transfer of B850-B850 is exciton or superexchange type depending on the excitation wavelength.

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434_3928.

FORWARD ELECTRON TRANSFER IN CHLOROBIUM REACTION CENTRES STUDIED BY TRANSIENT EPR

van der Est A., Scheller H.V., Hager-Braun C., Hauska G., Stehlik D.

Dept. of Physics, Free University Berlin, Berlin Germany
Dept. of Plant Biology, The Royal Veterinary and Agricultural University, Friedriksberg C, Denmark
Lehrstuhl fuer Zellbiologie und Pflanzenbiologie, Universitaet Regensburg, Regensburg Germany

Keywords:

electron transfer, EPR, green sulfur bacteria, heliobacteria, quinones

Transient EPR spectra of reaction centres (RC) of C. vibrioforme and C. tepidum are presented. The C. vibrioforme RC have been shown to contain 1.8 menaquinone-7 molecules/reaction centre, whereas the quinones are absent in the C. tepidum RC. The transient EPR spectra observed in a time window 100-200 ns following light excitation are assigned to the state P⁺Fx^- and are identical in the two samples. Since the polarization pattern depends on the magnetic properties and lifetime of the precursor states, it is concluded that these are the same in both samples which suggests that the quinone is not involved in forward electron transfer.

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442_21851.

EXCITON DYNAMICS IN GENETICALLY MODIFIED LH2 ANTENNAE OF PURPLE BACTERIA

Polívka T., Herek J.L., Fowler G.J.S., Pullerits T., Hunter C.N., Sundström V.

Chemical Physics, Lund University, Box 124, S-221 00, Lund, Sweden
Dept. of Molecular Biology and Biotech-nology, Sheffield University, U.K.

Keywords:

excited state dynamics, light harvesting complexes, time-resolved spectroscopy

Femto
Femtosecond transient absorption spectroscopy was used to study exciton dynamics in the B850 ring of the LH2 antenna of the purple bacterium Rb. sphaeroides. Genetic manipulation can be used to introduce different building units into the B850 rings resulting in significantly broader absorption spectra. The time-resolved experiments revealed an increase in inhomogeneity of the B850 rings of the mutants. In addition to fast decay components reflecting migration of the exciton through the B850 band, components with slower time constants were also found and correlated to of the higher degree of inhomogeneity in the genetically modified samples.

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445_26909.

PARALLEL MODE EPR STUDIES ON THE OXYGEN EVOLVING COMPLEX IN PHOTOSYSTEM II

Ghibaudi E., Rutherford A.W.

Département de Biologie Cellulaire et Moléculaire
Section de Bioénergétique
CEA/Saclay
91191 Gif-sur-Yvette (France)

Keywords:

EPR, manganese cluster, O2 evolution, OEC proteins

Water splitting takes place in Photosystem II (PSII) and is mediated by the oxygen-evolving complex (OEC), which consists of a tetranuclear Mn cluster, a tyrosine residue and Cl^- and Ca²⁺ cofactors. The enzyme cycles through 5 distinct kinetic states (S_i), which are characterized by different redox states of the Mn cluster [1]. EPR investigations on the OEC have shown that both S₂ and S₀ are paramagnetic; further, a light-induced split signal arises from the formal S₃ state of Ca²⁺ or Cl^--depleted PSII (in which the Mn cluster, in the redox state of S₂, interacts magnetically with an organic free radical, probably Tyr_Z·). Parallel polarization EPR has shown a large unresolved low-field signal associated to this state [2]. A correlation study on the EPR signals arising from this state of Ca²⁺-depleted/EGTA-treated PSII, detected by parallel and perpendicular mode EPR, has been carried out.
[1] Yachandra V. et al. (1996) Chem. Rev. 96, 2927-2950
[2] Mino H. et al. (1998) Biochem. 37, 2794-2799

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448_28385.

TWO CAROTENOID BIOSYNTHESIS PATHWAYS EXIST IN THE CYANOBACTERIUM ANABAENA PCC 7120

Mann V., Hirschberg J.

The Hebrew University of Jerusalem,
Department of Genetics,
Givat Ram, Jerusalem 91904, ISRAEL

Keywords:

biosynthesis, carotenoids, cyanobacteria, evolution

Bacteria and plants contain distinct pathways of carotenoid biosynthesis. Cyanobacterial genes for carotenogenic enzymes are conserved with the homologous genes from plants and algae. However, very little or no similarity exists between the plant-type and the bacterial-type genes. In bacteria phytoene is converted to lycopene by a single phytoene desaturase enzyme, CRTI. The same reaction in cyanobacteria and plants is carried out by two enzymes, phytoene desaturase, CRTP (PDS) and z-carotene desaturase, CRTQ (ZDS). We have found that Anabaena PCC 7120 contains both the plant-type genes crtP and crtQ, and the bacterial-type crtI. All three genes are expressed. Our data suggest that the coexistence in Anabaena of two pathways reflects a horizontal gene transfer that occurred in the course of evolution of this nitrogen-fixing cyanobacterium.

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453_3121.

PHOTOSELECTION EFFECTS IN EPR-DETECTED TRIPLET STATES OF PHOTOSYNTHETIC PIGMENT MOLECULES

Proskuryakov I.I., Klenina I.B., Borovykh I.V., Gast P., HoffA.J.

Department of Biophysics, Huygens Laboratory,
Leiden State University,
Postbus 9504, 2300 RA Leiden,
The Netherlands

Keywords:

bacterial reaction center, Bchl, EPR, P680, polarized spectroscopy, triplet states

Strong effects of photoselection with plane-polarised laser light are observed when the triplet states of pigment molecules, both in solution and within photosynthetic reaction centres, are detected with cw time-resolved EPR. Simulation of the spectra obtained with the light polarisation plane parallel and perpendicular to the magnetic field yields information on the relative orientation of the optical transition moment and triplet magnetic axes. The technique is essentially free from a severe drawback of conventional EPR magnetophotoselection, the latters dependence on spin-lattice relaxation, thus enabling studies in a wide temperature range. The following phenomena will be reported: 1.) Deviation of the ³P electronic structure from the molecular symmetry of P in bacterial RCs. 2.) Temperature-induced variations of the ³P properties in RCs of photosynthetic bacteria and plant PSII. 3.) Thermoactivated dynamics of triplet excitation in RCs.

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456_1542.

ELECTRIC FIELD MODULATED P⁺Q_A^- RECOMBINATION IN PHOTOSYSTEM II

de Wijn R., van Gorkom H.J.

Biophysics Department
Huygens Laboratory
Leiden University
P.O. BOX 9504
2300 RA Leiden
The Netherlands

Keywords:

charge recombination, chloroplast, electrogenic reactions, luminescence, photosystem 2

Electroluminescence can be used as a tool to study kinetic, thermodynamical and possibly structural aspects of photosynthetic electron transport. In order to obtain a better understanding of the underlying processes in photosystem II we have applied this technique to a suspension of Tris-treated, osmotically swollen, spinach choroplasts. By using 10 Hz repetitive excitation the secondary electron donor Y_Z is kept in the oxidized state, hence the charge pair P⁺Q_A^- in the presence of Y_Z⁺Q_B^- will be the precursor for charge recombination. This is confirmed by a strong dependence of the electroluminescence signal on the excitation rate. Photosystem I electroluminescence is excluded by using sufficiently low field strengths. In this way we obtain simplified conditions for charge recombination in photosystem II, facilitating analysis of the electroluminescence kinetics in terms of Marcus rate theory, which is in progress.

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459_1992.

ADAPTATION OF PHOTOSYNTHESIS TO LIGHT AND COLD IN GREEN OVERWINTERING LEAVES OF ALPINE PLANTS

Lütz C., Schönauer E., Neuner G.

GSF Research Centre for Environment and Health, Exposure Chamber Unit,
Ingolstaedter Landstr. 1,
D-85764 OBERSCHLEISSHEIM
Germany

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Keywords:	abiotic stress, carotenoids, environmental stress, flavonoids, fluorescence

Some alpine plants retain green leaves over the winter, well protected by snow. During snow melt in spring the leaves are suddenly exposed to markedly altered light and temperature conditions. Selected plants from altitudes above 2100m in the European Alps are studied for their adaptation of photosynthesis in response to spring and summer. Based on measurements of plastid pigments, Kautsky- and deep temperature fluorescence, on antioxidant and flavonoid compounds together with anatomical observations several adaptive mechanisms are described. Each plant possesses an own strategy to cope with the rapidly changing spring climate conditions. Photosynthesis and leaf structure remain remarkably stable when comparisons were made between samples covered by snow for months and those exposed to the climate in an open field above timberline.

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460_2023.

PHOTOSYNTHETIC DECLINE OF WHEAT FLAG LEAVES IN RESPONSE TO ELEVATED ATMOSPHEREIC CO₂

Sicher R.C., Bunce J.A.,

Climate Stress Laboratory, USDA-ARS
Beltsville Agricultural Research Center
10300 Baltimore Ave.
Beltsville, MD. 20705 USA

Keywords:

acclimation, elevated CO2 concentration, Rubisco, senescence

Net CO₂ exchange (NCE) rates were measured on flag leaves of wheat plants (Triticum aestivum L.) grown in small field chambers at either 350 or 700 m l l^-1 CO₂ between 1995 and 1997. Net photosynthetic rates in response to growth at elevated CO₂ were decreased 22% on average when NCE rates were averaged for 1995 through 1997 (P£0.0001). Mean soluble protein, Rubisco activity, a-amino N and Chl concentrations were decreased (P£0.0001) in elevated compared with ambient CO₂-grown wheat flag leaves. Starch and sucrose were increased but hexose levels and acid proteinase activity were unaffected by the elevated CO₂ treatment. The above results suggested that premature senescence contributed to the photosynthetic decline observed in wheat flag leaves during growth at elevated CO₂. Changes of a-amino N were correlated with photosynthetic decline but acid proteinase activity probably was under endogenous control.

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464_10421.

PARTITION OF FERREDOXIN-NADP⁺ REDUCTASE BETWEEN PHOTOSYNTHESIS AND ANTIOXIDANT METABOLISM

Carrillo N.

Molecular Biology Division, PROMUBIE, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.

Keywords:

electron transfer, environmental stress, FNR, free radicals, oxidative stress

The chloroplast flavoprotein ferredoxin-NADP reductase (FNR) catalyzes the final step of the photosynthetic electron transport chain, namely, the reversible electron transfer between reduced ferredoxin and NADP, generating NADPH for CO₂ assimilation. In heterotrophic tissues and organisms, the reaction is driven in the opposite direction, providing reduced ferredoxin for a variety of redox metabolisms, and highlighting the versatility of FNR functions. This plasticity is also evident at the structural and biochemical levels, as reflected by the ability of FNR to functionally interact with a wide repertoire of redox partners besides ferredoxin. Recent data indicate that, in addition to their specific metabolic roles, FNR proteins are involved in the cellular antioxidant response. We provide evidence indicating that the unique properties of these flavoenzymes make them functional as general purpose antioxidants, through the regulation of the NADPH/NADP homeostass in aerobic tissues and organisms. In chloroplasts, the FNR response toward an oxidative challenge would result in progressive suppression of photosynthesis, and a concomitant increase of NADPH oxidation in the plastid stroma.

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467_27967.

PINE SEEDS GENOME CHANGES AFTER IRRADIATION

Isaenkov S.V., Sokolov N.V., Sorochinsky B.V.

Institute of Cell Biology & Genetic Engineering Ukranian Academy of Sciences Department of Radiobiology & Biophysic Zabolotnogo str.148, Kiev, 252143, Ukraine.

Keywords:

chromatography, environment, repair mechanism, Ionizingradiation, pine

The aim of this studies is the development of methods for the detection of hidden DNA damages in irradiated pine seeds. Since the pine tree is the main forestry species at the Chernobyl region it is necessary to investigate the quality of seeds used for new forest planting. Seeds from different part of the Chernobyl zone have been germinated for 10 days and protoplasts from shoots were isolated. The protoplasts were exposed to additional external irradiation and then DNA was analyzed by pulsed-field electrophoresis and hydroxyapatite chromatography as well. DNA damages have been monitored after different time intervals between irradiation and cell lysis in order to analyze the efficiency of the DNA repair systems. An experimental system based on pulse-field electrophoresis of DNA from single cells have been developed. The analysis has revealed a non-linear dose-dependent effects, the seeds irradiated with dose of 0.03 Gy were more susceptible to the DNA damage than seeds irradiated with 3 Gy of chronc irradiation. These results may be explained by presence of different DNA repair systems - constitutive and inducible. We conclude that the irradiation dose of 3 Gy is most effective in the induction of DNA repair systems, which afterwards defend the DNA in cell from further damage by new irradiation.

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468_2032.

PHOTOSELECTION IN ESP SPECTRA OF PHOTOSYNTHETIC REACTION CENTERS

Borovykh I.V., Klenina I.B., Gast P., HoffA.J., Proskuryakov I.I.

Department of Biophysics, Huygens Laboratory, Leiden State University, Postbus 9504, 2300 RA Leiden, The Netherlands

Keywords:

bacterial reaction center, electron transfer, EPR, polarized spectroscopy, radical pair

The first relatively stable stage of photoinduced electron transfer in reaction centres of many photosynthetic bacteria is P⁺Q_A^-. Time resolved EPR is well-suited for the detection of an electron spin-polarised (ESP) signal of P⁺Q_A^-. Important structural and functional properties of the radical pair can be obtained from such data.
The shape of the P⁺Q_A^- >^- signal in Zn²⁺-substituted RCs of Rb. sphaeroides R26 strongly depends on the wavelength of the excitation laser flash. The observed dependence of the ESP signal of P⁺Q_A^- arises due to the effect of photoselection by plane-polarised laser light, as proved by rotating its polarisation plane parallel and perpendicular to the magnetic field of the spectrometer. Simulation of such spectra obtained with 90^o-rotation of the polarisation plane yields the relative orientation of the P⁺ and Q_A^- cofactors with high reliability and precision. The photoselection-ESP enables studies on structural organisation of systems other than bacterial RCs.

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469_10203.

FEEDBACK REGULATION OF HIGHER PLANTS PHOTOSYNTHETIC ELECTRON TRANSPORT

Johnson G.N., Ott T.

School of Biological Sciences, University of Manchester, 3.614 Stopford Building, Oxford Rd,Manchester, M13 9PT

Keywords:

abiotic stress, electron transport, Mehler reaction, oxidative stress, P700, photoprotection, sucrose

We have investigated the effects of sucrose feeding on the kinetics of photosynthetic electron transport in leaves of Silene dioica L. Leaves were feed through the petiole with sucrose concentrations in the range 0-600 mM. The kinetics of re-reduction of P700 were measured following a light to dark transition. Feeding leaves with sucrose resulted in a reduction in the rate constant from around 60 ms^-1 at 0 mM sucrose to 30 ms^-1 at 600 mM at 1300 µEm^-2s^-1. Measurements of chlorophyll fluorescence indicate that, whilst there is an decrease qP over this range of sucrose concentrations, there is no significant change in non-photochemical quenching, possibly indicating that no change in lumenal pH is occurring. The results are discussed in the light of our understanding of regulation of electron transport and the role of sucrose as a feedback inhibitor of photosynthesis.

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470_21492.

TEMPERATURE REGULATION OF HIGHER PLANTS PHOTOSYNTHETIC ELECTRON TRANSPORT

Ott T., Johnson G.N.

School of Biological Sciences, University of Manchester, 3.614 Stopford Building, Oxford Rd,Manchester, M13 9PT

Keywords:

abiotic stress, electron transport, temperature acclimation, temperature dependence

We have measured the effects of varying temperature on the kinetics of electron transport in the plant Silene dioica L. As, expected, the rate constant for reduction of P700 following a saturating-light-to-dark transition increases with increasing temperature. When data are presented as an Arrhenius plot, a clear two phase relationship is seen with a break at around 20^oC, the growth temperature. Growing plants at 12^oC shifts this break point to around 12^oC. Supplying plants with saturating rather than ambient CO₂ increases the rate constant for electron transport at temperatures but not at temperatures below the growth temperature. Results are discussed in relation to the optimisation of the photosynthetic apparatus to growth temperature and the avoidance of oxidative stress.

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478_16651.

TOPOLOGY OF CHROMOPHORES AND COFACTORS IN PHOTOSYSTEM II: FINE ANALYSIS OF DIFFERENCE SPECTRA BY POLARIZED SPECTROSCOPY

Ahlbrink R., Junge W.

Biophysik, Universitaet Osnabrueck, D-49069 Osnabrueck, Germany

Keywords:

electrochromism, lineardichroism, P680, photosystem2

To elucidate the topology of the cofactors in PS II, we recently used the electrochromic response of core chromophores to probe the location of uncompensated electric charges during partial reactions of water oxidation (1). The deconvolution of these electrochromic difference spectra in terms of Gaussians is inherently ambiguous. We extend the analysis by polarized laser flash spectroscopy in analogy to previous work on PS I (2). A prerequisite of a fine analysis of difference spectra by dichroitic tags, the anisotropy of the antennae system of oxygen evolving core particles, is demonstrated.
(1) Mulkidjanian, A.Y., Cherepanov, D.A., Haumann, M. and Junge, W. (1996), Biochemistry 35, 3093-3107.
(2) Schaffernicht, H. and Junge, W. (1982), Photochem. Photobiol. 36, 111-115.

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483_1131.

D13C OF RESPIRED CO₂ AND LEAF CARBOHYDRATES IN BEAN PLANTS UNDER PROGRESSIVE DROUGHT

Duranceau M., Ghashghaie J., Badeck F., Deleens E., Cornic G.

Laboratoire d'Ecophysiologie Végétale, CNRS, URA 2154, Université de Paris-Sud, F-91405 Orsay

Keywords:

carbon isotope, gas exchange, leaf physiology, water stress, respiration

The variations of d13C in both leaf carbohydrates and CO₂ respired in the dark in cotyledonary leaves of Phaseolus vulgaris L. were investigated during plant dehydration. As expected, sucrose and starch became 13C-enriched with decreasing stomatal conductance during the first half of the dehydration cycle. But, during the second half, when stomata remained closed and leaf net photosynthesis approched zero, the carbohydrates became 13C-depleted. The variations of modeled discrimination with changes in pi/pa paralleled those of sucrose d13C, suggesting that the observed variation in d13C of sucrose was due to stomatal closure. d13C of CO₂ respired in the dark was correlated with sucrose d13C, respired CO₂ being always 13C-enriched compared to sucrose by about 6‰. This suggests that a discrimination against 12C occurs during dark respiration processes, releasing 13C-enriched CO₂.

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484_6601.

PHOTOSYNTHETIC ACCLIMATION OF SILENE DIOICA TO GROWTH IN FLUCTUATING LIGHT

Yin Z.-H., Johnson G.N.

School of Biological Sciences,
University of Manchester
3.614 Stopford Building
Oxford Road
Manchester, M13 9PT
UK

Keywords:

abiotic stress, fluorescence, light acclimation, pigments, Rubisco

Photosynthetic acclimation of Silene dioica to growth in fluctuating light has been investigated using a leaf oxygen electrode, chlorophyll fluorescence and analysis pigments. O₂ evolution results clearly indicate that S. dioica optimises its phoptimises its photosynthetic apparatus to different light environments. Under fluctuating light conditions, the frequency of light cycle affected significantly this optimisation. S. dioica does not optimise its photosynthetic apparatus to the integrated daily light intensity. The photosynthetic apparatus in S. dioica plants appear to be switched between either a high or a low light state, for which there seems to be a light threshold. Changes in the rate of PSII photochemistry measured under ambient CO₂ concentration give further evidence indicating significant acclimative responses of S. dioica to fluctuating light. There were significant changes in Rubico protein, but not in pigment composition. It seems that not all components of photosynthetic apparatus were regulated at the same way during the acclimation to fluctuating light.

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486_27006.

EXCITONIC INTERACTIONS AMONG CHLOROPHYLLS IN PLANT LHC II REVEALED BY NON-LINEAR LASER SPECTROSCOPY AND COMPARISON TO IN-VITRO AGGREGATES

Schubert A., Stiel H., Ehlert J., Leupold D., Lokstein H.

Institut fuer Biologie, Humboldt-Universitaet, Unter den Linden 6, D-10099 Berlin, Germany
Max-Born-Institut, Rudower Chausse 6, D-12489 Berlin, Germany

Keywords:

aggregates, excited state dynamics, laser spectroscopy, LHC II, polarized spectroscopy

Chlorophyll (Chl) aggregates can serve as model systems to study excitonic interactions between such pigments and their possible significance for spectral properties of photosynthetic pigment-protein complexes. Aggregates of Chl a were formed in an aqueous DMSO environment and investigated by non-linear absorption and non-linear polarization spectroscopy (frequency domain) in the Q_y-absorption band. Most significant are a considerable excited state lifetime shortening and an increased (with respect to monomeric Chl) ground and excited state absorption. These data are compared to those obtained with isolated trimeric light-harvesting complex (LHC) II. The results prove the significance of extensive excitonic coupling among Chls in LHC II. Implications for excited state dynamics and energy transfer mechanism(s) in antenna complexes are discussed.

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487_5953.

QUANTUM CHEMICAL COMPUTATIONS OF HEME MODEL SYSTEMS IN CYTOCHROMES

¹Zaric S., ²Knapp E.W.

¹Department of Chemistry, University of Belgrade, Studentski trg 16, 11001 Beograd, Yugoslavia
²Institut fuer Kristallographie, FB-Chemie, Freie Universitaet Berlin, Takustrasse 6, D-14195 Berlin, Germany

Keywords:

cytochromes, electron transfer, porphyrins, redox potential

Different types of cytochromes are involved in the various parts of the electron-transport chain, of photosynthetic processes. The functional groups of the cytochromes are the hemes. A variation of the redox potential of the heme in different cytochromes is due to specific covalent ligation primarily by ligands in axial positions and non-covalent interactions of the heme with the surrounding protein. Hence, quantum chemical calculations on the heme can help to explain properties and behavior of cytochromes. The heme is a complex with a transition metal where the choice of a suitable theoretical method can be very critical. Density functional theory (DFT) has been used very successfully for transition metal complexes. Using DFT methods calculated geometries of iron porphyrin complexes are in good agreement with available experimental data (1). We are using DFT methods to calculate properties of the heme in cytochromes like geometry, redox potential and the energetics of the different spin states under various conditions.
1. Rovira, C.; Ballone, P., Parrinelo, M. (1997) Chem. Phys. Lett. 271, 247-250.

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488_13461.

PHOSPHORIBULOKINASE: MUTAGENESIS OF THE MOBILE LID AND "P-LOOP"

Runquist J.A., Koteiche H.A., Harrison D.H.T., Miziorko H.M.

Department of Biochemistry
Medical College of Wisconsin
Milwaukee, WI 53226
USA

Keywords:

C metabolisms, Calvin-cycle, enzymes, site-directed mutagenesis, sugar phosphates, phosphoribulokinase

The phosphoribulokinase (PRK) lid domain contains 3 invariant arginines (R168, R173, R187). Precedent for function of lid arginines in phosphotransferases prompted substitution of glutamine for each invariant argininine. These PRK mutants retain structural integrity, as indicated by their stoichionetric binding of a fluorescent alternative substrate (TNP-ATP) and the allosteric effector (NADH). Kinetic studies indicated >10,000-fold diminution in V/K for R168Q, attributable to a >300-fold decrease in Vm and an increase (50-fold) in Km for Ru5P. For R173Q, a 15-fold diminution in Vm and 100-fold increase in Km for Ru5P was observed. These observations suggest new components of PRK's Ru5P binding site and confirm that the lid domain is part of the active site. R186Q and R187Q mutants suggest that these arginines influence substrate binding cooperativity. PRK C194A (which lacks any cysteines but functions like WT-PRK) was mutated to replace the P-loop's A16 with cysteine (the residue found in eukaryotic PRKs). A16C substitution results in >1000-fold drop in Vm but little change in substrate binding or activation properties.
USDA-NRICGP/Photosynthesis

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490_21074.

ELECTROSTATIC INTERACTIONS MODULATE THE AFFINITY OF CHLOROPLAST FRUCTOSE-1,6-BISPHOSPHATASE FOR THIOREDOXINS

Mora-Garcia S., Duek P., Wolosiuk R.,

Instituto de Investigaciones Bioquimicas
Av. Patricias Argentinas 435
1405-Buenos Aires
Argentina

Keywords:

Calvin-cycle, electrostatic interaction, thioredoxins, chloroplast Fructose-1,6-bisphosphatase, thiol/disulfide exchange

Thioredoxins (Trx) modulate enzyme activity by controlling the redox state of regulatory disulfide bridges. Two phylogenetically distant Trxs coexist in the stroma of plant chloroplasts, Trx-m and Trx-f. The latter selectively activates by reduction a key enzyme of the Benson-Calvin cycle, Fructose-1,6-bisphosphatase (CFBPase). We set out to identify factors important for the discrimination between both Trxs. E. coli Trx and Trx-m are less proficient in the activation of CFBPase than Trx-f. Mutations that introduced positive charges on the active site surface enhanced the ability of E. coli Trx to activate the enzyme; the effect of individual mutations was additive. Moreover, affinity of Trxs for the enzyme was sensitive to the ionic strength of the medium. At high salt concentrations, all Trxs displayed similar activation proficiencies, seemingly dependent upon non-charged surface features. These results show that intermolecular electrostatic interactions play a key role in the discriminat in the discrimination of Trxs by target proteins.
This work was supported by grants from CONICET/FONCYT, Argentina.

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492_14820.

COMBINATORIAL/REPLACEMENT MUTAGENESIS OF THE CD LOOP IN D1 AND D2 OF PHOTOSYSTEM II

Vermaas W., Ermakova-Gerdes S.Y., Vavilin D., Keilty A.

Dept. of Plant Biology and Photosynthesis Center, Arizona State University, Box 871601, Tempe, AZ 85287-1601, USA

Keywords:

cyanobacteria, D1 protein, D2 protein, photosystem 2, site-directed mutagenesis

The lumenal cd loop in the D1 and D2 proteins of PSII connects the Y_Z/Y_D-containing helix C with helix D. In this loop, (1) His (H189/H190) serves as proton acceptor upon Tyr oxidation, (2) Asp170 of D1 is important for Mn ligation or assembly of the water-splitting complex, (3) Arg180 of D2 affects Y_D and P680 function, and (4) based on the purple bacterial structure, it may be involved in binding an accessory chlorophyll. The questions to be addressed are (1) which amino acid sequences can function in the cd loop, (2) which residues contribute to the functional difference of D1 and D2, and (3) what are the functional effects of regional sequence alterations. We approach this by replacing regions of 8 codons by random sequences or by the homologous D1/D2 sequence, followed by selection for photoautotrophy. Sequence comparisons of sets of mutants have yielded the following insights: (1) Arg180 of D2 is required unless an Arg is present at position 184; then Gly or Val suffices at position 180. (2) Positions 166 and 169 of D2 (S, F) are highly conserved. (3) D1 regions can be functionally accommodated in the same region of D2, but not the other way around. Other functionally required sequence patterns in the cd loop and an initial characterization of selected combinatorial mutants will be presented.

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494_5677.

SINGLET ENERGY TRANSFER FROM CAROTENOIDS TO TETRAPYRROLES IN BIOMIMETIC MODELS FOR PHOTOSYNTHETIC ANTENNA FUNCTION

MacphersonA.N., Gillbro T., Tatman D., Liddell P.A., Mariño-Ochoa E., Gust D., Moore T.A., Moore A.L.

Umea University S-901 87 Umea, Sweden and Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA.

Keywords:

antennae, carotenoids, spectroscopy

The rate and mechanism of the singlet-singlet energy transfer from carotenoids to tetrapyrroles have been examined in covalently linked dyads consisting of a carotenoid linked to a purpurin or a phythalocyanine. In the case of a carotenopurpurin, transient fluorescence measurements demonstrate that the fluorescent excited state of a carotenoid model has a lifetime of 161 fs while the corresponding excited state of the carotenoid in the carotenopurpurin dyad is quenched to 45 fs. The observed quenching in the lifetime of the donor state corresponds to ca 70% efficiency of singlet-singlet energy transfer. This result matches the quantum yield obtained by steady state fluorescence excitation measurements. Concomitant with the decay of the carotenoid excited state, a rise of the excited S₁ state of the purpurin moiety is detected at 694 nm. This signal rises monoexponentially with a time constant of 62 fs. Because there is no component of the rise time that corresponds to the S₁ state lifetime of the carotenoid (7-8 ps), the S₂ state of the carotenoid must be the sole donor state in this efficient singlet-singlet energy transfer process.

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495_15154.

MEASUREMENTS OF RIBULOSE 1, 5-BISPHOSPHATE CARBOXYLASE/OXYGENASE ACTIVITIES BY NMR

Wang Z.-Y., Luo S., Sato K., Kobayashi M., Nozawa T.

Department of Biomolecular Engineering, Faculty of Engineering,
Center for Interdisciplinary Science, Tohoku University,
Sendai 980-8579, Japan

Keywords:

C metabolisms, NMR, Rubisco

High resolution NMR spectroscopy has been demonstrated to be capable of measuring the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities. The carboxylase reaction can be monitored in situ by ¹H- and ³¹P-NMR. The incorporated carbon atom is unequivocally identified as the C-1 carbon of 3-phosphoglycerate from ¹³C spectrum. ¹H spectra obtained with H₂O as solvent provide the most efficient quantitative measurement of the reaction products. ³¹P spectra give essentially the same result as ¹H-NMR but have advantage of showing the degree of reaction at any time during the reaction without the need for complete consumption of ribulose bisphosphate or removal of it from the reaction mixture. Both carboxylase activity and CO₂/O₂ specificity factor are determined for three enzymes from different sources containing higher plant and photosynthetic bacteria, and are in agreement with those measured by other methods.

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496_7141.

MEMBRANE LIPID COMPOSITION OF THE CYANOBACTERIUM NOSTOC PUNCTIFORME UNDER VARIOUS CONDITIONS OF CARBON NUTRITION

Mikhaylenko N.F., Skorokhod T.Ph.

N.G. Kholodny Botany Institute,
National Academy of Sciences of Ukraine
2 Tereshchenkovskaya St.,
Kiev, 252601
Ukraine

Keywords:

adaptation, cyanobacteria, glucose regulation, glycerolipids, herbicide resistance

Nostoc punctiforme was cultivated at the presence of 0,1% glucose, at photoheterotrophic (with glucose and 10 mkM diuron) and chemoheterotrophic (with glucose in the darkness) conditions. Adding of glucose resulted in increase of digalactosyl diacylglycerol (DGDG) and phosphatidylglycerol (PG) content but always caused the termination of sulfolipid synthesis. In the presence of diuron only, PG content lowered markedly and DGDG completely disappeared while the levels of monogalactosyl diacylglycerol and sulfoquinovosyl diacylglycerol increased. The changes of lipid composition under photoheterotrophic conditions varied amongst the different strains of this cyanobacterium. During chemoheterotrophic growth the levels of galactolipids and PG increased.

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501_16989.

INVESTIGATION OF CONFORMATIONAL DYNAMICS OF ATPSYNTHASE BY "TRITIUM MAPPING" METHOD

Podorvanov V.V., Tereshchenko A.F., Zolotareva E.K.

N.G. Kholodny Botany Institute,
National Academy of Sciences of Ukraine
2 Tereshchenkovskaya St.,
Kiev, 252601
Ukraine

Keywords:

ATP synthase, chloroplast, polypeptide composition, radioactive isotopes

D>
It was studied tritium label incorporation into polypeptides of chloroplast ATPase in the process of dark incubation of isolated CF1 factor or chloroplasts at -20^oC for 14 days in the presence of various concentration of tritium water and ³²P-ATP. Tritium incorporates into surface parts of CF1 polypeptides due to free-radical reactions which are induced by b-particles formed in the course of radioactive disintegration of tritium atoms. If ³²P-ATP was present in the reaction medium tritium incorporated mainly in the parts of polypeptides near by ATP-binding sites because of high energy of b-particles formed in the process of radioactive disintegration of ³²P.

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505_18126.

IN VIVO MUTAGENESIS OF THE psaC SUBUNIT: INTERACTION BETWEEN SOLUBLE FERREDOXIN AND PHOTOSYSTEM I

Fischer N., Sétif P., Hippler M., Rochaix J.D.

Dept. of Molecular Biology and Plant Physiology
University of Geneva
30, quai Ernest-Ansermet
CH-1211 Geneva, Switzerland

Keywords:

electrostatic interaction, ferredoxins, Fe-S centers, mutational analysis, photosystem 1

The PsaC subunit of photosystem I binds the two terminal electron acceptors FA and FB. PsaC resembles bacterial (4Fe-4S) ferredoxins but contains a C-terminal extension as well as an additional 10 amino acid internal stretch. We have used an in vivo mutagenesis approach to investigate the function of several residues located close to the iron-sulfur clusters as well as the role of the internal extension of PsaC. Mutated psaC genes generated either by site-directed mutagenesis or by oligonucleotide-directed random mutagenesis, were introduced into the chloroplast genome of Chlamydomonas reinhardtii via biolistic transformation. Analysis of the transformants, biochemical and biophysical characterization of isolated PSI complexes show that PsaC directly participates in the electrostatic docking of soluble ferredoxin to PSI and that this interaction is crucial for fast electron transfer. Implication of the results obtained with different mutants with respect to the orientation of PsaC within PSI will be discussed.

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509_18415.

BIOCHEMICAL GENETICS OF THE STRUCTURE, FUNCTION AND BIOGENESIS OF BACTERIAL CYTOCHROME BC₁ COMPLEX

Daldal F.,

University of Pennsylvania
Plant Science Institute
204 Mudd Bldg.
Phila, PA 19104-6019

Keywords:

bacterial photosynthesis, biogenesis, cytochrome complexes, electron transfer, phototrophs, site-directed mutagenesis

The cyt bc₁ complex in bacteria and mitochondria, and its analog the cyt b₆f complex in chloroplasts, is a multisubunit membrane enzyme essential for both respiration and photosynthesis. It is a major contributor of the cellular proton motive force that drives ATP synthesis. In the facultative phototroph Rhodobacter, intensely studied as a model system for eukaryotic organelles, the cyt bc₁ complex is found in its simplist form, constituted of three subunits (cyt b, cyt c₁ and the FeS protein) that contain four redox centers (two b hemes-b_H and b_L, a c heme and a [2Fe2S] cluster). These components form two quinone processing domains (Qo and Qi) which catalyze intramembrane electron movement and across membrane proton uptake and release. Tremendous progress in respect to its structure, function and biogenesis was accomplished in recent years using multidisciplinary approaches including molecular genetics, biochemistry/biophysics and crystallography. This knowledge has now set the stage for 3D-structure-directed molecular engineering initiatives aimed at converting the bacterial bc₁ complex to other related complexes of different origins, such as chloroplast-like b₆-c₁ complex, in order to rigorously correlate the structural and functional similarities and differences between the members of the extremely well conserved ubihydroquinone: cyt c oxidoreductases.

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510_7791.

NO₂^- UPTAKE BY A NO₂^- TRANSPORTER OF CHLOROPLAST ENVELOPE

Takahashi M., Haruki H., ^*Sugiura M.

Department of Applied Biological Chemistry, Osaka Prefecture University, Sakai, 599-8531, Japan
^*RIKEN, Wako, 351-0198, Japan

Keywords:

metabolite transport, N metabolism, proton transport, chloroplast envelope

NO₂^- influx into chloroplasts is evidenced as a multiphasic process which is complised of a saturable H⁺/NO₂^- symport and simple diffusion of HNO₂. Inhibition by protein chemical modifier and H⁺ conductor indicates participation of NO₂^- transporter in the active transport of NO₂^- into chloroplast. A putative NO₂^- transporter cDNA (Nitr1) was cloned from greening cucumber cotyledons. It encodes 484-amino acid membrane protein with 10 transmembrane domains showing sequence homology to nitrate and peptide transporters. Nitr1 mRNA was present in green tissues but not in roots and etiolated seedlings. Nitr1 protein was localized in chloroplast fractions especially in inner and outer envelope membranes with higher presence in the inner envelope. Heterologous expression modifies the permeability of yeast cells to NO₂^-.

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516_31788.

PYRUVATE, p-DIKINASE OF CAM PLANTS: SPECIES VARIATION IN THE SUBCELLULAR LOCALIZATION AS REVEALED BY IMMUNOELECTRON MICROSCOPY

Kondo A.^1,3, Nose A.¹, Yuasa H.², Ueno O.<sup>3

1</sup>Fac. Agri., Saga Univ., Honjo, Saga 840-8502, Japan,
²Res. Inst. Evol. Biol., Setagaya, Tokyo 158-0097, Japan,
³Dept. Plant Physiol., Natl. Inst. Agrobiol. Resour., Tsukuba, Ibaraki 305-8602, Japan.

Keywords:

CAM, PPDK localization, immunogold electron microscopy

In ME-CAM, pyruvate, p-dikinase (PPDK) is involved in the conversion of pyruvate to PEP, and is considered to be located in chloroplasts. In recent our study, however, PPDK has been found not only in the chloroplasts but also in the cytosol of the mesophyll cells in some ME-CAM plants. In this study, we investigated the intracellular localization of PPDK for 15 species in 8 genera of 5 families by immunogold electron microscopy. It was revealed that the intracellular patterns of PPDK localization are varied among species and are divided into three types: Chlt type which accumulates PPDK only lates PPDK only in the chloroplasts, Chlt-Cyt type which accumulates in both the chloroplasts and cytosol, and Cyt type which accumulates only in the cytosol. Mesembryanthemum crystallinum (Aizoaceae) and Crassula argentea (Crassulaceae) represent the Chlt type, and Schlumbergera russelliana (Cactaceae) does the Cyt type. Other species including Kalanchoe are classified into the Chlt-Cyt type. Even within this type, the accumulation levels of PPDK in the chloroplasts were varied among species. Although the biochemical significance of species variation in PPDK localization is unclear, the results might give a clue to elucidate the molecular evolution of PPDK in CAM.

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517_28247.

STRUCTURE OF THE Mn-CLUSTER IN PHOTOSYNTHETIC OXYGEN EVOLVING COMPLEX EVALUATED BY MAGNETIC PROPERTIES OF S₂-STATE MULTILINE SIGNAL

OnoT.-A., Kusunoki M., Hasegawa K.

Photosynthesis Research Laboratory, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0192, Japan

Keywords:

EPR, manganese cluster, O2 evolution, photosystem 2, S-states

Effective hyperfine parameters and Euler Angles of Mn ions of the multiline S₂-state Mn-cluster, and system parameters of the cluster were determined by the simulation of the multiline EPR signal with an anithotropic model in oriented photosystem II membranes. The obtained parameters indicate that the O-O vector of the putative di-m-oxo bridged Mn(III)-Mn(IV) dimer unit in the S₂-state Mn-cluster (III-IV,IV,IV) tilts by 43^o to 56^o with respect to the normal of thylakoid membrane. Furthermore, spin-exchange structures of the S₂-state cluster were numerically evaluated by taking into account the magnetic properties of the multiline signal including the effective ⁵⁵Mn hyperfine constants determined by the simulation. The results indicate that the cluster has one strongly-antiferromagnetic coupling (J>170 cm^-1) and other exchange constants are lower than 150 cm^-1 at most.

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520_32736.

ENERGY TRANSDUCTION IN LEAVES STUDIED BY MILLISECOND DELAYED LIGHT EMISSION

Shen Y.-K., Shi X.-B., Li D.-Y., Ye J.-Y.

Shanghai Institute of Plant Physiology, the Chinese Academy of Sciences,
Shanghai 200032, China

Keywords:

ATP, energy transduction, NADP, photophosphorylation, photosystem 2, Milisecond Delayed Light Emission

It is generally considered that millisecond delayed light emission (msDLE) is originated from the back reaction of light induced charge separation in PSII, which is stimulated by the proton motive force to provide the energy to overcome the activation energy barrier of the recombination process. Therefore, the msDLE may be used as an useful indicator of energy transduction.
In our previous papers, the work studying the effect of uncouplers on the msDLE and photosynthesis in leaves showed that what people knew from in vitro research about photophosphorylation could in many aspects coincide with its role in vivo and on changing the carbon dioxide concentration in air, an oscillation of msDLE occurred which reflected the regulation of the assimilation power. In this paper, we further study this problem by adding methyl viologen into leaves through petiole. Methyl viologen can act as electron acceptor instead of NADP⁺. When it was present in leaves the proton motive force and ATP formation still took place, but NADP⁺ reduction and carbon assimilation were almost stopped. In such case, the msDLE reached the steady state slower and remained much higher than those in control leaves, and on changing the carbon dioxide concentration in air the phenomenon of oscillation in msDLE nearly disappeared. The above results confirm our previous conclusion and give us more valuable insight into the dynamics of the energy transduction in leaves under changing environment.

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523_932.

SPIN RELAXATION ENHANCEMENT OF THE Mn CENTRE OF PHOTOSYSTEM II

Kuzek D.A., Pace R.J.

Department of Chemistry
The Faculties
The Australian National University
Canberra ACT 0200
Australia

Keywords:

EPR, manganese cluster, photosystem 2, spin relaxation enhancement

A widely proposed model for the organization of the manganese center in the Oxygen Evolving Center (OEC) of Photosystem II (PSII) is that of a tetranuclear cluster. Interpretations of the hyperfine coupling of the multiline signal and also the extended X-ray fine structure (EXAFS) pattern (especially the proposed identity of the 3.3Ĺ distance) have been used to support this. There is however an ever increasing body of evidence which suggest a dimer of dimers organization of the manganese center; both from work within our group, as well as from the recent confirmation of the S₁ parallel polarization signal.
Spin relaxation enhancement studies on three of the EPR signals associated with the S₂ state of the OEC have been conducted. Each of these signals is thought to arise from either one of the dimers in the OEC. Relaxation enhancement studies are able to probe each of these sites responsible for the appropriate signal.
The results from the relaxation enhancement study strongly suggest that the signals in the S₂ state arise from centers which are not magnetically exchange coupled. From these results we would exclude the possibility that the four Mn form a closely coupled tetrameric cluster, but the results support the model of a pair of magnetically isolated dimers.

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527_1780.

RAPID ISOLATION AND CHARACTERIZATION OF HIS-TAGGED PS II CORE COMPLEX FROM CHLAMYDOMONAS

Sugiura M., Inoue Y., Minagawa J.

Photosyn. Res. Lab., Inst. of Phys. and Chem. Res. (RIKEN), Wako, Saitama 351-0198, Japan

Keywords:

chromatography, D2 protein, O2 evolution, photosystem 2, Chlamydomonas

We have developed a simple and rapid procedure to isolate an oxygen-evolving PS II core complex from Chlamydomonas reinhardtii. A His-tag made of six consecutive histidine residues was genetically attached at the carboxy terminus of D2 protein to create a metal binding site on the PS II supramolecular complex. The recombinant cells producing the His-tagged variant of D2 protein grew photoautotrophically as well as the wild type cells. Characterization of the oxygen evolution and the thermoluminescence properties revealed that the His-tagging did not affect the functional integrity of the PS II reaction center. A PS II core complex was isolated from the detergent-solubilized thylakoids of the recombinant cells in 4 hours by a single one-step Ni²⁺ affinity column chromatogty column chromatography. This preparation consists of D1, D2, CP43, CP47, 33 kDa, and a few low molecular weight proteins, and retains a high rate of oxygen-evolving activity [>2,000 µmol (mg of Chl)^-1 h^-1]. The complex contained approximately 50 Chl per Y_D⁺ or 2 molecules of cytb₅₅₉, equivalent to 4 Mn atoms per reaction center.

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529_2326.

PHOTOSYNTHESIS AND PRODUCTIVITY RESPONSE TO CHANGES IN BLUE/RED LIGHT RATIO IN DUNALIELLA BARDAWIL UNDER FIELD CONDITIONS

Ashkenazi R., Yakir D., Yogev A.

Department of Environmental Sciences and Energy Research
Weizmann Institute of Science, Rehovot Israel.

Keywords:

bioreactors, blue light, diurnal changes, green algae, light acclimation

D. bardawil was grown under field conditions in a novel optical bioreactor that allowed the manipulation of sunlight spectrum and intensity. Reducing blue/red (480 nm/710 nm) ratio induced low-light photosynthetic response. Relative to control, antennae size was doubled, Chl a/Chl b ratio decreased by 11%, total chlorophyll concentration increased 2.7 times, and a 10 fold increase in xanthophyll epoxidation state was accompanied by decreasing levels of Cbr (Carotene biosynthesis related protein; consistent with coupling between Cbr and Zeaxanthin). Total cells density increased by 60% and b-carotene concentration (per ml.) increased by 15%. Increasing blue/red light ratio of the sunlight, induced high-light response. Chl a/Chl b ratio increased by 1.5 fold and antennae size decreased by 30%. However, no significant change in total chlorophyll concentration or in cells density was observed and b-carotene concentration decreased (unexpectedly) by 40%. We conclude that photosynthetic and pigments productivity in D. bardawil can be increased by manipulating sunlight blue/red ratio and intensity.

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531_2294.

WIDESPREAD OCCURRENCE OF NON-UNIFORM CHLOROPHYLL FLUORESCENCE QUENCHING DURING PHOTOSYNTHETIC INDUCTION IN WILTED LEAVES OBSERVED WITH A FIELD-PORTABLE IMAGING SYSTEM

Osmond C.B., Büchen-Osmond C., Daley P.F.

Photobioenergetics Group
Research School of Biological Sciences
Australian National University
P.O.Box 475, Canberra, ACT 2601, Australia

Keywords:

Chl fluorescence induction, stomata, water stress, Wilted

A field-portable instrument for observing chlorophyll fluorescence quenching, developed by Daley (1995), was used to determine whether patchy fluorescence quenching was widespread in wild plants. The properties of the imaging system will be summarised, and examples of patchy fluorescence quenching captured on tape with this system will be presented. In most species examined complex patchiness was observed in the first 10-30s of Kautsky fluorescence transients. Two main forms of patchiness at the sub millimetre scale were observed; vein defined (i.e., areas between veins quenched more slowly) or vein associated (i.e., either or both sides of major veins quenched more slowly). These early quenching events most probably reflect effects of localised differences in tissue water potentials on processes of photosynthetic electron transport pathway adjustment, prior to induction of CO₂ assimilation.
Daley, P.F. (1995) Chlorophyll fluorescence analysis and imaging in plant stress and disease. Canadian J. Plant Pathology 17: 167-173.

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535_10777.

IMMOBILIZATION OF CHLOROPLASTS: PHOTOBIOREACTOR WITH P450 MONOOXYGENASE

Hara M.^1,3, Iazvovskaia S.¹, Ohkawa H.², Asada Y.³, Miyake J.<sup>1,3

1</sup>NIBH, AIST, MITI, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan,
²Dept. of Biological and Enviromental Science, Faculty of Agriculture, Kobe University, Rokkodai-cho 1, Nada-ku, Kobe, Hyogo 657-0013, Japan,
³NAIR, AIST, MITI, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan

Keywords:

bioreactors, chloroplast, coupling/uncoupling, NADP, NADPH

We want to construct the light-driven bioreactor using the coupling reaction of P450 monooxygenase and photosynthetic electron transfer reactions in isolated chloroplasts. Spinach and Cactus chloroplasts are suitable materials a regenerator of NADPH. Under illumination, NADPH was formed form NADP and then used for P450 monooxygenase reaction (7-ethoxycoumarin w-deethylation) in the mixure consisting of isolated crude chloroplasts and microsomes in which the fused enzyme was expressed. Cactus chloroplasts showed higher thermostability than Spinach one. We also immobilized both the fusion enzyme between rat CYP1A1 and yeast P450 reductase, and chloroplasts to work as a light-driven bioreactors. Entrapment in agarose gel gave a good results to retain the enzymatic activity as far as we tried various methods for immobilization. [Reference: Hara, M., et al. (1997) J. Ferment. Bioeng. 84, 324-329 (1997)].

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536_11782.

STRUCTURE AND EXPRESSION OF Lhcb GENES FROM NICOTIANA SYLVESTRIS

Hasegawa K., Yukawa Y., Niwa M., Sugita M., Sugiura M.

Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan

Keywords:

higher plants, LHC II, light harvesting complexes, nuclear genes, photosystem 2

Type I light-harvesting chlorophyll a/b-binding proteins of photosystem II (LHCII) are encoded by a Lhcb1 gene family of 3-16 members in higher plants. In this study, we have cloned and sequenced nine members of the Lhcb1 gene family from N. sylvestris and measured the steady-state mRNA levels for each of the members in various tissues. Four genes, designated Lhcb1*1-Lhcb1*4, are organized in a tandem array with 10 kb, whereas the two genes, Lhcb1*5 and Lhcb1*6, are separated by 1.7 kb and transcribed oppositely. The other members are present as single genes. RNase protection assay using a gene specific probe indicated that Lhcb1 mRNA accumlated by tissue specific mannar. However, Lhcb1*2 mRNA accumlated in different pattern from other members.

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540_26772.

INCREASE OF INITIAL CHLOROPHYLL FLUORESCENCE Fo LEVEL AND ITS POSSIBLE MECHANISM IN SOYBEAN LEAVES

HongS.-S., Xu D.-Q.

Shanghai Institute of Plant Physiology
The Chinese Academy of Sciences
300 Fenglin Road
Shanghai 200032, China

KeywordTOP>

Keywords:

D1 protein, fluorescence, soybean, inactivation, lincomycin

Using a portable PAM-2000 fluorometer it was found that in response of initial chlorophyll fluorescence Fo level to strong light there were different cases in various plant species examined. When the photochemical efficiency of photosystem II, Fv/Fm, declined, Fo increased significantly in leaves of some plants such as soybean and cotton while Fo decreased remarkably in leaves of other plants such as wheat and barley. In order to explore the mechanism of the increase in Fo in soybean leaves the change in D1 protein content and the effects of Lincomycin and far-red light on these fluorescence parameters were observed by SDS-PAGE and chlorophyll fluorescence analysis. The following results were obtained: (1) the amount of inactive PS II reaction centers increased under strong light and decreased during subsequent dark recovery; (2) no net loss of D1 protein occurred after strong light treatment; (3) Lincomycin entered through petioles following strong light treatment had no effect on the decay of Fo in the dark and D1 protein level; and (4) far-red light could prevent the increase in Fo under strong light and accelerate the dark decay of increased Fo after strong light treatment. Based on these results, it is deduced that the increase of Fo under strong light is due to the reversible inactivation of part of PS II reaction centers rather than the net loss of D1 protein.

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542_14331.

STUDIES ON CARBON DIOXIDE EXCHANGE OF SCOTS PINE NEEDLES USING CUVETTE FIELD MEASUREMENTS AND A THREE-DIMENSIONAL STOMATAL MODEL INCLUDING GASEOUS PHASE AND LEAF MESOPHYLL

Aalto T., Vesala T., Mattila T., Hari P.^*, Juurola E.^*

Dept. of Physics, P.O.Box 9, 00014 University of Helsinki, Finland,
^*Dept. of Forest Ecology, P.O.Box 24, 00014 University of Helsinki, Finland

Keywords:

CO2 uptake, gas exchange, intercellular CO2 concentration, modelling, stomata

The dependences of CO₂ exchange of Scots pine needles on environmental variables and needle morphology is discussed. The temperature and light response of Scots pine shoots was studied in northern and southern Finland using cuvette field measurements and a biochemical model. The measured CO₂ fluxes in northern Finland indicated relatively weak temperature dependence at cool temperatures (<18^oC). A 3-dimensional model was constructed for studying the diffusion of CO₂ molecules from air into stomatal air spaces and futher into leaf mesophyll cells, where the local assimilation rate of CO₂ was calculated according to the biochemical model. The amount of CO₂ absorbing surface inside stoma and different diffusivities for CO₂ in the leaf mesophyll had a clear effect on the net CO₂ flux. Changes in the temperature-dependent solubility of CO₂ in the cell walls also altered the resulting flux and CO₂ concentrations in the leaf mesophyll.

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545_8163.

COMPARISON OF THE STRUCTURE AND FUNCTION OF CF1 e SUBUNIT FROM DIFFERENT PLANT CHLOROPLASTS

Wei J.-M., Shi J., Gu Y., Yang Y.-Z., Shen Y.-K.

Shanghai Institute of Plant Physiology, the Chinese Academy of Sciences. Shanghai 200032, China

Keywords:

ATP synthase, chloroplast, mutants, photophosphorylation, site-directed mutagenesis

Previously, it was found in our lab that changing the CF1 between broad bean and spinach chloroplasts showed enhancement in photophosphorylation activities. Now in comparative study of structure and function of chloroplast-ATPase from spinach and broad bean, we found that the activity of Mg²⁺-ATPase of chloroplasts activated by methanol from broad bean was higher than that from spinach, but the Mg²⁺-ATPase activity of spinach chloroplasts activated by DTT + light was higher than that from broad bean. The SDS-PAGE map showed that the molecular weight of e subunit of broad bean CF1 was smaller than that of spinach CF1.
We cloned atpE gene of chloroplast of broad bean and determined its DNA sequence. The amino acid sequence of e subunit of broad bean CF1 induced from DNA sequence, shows 84% and 74.4% homology respectively when compared with that of spinach sequence and maize sequence, but the degree of inhibition by adding e subunit of maize CF1 on the ATPase activity of spinach CF1(-e) was higher than that of broad bean.
After changing Thr-42 of e subunit to Cys, Arg, Ile and Pro by site-directed mutagenesis, which were named mutant eT42C, mutant eT42R, mutant eT42I, mutant eT42P respectively, the mutant eT42I showed much stronger inhibition on the Ca²⁺-ATPase activity than that of wild-type e subunit, while mutant eT42C and eT42R only slight inhibition.

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548_8630.

b-CAROTENE TO ZEAXANTHIN CONVERSION DURING ENHANCED D1 PROTEIN TURNOVER IN CHLAMYDOMONAS REINHARDTII

Jahns P., Depka B., Trebst A.

Plant Biochemistry, Heinrich-Heine-University Düsseldorf (P.J.) and Ruhr-University Bochum (B.D, A.T.), Germany

Keywords:

carotenoids, D1 turnover, herbicides, light stress, xanthophyll cycle

The D1 protein content and carotenoid composition was investigated in Chlamydomonas reinhardtii during exposure to high light. After 2h of high light no loss of the D1 protein was detectable, while the b-carotene content was significantly reduced. In parallel an increase of the zeaxanthin (Zea) content was found, which was higher than can be accounted for by the de-epoxidation of violaxanthin (Vio). Inhibitors of carotene biosynthesis led to the loss of D1 protein, indicating the requirement of b-carotene synthesis for the reassembly of photosystem II (PSII). The increase of the Zea content, however, was much less effected by the inhibitors. This implies that the additional Zea is not derived from newly synthesized carotenoids. We therefore assume that b-carotene which is bound to PSII is hydroxylated to Zea under high light stress. The portion of Zea formed via this path was convertible to Vio during a following low light period.

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549_9144.

THREE-DIMENSIONAL STRUCTURE OF PHOSPHOENOLPYRUVATE CARBOXYLASE FROM ESCHERICHESCHERICHIA COLI AT 2.8Å RESOLUTION

Kai Y., Matsumura H., Inoue T., Terada K., Nagara Y., Yoshinaga T., Kihara A., Izui K.

Department of Materials Chemistry, Graduate School of Engineering, Osaka University, Suita, 565-0871, Japan
Department of Public Health, Graduate School of Medicine; Department of Chemistry, Graduate School of Science; and Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan

Keywords:

CO2 concentrating mechanisms, X-ray diffraction, crystal structure

The first three-dimensional structure of phosphoenolpyruvate carboxylase (PEPC) was determined by X-ray diffraction method at 2.8Å resolution for the E. coli PEPC in the form of aspartate complex. As the aspartate is the inhibitory allosteric effector molecule of the enzyme, the present structure corresponds to the inactive state of PEPC. The active site was specified on the C-terminus side of the eight-stranded b-barrel, which is the main structural motif of the enzyme. The relative locations of catalytically important amino acid residues revealed will give fundamental structural bases for the design of a new plant with improved efficiency for CO₂ assimilation.

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553_18032.

LEAF PHOTOSYNTHESIS IN RICE PLANTS TRANSFORMED WITH THE ANTISENSE OR SENSE GENE TO THE SMALL SUBUNIT OF RUBISCO

Makino A.¹, Nakano H.¹, Mae T.¹, Shimada T.², Matsuoka M.³, Shimamoto K.⁴, Yamamoto N.<sup>5

1</sup>Department of Applied Biological Chemistry, Tohoku University, Sendai 981-8555, Japan;
²The Ishikawa Agricultural College, Ishikawa 921, Japan;
³Bioscience Center, Nagoya University, Nagoya 464-01, Japan;
⁴Nara Institute of Science and Technology, Ikoma 630-01, Japan;
⁵National Institute of Agrobiological Resources, Tsukuba 305, Japan

Keywords:

elevated CO2 concentration, gas exchange, Rubisco, transgenic plants, rice

Rice was transformed with the antisense or sense gene to rbcS and the rbcS promoter. The transformants with reduced (>35% wild-type) and enhanced (<135% wild-type) N allocation to Rubisco were obtained with the antisense and sense genes, respectively. The A:Ci curve studies showed that the CO₂-saturation point in the antisense plants was enhanced up to >100 Pa, although that in the sense plants was around normal CO₂ (36 Pa). Most of the antisense plants were fertile, but all the sense plants with enhanced Rubisco were sterile. The antisense plants with 65% wild-type Rubisco showed 5-15% higher rates of photosynthesis at 100 Pa CO₂ than the wild type plants for the same leaf N content, although they had 20% lower photosynthesis in normal air (Makino et al., 1997, Plant Physiol. 114: 483-491). However, the biomass of these plants was not greater than that of the wild type plants even when they were grown in 100 Pa CO₂.

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559_5188.

DOES THE PRE-TREATMENT OF SUNFLOWER SEEDLINGS WITH Cd INFLUENCE THE EFFECTS OF OZONE ON PHOTOSYNTHESIS?

Di Cagno R.¹, Andreucci A.¹, Guidi L.², Stefani A.¹, Soldatini G.F.<sup>2

1</sup>Scuola Superiore di Studi Universitari e di Perfezionamento "S. Anna", Via Carducci, 40 - 56100 Pisa (Italy)
²Dipartimento di Chimica e Biotecnologie Agrarie, Via del Borghetto, 80 - 56124 Pisa (Italy)

Keywords:

abiotic stress, adaptation, C3, Chl fluorescence induction, CO2 uptake, environmental stress, fluorescence, gas exchange, heavy metals, intercellular CO2 concentration, net photosynthesis, non-photochemical quenching, ozone, photochemical quenching, quantum yield, quenching

Among environmental pollutants, ozone and trace elements have became increasingly harmful to natural ecosystems. The aim of this work was to study the effects of acute ozone fumigation (120 nL L-1 for 2h) on photosynthetic process of Helianthus annuus L. seedlings grown for 7 days in the nutrient solutions containing 20 µM Cd²⁺.
The effects on the photosynthetic process were studied using gas exchange measurements and chlorophyll a fluorescence. The possible interactions between cadmium and ozone are discussed.

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564_28447.

CHLOROPHYLL a - PHOTOACTIVE SPECIES IN PHOTOVOLTAIC AND PHOTOELECTROCHEMICAL DEVICES

Tugulea G.L., Antohe S., Dulcu I.

Faculty of Physics - University of Bucharest
P.O.Box MG-11, Bucharest-Magurele, 76900 Romania

Keywords:

electron transfer, electronic structure, light activation, modelling, photoconversion

The photoelectrochemical behaviour of the chlorophyll a species P740 (a water adduct of chlorophyll a, absorbing at 740 nm) was investigated under potentiostatic conditions, in different electrolytes. Thin films of P740 chlorophyll, of different thickness, have been obtained by electrodeposition on SnO₂ coated glass. The photocurrent action spectrum for P740 chlorophyll deposited on SnO₂ electrode showed a dependence on both chlorophyll a film characteristics and electrolyte properties. Photovoltaic devices of sandwich type: (SnO₂electrode/P740chla/Me) have been prepared and studied. I-V characteristics of the photovoltaic devices, in dark and under illumination, have been performed. The obtained data, on both electrochemical and photovoltaic devices using P740 chlorophyll a in thin films, lead to the design of the energetic diagrams of chlorophyll a in these systems and to the explanation of chlorophyll a behaviour in the process of charge generation under illumination and in the transfer of electrons.

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571_19791.

QUENCHING OF CHLOROPHYLL a FLUORESCENCE BY b-CAROTENE

Doina G.M.¹, Iorga B.², Balan P.<sup>1

1</sup>Faculty of Physics - University of Bucharest, P.O.Box MG-11, Bucharest-Magurele, 76900 Romania
²Faculty of Chemistry, University of Bucharest, Romania

Keywords:

carotenoids, fluorescence, pigments, quenching

In the last time an additional carotenoid function has been proposed which involves a specific carotenoid-chlorophyll interaction in their singlet excited states, leading to a reduction in chlording to a reduction in chlorophyll fluorescence. The present work reports the quenching of chlorophyll a fluorescence by trans b-carotene, in benzene. The fluorescence intensity was measured at 670 nm, at different excitation wavelengths: 380, 430, 618 and 650 nm. The trans b-carotene concentrations ranged from 10^-6M to 4·10^-5M. When the samples were excited at 615 nm and 650 nm, the plots that are linear in a Stern-Volmer sense show that the quenching process has high rate constants. b-carotene appears to be a good quencher of chlorophyll a fluorescence. It is possible that two different types of quenching processes should exist.

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574_18423.

IDENTIFICATION OF A NOVEL GENE INVOLVED IN HIGH-LIGHT RESISTANCE OF SYNECHOCYSTIS SP. PCC 6803

Kamei A., Ogawa T., Ikeuchi M.

Department of Life Sciences (Biology)
University of Tokyo
Komaba 3-8-1
Meguro, Tokyo 153-8902
Japan

Keywords:

acclimation, cyanobacteria, light stress, mutants, signal transduction

A glucose-tolerant strain (GT strain) was derived from the type strain of Synechocystis sp. PCC 6803 (PCC strain), which was originally isolated from a natural habitat. To study the high light resistance of photosynthetic organisms, we compared the growth properties of these two strains at high light (400 µE/m²/s). PCC strain grew for a while but then was bleached, whereas GT strain could grow, like under low light (20 µE/m²/s). This suggests that GT strain obtained the high-light resistance by genetic modification of PCC strain. Ogawa et al. (1995) reported that GT strain lacks a DNA sequence in genome of PCC strain, which partly corresponds to a putative gene, slr2031. Deduced amino acid sequence of slr2031 is homologous to Ser/Thr-protein phosphatase type 2C. We disrupted this gene in PCC strain and found that the mutant is resistant against high light or high temperature/light like GT strain. The mutant also showed higher efficiency of transformation and lack of motility like GT strain. These indicate that a novel gene, slr2031, is responsible for the multiple phenotypes of PCC strain, which may be dispensable for survival under laboratory conditions. Possible roles of slr2031 in Synechocystis sp. PCC 6803 will be discussed.

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579_31455.

PHOTOSYNTHESIS AND CARBON STORAGE BETWEEN TIDES IN A BROWN ALGA, FUCUS VESICULOSUS L.

Kawamitsu Y.^*, Boyer J.S.

College of Marine Studies, University of Delaware, Lewes, DE 19958, USA
^*Present address: College of Agriculture, University of the Ryukyus, Okinawa 903-0213, Japan

Keywords:

C3, C4, CAM, marine algae, O2 evolution

Intertidal macroalgae may spend a significant part of their lives in air. During photosynthesis (Ps), they encounter much lower concentrations of inorganic C (C_i) than in seawater. Because they accumulate C_i from seawater, we investigated whether they similarly accumulate it from air. We measured Ps in F. vesiculosus with gas phase O₂ electrode or CO₂ exchange apparatus in air and with a liquid phase O₂ electrode in seawater. Maximum rates were rapid and similar in air and seawater regardless of the method. Tissue from seawater could carry on Ps in CO₂-free air indicating that C was stored in the tissue. After 2h, this store was depleted and Ps ceased. Supplying CO₂ in air replenished the store. Under identical conditions, terrestrial C3 and C4 showed no evidence of this store, but a CAM species did. However, Fucus did not store CO₂ significantly in the dark. We found a small acid-releasable pool of C in the tissue that disappeared as Ps depleted the C store. However, the pool was too small to account for the total C stored. While CO₂ was being acquired or released from the store in the light, Ps was not inhibited by 21% O₂. These results indicate that there are two parallel paths for the supply of CO₂ to Ps. Ps in Fucus thus appears to be C3-like in its rapid fixation of CO₂ from a small inorganic pool into PGA. However, it is C4-like in its pre-fixation of C in an organic pool in the light, and CAM-like in its ability to slowly use this pool as a sole source of CO₂.

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580_14396.

CRYSTALLIZATION OF LIGHT-HARVESTING COMPLEX II FROM VICIA FABA (FABACEAE)

Nickel R., Wild A.

Institute for General Botany, Johannes Gutenberg University, D-55099 Mainz-Germany

Keywords:

2D crystals, crystallization, light harvesting complexes, pigment-protein complexes

Even the structure of LHC II of higher plants has been determined by electron crystallography to 3.4Å, the high-resolution structure based on x-ray crystallography is still missing. We investigated the possibility to get highly ordered 3-D crystals by varifying standard purification systems, including chromatographic methods. The highly purified LHC II was analyzed according to lipid composition, spectral properties and oligomeric state. Although the trimeric pigment-protein complex was increasingly delipidated during the purification procedure, we could induce the formation of 2-D crystals, which were analyzed by cryo-electron microscopy. Our attempts to reach highly ordered 3-D crystals, which are suitable for high-resolution x-ray analysis, lead to hexagonal as well as octaedric crystals. Both crystal forms are still under investigation according their suitability to x-ray analysis.

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581_6378.

ANALYSIS OF THE PSI-N SUBUNIT OF PSAN DEFICIENT TRANSGENIC ARABIDOPSIS

Haldrup A., Naver H., Scheller H.V.

Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.

Keywords:

electron transport, P700, plastocyanin, transgenic plants, PSI-N

The PSI-N subunit, encoded by the nuclear gene psaN, is the only PSI subunit located entirely in the thylakoid lumen. The PSI-N subunit is only present in PSI of higher plants. The role of the subunit in the PSI complex was investigated in transgenic arabidopsis plants which were generated using antisense and co-suppression strategies. Several lines without detectable levels of PSI-N were identified. The psaN mutants still assembled a functional PSI complex and were capable of photoautotrophic growth. The plants had a slightly increased PSI/PSII ratio but no difference in the PSI antenna was detected in 77K fluorescence emission spectra or by in vivo light saturation ht saturation studies of P700 oxidation. Flash induced absorption changes at 834 nm in the presence of known amounts of plastocyanin revealed that the PSI-N less plants have a decreased ability to accept electrons from plastocyanin.

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583_7726.

PHOTOSENSITIZED OXYGEN UPTAKE BY DISORGANIZED CHLOROPHYLL

Caspi V., Raskin V.I., Malkin S., Marder J.B.

Dept. Agric. Botany, The Hebrew University, Rehovot, Israel (VC, VIR, JBM)
Dept. Biological Chemistry, The Weizmann Institute, Rehovot, Israel (SM)

Keywords:

fluorescence, oxidative stress, singlet O2, triplet states, chlorophyll organization

The photoexcited singlet state of chlorophyll is normally used efficiently (>80%) for photosynthesis. Alternatively, and particularly under conditions where chlorophyll is disorganized, relaxation occurs via the triplet state and generates singlet oxygen, leading to oxidative damage. We have examined chlorophyll photodynamics using chlorophyll solutions as a model for disordered chlorophyll. Light dependent oxygen uptake was amplified by adding oxidisable substrates e.g. linoleic acid, ascorbate and tryptophan. The rate increased linearly with light intensity and was decreased by quenchers of singlet oxygen or excited chlorophyll. The early stage of greening of etiolated barley provides potential conditions for photodamage: poorly organized chlorophyll, light and oxygen. Chlorophyll disorder is indicated by high level of F₀ and long fluorescence lifetime (~5.6 ns). Certain compounds, such as aminolevulinic acid and cadmium, exacerbate this condition. The extent to which these conditions promote photosynthesized oxygen uptake is under study.

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587_25668.

SPECIFIC FEATURES OF PHOTOSYNTHETIC APPARATUS OF PLANTS IN HIGH MOUNTAIN DESERTS OF THE PAMIRS UNDER ECOLOGICAL STRESS

Kondratchouk A.V., Pyankov V.I.

Department of Plant Physiology, the Urals State University, Lenin Avenue, 51, 620083, Ekaterinburg, Russia

Keywords:

abiotic stress, structural changes

The investigations were carried out in the natural alpine dry conditions of the East Pamir mountains in altitudinal range 3500-4700m above sea level. The studies were focused on the changes of quantitative mesophyll structure in the same species, inhabiting in different elevations and soil conditions.
This investigation permits us to determine the common traits in adaptation of plants with elevation on the level of assimilating apparatus, which express in microfillio of plants, increase thickness of leaf blade and specific leaf weight (1/SLA), development of palisade mesophyll, formation of high number of chlorenchime cells and chloroplasts per unit of leaf area. Probably this peculiarities tends to the development of internal assimilation surface (Amez/A and Achl/A) and decrease of mesophyll resistance to CO₂ diffusion in stress conditions of Pamir mountains.
We were supported by grant RFBR No.97-04-49900.

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589_29947.

MUTANTS OF C4 PHOTOSYNTHESIS

Leegood R.C.¹, Bailey K.J.¹, Dever L.V.², Lea P.J.<sup>2

1</sup>Robert Hill Institute and Department of Animal and Plant Sciences, University of Sheffield, S10 2TN, Sheffield, UK
²Department of Biological Sciences, Lancaster University, LA1 4YQ, Lancaster, UK

Keywords:

C metabolisms, C4, CO2 concentrating mechanisms, enzymes, metabolic processes, mutants

Photosynthetic mutants of the C4 plant, Amaranthus edulis, have been isolated using a CO₂ screen. These plants comprise mutants that contain less than 10% of the wild-type activity of (i) PEP carboxylase (PEPC) or (ii) NAD-malic enzyme (NAD-ME) or (iii) which accumulate photorespiratory glycine. Heterozygous plants of both PEPC and NAD-ME mutants have been used to study the control of photosynthetic carbon assimilation. PEPC exerted considerable control in the wild-type, with a relatively high control coefficient (0.35) in saturating light and ambient CO₂. The flux control coefficient was increased in low CO₂, in high light or in plants containing lower PEPC activity. The rate of CO₂ assimilation decreased down to about 55% PEPC, but there was an upturn in the light-saturated rate as PEPC was further reduced, indicating the operation of a compensating mechanism. This compensation was not due to changes in phosphorylation state, but may have been due to the accumulation of photorespiratory glycine and serine. In contrast, NAD-ME exerted very little control over the rate of CO₂ assimilation.

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593_15754.

ANTISENSE APPROACH USED TO STUDY THE FUNCTION OF PSI-E IN BARLEY

Nielsen H.L., Scheller H.V., Gilpin M.J., Jensen A.M., Moller B.L.

Plant Biochemistry Laboratory, Department of Plant Biology,
Royal veterinary and Agricultural University, 40 Thorvaldsensvej
DK-1871 Frederiksberg C, Copenhagen, Denmark

Keywords:

abiotic stress, down regulation, gene expression, mutational analysis, nuclear genes, regulatory processes, Cyclic electron transport

PSI-E is one of the proteins in Photosystem I, located on the stromal surface partly covering PSI-C. Studies of Synechococcus mutants suggest that PSI-E is involved in cyclic electron transport around PSI. In order to investigate the role of PSI-E in vivo, barley was transformed using an antisense approach. A partial and a full-length antisense PsaE construct were made using the rice actin promoter and the rubisco terminater. The bar gene was used as selectable marker. The plasmid DNA was introduced into to immature barley embryos by particle bombardment. Bialaphos resistant calli were obtained, and plants regenerated. Transformed plants were analyzed by PCR, Western and Southern blotting. Characterization of the second generation of transformants, will be presented.

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596_29382.

COSUPPRESSION OF PHOTOSYSTEM I SUBUNIT PSI-H IN ARABIDOPSIS. PSI-H IS IMPORTANT FOR ELECTRON TRANSFER AND STABILITY OF PHOTOSYSTEM I

Naver H., Haldrup A., Scheller H.V.

Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, 40, Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark.

Keywords:

electron transport, photosystem 1, transgentem 1, transgenic plants, Photosystem I subunit PSI-H

A cDNA clone for the psaH was transformed into Arabidopsis thaliana in sense orientation in order to obtain plants with low levels (cosuppression) of the PSI-H subunit of photosystem I (PSI). Four independent transformant lines with less than 3% PSI-H compared to wild-type were obtained. The extent of cosuppression can not be correlated with the copy number of the inserted psaH gene as determined by Southern blot analysis. Plants lacking PSI-H also have decreased PSI-L levels shown by western blot analysis of thylakoid preparations. The NADP⁺ photoreduction activity of the PSI complex without PSI-H was only 61% of the wild-type level. Further, the PSI-H less PSI complex is unstable compared to the wild-type as shown by urea treatment of thylakoid preparations followed by flash photolysis. In conclusion, PSI-H is shown to be important for: 1) interaction of subunit PSI-L, 2) efficient electron transfer and 3) stability of the PSI complex.

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598_22081.

ROLE OF ATP AND POLYPHOSPHATES IN GREEN ALGAE BIOENERGETIC

Forti G., Finazzi G., Foietta A.

Centro CNR Biologia Cellulare e Molecolare delle Piante, via Celoria 26, 20133 Milano Italy

Keywords:

ATP, microalgae, polyphosphate

It has previously been found that, in whole cells of green algae, a deep interconnection between respiration and photosynthesis allows the establishment of a high ATP/ADP ratio and the generation of a permanent electrochemical proton gradient across thylakoids membranes, due to ATP hydrolysis.
We have investigated the metabolic connection between the respiratory and photosynthetic activities in whole cells of Chlorella and Chlamydomonas. This has been done through measurement of respiratory and photosynthetic O₂ changes in the absence and presence of uncouplers and the variations of ATP, ADP and polyphosphates. The respiratory activity is more sensitive to uncoupling than the photosynthetic activity. Uncoupling in the dark resulted in a decrease of the ATP/ADP ratio, which remains high even in fully uncoupled conditions. We observed that the changes of polyphosphates in the cells was opposite to those in the nucleotides pool, suggesting a rather fast equilibration of these two energy sources.

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601_26260.

ACCLIMATIVE PROTEOLYSIS OF THE MAJOR LIGHT-HARVESTING CHLOROPHYLL a/b PROTEIN OF PHOTOSYSTEM II (LHCII)

Yang D.-H., Lindahl M., Webster J., Andersson B.

Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden

Keywords:

higher plants, LHC II, light acclimation, photosystem 2, proteolysis

The long term regulation of LHCII is reflected by a decrease in antenna size in plants exposed to high light. We have identified a proteolytic activity degrading LHCII during acclimation of spinach from low to high intensity light. This ATP-dependent proteolytic activity of serine or cysteine type is associated with the outer membrane surface of the stroma-exposed thylakoids. The identity of the protease is not known, but it does not correspond to any of the recently identified chloroplast ATP-dependent proteases, Clp or FtsH, which are homologues to bacterial enzymes. The acclimative response shows a delay of two days following transfer of the plants to high light. This delay was shown to be attributed to expression or activation of the protease. Furthermore, the LHCII degradation was shown to involve reversible protein phosphorylation and migration of LHCII from the grana to the stroma-exposed thylakoid regions. However, dephosphorylation of LHCII was required to initiate its degradation. Identification and purification of the thylakoid bound protease are currently under investigation. Preliminary observation suggests also the involvement of an endogenous protease inhibitor as a regulatory element in the acclimation of the LHCII antenna.

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604_32738.

REGULATION OF PHOTOSYNTHESIS AND TRANSLOCATION OF PHOTOSYNTHATES BY ENDOGENIOUS PHYTOGORMONS IN LEAVES DURING ONTOGENESIS

Kiselyova I.S., Borzenkova R.A.

Department of Plant Physiology, the Urals State University, Lenin Avenue, 51,Ekaterinburg, 620083, Russia

Keywords:

C partitioning, hormones, leaf physiology, ontogenesis, whole leaf

Changes in growth rate, photosynthesis, hormonal status and export function were studied in leaves of barley (flag and preflag) and potato (7-th, 11-th, 15-th) during ontogenesis. The higher photosynthesis, absolute growth rate and lower source activity were revealed in leaves, appeared earlier - lower ones in comparison with upper. The increase of CO₂ assimilation in juvenile leaves was accompanied by the raising of their source activity, incorporation of assimilates mainly to insoluble carbohydrates (starch, cellulose and hemicellulose) and correlated with the increase of IAA and cytokinine concentration. Further intensification of export function coincided with the decrease in concentration of these hormones, increase of ABA and photosynthate incorporation mainly to sucrose. Development of source activity correlated also with the ratio cytokinine/ABA (r=-0.86) and IAA/ABA (r=-0.93).
Our research was supported by grant RFBR#97-04-48374.

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608_499.

THE FMO COMPLEX OF GREEN SULFUR BACTERIA: EXPERIMENTS AND SIMULATIONS

Vulto S.I.E., de Baat M.A., Louwe R.J.W., PermentierH.P., Neef T., Miller M., Aartsma T.J.

Biophysics Department
Huygens Laboratory
Leiden University
P.O.Box 9504
2300 RA Leiden
The Netherlands

Keywords:

electronic structure, excited state dynamics, green sulfur bacteria, light harvesting complexes, time-resolved spectroscopy

We have simulated the electronic structure of the FMO complexes from P. aestuarii and C. tepidum considering only one subunit. The lowest exciton state is dominated by the contribution of a single molecule, BChl 3. From the simulations it is clear that the spectra are mainly determined by variations in site energies rather than by the dipolar coupling. Comparing the two FMO complexes, we can relate the spectroscopic differences to the structural features. We will also report on simulations of the excited state dynamics in those systems. Energy transfer within one subunit will be discussed on the basis excitonic coupling. Energy transfer between subunits requires a Förster mechanism, since anisotropy measurements at 825 nm show a decay of ~100 ps, suggesting slow energy transfer between subunits.
This work was supported by the EC (FMR X-CT96-0081).

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621_11052.

PHOTOSYNTHESIS AND THE WORLD WIDE WEB

Govindjee

University of Iliinois at Urbana-Champaign, 265 Morrill Hall, 505 South Goodwin Avenue, Urbana, IL 61801-3707, USA

Keywords:

atomicmodel, Chlfluorescenceinduction, cytochromecomplexes, energytransduction, primaryprocesses, reactioncenters, WorldWideWeb

The World Wide Web is an important resource for public awareness and for educating all citizens of the World including its political leaders, granting agencies, pre-university and university students, as well as teachers and researchers in all fields of photosynthesis. The first and most complete site, related to photosynthesis, is at: http://photoscience.la.asu.edu/photosyn (maintained by L. Orr).
My site is at: http://www.life.uiuc.edu/govindjee/; this site has some slides that can be down-loaded; a Chl fluorescence (Kautsky Effect) clip; an introductory article "Photosynthetic Process"; and "Photosynthesis and Time", etc.
A.R. Crofts has an excellent site for a course on "Biological Energy Transduction": http://arc-gen1.life.uiuc.edu/Bioph354/ and another for cytochrome complexes: http://arc-gen1.life.uiuc.edu/bc-complex_site.
J. Marder has a nice RC tutorial at: http://www.agri.huji.ac.il/~marder/rc_view.
And, W.-D. Schubert has a site for PSI atomic model: http://www.chemie.fu-berlinde/~phosys.
J. Allens site is at: http://plantcell.lu.se/ltm/; it has an interesting discussion on the primary events of photosynthesis and light-harvesting Complex II.
Most labs. have sites, but I mention only those that were provided to me by E-mail:
http://www.biosci.ohio-state.edu/~rsayre, http://www.life.uiuc.edu/pru and http://130.126.50.91/vlad/.
E-mail:gov@uiuc.edu

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622_16401.

CHLOROPHYLL-FLUORESCENCE MEASUREMENTS DURING DRYING AND REMOISTENING OF THE DESICCATION-TOLERANT LEAFY LIVERWORT PORELLA PLATYPHYLLA

Marschall M.¹, Proctor M.C.F.², Molnár I.<sup>1

1</sup>Dept. of Plant Physiology, Eszterházy K. T. T. College, H-3301 Eger, Pf. 43
²Dept. of Biol. Sciences, University of Exeter, EX4 4PS, UK

Keywords:

Chl fluorescence induction, dessiccation, drought stress, quenching, water stress

As shoots of P. platyphylla dried, Fv/Fm fell to about half of its full-turgor value at water content 37% DW; EQY showed a similar response. The parameter qP did not change significantly over a same range, but there was some indication of an increase in qN and NPQ.
On remoistening material kept air-dry for 1 week (water content 13% DW corresponding to cell RWC < 0.1), Fv/Fm, qP and EQY rose quickly, reaching steady pre-desiccation levels within 2-3h (> 75% within 1h). Material recovering in continuous actinic light (100 µmol m^-2 s^-1 PPFD) showed much higher qN and NPQ than material in the dark. Down-regulation of Fm' (as measured by qI) was much more strongly marked and persistent in material kept in continuous light.

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623_8773.

PHOTOINHIBITION OF PEA LEAVES BY ACCUMULATION OF OVER-SATURATING LIGHT PULSES IN DARKNESS

Apostol S.L., Briantais J.M., Moya I.

LURE-CNRS, Bat.209D, Centre Universitaire Paris Sud, 91405 Orsay, France

Keywords:

Chl fluorescence induction, photoinhibition, whole plants, saturating pulses, diurnal cycle

In this study the dependence of PSII photoinactivation as a function of cumulative high-intensity light pulses (1s, 10,000 µmol m^-2s^-1) in the dark was investigated, during a diurnal cycle, in pea leaves.
The status of the photosynthetic apparatus was monitored by changes in the photochemical efficiency of PSII as estimated by the variable chlorophyll fluorescence measurements, performed using a new fluorimeter (Frequency Induced Pulse Amplitude Modulation) allowing to measure at distance (1 meter) by the saturating pulse method the PSII electron transfer rate, during a diurnal cycle. Gas exchange measurements were realized simultaneously with fluorescence measurements. The accumulation of over-saturating pulses in darkness is accompanied by a progressive decline of PSII quantum yield, that is revealed during the daylight, starting early in the morning. A remarkable effect of protection against this photoinactivation by over-saturating pulses treatment during night was observed in the presence of continuous low light backgrounds. Besides, treatments of leaves by various chemicals known to influence state of PSII give evidences of a photoinhibitory effect induced by the over-saturating pulses applied in dark, on the leaf.

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625_13265.

PHOTOSYNTHETIC EFFICIENCY AND PHOTOPROTECTION IN BARLEY MUTANTS LACKING LHCII

Augusti A., Scartazza A., Brugnoli E.

CNR, Istituto per l’Agroselvicoltura Via Marconi 2, 05010 Porano (TR), Italy

Keywords:

antennae, non-photochemical quenching, photoinhibition, photoprotection, xanthophyll cycle

The role of PSII light harvesting complex (LHCII) in light energy capture, utilisation and dissipation was studied in barley mutants deficient in chl b and in LHCII. The quantum yield of PSII electron transport (QY), non-photochemical fluorescence quenching (NPQ) and pigment content and composition were measured in the mutant and in the wild type (WT). Plants were grown either at optimal (23°C) or at low (11°C) temperature, to investigate possible acclimation responses to contrasting excitation pressures. At low temperature, the mutant showed a slight down-regulation of PSII, not evident in WT. The mutant showed a generally lower pigment content compared to WT. However, the contents of ß-carotene and of xanthophyll cycle components, expressed on a chl basis, were higher in the mutant than in WT. The mutant had higher QY in the light and lower NPQ compared to WT, indicating a lower photoprotectioncapacity. On the other hand, mutants had always a higher deepoxidation state and xanthophyll content with respect to WT. Hence, NPQ and zeaxanthin were not univocally correlated.

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634_20880.

FUNCTIONAL AND SPECTRAL ASSIGNMENT OF CHLOROPHYLLS IN THE LIGHT HARVESTING COMPLEX II OF HIGHER PLANTS

Trinkunas G.², Müller M.G.¹, Martin I.¹, Valkunas L.², Holzwarth A.R.<sup>1

1</sup>Max-Planck-Institut fur Strahlenchemie, Stiftstr. 34-36, Mülheim a. d. Ruhr, D45470, Germany
²Institute of Physics, A. Gostauto 12, Vilnius 2600, Lithuania

Keywords:

antennae, higher plants, LHC II, light harvesting complexes, photosystem 2

We report the results of the excitation dynamics modeling for the recently resolved trimeric LHC II complex containing 7 Chls a and 5 Chls b. Femtosecond transient absorption data at 4-5^oC with pulses of 60 fs duration were obtained for excitation at 11 different wavelengths between 640 to 690 nm and probed in the interval of 620-740 nm. Assuming an incoherent excitation energy transfer mechanism the 12 Chl spectral forms, generated by the deconvolution of the absorption spectrum, are attributed to the Chl sites in the structure. The simulated data well reproduce the kinetic and steady state spectral data. From this comparison we obtain the so far missing spectral and orientation assignment for Chl molecules in the LHC II structure. One of the most striking conclusions from our study is the assignment of four Chls b in close proximity to the central luteins.

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635_20256.

EXTRACELLULAR ASCORBATE MEDIATES OZONE TOLERANCE IN BROAD BEAN?

Turcsanyi E., Barnes J.D.

Department of Agricultural & Environmental Science
Ridley Building, University of Newcastle
Newcastle upon Tyne, NE1 7RU, UK

Keywords:

abiotic stress, ascorbate, modelling, oxidative stress, ozone

This poster reports the relationship between ASC content of apoplast/symplast and O₃ resistance in leaves of broad bean (Vicia faba L.). Major findings include:
1. Potentially damaging O₃ concentration cause a significant decline in the concentration of ASC in the apoplast.
2. The redox state of apoplastic ASC remains unchanged under the influence of O₃, indicating that the rate of replenishment of apoplastic ASC is sufficient to keep pace with the rate at which it is oxidised.
3. A linear relationship exists between the concentration of ASC in the apoplast and symplast, suggesting that diffusion-limited processes constitute the predominant mechanism controlling the replenishment of apoplastic ASC.
4. Model calculations reveal that the concentration of ASC in the apoplast is sufficient to detoxify the major part of O₃ entering into the leaf at environmentally-relevant O₃ concentrations.

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637_25222.

SELENITE STRESS INDUCES A MODIFICATION OF THE CYTOCHROME COMPOSITION BY THE PURPLE BACTERIA RHODOSPIRILLUM RUBRUM

Kessi J., Vasserot M., Bachofen R.

Inst. of Plant Biology
University of Zürich
Zollikerstr. 107
CH-8008 Zürich
Switzerland

Keywords:

alternative electron transport, cytochromes, purple bacteria, Selenite stress

R. rubrum was grown photosynthetically (anaerobically) in the presence of 0.5 mM selenite. Under these conditions the bacteria reduce selenite to metallic selenium in a reaction going nearly to completion. The presence of selenite in the culture is accompanied by modifications in the absorption spectra of cytoplasma, chromatophores and cell debris obtained after sequential centrifugation of the disrupted cells. As shown on polyacrylamide gel electrophoresis specifically stained for cytochrome detection, these proteins are also largely modified when the cells are growing in the presence of selenite. This suggests the induction of an alternative electron transport system by these stress conditions. We are presently studying cytochromes from the chromatophores to compare the selenite induced cytochromes with those of reference cultures grown in the absence of selenite.

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639_22623.

IDENTIFICATION OF A SIXTH LOCUS INVOLVED IN C-TYPE CYTOCHROME BIOSYNTHESIS

DreyfussB.W., Merchant S.

Department of Chemistry and Biochemistry, UCLA, Box 951569, Los Angeles, CA 90095-1569

Keywords:

algae, biosynthesis, cytochromes, heme

Chloroplast contain two c-type cytochromes, membrane-anchored cyt f of the cyt b₆ f complex and soluble cyt c₆. Five loci, one chloroplastlocus, ccsA, and four nuclear CCS loci are required for the formation of holocytochromes f and c₆. Mutants in all five loci display a pleiotropic deficiency in both c-type cytochromes and are specifically blocked in the attachment of heme to the apocytochromes. To facilitate the cloning of the genes defined by these loci, insertional mutants were generated by glass bead transformation of an arginine auxotrophic strain with pARG7.8f3. Mutants were identified that displayed the characteristic cyt f and cyt c₆ deficient phenotype. Genetic complementation analysis suggests that one of the mutants defines a new locus involved in c-type cytochrome biogenesis, designated CCS5 for the fifth nuclear locus in this pathway. The inserting pARG7.8f3 DNA segregates with the mutant phenotype during genetic crosses of strain ccs5-1::ARG7, confirming that the CCS5 locus is tagged. A unique 15 kb SalI genomic DNA fragment containing Arg7 sequences is observed in ccs5-1::ARG7 by DNA hybridization, and this will be exploited for cloning DNA flanking the site of insertion, which in turn will be used to identify the Ccs5 gene.

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641_22586.

FLUORESCENCE MEASUREMENTS OF A COUPLING FACTOR ARABIDOPSIS MUTANT DISPLAY CHANGES IN NON-PHOTOCHEMICAL QUENCHING

Spilotro P., Govindjee

Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801-3707, USA

Keywords:

energy dissipation, fluorescence, light stress, pH-gradients, quenching, xanthophyll cycle, Arabidopsis

The Coupling Factor (CF) complex within the thylakoid membrane uses the proton motive force created by the electron transfer chain for enzymatic activity. In plants, the redox state of the g-subunit of the CF complex is thought to be complex is thought to be involved in its activity. Further, it has been suggested that plants have a photoprotective mechanism that is initiated by the use of the excess DpH in high light that leads indirectly to "non-photochemical quenching" (NPQ) of Chl a fluorescence as violaxanthin is converted to the quencher zeaxanthin. In order to examine the relationship between the two processes, we have measured Chl a fluorescence, both by Hansatech PEA and Walz PAM-2000 fluorometer, on the leaves of wild-type Arabidopsis thaliana and a mutant of the g-subunit, called cfq (Gabrys et al., 1994, Plant Physiol. 104:769-776). This mutant, as a result of its continually oxidized g-subunit, is expected to require a larger DpH for CF activation. Our measurements have demonstrated unique differences in NPQ between the mutant and the wild type at varying light intensities.
We thank Prof. Don Ort for the seeds of the mutant, Dr. Pete Kelly and Ms. Carly Thomas for their help, and to the IPTRG from the NSF (DBI96-02240).

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642_23388.

CHARACTERIZATION OF AN S1-STATE SPECIFIC EPR MULTILINE SIGNAL OF THE PHOTOSYSTEM II MANGANESE CLUSTER

Campbell K.A., Gregor W., Pham D.P., Peloquin J.M., Debus R.J., Britt R.D.

Dept. of Chemistry, Univ. of California, Davis, CA95616,
Dept. of Biochemistry, Univ. of California, Riverside, CA92521

Keywords:

EPR, manganese cluster, OEC proteins, photosystem 2

Following the report of a parallel polarization mode EPR multiline signal (indicative of an integer spin system) of a PSII core from Synechocystis 6803 in the dark-stable S1 state, we report a similar signal obtained from salt-washed PSII membranes (BBY particles) and cores solubilised with octylglucoside (both from spinach), but not from native PSII membranes or cores solubilised with octylthioglucoside. Since the formation of this signal, but not the S2 state signal thus correlates with the removal of the 23 and 17 kD extrinsic proteins (which are lacking in cyanobacterial PSII), we infer modulation of the magnetic properties of the Mn cluster in the S1 state by these proteins. The signal displays 16-18 hyperfine lines, indicative of a multinuclear Mn cluster. Illumination at 196K abolishes 60-70% of the signal. A Curie-law behaviour of the signal intensity favors a ground or low-lying excited state.

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646_23249.

EXPRESSION OF Na⁺/H⁺ ANTIPORTER FROM VIBRIO ALGINOLYTICUS IN A FRESH WATER CYANOBACTERIUM SYNECHOCOCCUS SP. PCC 7942

Kaku N., Okuda K., Hibino T., Tanaka Y., Nakamura T., TakabeTet., TakabeTer.

Research Institute, Meijo University
Faculty of Science and Technology, Meijo University
Faculty of Pharmacy, Chiba University
BioScience Center, Nagoya University

Keywords:

environmental stress, salt stress, salt tolerance

Na⁺/H⁺ antiporter from Vibrio alginolyticus was expressed in a fresh water cyanobacterium Synechococcus sp. PCC 7942 by using a shuttle plasmid. The expression was confirmed by Northern analysis and quenching of the fluorescence upon the addition of NaCl. Effects of expression of the Na⁺/H⁺ antiporter on the growth rate, photosynthetic activity, ion contents, and ATP levels were examined, and its role for the salt tolerance was discussed.

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650_2174.

MOLECULAR CHAPERON DnaK FROM A HALOTOLERANT CYANOBACTERIUM APHANOTHECE HALOPHYTICA

Hibino T., Sugino M., Tsujimura K., Nii N., Ishikawa H., Yoshikawa H., TakabeTer.

Research Institute, Meijo University
Faculty of Science and Technology, Meijo University
Faculty of Agriculture, Meijo University
Department of Bio-science, Tokyo University of Agriculture

Keywords:

chaperone proteins, environmental stress, salt stress, salt tolerance

A genomic locus encoding a distinct member of the DnaK/Hsp70 family of molecular chaperones, dnaK, was cloned from the halotolerant cyanobacterium A. halophytica which can grow at high external salinities up to 2M NaCl. Co-expression of dnaK with a plant plastocyanin precursor in E. coli resulted in a dramatic increase in the solubility of the plant protein. Refolding activitties of LDH and plastocyanin were examined by using the DnaK proteins from A. halophytica and a flesh water cyanobacterium Synechococcus sp. PCC 7942.

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651_28039.

cDNA CLONING AND TISSUE SPECIFIC EXPRESSION OF MAIZE SUCROSE TRANSPORTER

Aoki N.¹, Hirose T.², Ohsugi R.<sup>3

1</sup>Japan International Research Center for Agricultural Sciences, Tsukuba 305-8686, Japan
²Hokuriku National Agricultural Experiment Station
³National Institute of Agrobiological Resources

Keywords:

C4, cDNA cloning, sucrose transport

Metabolism and transport of photoassimilate in C4 plants have still been disputable. To elucidate sucrose transport mechanism in a C4 plant, maize, we isolated a cDNA clone of sucrose transporter from maize leaves. The cDNA clone (ZmSUT1) encoded an open reading frame of 1,566 bp (521 amino acids) and showed 82% identity at the amino acid level to rice sucrose transporter (T. Hirose et al. (1997) Plant Cell Physiol. 38: 1389). ZmSUT1 was expressed in green leaf and germinating seed whereas little or no expression was observed in root and etiolated shoot, suggesting that the sucrose transporter would play a role in phloem loading of sucrose in source organs.

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653_7315.

PHOTOSYSTEM II CARBONIC ANHYDRASE ACTIVITY DEPENDS ON CHLORIDE AND CALCIUM

Stemler A.J.

Section of Plant Biology
University of California-Davis
Davis, CA, 95616 USA

Keywords:

anions, bicarbonate, carbonic anhydrase, manganese cluster, O2 evolution

Photosystem II carbonic anhydrase activity and oxygen evolution in various chloroplast membrane preparations were compared as a function of added chloride and calcium concentrations. In thylakoids, both carbonic anhydrase activity and oxygen evolution showed an optimum of 10 mM or less for chloride. In photosystem II-enriched membranes that were salt-washed to remove the 24 and 17 kDa extrinsic proteins, the optimum chloride concentration for both activities shif activities shifted to 30 mM. In photosystem II membranes washed with calcium chloride to remove all three extrinsic proteins, the optimum chloride concentration for both oxygen evolution and carbonic anhydrase activity shifted further, to over 400 mM. In calcium-depleted photosystem II membranes, stimulation of both carbonic anhydrase activity and oxygen evolution by increasing calcium concentration appeared biphasic. Both activities showed a low concentration response (0.01-1 mM) and another increase in the range 2-5 mM. The parallel response of carbonic anhydrase activity and oxygen evolution to chloride and calcium implies that the carbonic anhydrase activity is closely associated with the oxygen-evolving mechanism. A possible explanation is that the carbonic anhydrase supplies bicarbonate required for photosystem II activity on the donor side (Klimov et al., FEBS Lett. 363: 251-255, 1995).

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654_13068.

CROSS-RECONSTITUTION OF FOUR EXTRINSIC PROTEINS FROM A RED ALGA WITH HIGHER PLANT AND CYANOBACTERIAL PSII

Enami I., Yoshihara S., Ohta H., Shen J.-R.

Dept. Biol., Fac. Sci., Sci. Univ. of Tokyo, Tokyo 162-8601
RIKEN Harima Inst., Hyogo 679-5143, Japan

Keywords:

O2 evolution, OEC proteins

Photosystem II (PSII) from a red alga, Cyanidium caldarium, contains four extrinsic proteins of 33, 20, 12 kDa and cyt c-550 [Enami et al. (1995) B.B.A. 1232, 208-216]. Among these four components, the 33 kDa protein is commonly found in PSII from cyanobacteria to higher plants and can bind to PSII completely by itself, whereas cyt c-550 and the 12 kDa protein are found only in cyanobacteria, red algae, and perhaps diatoms but not in higher plant PSII, and their binding in the red algal PSII requires other extrinsic proteins. Furthermore, the 20 kDa protein is unique to the red algal PSII and absent in either cyanobacterial or higher plant PSII. This protein does not seem to function directly in oxygen evolution but is required for the maximal binding of cyt c-550 and the 12 kDa protein to sustain the optimal activity of oxygen evolution [Enami et al. (1998) Biochemistry 37, 2787-2793]. In order to compare the binding and functional properties of the red algal extrinsic proteins with those of the higher plant or cyanobactrial extrinsic proteins, we performed cross-reconstitution experiments with extrinsic proteins from the red alga and PSII from higher plant or cyanobacterium, or extrinsic proteins from higher plant or cyanobacterium and PSII from the red alga. Our results suggested that the red algal extrinsic proteins play an important role during evolution of oxygen-evolving system.

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655_7700.

EFFECTS OF A MAIZE SPS GENE EXPRESSED IN RICE ON ITS ACTIVITY AND CARBON PARTITIONING

Ono K., Furukawa K.^*, Murata T.^*, Ishimaru K., Ozawa K., Ohkawa Y., Ohsugi R.

Natl. Inst. Agrobiol. Resources, Tsukuba, Ibaraki, 305-8602, Japan,
^*Fac. Agric. Iwate Univ., Morioka, Iwate, 020-0066, Japan

Keywords:

C partitioning, carbohydrate, enzymes, higher plants

Transgenic rice (Nipponbare) plants expressing a maize sucrose-phosphate synthase (SPS) gene have been produced. Transformants were identified using PCR or northern hybridization. We obtained transformants with various amount of maize SPS protein and those with very small amount of rice SPS protein. There was a positive correlation between the amount of maize SPS protein and the SPS activity. We also found a negative correlation between SPS activity and the starch content of the flag leaves.

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659_12441.

CHLOROPHYLL FLUORESCENCE INDUCTION IN ANNUAL AND PERENNIAL LEAVES

Force L.E., Critchley C., van Rensen J.

Department of Botany
The University of Queensland, Qld. 4072
Australia

Keywords:

Chl fluorescence induction, fluorescence, photosystem 2, plastoquinone pool

Annual and perennial species differ in rates of photosynthesis, respiration-transpiration and growth. The efficiency of assimilate conversion into new growth is greater in the annual plants than in the perennials, with the highest proportion of the biomass being allocated to the annual's leaves while it can be directed to the roots in perennials. The photosynthetic capacities are higher and the growth rates faster in annual species compared to perennial species.
We have conducted experiments to investigate differences in the O-J-I-P chlorophyll a fluorescence transient between annual and perennial species. The results show that annual and perennial species differ in their reduction of the plastoquinone pool represented by the J to I fluorescence rise of the Kautsky curve. This area of the curve was significantly correlated with the variable fluorescence and Fv/Fm ratio. We suggest that the photosynthetic efficiency of Photosystem II as determined by the flow of electrons out of the photosystem and into the plastoquinone pool is higher in annuals.

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661_16362.

CHARACTERIZATION OF CHLAMYDOMONAS TRANSFORMANTS WITH IMPAIRED PROCESSING OF A PRECURSOR D1 AND OXYGEN-EVOLVING ACTIVITY OF PHOTOSYSTEM II

Hatano A.¹, Minagawa J.², Inoue Y.², Takahashi Y.<sup>1

1</sup>Department of Biology, Faculty of Science, Okayama University, Tsushima-naka, Okayama 700-8530, Japan;
²The Institute of Physical and Chemical Research, Saitama, Japan

Keywords:

algae, chloroplast transformation, D1 protein, photosystem 2

The D1 of PSII is synthesized as a precursor form (pD1) and then processed between Ala-344 and Ser-345 in C. reinhardtii. We have generated chloroplast transformants of Chlamydomonas in which Ala-344 or Ser-345 of the pD1 was substituted by Pro (A344P or S345P) to elucidate relationship between the processing of the pD1 and the assembly of O₂-evolving complex. In contrast to the S345P mutant of Synechocystis 6803 that is completely deficient in the processing (1), the corresponding Chlamydomonas mutant showed the processing albeit at considerably reduced rate and the concomitant assembly of functional O₂-evolving complex. However, Chlamydomonas A344P mutant was completely deficient in the processing and O₂-evolution. These results confirmed that the processing of pD1 is prerequisite to the assembly of functional Mn cluster and suggested that Ala-344 is more important for the processing than Ser-345.
(1) Nixon et al., Biochemistry (1992) 31, 10859-10871

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663_17344.

ANTIOXIDANT ACTIVITY OF CHLOROPHYLLS

Shioi Y., Hoshina C., Tomita K.

Dept. of Biol. & Geosci., Fac. of Sci., Shizuoka Univ. 836 Ohya, Shizuoka 422-8529, Japan

Keywords:

Chl biosynthesis, Chl degradation, chloroplast development, enzymes

Chlorophylls (Chls) and their derivatives protect linolenic acid from peroxyl radical induced oxidation in vitro. Chls in the chloroplast membrane is bleached at the same rate as peroxyl radical formed. Bleaching of Chl is also observed in the xanthine-xanthine oxidase system which produces superoxide radical. These results show that Chls are bleached by the nonenzymatic reaction with hydroxyl or superoxide radical. The data also indicate that Chls can function as a physiological antioxidant in chloroplast membrane.

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664_23590.

STRUCTURAL MODEL FOR THE Mn CENTRE WITHIN PHOTOSYSTEM II

ÅhrlingK.A., Smith P.J., Razeghifard M.R., Pace R.J.

Department of Chemistry
Faculties
The Australian National University
Canberra 0200, ACT
Australia

Keywords:

EPR, manganese cluster, O2 evolution, photosystem 2

A structural model for the Mn organisation in the oxygen evolving complex (OEC) of photosystem II is described, based on a unified interpretation of the nature and behaviour of the Mn-related EPR signals from the functional, uninhibited enzyme. There are presently at least seven such signals, which are well characteristed. The model assumes that the four Mn of the OEC are arranged as two exchange-coupled pairs, which do not magnetically interact, but are sufficiently close (<10Ĺ) for rapid electron transfer to occur between them. One pair (responsible for the multiline and related EPR signals) undergoes the S-state cycling, while the other is associated with Y_z and performs an electron transfer function. The probable oxidation states of the various components of the system during the catalytic cycle are discussed, as well as the structural assignments in terms of the known EXAFS features of the OEC. The likely water oxidation mechanism, consistent with recent fast kinetic EPR measurements of O₂ release by ourselves and electrochromic studies by others will be presented.

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668_24373.

THE S₀ STATE EPR SIGNAL FROM THE Mn CLUSTER ARISES FROM AN ISOLATED GROUND STATE

ĹhrlingK.A., Peterson S., Styring S.

Department of Biochemistry
Center for Chemistry and Chemical Engineering
Lund University
PO Box 124
S-221 00 LUND, Sweden

Keywords:

EPR, manganese cluster, O2 evolution, photosystem 2, S-states

During photosynthesis, the Mn-containing oxygen evolving complex in photosystem II cycles through five oxidation states, S₀-S₄. Two of these, S₀ and S₂ are paramagnetic and give rise to EPR signals. In S₂, the wide, complex signal called the multiline signal (centered around g=2) and the broad featureless signal around g=4 have been the focus of much study over the years. Recently the S₀ state signal from the Mn cluster was discovered [Ĺhrling, et al., (1997) Biochemistry 36, 13148-13152; Messinger et al., (1997) J. Am. Chem. Soc. 119, 11349-11350]. It is an even wider, structured signal, centered around g=2 and appears to span 2500G. Here we present a study of the temperature dependence of this signal and compare it with the temperature dependence of the S₂ state multiline signal. Both signals are found to arise from S=1/2 ground states. In the case of the S₀ state signal, this is an isolated ground state with a gap to the next excited state of at least 30 cm^-1. Consequently, we have not observed any signals from thermally populated states in S₀ (similar to the g=4 state in S₂).

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674_26322.

IN VITRO TRANSCRIPTION ANALYSIS OF THE RBCS GENE

Yukawa Y., Sugita M., Sugiura M.

Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan

Keywords:

chloroplast genes, gene regulation, higher plants, RNA polymerase, Rubisco

We previously developed an RNA polymerase II-dependent in vitro transcription system from nuclei of non-photosynthetic tobacco culture cells (BY-2). We refined all steps of preparation procedures of the nuclear extract, so that the transcriptional activity increased about ten-fold. The transcription of tomato and tobacco rbcS genes were assayed using this imprroved system. Transcription of rbcS was detected using its "naked" templates (circular recombinant plasmid DNAs). Consequently, the regulation of rbsS transcription is suggested to occur at a higher-order configuration of DNA, or at a nucleosome level. Next, we analyzed basal promoter elements of the rbcS gene. Progressive deletion of the 5' flankiung sequence was made from the tomato rbcS gene followed by in vitro assay showed that the 55 bp upstream sequence (including the TATA element) was enough for basal rbcS transcription.

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676_26761.

A NOVEL GENE, PMGA, SPECIFICALLY REGULATES PHOTOSYSTEM STOICHIOMETRY IN A CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803 IN RESPONSE TO HIGH LIGHT.

Hihara Y., Sonoike K., Ikeuchi M.

Dept. Life. Sci. (Biology), Univ. Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan

Keywords:

cyanobacteria, light acclimation, light stress, mutants, photosystem 1

We have obtained results implying that a novel gene pmgA is involved in the regulation of the PSII/PSI ratio, which is the well-known acclimation response to high light in cyanobacteria. Chlorophyll fluorescence spectra of cells at 77K suggested that the ratio of PSII/PSI increased in wild type upon high light illumination, whereas it remained almost constant in pmgA mutants. Consistently, direct measurement of photosystem content revealed that pmgA regulates photosystem stoichiometry by selective suppression of PSI accumulation. Single and mixed culture experiments of wild type and pmgA mutants in high light revealed that the mutants could not tolerate prolonged stress of high light. Surprisingly, the cellular PSI activity of the mutants increased drastically in the course of suppression of growth in high light. We analyzed gene expression of pmgA by RT-PCR and found that pmgA transcript increased greatly after the shift to high light. All these data suggest that regulation of photosystem stoichiometry mediated by pmgA is induced upon the shift to high light in order to avoid the high light stress.

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678_11532.

ISOLATION OF A BARLEY GENE ENCODING BETAINE ALDEHYDE DEHYDROGENASE THAT IS NOT LOCALIZED IN MICROBODY

Nakamura T., Nomura M., Ku M.S.B., Takabe T.

Bioscience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan (Nakamura & Takabe)
Faculty of Agriculture, Kagawa University, Miki, Kita, Kagawa 761-0795, Japan (Nomura)
Botany Department, Washington State University, Pullman WA 99164, USA (Ku)

Keywords:

adaptation, osmoregulators, salt stress

Many halophytes are known as glycinebetaine accumulators. Glycinebetaine is a compatible solute and acts as an excellent osmoprotectant. In higher plants, betaine aldehyde dehydrogenase (BADH) catalyzes the last step of glycinebetaine synthesis. BADH was well characterized in various plants. Existence of BADH isozyme was suggested in spinach and sorghum. By Western blot analysis, we also detected BADH like isozyme in barley. We screened barley cDNA library and obtained an isozyme gene. From analysis of deduced amino acid sequence, it is likely that intracellular localization of the isozyme is different from a barley microbody-type BADH cloned previously [Ishitani M., Nakamura T. et al., (1995) Plant Mol. Biol. 27, 307; Nakamura T. et al., (1997) Plant J. 11, 1115]. We performed Northern blot analysis using total RNA extracted from barley leaves under various stresses. We found that the expression pattern differed in both BADH genes.

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685_9110.

TRANSLATIONAL CONTROL OF PHOTOSYNTHETIC GENES IN TOBACCO PLASTIDS

Sugiura M., Kusumegi T., Sugishita H., Murakami K., Ideue T., Hirose T.

Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan

Keywords:

chloroplast genes, gene expression, post-transcription, RNA editing, RNA processing

Translation is one of the key control steps of photosynthetic gene expression in plastids. To analyze the molecular process and control of translation in plastids, we have developed an in vitro translation system from tobacco green chloroplasts (1). All mRNA templates used were made by T3 RNA polymerase on relevant tobacco plastid genes fused with the lacZ gene towards 3 sides.
The 5UTR of rbcL mRNA contains a Shine-Dalgarno (SD, typically GGAGG) sequence at a proper position (~10 nt upstream from the start codon). In vitro assay showed that the SD is essential for its translation. However, one-third of tobacco protein-coding genes, such as psbA and atpB, contain no SD-like sequences at proper positions. Our in vitro analysis revealed that three novel cis-elements are required for translation of psbA mRNA (1) and a U-rich sequence is critical for that of atpB. Both mRNAs were suggested to require specific trans-factors for their translation. Therefore, tobacco plastids have multiple mechanisms for translational initiation.
The psaC and ndhD genes are co-transcribed but the resultant di-cistronic mRNA was found to be inactive. Inter-cistronic cleavage was found to be required for translation of both cistrons. Furthermore, translation of the ndhD mRNA could only be started with creating the AUG start codon from ACG by RNA editing whose extent depends on developmental and enviromental conditions (2). Hence, RNA editing is an additional control step for the expression of ndhD.
(1) T. Hirose and M. Sugiura, EMBO J. 15: 1687 (1996)
(2) T. Hirose and M. Sugiura, EMBO J. 16: 6804 (1997)

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686_21422.

CAPACITY FOR 5-AMINOLEVULINIC ACID SYNTHESIS IN BLACK PINE SEEDLINGS

Drazic G.D., Bogdanovic M.T., Mihailovic N.L.J.

Belgrade University Institute for the Application of Nuclear Energy Department for Plant Physiology Banatska 31b 11080 Zemun, Belgrade Yugoslavia

Keywords:

Chl biosynthesis, cytokinins, light regulation, regulatory processes, gymnosperms

Chlorophyll synthesis is initially regulated at the level of 5-aminolevulinic acid (ALA) synthesis which is strongly stimulated by light and repressed in etiolated tissues of angiosperms. Gymnosperms are capable of synthesising chlorophyll when seeds are germinated in the dark. ALA-synthetic activity has been monitored in black pine seedlings during germination in the light and in the dark in order to show whether ALA-formation rate is the limiting step within chlorophyll biosynthesis of gymnosperm seedlings. ALA-synthetic activity is increased during the germination (from 1.28 mmoll/gFW on the 3rd day of germination to 6.13 on the 10th day in the light; from 1.64 to 5.74 mmoll/gFW in the dark). ALA-synthetic activity is about three times higher in the cotyledons than in the root. Cytokinin (10-5 M benzyl adenine), that induces a 20% increase of chlorophyll accumulation, decreases ALA-synthetic activity three times, both in the dark and in the light.

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691_9551.

STRUCTURAL CHANGES UPON BINDING OF PHOSPHATIDYLGLYCEROL TO LIGHT-HARVESTING COMPLEX II OF HIGHER PLANTS, IDENTITY OF LARGE-SCALE ORGANISED STRUCTURES IN LIPID-PROTEIN COMPLEXES

^1,2Veverka V., ²Motejl M., ^1,2Hrabal R., ^1,2Stys D.

Laboratory of Biomembranes, Faculty of Biological Sciences, University of South Bohemia, Braniovská 31, 370 05 České Budějovice, Czech Republic, tel. +420 38 777 5542, fax. +420 38 45985, e-mail: stys@entu.cas.cz
¹NMR laboratory, Institute of Chemical Technology, Technická 5, CZ 166 28 Praha 6, Czech Republic, tel. +420 2 2435 3805, fax +420 2 311 6109
²Department of Autotrophic Microorganisms, Institute of Microbiology, CAS, CZ 379 81 Trebon - Opatovicky Mlyn, Czech Republic.

Keywords:

2D crystals, complex formation, light harvesting complexes, lipid-protein interactions

The peptide fragment of the light harvesting complex II comprising the trimerisation site binds strongly and selectively to phosphatidylglycerol and undetectably to the other LHCII-bound lipid digalactosysldiacylglycerol. Detailed analysis of the interactions of the N-terminal domain of the protein with phosphatidylglycerol by NMR spectroscopy confirms the identity of the trimerisation region with the phosphatidylgycerol binding site. The CD spectroscopy is dominated by unusual signal of the lipid-peptide complex, which is identical to that observed in trimers of LHCII. The LHCII trimers exhibit tendency to form larger organised structures whose minimal unit are hexamers of basic trimeric subunit. The comparison with measurements in intact thylakoid membranes indicates that the partly and loosely organised arrays or supercomplexes of LHCII exist in thylakoids in ion-free solutions or at elevated temperatures.

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696_24151.

TWO MEMBRANE-BOUND C-TYPE CYTOCHROMES COUPLE QUINOL OXIDOREDUCTASE TO THE P840 REACTION CENTER IN CHLOROBIUM

Oh-oka H., Iwaki M., Itoh S.

Dept. Biol., Grad. Sch. Sci., Osaka Univ., Toyonaka 5600043, Natl. Inst. Basic Biol. Okazaki 4448585, Japan

Keywords:

bacterial reaction center, cytochrome complexes, electron transfer, green sulfur bacteria, Q-cycle

Photosynthetic reaction center (RC) of green sulfur bacteria seems to share a common ancestor with the photosystem I RCs of plants and cyanobacteria. We report the mediation of eletron transfer between the quinol oxidoreductase and P840 RC complex in Chlorobium by the two distinct membrane-bound c-type cytochromes, which could be discriminated by the heme-staining on the gel and low-temperature redox difference spectra. We measured the electron transfer activity between both complexes in the chlorosome-depleted membranes isolated from C. tepidum. Rapid oxidations of two c-type cytochromes were detected after the flash excitation. Their re-reductions occurred in parallel with the reduction of cytochrome b in the presence of antimycin A, while the reductions of both cytochromes c and b were suppressed by the addition of stigmatellin. These results suggest the operation of the typical bc complex and its tight coupling to P840 RC with the membrane-bound cytochromes c.

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704_19835.

INVESTIGATION OF THE PLASTOQUINONE POOL SIZE AND FLUORESCENCE QUENCHING IN PHOTOSYSTEM II (PS II) MEMBRANE FRAGMENTS

Kurreck J., Reifarth F., Renger G.

Max-Volmer-Institute for biophysical chemistry and biochemistry, Technical University Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany

Keywords:

fluorescence, photosystem 2, plastoquinone pool

The plastoquinone pool size of PS II membrane fragments is diminished compared to that of intact thylakoids. A recently developed method [Kurreck et al., (1995) Biochemistry 34, 15721-15731] which comprises purified Plastoquinone-9 and a special sonication procedure enabled us to prepare PS II membrane fragments with different pool sizes. Even acceptor capacities exceeding those of the natural pool consisting of 5 to 7 plastoquinone molecules per PS II were achieved. The reconstituted samples were used to analyse the non photochemical fluorescence quenching of the oxidized plastoquinone pool: By measuring fluorescence induction curves of DCMU-inhibited samples, a linear Stern-Volmer-type relationship between fluorescence quenching and pool size was obtained. In PS II membrane fragments the quenching effect of the pool is much stronger than in thylakoids. Measurements of the flash-induced changes of the fluorescence quantum yield led to similar results. The underlying mechanisms ofpool quenching will be discussed.

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708_11026.

EXPRESSION OF CHROMATIUM VINOSUM RUBISCO IN THE CARBOXYSOME DEFICIENT MUTANT OF CYANOBACTERIUM SYNECHOCOCCUS PCC 7942

Kojima K., Haranoh K., ^*Kobayashi K., Iwaki T., Wadano A.

Department of Applied Biochemistry, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
^*Laboratory of Plant Cell Technology, University of Shizuoka, 52-1 Yada, Shizuoka, 422, Japan

Keywords:

CO2 concentrating mechanisms, cyanobacteria, Rubisco

RuBisCO of Chromatium vinosum was expressed in cyanobacterium Synechococcus PCC 7942 by the transformation with the bidirectional expressing vector. In order to search strong promoters, we examined lac promoter, trc promoter, Chromatium vinosum RuBisCO promoter, Synechococcus PCC 7942 psbAI and bc promoter, Synechosystis PCC 6803 psbAII and rpoD1 promoter by luciferase reporter system. The results of luciferase assay showed that the activity of Synechococcus PCC 7942 psbAI promoter was more than 10 times as strong as that of lac promoter. Synechococcus PCC 7942 psbAI promoter was used for expressing RuBisCO protein of Chromatium vinosum in Synechococcus PCC 7942, and the promoter made a high activity of Chromatium vinosum RuBisCO in the cyanobacterium, as confirmed with the Western blotting and measurement of RuBisCO activity. The stability of the shuttle vector in Synechococcus PCC 7942 was checked by the plasmid rescue method. Southern analysis showed that the vector was not incoporated into the cyanobacterial genome.

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711_32235.

INORGANIC-N SIGNAL TRANSDUCTION FOR EXPRESSION OF MAIZE C4PPC1

Sugiyama T., Sakakibara T., Taniguchi T.

Dept. Applied Biol. Sci., Sch. Agric. Sci., Nagoya Univ., Nagoya 464-8601, JAPAN

Keywords:

cytokinins, gene regulation, nutrients, PEPC

Inorganic N-sources function as substrates for assimilation as well as signals for gene expression, requiring cellular, intercellular, and organ-to-organ communication. We recently identified an early responsive gene to cytokinin, ZmCip1, which encodes a response regulator homolog in maize. Transcription of ZmCip1 is induced by cytokinins in the detached leaves of N-starved plants, whereas its transcription is induced in leaves by inorganic N-sources in the whole plants. Active cytokinins for the induction of the gene accumulate in roots in response to external N-source. Induction of ZmCip1 by the signals precedes that of C4Ppc1 which is a possible targeting gene of ZmCip1. Based on the results we discuss cytokinins as a root-to-leaf signal for external inorganic N-availability for the induction of C4Ppc1 and its signal transduction mediated by ZmCip1.

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712_4566.

LIGHT-DEPENDENT FRAGMENTATION OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE IN CHLOROPLASTS ISOLATED FROM WHEAT LEAVES

Ishida H., Shimizu S., Makino A., Mae T.

Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi Aoba-ku, Sendai 981-8555, Japan

Keywords:

active oxygen, degradation pathways, photooxidation, Rubisco

The large subunit (LSU) of Rubisco is directly degraded into an N-terminal side fragment of 37 kDa and a C-terminal side fragment of 16 kDa by active oxygen, probably hydoxyl radical, generated in the lysates of chloroplasts in light (H. Ishida et al., 1997, Plant Cell Physiol. 38&#iol. 38: 471-479). In the present study, we demonstrate that this fragmentation of the LSU also occurs in the same manner in intact chloroplasts. The fragmentation of the LSU was observed when intact chloroplasts from wheat leaves were incubated at 4^oC under illumination in the presence of KCN or NaN₃, which is a potent inhibitor of active oxygen-scavenging enzyme(s). The properties, such as molecular masses and cross-reactivities against the site-specific anti-LSU antibodies, of the fragments found in the chloroplasts were the same as those found in the lysates. The fragmentation was completely inhibited by DCMU, and only partially inhibited by methyl viologen in the lyates. The addition of hydrogen peroxide to the lysates stimulated LSU fragmentation in light, but did not induce any fragmentation in darkness. Thus, we conclude that both production of hydrogen peroxide and generation of the reducing power at thylakoid membranes in light are essential requirements for fragmentation of the LSU.

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713_11632.

THE EFFECT OF PHOTOSYSTEM II INHIBITORS DCMU AND BNT ON THE HIGH-LIGHT INDUCED D1 TURNOVER IN TWO CYANOBACTERIAL STRAINS

Komenda J., Masojídek J.

Laboratory of Photosynthesis, Institute of Microbiology, Opatovický mlýn, 37981 Tøeboò, Czech Republic

Keywords:

cyanobacteria, D1 turnover, herbicides, photoinhibition, photosystem 2

The high-light induced D1 protein turnover was studied in whole cells of two cyanobacterial strains Synechocystis PCC 6803 and Synechococcus PCC 7942 in the presence of PSII inhibitors DCMU and BNT. In Synechocystis, the D1 turnover and PSII photoinactivation were slowed down to a similar extent in the presence of either inhibitor as compared with control cells. The ongoing D1 synthesis in the presence of inhibitors was confirmed by both the stable PSII activity and the constant steady-state level of D1. In Synechococcus cells, both DCMU and BNT blocked the turnover of the "low-light" D1:1 form but did not prevent the exchange of the "high-light" form D1:2 for D1:1. Similar effect of both herbicides on the D1 exchange was in contrast with their influence on the rate of PSII photodamage. DCMU exhibited the pronounced protective effect, while BNT significantly accelerated PSII photoinactivation. Possible reasons for the differences between the strains are discussed.

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714_17116.

THE STRUCTURE OF THE BILIPROTEIN AGGREGATES OF THE Chl d-CONTAINING PROKARYOTE ACARYOCHLORIS MARINA

Marquardt J.¹, Hu Q.², Miyashita H.², Mörschel E.¹, Miyachi S.<sup>2

1</sup>Department of Biology/Botany, Philipps University, Karl-von-Frisch-Str., 35032 Marburg, Germany
²Marine Biotechnology Institute, Kamaishi Laboratories, Heita, Kamaishi City, Iwate 026, Japan

Keywords:

antennae, cyanobacteria, phycobiliproteins, procaryote, prochlorophytes

Acaryochloris marina, a photosynthetic prokaryote containing predominantly Chl d, contains phycocyanin (PC), allophycocyanin (AP) and traces of phycoerythrin (PE) as additional antenna pigments. In isolated biliprotein aggregates several biliprotein subunits with apparent molecular masses between 16 and 20 kDa were found. They cross-reacted with antibodies against PC and/or AP from a red alga. The biliproteins are not organized as hemidiscoidal or hemiellipsoidal phycobilisomes as in most cyanobacteria, but as rod-shaped structures of 26.0 x 11.3 nm. Under low ionic strength conditions these aggregates dissociate into smaller particles, confirmed to represent trimeric (a/b)x3 and hexameric (a/b)x6 biliprotein units. The entire isolated aggregates obviously consist of 4 hexamers. Some hexamers contain PC alone, others in combination with AP. The organization of PE is still unclear.

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716_18748.

ISOLATION OF SALT-INDUCED cDNA CLONES IN BARLEY LEAVES USING DIFFERENTIAL DISPLAY

Muramoto Y., Nakamura T., Nakase A.A., Takabe T.

Bioscience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan

Keywords:

higher plants, salt stress

When undergo salt stress, higher plants respond at molecular level. It is assumed that salt-induced genes are correlated with stress tolerance. By investigating salt-induced genes, it can be clarified how plants respond and adapt to high-salinity condition, and we will be able to develop the genetic engineering for crop improvement. We have used differential display method to identify up-regulated genes in barley leaves under salt stress. The technique is a highly sensitive system to detect the differentially expressed genes. We isolated several salt-induced genes such as yeast Ssf1,2 gene homologue which may play a role in mating signaling pathway in yeast. We discuss about roles of these gene products under salt stress.

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722_31177.

INTERACTION OF PHOTOSYNTHETIC PIGMENTS WITH VARIOUS ORGANIC SOLVENTS AS REVEALED FROM MAGNETIC CIRCULAR DICHROISM MEASUREMENTS

Umetsu M., Wang Z.-Y., Kobayashi M., Nozawa T.

Department of Biomolecular Engineering,
Graduate School of Engineering,
Tohoku University,
Aobayama 07 Aoba-ku,
Sendai 980-8579,
Japan

Keywords:

Bchl, circular dichroism, pigment binding, pigments

The central metal atom of photosynthetic pigments in vivo and in vitro is coordinated by ligands. The resonance Raman spectra in the 1620-1510 cm^-1 region are well used to distinguish between the five- and six-coordinate species of the metal atom in pigments. In this study, magnetic circular dichroism spectra were measured on Mg chlorin derivatives in various hydrophilic organic solvents. The MCD intensity of Q_y(0-0) transition was influenced dramatically by the coordination state of the central Mg atom. When the five-coordinate species changed to six-coordinate species, the MCD intensity of Q_y(0-0) transition became stronger, and the difference in energies between Q_y(0-0) transition and the second longest wavelength transition [Q_x(0-0) transition] decreased. Moreover, this change also depended on the interaction strength of the ligands to the Mg atom. We report that the correlation between the MCD intensity of Q_y(0-0) transition and the energy difference can be used as a new measure for characterizing the coordination number of the Mg atom and interaction strength of the Mg atom with ligand.

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736_14175.

PRIMARY CHARGE SEPARATION AT LOW TEMPERATURES IN D1-D2 REACTION CENTERS, STUDIED BY PHOTON ECHO AND PUMP-PROBE SPECTROSCOPY

Prokhorenko V.I., Holzwarth A.R.

Max-Planck-Institut fuer Strahlenchemie
Stiftstrasse 34-36
D-45470, Muelheim a.d.Ruhr
Germany

Keywords:

charge separation, femtosecond spectroscopy, photon echo, photosystem 2, reaction centers

We report experimental and theoretical photon echo and pump-probe studies of the primary charge separation in photosystem II reaction centers (RC) at 1.33K. Experiments were carried out at low excitation intensities with spectral and time resolution of 1 nm and 500 fs, respectively. By comparison of measured and theoretically calculated photon echokinetics we conclude that the accessory chlorophyll in the active branch of the RC core is the primary electron donor. The charge separation occurs with a time constant of »1.5 ps, in good agreement with previously published data. Our conditions are based on the structural model of Svensson et al. (1). Comparison of pump-probe transient absorption kinetics with the calculated data the primary electron acceptor in the RC core was also determined.
1. Svensson B., Etchebest C., Tuffery P., Kan P., Smith J. and Styring S. Biochemistry, 1996, 35, 14486.

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738_18246.

GENOME ANALYSIS OF A CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803

Tabata S.

Kazusa DNA Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan

Keywords:

cyanobacteria, gene expression, mutants, Genome

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 is completely sequenced, and information on the structures of the 3,168 potential protein-coding genes is available. Having such information in hand, it is now possible to take new approaches for investigation of gene function and gene regulation on a genome-wide level. High-density DNA array covering the entire cyanobacterial genome has been developed. Hybridization experiments using probes representing the total mRNA species in the cells under different growth conditions or with different mutations would be extremely useful in monitoring gene expression and in studying a regulation network. A systematic gene disruption project is in progress by a consortium of Japanese laboratories, and a new web database, CyanoMutants [http://www.kazusa.or.jp/cyano/mutants/], was created to post mutant information and to facilitate communication among the community. These new approaches would accelerate understanding of photosynthetic processes in cyanobacteria.

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740_12089.

ISOLATION OF A HIGHLY ACTIVE PSII-LHCII SUPERCOMPLEX BY A DIRECT METHOD

Eshaghi S.^1,2, Andersson B.¹, Barber J.<sup>2

1</sup>Department of Bioichemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden
²Department of Biochemistry, Imperial College of Sceince, Technology and Medicine, 2AY SW7, London, U.K.

Keywords:

OEC proteins, photosystem 2, oxygen evolution

PSII is a multi-subunit protein complex, which contains at least 25 polypeptides, including integral membrane proteins, soluble proteins as well as light harvesting Chl a/b binding proteins. To isolate this protein complex, in an intact form, has been a difficult problem. A series of PSII isolation procedures have been applied, which involve several purification steps. However, all these steps originate from PSII enriched membranes, so called BBYs, which already have been treated with detergent. The result of these isolation steps is loss of functional intactness, usually by releasing the 23 kDa and especially the 16 kDa extrinsic proteins of the oxygen evolving complex (OEC). In this work, we have succeeded to isolate a highly pure PSII-LHCII supercomplex directly from thylakoid membranes, with a very gentle treatment. This complex contains all the proteins of OEC and the intactness of both the donor side and acceptor side have resulted in the very high oxygen evolution activity of about 1.8 mmol O₂/mgChl·h.

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741_3999.

THE FORMYLAMINO GROUP OF ANTIMYCIN IS ESSENTIAL IN SPECIFIC INHIBITION OF CYCLIC ELECTRON TRANSPORT

Teicher H.B., Motawia M.S., Scheller H.V., Møller B.L.

Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Denmark.

Keywords:

electron transport, inhibitors, antimycin, cyclicelectrontransport

Antimycin inhibits ferredoxin-dependent cyclic electron transport in chloroplasts. While this has been well established for many years, the protein that binds antimycin has never been identified. We have investigated the interaction between antimycin and antimycin-binding proteins in chloroplasts by using antimycin analogs. Nitro- and formylamino-analogs of antimycin were synthesized chemically, using octadecylamine as the lipophilic part of the molecule. Both native antimycin and the antimycin analogs were able to inhibit ferredoxin-dependent cyclic electron flow at concentrations in the range 10-40 µM, while only the nitro- analog inhibited linear electron flow, from H₂O to methyl viologen. These results indicate that altering the formylamino group at carbon 3 of the salicyl moiety of antimycin leads to a loss of binding specificity, while apparently having no effect on the ability of the molecule to inhibit ferredoxin- dependent cyclic electron flow.

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744_2461.

THE OXIDATION BEHAVIOR OF BACTERIOCHLOROPHYLL A IN PURPLE BACTERIAL REACTION CENTERS AND ANTENNA COMPLEXES

Kropacheva T.N., HoffA.J.,

Department of Chemistry
Udmurt State University
Izhevsk
Russia

Keywords:

bacterial reaction center, light harvesting complexes, optical properties, purple bacteria, redox potential, electrochemistry

The oxidative electrochemistry of bacteriochlorophyll (BChl) in the photosynthetic apparatus is expected to be different from that in organic solvents due to interactions with the protein matrix and/or with other pigment molecules. Thin-layer spectroelectrochemistry was used to study the oxidation of BChl in vivo in the presence of several redox mediators. For Rb. sphaeroides R-26 reaction centers reaction centers about one-half of the accessory BChl can be reversibly oxidized with the one-electron midpoint potential (E_m) of 780 mV (vs. NHE), that is much higher than that for the primary donor P860 (E_m=490 mV). Unlike for P860, reversibility of accessory BChl oxidation strongly depends on the applied potential and the reaction time. The LH1 complex of a RC-less mutant of Rb. sphaeroides contains at least two BChl components differing in their oxidation behavior. Full reversibility is observed for about one-third of the total B880 absorbance with E_m=600 mV (n=1). In LH2 complexes of Rps. acidophila both B800 and B850 components are subject to degradative oxidation at potentials above ~500 mV and ~600 mV, respectively.

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747_28694.

ATPASE AND PHOSPHATE EXCHANGE ACTIVITIES IN Mg-CHELATASE SUBUNITS OF CHLOROBIUM AND SYNECHOCYSTIS

Petersen B.L., ^*Kannangara G.C., Henningsen K.W.

Royal Veterinary and Agricultural University, 1871 Frederiksberg C,
^*Carlsberg Laboratory 2500 Valby, Denmark.

Keywords:

ATP, Chl biosynthesis, cyanobacteria, green sulfur bacteria, purple bacteria

The Mg-chelatase subunits D, H and I of Chlorobium vibrioforme and Synechocystis PCC6803 were over expressed in E. coli, purified and tested for ATPase and phosphate exchange activity. The obtained activities were compared with the activities of the corresponding subunits of Rhodobacter sphaeroides. The Synechocystis I and the C. vibrioforme H and I subunits hydrolysed ATP at the rates of 2.0, 1.8 and 0.16 nmoles ^. mg protein^{-1 .} min^-1. The ATPase activity of C. vibrioforme H was comparable to that of R. sphaeroides H. Synechocystis H failed to hydrolyse ATP. The Synechocystis and C. vibrioforme I catalysed a transfer of PO₄ from ATP to ADP at the rate of 1.7±0.2 nmoles ^. mg protein^{-1 .} min^-1. The exchange activities were correlated with a conserved sequence motif GXRGTGKSTXVRALA in the primary structure of the I subunits. Mg-chelatase activity was reconstituted by combining the three subunits of the same bacterium. Heterologous subunit combinations, however, resulted in low or no Mg-chelatase activity, probably due to the diversity in ATPase activity observed in the subunits.

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748_17190.

STATE TRANSITION IN SYNECHOCOCCUS PCC 7942. MOBILE ANTENNA OR SPILLOVER?

Koblizek M., Komenda J., Masojídek J.

Instute of Microbiology Laboratory of Photosynthesis 379 81 Trebon Czechia

Keywords:

cyanobacteria, fluorescence, light acclimation, quenching, statetransition

Cyanobacteria posses the effective mechanism of redistribution of excitation energy called state transition. The energy is redistributed from phycobilisomes (mobile antenna model) or among chlorophyll antennae (spillover model). To discriminate between these mechanisms, chlorophyll fluorescence of the cyanobacterium Synechococcus PCC 7942 was measured using the fluorometer PAM 101 with three different emitters. The 'blue' emitter (450 nm) excites chlorophylls, the 'orange' emitter (625 nm) phycocyanins and the 'red' emitter (655 nm) allophycocyanins and chlorophylls. The actual photochemical yield and non-photochemical quenching were determined during State 2 ® State 1 transition. Significant alternations of these parameters were observed with all the emitters but the most pronounced changes were seen under the 625 nm excitation. We propose the combined model of state transition with the major role of spillover.

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752_3928.

WATER STRESS EFFECTS ON VARIATIONS OF STEADY STATE CHLOROPHYLL FLUORESCENCE (FS) IN RESPONSE TO LIGHT

Flexas J., Escalona J.M., Medrano H.

Institut Mediterrani d'Estudis Avançats (IMEDEA)-Universitat de les Illes Balears (UIB).
Cra. de Valldemossa, Km. 7,5. 07071 Palma de Mallorca. Spain

Keywords:

drought stress, fluorescence, photochemistry, quenching

Steady-state chlorophyll fluorescence (Fs) is generally not used as single parameter but for calculation of other derived parameters. However when diurnal time courses of this parameter are followed, under irrigation and water stress, marked differences are observed. Non-stressed plants show a pattern, which approximately follows that of light intensity whereas, for water-stressed plants strong midday depression is observed, often achieving values below pre-dawn Fo.
Data on laboratory, greenhouse and field conditions in grapevine leaves are analysed to show that these patterns are present for all studied environmental conditions. Relationships between Fs and other chlorophyll fluorescence signals, carbon assimilation rate as well as water relations’ parameters are shown. The interest and significance of this parameter as putative indirect indicator of plant water stress is discussed.

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754_8122.

CONTINUOUS CHLOROPHYLL FLUORESCENCE AND GAS-EXCHANGE MEASUREMENT AS A NEW APPROACH TO STUDY WATER STRESS EFFECTS ON LEAF PHOTOSYNTHESIS

Flexas J., Briantais J.M., Medrano H., Moya I.

Institut Mediterrani d'Estudis Avançats (IMEDEA)-Universitat de les Illes Balears (UIB).
Cra. de Valldemossa, Km. 7,5. 07071 Palma de Mallorca. Spain

Keywords:

drought stress, fluorescence, gas exchange, photochemistry, quenching

Diurnal time courses of chlorophyll fluorescence were followed continuously and at distance during the development of water stress using a new fluorometer (FIPAM) developed at LURE. Together with fluorescence measurements gas-exchange rates of the same leaf were recorded over twenty days. Under drought conditions a good correspondence between net photosynthetic rate (A) and the rate of electron transport (ETR) before noon, found in irrigated plants, was lost: when A almost vanished, the ETR was still about 50% of the control value. It is suggested that, under the studied conditions, diverse mechanisms consuming electrons from electron transport chain, such as photorespiration, increased.
In spite of the little effect of drought on electron transport rate, a simple fluorescence parameter, F_s, accurately reflected plant status. The variations of this parameter showed a direct correlation with irradiance under irrigation conditions, while an inverse relation was developed with increasing water stress.

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755_21623.

NTENT='post'> INVOLVEMENT OF GTP IN THE PRIMARY PROTEOLYSIS OF THE D1 PROTEIN DURING PHOTOINHIBITION OF PHOTOSYSTEM II

Spetea C.¹, Hundal T.¹, Lohmann F.², Andersson B.<sup>1

1</sup>Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden;
²Free University of Berlin, Germany.

Keywords:

ATP, D1 protein, photoinhibition, photosystem 2, proteolysis, GTP

Photosystem II (PSII) is the main target for light-induced inactivation of photosynthetic electron transport, leading to a proteolytic degradation of the reaction centre D1 protein. In order to obtain information on the requirements of the proteolytic system, spinach thylakoid membranes and oxygen-evolving PSII core complexes were photoinactivated at 0^oC and subsequently incubated in the dark at room temperature in the absence or presence of various nucleotides. Addition of ATP to the photoinactivated thylakoids did not affect the time course for the initial steps of D1 protein degradation. Surprisingly, addition of GTP however accelerated the process, leading to accumulation of a 23 kDa N-terminal fragment. Non-hydrolysable GTP analogs inhibited the degradation implying requirement for GTP hydrolysis as well as the existence of tightly bound GTP to the thylakoid membrane. The GTP effect on the primary proteolysis of the D1 protein was also observed in photoinactivated PSII core complexes. It is suggested the involvement of a GTP-binding protein in the primary cleavage of the D1 protein.

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757_5140.

ISOLATION AND CHARACTERISATION OF PHOTO-SYSTEM II COMPLEXES FOR CRYSTALLIZATION

Büchel C., Morris E., Barber J.

Department of Biochemistry, Imperial College, London SW7 2AY, UK

Keywords:

2D crystals, antennae, crystallization, mass spectrometry, photosystem 2

One of the inner antenna proteins of PSII, CP43, was isolated using ion exchange chromatography. Intactness and purity was checked spectroscopically and by using SDS-PAGE and mass spectroscopy, respectively. 3D crystals were grown, which diffracted to 7Ĺ, but showed a high mosaicity. As CP43 is known to be quite unstable this might have been caused by heterogeneity due to breakdown of the apoprotein. To check this, further MALDI and Electronspray Mass Spectrometry was employed in order to map the native protein and to characterise breakdown fragments. To avoid the long crystallisation times required for 3D crystallisation, essentially the same conditions were used to grow 2D crystals directly on electron microscopy grids and the images were subjected to image analysis. The results have been compared with that obtained from crystals of the CP47-reactioncentre complex.

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759_2425.

CHLOROPHYLL FLUORESCENCE IMAGING OF HEAVY METAL TREATED PLANTS

Ciscato M., Valcke R.

Laboratory of Botany - Dept. SBG
Limburgs Universitair Centrum
Universitaire Campus building D
B-3590 Diepenbeek
Belgium

Keywords:

abiotic stress, fluorescence, fluorescence imaging, heavy metals, imaging

Chlorophyll fluorescence has been widely applied as a powerful non-invasive technique for in-vivo analysis of plant stress. In the last years a new approach, the two-dimensional image analysis of the fluorescence signal, has been found very promising. Main advantage of this new system is the possibility to highlight differences in the spatial distribution of the fluorescence emission, which cannot be resolved with ordinary fluorometers. Furthermore, the distribution of the photosynthetic activity can be estimated from the fluorescence images.
We applied a fluorescence imaging system, which is under continuous development in our laboratory, to metal treated plants. We tested the possibility to use fluorescence imaging to follow in time the metal uptake by the leaf, by following related changes in fluorescence emission. Cu-treated tobacco leaves showed time dependent changes in the fluorescence signal in areas close to the veins. These results will be discussed in relation to the physiological status of the leaf.

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761_5194.

SPECTRAL PROPERTIES OF LONG-WAVELENGTH CHLOROPHYLLS IN BARLEY PHOTOSYSTEM I DEPEND ON INTIMATE INTERACTION BETWEEN Lhca1, Lhca4 AND THE REACTION CENTRE

Bossmann B., Grimme L.H., Knoetzel J.

Institute of Cell Biology
University of Bremen
Leobener Strasse NW2
D-28359 Bremen
Germany

Keywords:

antennae, fluorescence, higher plants, photosystem 1

Using 77K fluorescence emission spectroscopy on intact leaves of chlorina barley mutants lacking individual LHCI-730 proteins, two chlorophyll (Chl) spectral forms with emission peaks at 732 nm and 742 nm could be assigned to photosystem I (PSI) Chl a/b-proteins Lhca1 and Lhca4, respectively.
From the analysis of viridis-zb⁶³ which is almost devoid of PSI reaction centres, and the comparison with fluorescence emission characteristics of isolated LHCI-730 and in vitro reconstituted Lhca1/4, it could be concluded that the formation of the longest wavelength emitting chlorophylls in plants depends on the association of LHCI-730 with the reaction centre.
Support from the Deutsche Forschungsgemeinschaft (Kn 276/3-3) is acknowledged.

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763_8902.

ATP- AND ZINC-DEPENDENT SECONDARY PROTEOLYSIS OF THE D1 PROTEIN DURING PHOTOINHIBITION OF PHOTOSYSTEM II - POSSIBLE INVOLVEMENT OF THE FtsH PROTEASE

Hundal T.¹, Spetea C.¹, Lohmann F.², Andersson B.<sup>1

1</sup>Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden;
²Free University of Berlin, Germany.

Keywords:

ATP, D1 protein, photoinhibition, photosystem 2, proteolysis, FtsH, GTP

Following light-induced inactivation of photosystem II (PSII) electron transport, the reaction centre D1 protein is degraded, generating a 23 kDa N-terminal and a 10 kDa C-terminal fragments, which are further cleaved to smaller peptides. In this work, the requirements of the secondary proteolysis of the D1 protein were investigated by monitoring the amount of the 23 kDa fragment in photoinactivated thylakoids. Addition of ATP promoted the disappearance of the fragment without affecting the primary cleavage of the D1 protein, which indeed required GTP. The effect of ATP was accelerated by the addition of Zn²⁺. Non-hydrolysable ATP analogs inhibited the secondary cleavage. ATP and Zn²⁺ did not stimulate the degrada not stimulate the degradation of the fragment in photoinhibited PSII core complexes. These results suggest the involvement of an ATP- and Zn²⁺-dependent proteolytic activity in the secondary cleavage of the D1 protein, possibly related to the FtsH protease. Thus, D1 protein degradation is a multistep process requiring different regulators.

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764_10579.

NATURE OF LOW-FREQUENCY VIBRATIONAL MODES IN BACTERIAL RCS

Rischel C.¹, Spiedel D.², Ridge J.P.², Jones M.R.², Breton J.³, Lambry J.-C.¹, Martin J.-L.¹, Vos M.H.<sup>1

1</sup>INSERM U451, ENSTA, F-91761 Palaiseau Cedex, France,
²Dept. Mol. Biol. and Biotechnology, Univ. of Sheffield, Sheffield S10 2UH, UK,
³SBE/DBCM, CEA-Saclay, F-91191 Gif-sur-Yvette Cedex, France

Keywords:

bacterial reaction center, femtosecond spectroscopy, mutants, vibrational spectroscopy

The spectrum of low-frequency vibrational modes activated by P^* formation, and identified by femtosecond coherence spectroscopy, was studied for mutant R. sphaeroides RCs with an altered pattern of hydrogen bonding between the P Bchls and the surrounding protein. It varied strongly between the different RCs, including between different mutants with the same pattern of hydrogen bonds. We argue that these variations are not due to changes in the selection of the ensemble of activated modes, but to changes in the nature of the individual modes. This indicates that they are not localized in the Bchls, but strongly involve the protein. Furthermore, the possibility of oscillating charges in the P⁺HL^- state was studied by a detailed analysis of the kinetics of the B electrochromic shift in WT and YM210L (P^* only) RCs.

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766_9504.

CONDITIONAL BARLEY MUTANT CHLORINA-104 LACKS A HSP70 HOMOLOGUE OF THE CHLOROPLAST PROTEIN IMPORT COMPLEX UNDER RESTRICTIVE GROWTH CONDITIONS

Knoetzel J., Grimme L.H.

Institute of Cell Biology
University of Bremen
Leobener Str. /NWII
D-28359 Bremen
Germany

Keywords:

biogenesis, chaperone proteins, mutants, protein import, temperature dependence

Under restrictive growth conditions, chlorina-104 lacks the LHCI-680B-constituting polypeptide Lhca2. In organello import assays have shown that the mutant fails to import Lhca2 precursor protein into chloroplasts.
Immunoblot analyses revealed that one protein component of the chloroplast outer envelope import machinery is missing under restrictive growth conditions. This protein was identified as a HSP70 homologue. Reconstitution of protein import into chloroplasts could be achieved when recombinant cytosolic HSP70 was added to the in vitro translation and the import assay.
Support from the Deutsche Forschungsgemeinschaft (Kn 276/3-3) is acknowledged.

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772_19785.

IN VITRO RECONSTITUTION OF BARLEY Lhca1 AND Lhca4, THE PROTEINS OF PHOTOSYSTEM I ANTENNA SUBCOMPLEX LHCI-730

Klimmek F., Grimme L.H., Knoetzel J.

Institute of Cell Biology, Biochemistry and Biotechnology
University of Bremen
Leobener Str. /NWII
D-28359 Bremen
Germany

Keywords:

higher plants, light harvesting complexes, photosystem 1, reconstitution, LHCI

LHCI-730 consists of a heterodimer of Lhca1 and Lhca4 as has clearly been shown from in vitro reconstitution experiments (Schmid et al. (1997) Proc. Natl. Acad. Sci. USA 94, 7667-7672). In order to supplement stroma membranes of barley chlorina mutants lacking Lhca1 and/or Lhca4, we have cloned Lhca1 and Lhca4 from barley and ligated the cDNAs into pDS expression vectors. The proteins were reconstituted with pigments, biochemically and spectroscopically characterized.
Support from the Deutsche Forschungsgemeinschaft (Kn 276/3-3) is acknowledged.
We would like to thank Prof. H. Paulsen and Dr. V. Schmid (University of Mainz, Germany) für providing us with the expression vectors.

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780_20134.

REDOX- AND PUMP INTENSITY DEPENDENCE OF ENERGY TRANSFER IN CHLOROSOMES OF CB. PHAEOBACTEROIDES

Aschenbrücker J., MaY.-Z., Miller M., Gillbro T.

Jochen Aschenbruecker
Department of Physical Chemistry
University of Umea
S-90187 Umea
Sweden

Keywords:

chlorosomes, electronic excitation transfer, green sulfur bacteria, quenching, ultrafast spectroscopy

Excitation energy transfer in the BChle containing chlorosomes of Cb. phaeobacteroides was studied by two-colour femtosecond transient absorption technique. The kinetics were probed between 500 and 820nm under oxidising and reducing conditions using excitation at 425, 490 and 530nm. No dependence of the kinetics on the pump wavelength could be observed. The kinetics in the Q_y region of BChle exhibit multi-exponential decays with major components of (4±1)ps and (20±3)ps. The amplitude of the faster component is increased by oxidising conditions and by using higher pump intensities, indicating that quenching in the presence of oxidised species and annihilation occur with similar time constants. Corresponding lifetimes were also obtained in the rise of the transient signal of baseplate BChla.
This work is supported by the EU TMR project Green Bacterial Photosynthesis.

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781_22908.

X-RAY CRYSTAL STRUCTURE REVEALS CARDIOLIPIN ASSOCIATED WITH THE REACTION CENTRE FROM RHODOBACTER SPHAEROIDES

Fyfe P.K.¹, McAuley-Hecht K.E.², Ridge J.P.¹, Isaacs N.W.³, CogdellR.J.², Jones M.R.<sup>1

1</sup>Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S10 2UH, United Kingdom
²Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow, G12 8QQ, United Kingdom
³Department of Chemistry, University of Glasgow, Glasgow, G12 8QQ, United Kingdom

Keywords:

bacterial reaction center, lipid-protein interactions, mutants, X-ray diffraction

X-ray diffraction data collfraction data collected from crystals of a single mutant (AM260W) of the Rhodobacter sphaeroides reaction centre (RC), reveals the presence of a well resolved lipid molecule associated with the transmembrane region of the complex. This lipid has been identified as cardiolipin. Analysis of previously determined RC crystal structures suggests that this lipid may have been present throughout, but only with the increase in quality and resolution of the data can it now be assigned. The binding of the cardiolipin molecule and its possible roles in the RC will be discussed.

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782_4987.

THREE-PULSE PHOTON ECHO PEAK SHIFT MEASUREMENTS ON THE ACCESSORY PIGMENTS IN THE REACTION CENTER OF RB. SPHAEROIDES

Groot M.-L.¹, Yu J.-Y., Agarwal R.², Norris J.R., Fleming G.R.²

University of Chicago, Chicago, IL, USA, Perm. address: ¹INSERM U451, ENSTA, F-91761 Palaiseau Cedex, France, ²University of California, Berkeley, USA

Keywords:

bacterial reaction center, femtosecond spectroscopy

Frequency-resolved, 3-PEPS and transient gratings were recorded to study the ultrafast pigment-protein dynamics of the accessory pigments. For a qualitative understanding of the data, which includes an increase in rephasing capability after 1 ps, it is necessary to take into account the influence of the primary electron donor P on the 800-nm band and direct electron transfer from the excited accessory BA. The peak shift curves and the absorption spectrum are simulated via the time correlation function of the optical transition frequency. The bath correlation time is longer, approximately 90 fs, at 810 nm than at 790 nm, where it is around 60 fs. The energy transfer times are 80 fs at 810 nm and 130 fs at 790 nm, this indicates that around 810 nm no significant nuclear relaxation takes place prior to energy transfer.

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786_31167.

ELECTRON SPIN POLARISATION IN QUINONE RECONSTITUTED PHOTOSYNTHETIC REACTION CENTERS

Hulsebosch R.J., Gast P., HoffA.J.

Dept. of Biophysics, Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands.

Keywords:

bacterial photosynthesis, Qa, quinones, reaction centers, Electron Spin Polarisation EPR

Secondary radical pair Electron Spin Polarised (ESP) X-band EPR spectra of quinone-reconstituted and Zn-replaced reaction centers (RCs) of Rb. sphaeroides R26 have been investigated. Replacement of the native ubiquinone by e.g. benzoquinones or anthraquinones may lead, besides different magnetic interactions between the primary electron donor bacteriochlorophyll and the acceptor quinone of the secondary radical pair, to different lifetimes of the primary radical pair. Prolongation of the primary radical pair lifetime will induce significant transfer of spin-correlation between both radical pairs resulting in different ESP EPR spectra, as has been shown by Thurnauer et al. in Fe-containing RCs [1]. A theoretical description of the transfer of electron spin-correlation from one radical pair to another, presented by Hore [2], has been used to simulate the measured ESP spectra. From the simulations, new insights in the primary radical pair couplings have been obtained.
[1] M.C. Thurnauer et al., J. Phys. Chem. (1995) 99, 3854-3866.
[2] P.J. Hore, Mol. Phys. (1996) 89, 1195-1202.

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788_5259.

PIGMENT SYNTHESIS DURING THE FIRST PHOTOPERIOD OF GREENING (16 H) OF 2-D-OLD AND 10-D-OLD BEAN LEAVES

Schoefs B.¹, Bertrand M.², Lemoine Y.<sup>3

1</sup>Lab. Biomembranes, Univ. South Bohemia, Branisovska 31, CZ-37005 Ceske Budejovice
²INTECHMER/CNAM, BP 324, F-50103 Cherbourg Cedex.
³Plant Cytophysiology and Phycology (SN2), Univ. of Lille 1, F-59655 Villeneuve d’Ascq

Keywords:

chromatography, environmental stress, heavy metals, marine algae, pigments

The model usually used to study pigment synthesis in higher plants consists in old dark-grown leaves, the so-called etiolated leaves. However, this developmental stage is not encountered in nature since prolonged darkness is quite rare. In order to verify whether the results obtained with this model (10-d-old leaves) can be extended to a more natural leaf developmental stage (2-d-old leaves), we have followed the individual variations of carotenoids and chlorophyll(ide) during the first photoperiod of greening of leaves at these two developmental stages.
The chromatographic analysis demonstrates that 1) young leaves contain protochlorophyllide a and no protochlorophyllide a esters, 2) the carotenoid composition is very similar at the two developmental stages, 3) chlorophyll accumulation in young leaves presents a lag phase twice longer than the one in 10-d-old samples, 4) in young leaves, all the carotenoids, except antheraxanthin, increase after the lag phase. The b-carotene and violaxanthin increase is only transient (4h), 5) the cis/trans ratio of total carotenoids decrease during the light period simultaneously at both development stages.

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789_14540.

DETERMINATION OF THE CLEAVAGE SITE OF THE LARGE SUBUNIT OF RUBISCO BY ACTIVE OXYGEN SPECIES AND ITS INHIBITION BY CABP IN THE LYSATES OF WHEAT CHLOROPLASTS

Ishida H., Makino A., Mae T.

Department of Appried Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamati Aoba-ku, Sendai 981-8555, Japan

Keywords:

active oxygen, degradation pathways, photooxidation, Rubisco

The large subunit (LSU) of Rubisco is degraded into an N-terminal side fragment of 37 kDa and a C-terminal side fragment of 16 kDa by active oxygen species, probably hydroxyl radical, in the lysates of chloroplasts in light (Ishida et al., 1997, Plant Cell Physiol 38: 471-479). In the present study, we isolated the fragments and determined their terminal amino acid sequences. The C-terminal amino acid of the 37-kDa fragment was Ser³²⁸ and the N-terminal amino acid of the 16-kDa fragment was Thr³³⁰, indicating that the LSU was cleaved at Gly³³⁰ or its both ends. The fragmentation was completely blocked by the binding of CABP to the enzyme. The proposed mechanism of the site-specific fragmentation of the LSU by active oxygen is discussed in relation to the structure of the enzyme.

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791_14402.

FEMTOSECOND STUDIES OFD STUDIES OF CYTOCHROME C OXIDASES

Liebl U., Lipowski G., Martin J.-L., Vos M.H.

INSERM U451, ENSTA, F-91761 Palaiseau Cedex, France

Keywords:

cytochrome complexes, femtosecond spectroscopy, respiration

Cytochrome c oxidases are membrane-protein complexes catalyzing the reduction of molecular oxygen into water. We have isolated and purified the cytochrome c oxidases aa3 from Paracoccus denitrificans and cbb3 from the photosynthetic bacterium Rhodobacter capsulatus and used them for ultrafast optical studies. Multicolor femtosecond spectroscopy is a powerful tool to study heme electronic states as well as ligand and protein dynamics. Excitation is done in the a-bands of the respective heme proteins, using 50-fs pulses. The time course of population of a and a3 excited states and, upon excitation of CO-ligated a3, the unligated ground state, is compared to beefheart aa3 oxidase. A completely novel aspect is the real-time monitoring of nuclear motions associated with the formation of these states via oscillatory modulation of the transient spectra.

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794_22383.

MUTANTS OF THE RHODOBACTER SPHAEROIDES REACTION CENTRE LACKING THE LONG WAVELENGTH ABSORBANCE BAND OF THE PRIMARY DONOR BACTERIOCHLOROPHYLLS

Spiedel D.¹, van BrederodeM.E.², van Grondelle R.², Jones M.R.<sup>1

1</sup>Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S10 2UH, United Kingdom
²Department of Physics and Astronomy, Free University of Amsterdam, De Boelelaan 1081, HV 1081 Amsterdam, The Netherlands

Keywords:

bacterial reaction center, electron transfer, H-bond, site-directed mutagenesis, ultrafast spectroscopy, primarydonor

Four mutant reaction centre complexes have been constructed, with mutations in the vicinity of the bacteriochlorophylls which constitute the primary donor (P). Two of the mutations, FL167R and FL167H, were designed to introduce basic residues without disturbing the pattern of hydrogen bonds to P. The other two mutations combine these changes with the breakage of the hydrogen bond at the L168 position (FL167R/HL168F and FL167H/HL168F). At room temperature the Q_y transition of P is blue-shifted and markedly attenuated in the single mutants, and virtually eliminated in the double mutants. Results obtained clearly indicate that the bacteriochlorophylls of the primary donor are still present in the double mutants but that the optical properties of the dimer are strongly perturbed. Sub-picosecond absorption spectroscopy on the FL167R/HL168F RC at 77K with 796 nm excitation show a 200 fs relaxation of B^*, together with 1.3 ps and 97 ps components which describe reaxation of B bleaching and a limited amount of B and H bandshift formation.

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795_30104.

HEME BINDING TO AN APOCYTOCHROME B₆ FUSED TO MALTOSE-BINDING PROTEIN OVEREXPRESSED IN ESCHERICHIA COLI

Kroliczewski J., Johanningmaier U.^*, Szczepaniak A.

Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland
^*Lehrstuhl für Biochemie der Pflanzen, Ruhr-Universität, D-44780 Bochum, Germany

Keywords:

cytochromes, overexpression, hemeinsertion,

A cDNA for a cytochrome b₆ from spinach has been cloned in a bacterial expression vector, pMal c-2 (New England Biolabs). Bacteria transformed with this construct produced up to 20% their protein as a fusion protein: maltose-binding protein - apocytochrom b₆ (MBP-apocyt b₆) in a form of inclusion bodies. After lysing the cells, mildly solubilizing the membrane and repeatedly washing the pellet fraction, MBP-apocyt b₆ is 75% to 85% pure as judged from denaturing polyacrylamide gel electrophoresis. The fusion protein was purified to virtual homogeneity by gel filtration chromatography of dissolved inclusion bodies in a buffer containing urea. This purified MBP-apocyt b₆ was used in a heme isertion experiment. The spectrum of the oxidized reconstructed MBP-cyt b₆ displays a characteristic Soret band at 412 nm and broad band around 535 nm. Reduction of the MBP-cyt b₆ with sodium dithionate leads to a redshift of Soret (g-band) to 421 nm and to distinct a- and -bands at 562 and 535 nm, which are typical for bis-histidine ligated b-type cytochromes. The ratio of the absorbance of 5.6 at the g- and a-bands of the reduced MBP-cyt b₆ is also very close to that found for natural b-type cytochromes.

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798_2932.

INTERACTION OF TENTOXIN AND DERIVATIVES WITH THE CATALYTIC SECTOR OF CHLOROPLAST ATP-SYNTHASE

Santolini J., Sigalat C., André F., He X., Haraux F.

Section de Bioénergétique, CEA Saclay, F91191 Gif-sur-Yvette Cedex, France

Keywords:

ATP synthase, energy transduction, photophosphorylation, tentoxin

This lecture focuses on the mechanism by which the cyclic tetrapeptide tentoxin (TTX) inhibits and reactivates the F1 part of CF0CF1. Our studies use natural TTX as well as synthetic derivatives, radioactive or not. Two binding sites of different affinities were found to be enough to describe inhibition and reactivation. Some features of the tight site (inhibitory) could be deduced from the comparative efficiency of different TTX analogues. These analogues also allowed to discriminate binding on the loose site and reactivatory effect. We have shown that the interaction of TTX with the tight site does not depend on its dynamic state, whereas interaction of TTX with the loose site does depends on its dynamic state. A minimal model has been developed to describe the complex ATP-dependent interaction of TTX with the loose site. We are also interested in the cooperativity between the two TTX-binding sites and have shown that: 1) toxin binding on the tight site modifies the affinity of toxin for the loose site; 2) The activity of ternary complexes CF1·toxin1·toxin2 depends on the nature of toxins bound on both sites. We have studied the effect of TTX on membrane-bound CF0CF1 and have shown that the enzyme bearing two TTX molecules is competent for proton pumping. We are trying to identify the aminoacids of the binding site(s) of TTX in CF1. Our results will be discussed in the context of the rotatory mechanism of ATP synthesis.

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804_3463.

THE 1-DEOXY-D-XYLULOSE-5-PHOSPHATE PATHWAY FOR BIOSYNTHESIS OF CAROTENOIDS AND OTHER PLASTIDIC ISOPRENOIDS

Lichtenthaler H.K.

Botanisches Institut II
Universität Karlsruhe
Kaiserstr. 12
D-76128 Karlsruhe
Germany

Keywords:

algae, biosynthesis, carotenoids, chloroplast development, cyanobacteria

Isoprenoid biosynthesis in plants proceeds either via the cytosolic classical mevalonate pathway (sterols, sesquiterpenes, triterpenoids) or via the non-mevalonate 1-deoxy-D-xylulose-5-phosphate pathway for the biosynthesis of plastidic isoprenoids such as carotenoids, phytol (side-chain of chlorophylls), plastoquinone-9, isoprene, monoterpenes and diterpenes. Both pathways form isopentenyl diphosphate (IPP) from which all other isoprenoids are formed via head-tail condensation reactions. The recently detected plastidic 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway of IPP biosynthesis starts from glyceraldehyde-3-phosphate and pyruvate in a transketolase-type, thiamin-dependent reaction as catalysed by the DOXP-synthase. It yields DOXP as first intermediate. After a transposition step and further modifications IPP is formed. The novel DOXP pathway provides a new insight into chloroplast metabolism and explains many former odd research results. The DOXP-pathway is also present in algae and cyanobacteria (ß-carotene, phytol).

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808_445.

OVERPRODUCTION OF A SPINACH RIESKE POLYPEPTYDE FUSED TO MALTOSE-BINDING PROTEIN IN ESCHERICHIA COLI

Gubernator B., Johanningmaier U.^*, Szczepaniak A.

Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland
^*Lehrstuhl für Biochemie der Pflanzen, Ruhr-Universität, D-44780 Bochum, Germany

Keywords:

Fe-S centers, overexpression, Rieske protein

The Rieske protein of oxygenic photosynthesis is the electron acceptor for plastoquinon and donor for cytochrome f. Overexpression of the Rieske protein is a convenient way for isolation significant amount of apoprotein, and could help considerable for the understanding of structure/function relationship; it may also allow the insertion of iron-sulfur center and reconstitution Rieske protein and than whole active bf complex. In this work, we have overproduced in Escherichia coli of fusion, full-lenght, and truncated forms of the Rieske protein from Spinacia oleracea. The cloned cDNA’s were inserted downstream from malE gene of E. coli, which encodes maltose-binding protein (MBP), resulting in the expression of an MBP fusion protein. Overexpression of MBP fusion proteins yielded up to 10% of the total cell protein in both case as characterized by immunoblotting. Starting from 1 litre culture using affinity chromatography (amylose resin) we have isolated p to 15 mg pure fusion protein. The full-lenght or truncated Rieske apoprotein we are able to cleveage from MBP using factor Xa.

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818_12056.

PHOTOSYNTHETIC MACHINERY OF COTYLEDONS OF SESBANIA SESBAN PERFORMS BETTER UNDER HIGH OSMOTIC ENVIRONMENT

Puthur J.T., Sharmila P., Pardha Saradhi P.

Plant Physiology & Biotechnology Laboratory, Centre for Biosciences, Jamia Millia Islamia, New Delhi - 110 025, India.

Keywords:

abiotic stress, electron transport, fluorescence, photosystem 2, salt stress

Contribution of the developing cotyledons in harvesting light energy during the seed development is meagerly studied. Investigations were carried out to see photosynthetic efficiency of cotyledons of maturing embryo in developing seeds of Sesbania sesban (Fabaceae, a multipurpose, fast growing tree species). The photosystem II activity in terms of O₂ evolution and Fv/Fm of developing cotyledons, pod wall, cotyledonary leaves and leaflets from mature tree was measured in the presence of salt solutions of varying concentrations. With the exception of cotyledons from developing seeds all other plant parts showed sharp decline in PS II activity in the presence of salt above 100 mM. In contrast, in developing cotyledons peak PS II activity was noted in the presence of 250 mM salt. Our observations clearly pin point that the photosynthetic machinery in young cotyledons from developing seeds have inbuilt potential to photosynthesize efficiently under high osmotic environment. Here we hypothesize that the factors that optimize photosynthetic efficiency under high osmotic environment in developing cotyledons are lacking in other photosynthetic plant parts.

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819_23086.

THE SUBSTITUTION OF CHLORIDE IN THE OXYGEN EVOLVING COMPLEX OF PHOTOSYSTEM II

Wincencjusz H.M.¹, Yocum C.F.², van Gorkom H.J.<sup>1

1</sup>Department of Biophysics, Huygens Laboratorium, Leiden University, P.O.Box 9504, 2300 RA Leiden, The Netherlands
²Depts. of Biology and Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1048, USA

Keywords:

chloride, O2 evolution, photosystem 2

Photosystem II requires the inorganic cofactors Ca²⁺ and Cl^- to exhibit optimal activity. Cl^- can be functionally replaced by a small number of anionic cofactors (Br^-, NO₃^-, NO₂^-, I^-), but the rate of oxygen evolution is lower than with Cl^-. UV absorption difference spectroscopy was utilized as a probe to monitor donor side reactions in Photosystem II in the presence of Cl^- or surrogate anions. The rate of the final step of the water oxidation cycle was found to depend on the activating anion bound at the Cl^- site, but the kinetics of this step did not limit the light-saturated rate of oxygen evolution. Instead, the lower activity supported by surrogate anions appeared to result from an instability of the higher oxidation states of the oxygen evolving complex that was induced by addition of these anions. Reduction of these states is not related to the ability of the surrogate anions to bind at the Cl^- binding site.

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820_20918.

PERIDININ CHLOROPHYLL PROTEIN: STRUCTURE AND SPECTROSCOPY RELATED

Kleima F.J., Hofmann E., Gobets B., Peterman E.J.G., van Grondelle R., van Amerongen H.

Dept. of Physics and Astronomy and Inst. for Condensed Matter and Optical Physics, Vrije Univ. Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands and Fakultaet fuer Biologie, Univ. Konstanz, Box M656, D-78457 Konstanz, Germany.

Keywords:

carotenoids, dinoflagellates, excited state dynamics, fluorescence, light harvesting complexes, polarized spectroscopy

Peridinin Chlorophyll Protein (PCP), a water-soluble light-harvesting complex from dinoflagellates containing two Chlorophyll a (Chl a) molecules at a distance of 1.7 nm and 8 peridinins per monomer, has been studied using linear dichroism, circular dichroism, triplet minus singlet, polarized fluorescence and fluorescence line narrowing spectroscopy at different temperatures (4K-293K). The dynamics of excitation transfer is studied using time resolved fluorescence anisotropy measurements at room temperature. Due to the limited number of interacting Chl a molecules the complex is an excellent model system to relate spectroscopic parameters and structure (Hofmann E. et al., Science. 272 (5269): 1778-1791, 1996).

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828_31146.

LIGHT CONTROL OF CHLOROPLAST DEVELOPMENT: WHO HOLDS THE KEY?

Baynton C.E., Robertson S.E., Vinti G., Pyke K.A., Lopez-Juez E.

Biological Sc., Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK

Keywords:

gene expression, greening, light regulation, phytochromes, signal transduction

In angiosperms, the expression of nuclear-encoded genes (like Lhcb) involved in chloroplast development, requires both light perception (by phytochromes) and functional plastids. The analysis of Cab (Lhcb) underexpressor (cue) Arabidopsis mutants, obtained through a previously described screen (Li et al. 1995, Plant Cell 7, 1599), has led us to conclude that light and chloroplast signals are tightly interconnected. We propose that either both signals are co-required, or the chloroplast signal mediates phytochrome action, the plastids being the primary targets of the light signals. These two models are being tested through a variety of approaches. An Lhcb promoter-driven green fluorescent protein (GFP) reporter is being tested for potential expression and regulation in the complete absence of plastids, thanks to the occurrence of plastid-less guard cells in the plastid division mutants arc6 and arc12. cue mutants and a variety of chloroplast and etioplast-affecting treatments are being used to test whether the relationship between plastid and phytochrome signals is additive or interactive. We are also examining the plastid signal requirements for expression of a pine Lhcb promoter, previously shown to be expressed in tobacco in a light-independent fashion (Kojima et al. 1994, Plant J. 6, 591). Results to date will be presented.

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834_4872.

DEVELOPMENT OF "OXYGEN CLOCK" IN GREENING PEA LEAVES AS PROBED BY PERIOD FOUR OSCILLATIONS IN THE FLUORESCENCE AT 50 µs AND 2 ms AFTER PRE-FLASHES DURING OJIP TRANSIENT

Govindjee, Srivastava A., StrasserR.J.

University of Illinois at Urbana-Champaign, Department of Plant Biology, 265 Morrill Hall, 505 South Goodwin Avenue, Urbana, IL 61801-3707, USA
And, Department of Bioenergetics, University of Geneva, Ch-1254 Jussy-Lullier-Geneva, Switzerland

Keywords:

Chl fluorescence induction, chloroplast development, greening, higher plants, O2 evolution, photosystem 2, S-states

In dark adapted plants, the O₂ evolution per flash shows an oscillation of four as a function of the flash number. The development of this "oxygen clock" was probed by period 4 oscillations in Chl a fluorerscence yield measured at 50 µs (maxima after preflashes 2 and 6) and at 2 ms (minima after preflashes 2 and 6) during OJIP fluorescence rise (also see B. Strasser et al., this Congress). "Intermittent" (2 min light and 118 min dark) or "flashed" (1 ms flashes every 15 min) pea leaves, exposed to continuous light, showed increases in the amplitudes of the variable fluorescence (OJIP transient) and in the period 4 oscillations reflecting development of the "oxygen clock". Data will also be presented on changes in 77K emission spectra, thermoluminescence, O₂ evolution, and P700 absorbance during the development of the photosynthetic apparatus. These data will be compared with the quantum yield of O₂ evolution, DOD at 515 nm, pigment-protein complexes, and non-photochemical quenching, obtained in collaboration with F. Chow, A. Hope, C. Funk and A. Gilmore, respectively, at RSBS, Canberra, Australia.

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835_6624.

CRYSTALLOGRAPHIC ANALYSIS OF THE PHE M197 ® TYR MUTANT OF THE PHOTOSYNTHETIC REACTION CENTRE FROM RHODOBACTER SPHAEROIDES

Kuglstatter A., Fritzsch G., Michel H.

Max-Planck-Institut für Biophysik, Abteilung Molekulare Membranbiologie, Heinrich-Hoffmann-Straße 7, D-60528 Frankfurt/M., Germany

Keywords:

bacterial reaction center, Bchl, H-bond, mutants, X-ray diffraction

The site-directed mutant Phe M197 ® Tyr of the photosynthetic reaction centre (RC) from Rhodobacter (Rb.) sphaeroides has been crystallized and the structure has been determined by X-ray diffraction analysis at 2.7Ĺ resolution. Tyr M197 in the Rb. sphaeroides mutant RC is assumed to form a "viridis-like" H-bond, since the analogous Tyr in Rhodopseudomonas (Rps.) viridis RC is H-bonded to the C₂ acetyl carbonyl of D_B, the M-sided bacteriochlorophyll of the primary donor. Spectroscopic and redox studies with this mutant RC suggest the existence of the H-bond (Wachtveitl et al., Biochemistry 32, 12875, 1993). The new crystal structure does not solve the problem unambiguously. The geometry for H-bonding is nearly optimal, but X-ray crystallography at 2.7Ĺ cannot distinguish between the two alternative orientations of the acetyl group. Although the surroundings of Tyr M197 in the mutant RC and the analogous Tyr in the Rps. viridis wild-type RC are similar, the two phenol planes are almost perpendicular to each other. Compared to the Rb. sphaeroides wild-type RC the adjacent backbone of the mutant RC is shifted 0.5Ĺ away from the introduced hydroxy group. This study shows that the structure of the Phe M197 ® Tyr mutant RC is not in contradiction to the existence of the putative H-bond.
We thank D. Oesterhelt (MPI Biochemie, Martinsried) for providing us with the mutant strain.

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837_23946.

THE COUPLING REGIONS OF F0F1 ATP-SYNTHASE

Licher T., Kellner E., Lill H.

Abt. Biophysik, Universität Osnabrück, D49069 Osnabrück, Germany

Keywords:

ATP synthase, energy transduction, molecular biology, overexpression, photophosphorylation

Transfer of ions through F0 is coupled to hydthrough F0 is coupled to hydrolysis or synthesis of ATP by F1. Energy transfer is thought to be mediated mechanically via relative rotations of subunits within the complex. As with the elements of any motor, this implies a classification of the enzyme's subunits into stator and rotor elements in addition to the classical denomination as F1 and F0 subunits.
We have studied interactions occuring at interfaces between F0 and F1 in both, the stator and the rotor parts of the enzyme. The soluble regions of subunits b, b', and c have been expressed in Escherichia coli and purified with HisTags or as fusions to MalE. These tags were also used for immobilization of the soluble peptides and subsequent affinity chromatography with F1 or its single recombinant subunits. Additional Cysteines introduced at various positions of the primary sequences allowed fluorophore labelling of the recombinant peptides. Interactions of labelled peptides with F1 have then been monitored via Fluorescence Resonance Energy Transfer (FRET).

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838_7407.

ACCLIMATIZATION OF MICROPROPAGATED TOBACCO PLANTLETS

Kadlecek P., Ticha I.

Department of Plant Physiology, Charles University, Vinicna 5, CZ - 128 44 Praha 2, Czech Republic

Keywords:

acclimation, biotechnology, fluorescence, net photosynthesis

35-d-old tobacco (Nicotiana tabacum L. cv. Samsun) plantlets grown in vitro under 200 µmol photons m^-2 s^-1 and CO₂ saturation photoautotrophically (PA; without sucrose in the medium) or photomixotrophically (PM; with 3% sucrose) were transplanted into pots with soil and transferred to the greenhouse. After further 20 days the plants were transferred to open air. All the plants survived. After the 35 days of in vitro growth significant differences in chlorophyll a+b content, in photosynthetic capacity, in maximum photochemical efficiency and in carbohydrates content between the PA and PM grown plantlets were found. These differences disappeared during the first stage of acclimatization in the greenhouse. After the transfer to open air (higher irradiance) chlorophyll a+b content, photosynthetic capacity and carbohydrates content started to increase permanently and synchronously in the originally PA and PM grown plants. However, plant growth which was significantly enhanced by the sucrose pretreatment in vitro, continued to be increased (more leaves, larger leaf area and higher dry matter accumulation) in PM plants during the whole acclimatization period.

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839_18700.

CHLOROPHYLL FLUORESCENCE AS AN INDICATOR OF PHOTOSYNTHETIC BEHAVIOUR OF IN VITRO CHESTNUT DURING ACCLIMATIZATION AT HIGH CO₂ AVAILABILITY

Carvalho L., Osório L., Amâncio S.

IS Agronomia Dept. de Botânica e Eng. Biológica and Dept. de Eng. Florestal. 1399 LISBOA CODEX PORTUGAL

Keywords:

elevated CO2 concentration, fluorescence, light acclimation, photoinhibition, quantum yield

In vitro culture of woody plants is used in plant clonal propagation, with advantages when compared to in vivo propagation. A large number of woody species produced in vitro does not survive when transferred to field growth conditions obliging to an acclimatization period in controlled conditions. In chestnut, the manipulation of light availability gave rise to a reduction in the acclimatization time and an increase of survival rates although photooxidation was detected. Two CO₂ concentrations (A, 350 ppm and B, 700 ppm) at a light intensity of 300 µmol quanta m^-2·s^-1 during 6 weeks of acclimatization were tested. Comparisons were made through growth analysis and fluorescence parameters. Fv/Fm and Fv'/Fm' did not show significant differences between treatments, Genty parameter showed slightly higher values in treatment A, which means an increase in PS II yield. Plants acclimatized at 700 ppm CO₂ also presented higher values of total biomass, leaf area and eaf weight.

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842_19494.

ZEAXANTHIN RETENTION IN SINK-LIMITED PLANTS

Demmig-Adams B., Adams W.W. III, Ebbert V., Rosenstiel T.N.

EPOB Dept., Univ. of Colo., Boulder, CO 80309-0334, USA

Keywords:

carbohydrate, energy dissipation, photoinhibition, photosystem 2, xanthophyll cycle

Various environmental stresses induce retention of the deepoxidized forms of the xanthophyll cycle, zeaxanthin and antheraxanthin (Z+A), and sustained high levels of thermal energy dissipation in photosystem II (PSII). The possibility will be examined that this switch from rapidly modulated (Z+A)-dependent dissipation under favorable conditions to stress-induced retention of Z+A is related to a switch from non-sink-limited to sink-limited conditions at the whole plant level. Associations among Z+A retention, photoinhibition of PSII, photosynthetic downregulation, and carbohydrate retention in source leaves under stress will be demonstrated. The possible formation of xanthophyll-containing protein supra-complexes in thylakoids of (Z+A)-retaining leaves as well as a possible link to limited carbohydrate export will be discussed. Responses of plant species with different source/sink relationships to increased growth PFD or seasonal transition to winter will be contrasted.

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843_20593.

SITE-DIRECTED MUTANTS OF THE CHLOROPLAST psbE GENE IN CHLAMYDOMONAS REINHARDTII

Morais F., Barber J., NixonP.J.

Biochemistry Department, Imperial College of Science Technology and Medicine London SW7 2AY, UK

Keywords:

chloroplast genes, cytochrome b-559, D1/D2-cyt-b-559, site-directed mutagenesis, Chlamydomonasreinhardtii

One of the components of the PSII reaction centre is cytochrome b-559, a transmembrane haemprotein which is probably arranged in a heterodimer structure of the a and b subunits. These subunits are encoded by plastid genes, namely psbE and psbF, respectively. The function of this cytochrome is still uncertain but recent evidence suggests that it probably plays a role in protecting the reaction centre from photoinhibitory damage, by providing PSII with an alternative, possibly cyclic, route of electron flow. In order to test this hypothesis we describe the construction and characterisation of site-directed mutants constructed in the a subunit of cytochrome b-559 of Chlamydomonas reinhardtii by transformation of the chloroplast genome using biolistic technology.

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845_21573.

EFFECT OF Al ON MEMBRANE POLYPEPTIDES IN P-DEFICIENT SOYBEAN CHLOROPLASTS

Milivojevic D.B., Nikolic B.R., Drinic-Mladenovic S.D.

INEP, Banatska 31b, 11080 Belgrade, Yugoslavia
Pesticide and Environmental Research Centre, Banatska 31b, 11080 Belgrade, Yugoslavia
Maize Research Institute "Zemun Polje", S. Bajica 1, 11080 Belgrade, Yugoslavia

Keywords:

abiotic stress, D1 protein, LHC II, photosystem 1, photosystem 2, pigment-protein complexes

Inhibition of root growh is the primary symptom of Al-stress, while shoot growth inhibition is the secondary response to Al. We investigated the latter aspect of Al effect on photosynthetic processes and, to define the photosynthetic apparatus, we determined pigments and separated thylakoid polypeptides by SDS-PAGE. As Al-toxicity and P-deficiency often coincide, soybean plants were grown for 12 days on nutrient solutions with and without P. We added Al₂(SO₄)₃ in 0.5-1.0 mM concentrations into the nutrient solutions (pH=5 and 7) to exposed the plants to Al-toxicity and P-deficiency by the root. The analysis of soybean leaf pigments showed a decreased content of carotenoids under higher Al concentrations (0.7-1.0 mM) both in the acid and neutral nutrient solutions, with and without P. Chl a and b did not change their ratio. The analysis of thylakoid polypeptide content showed no difference between treated and untreated plants in the presence of P. P-deficiency in acid and neutral mediums raised the content of polypeptides bound to LHC-II proteins, and 32 kDa proteins of the PS-2 reaction centre, as compared to the PS-1 polypeptides. Al-stress and P-deficiency changed the integral components of thylakoid membranes.

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847_14333.

PHOTOPROTECTION IN HIGHER PLANT LEAVES INVESTIGATED BY DELAYED CHLOROPHYLL FLUORESCENCE AND PICOSECOND CHLOROPHYLL FLUORESCENCE DECAY MEASUREMENTS

Terjung F.

Carl von Ossietzky-University Oldenburg, Dept. of Physics, 26111 Oldenburg, Germany

Keywords:

adaptation, fluorescence lifetimes, light stress, xanthophyll cycle, delayed fluorescence

Picosecond chlorophyll fluorescence decay measurements were extended by additional light pulses for adaptation and/or saturation of the primary reactions of photosynthesis. Together with delayed chlorophyll fluorescence measurements it was possible to describe the photoprotective mechanisms in higher plant leaves under in vivo conditions. The most important adaptation mechanism was found to depend on the amount of zeaxanthin and an activating pH-gradient; an additional pH-gradient independent mechanism was observed in the presence of high amounts of zeaxanthin, clearly resolved by the additional light pulses in picosecond chlorophyll fluorescence decay measurements.

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849_8066.

CLONING OF CHLOROBIUM UROPORPHYRINOGEN DECARBOXYLASE GENE

Xu K., Ndivo V., Majumdar D.

Department of Biology Norfolk State University Norfolk, VA 23504 USA

Keywords:

Chl biosynthesisgreen sulfur bacteria, porphyrins, heme, coproporphyrin, Chlorobium

As a means to study the post uroporphyrin steps in chlorophyll biosynthesis in Chlorobium we attempted to clone the uroporphyrinogen decarboxylase (hemE) gene. Chlorobium DNA was partially digested and cloned into pBR322 and used to transform a restriction-minus Salmonella typhimurium strain by electroporation. Phage P22 was grown on a pool of the transformants. This phage library was then used to transduce a auxotrophic Salmonella hemE mutant (hemA^- hemE^-). By selecting for faster growing colonies we isolated 23 clones that complemented the hemE mutation. To confirm the nature of the cloned gene, a fresh batch of P22 was used to grow on each of the 23 clones and used to transduce the hemE mutant again. For each clone several hundred transductants were obtained. Growth of the transductants in LB medium was as fast as the wild type as measured at A600 at regular time intervals. One recombinant plasmid showed the insert to be about 4 Kb. Reverse phase HPLC analysis of expression of the hmE product, uroporphyrinogen decarboxylase, by direct assay of coproporphyrin in the cells as well as that made from externally added porphobilinogen to the cell extracts, will be presented.

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850_22788.

ELECTRON TRANSFER AND REDOX EQUILIBRIUM BETWEEN THE IRON-SULFUR CLUSTERS IN PS I: F_B IS THE TERMINAL ACCEPTOR

VassilievI.R., Shinkarev V.P.^*, Golbeck J.H.

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802 USA
^*Department of Plant Biology, University of Illinois, Urbana, IL 61801 USA

Keywords:

charge recombination, cyanobacteria, electron transfer, Fe-S centers, photosystem 1

The orientation of the PsaC subunit of PS I and the arrangement of its terminal acceptors, F_A and F_B, were studied using two approaches: 1) F_B was extracted by Hg-treatment of PS I isolated from Synechococcus sp. PCC 6301. Single-turnover flashes were used to promote a step-by-step electron transfer between electron carriers in control, F_B-less and F_B-reconstituted PS I, and the kinetics of P700⁺ reduction were analyzed after addition of ferredoxin, flavodoxin and methyl viologen. 2) Kinetics of electron transfer were analyzed using the distances between PS I acceptors estimated from X-ray data (Schubert et al., 1997) and the relationship between the electron transfer rate and distance (Moser et al., 1992). These analyses support the assignment of F_A as the cluster proximal to F_X, and the sequence of electron transfer on the acceptor side of PS I as F_X ® F_A ® F_B ® ferredoxin and enable estimation of the equilibrium constants of electron transfer between the iron-sulfur clusters.
Funded by NSF (MCB-969617).

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853_30496.

CHEMICAL RESCUE OF SITE-MODIFIED LIGANDS TO THE IRON-SULFUR CLUSTERS OF PsaC IN PHOTOSYSTEM I

Antonkine M.L., Falzone C.^*, Yang F., Golbeck G.H.

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802
^*Department of Chemistry, The Pennsylvania State University, University Park, PA 16802

Keywords:

EPR, Fe-S centers, mutants, NMR, photosystem 1

Cysteine 14, which ligates F_B, and cysteine 51, which ligates F_A, were changed to serine, aspartate, alanine and glycine in unbound PsaC. We proposed that 2-mercaptoethanol, a reagent used for iron-sulfur reconstitution, serves as an external rescue ligand in the absence of a biological ligand to the fourth iron. Instead of the expected [3Fe-4S] cluster, chemical rescue allows for formation of a [4Fe-4S] cluster in the alanine and glycine mutants. To support this hypothesis, we reconstituted C14G/C34S using 4-fluorothiophenol as the rescue ligand to F_B. NMR studies show a down-field hyperfine shifted resonance of the F¹⁹, which suggests that the aryl thiolate ligates a paramagnetic iron in the site-modified [4Fe-4S] cluster. EPR studies show g values of 1.91, 1.93 and 2.04 from the unmodified, reduced cluster, which implies that the modified, reduced cluster retains the S=3/2 spin state that is similarly found when 2-mercaptoethanol serves as the external rescue ligand. These results indicate that aryl or acyl thiolates can both serve as external rescue ligands to the F_B cluster of PsaC.
Supported by NSF MCB-9723661.

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855_29594.

MUTATIONS IN THE PHYLLOQUINONE BIOSYNTHETIC PATHWAY AFFECT THE PROPERTIES OF A₁ IN PHOTOSYSTEM I

Zybailov B., Shen G., Vassiliev I., Johnson W.^*, Reategui R.^*, Chitnis P.^*, Bryant D., Golbeck J.H.

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802
^*Department of Biochemistry and Biophysics, Iowa State University, Ames, IA

Keywords:

cyanobacteria, EPR, mutants, photosystem 1, phylloquinones

By analogy with the menaquinone biosynthetic pathway in Escherichia coli, the biosynthesis of phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone) in cyanobacteria is proposed to occur by participation of the MenB (naphthoate synthase) and MenA (phytyl-transferase) gene products and a methyl transferase. Insertional inactivation of the Synechocystis sp. PCC 6803 menA gene with an antibiotic resistance cartridge produced a mutant unable to grow photoautotrophically and sensitive to high light intensities when grown heterotrophically on glucose. The flash-induced charge separation in PS I complexes recombines with a 3-ms half-time, and is replaced by a 7 µs half-time after addition of sodium dithionite. Chemical extraction of the menA^- mutant confirms the absence of phylloquinone, but X-band EPR studies of living cells and Q-band photoaccumulation studies of PS I complexes show the presence of a quinone-like molecule in the A₁ site. We propose that a substitute quinone, one capable of accepting electrons from A₀^-, has been recruited into the A₁ binding site in the menA^- mutant.
Supported by NSF grants MCB-9723661; MCB-9723469; MCB9723001.

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862_13179.

PHYSIOLOGICAL IMPACTS OF CHILLING TEMPERATURE IN THE SUSCEPTIBILITY OF YOUNG COFFEE PLANTS (COFFEA ARABICA L.) TO PHOTOINHIBITION

Oliveira J.G., Magalhaes A.C.

University of Campinas
Institute of Biological
Department of Plant Physiology
P.O.Box: 6109
Zipe Box: 13083-970
Campinas, SP, Brazil

Keywords:

chilling, Chl fluorescence induction, fluorescence, light stress, photoinhibition

The species C. arabica is sensitive to low-above zero temperatures, and is subjected to many injuries under chilling temperatures. Actually, the plants had their photosynthetic activity particularly affected by chilling temperature, presenting irreversible damage under these conditions. This work aimed to evaluate the extention and the recovery capacity of young coffee plants when plants where exposed to high irradiance (~2500 µmol·m^-2·s^-1) in different times (5, 30 and 60 min.), compared to the effects shown under low temperature (10°C). Photosynthetic activity was measured from the data obtained with a portable fluorometer. The results were interpreted according to the polyphasic curve proposed by Govindjee (Photoch. and Photobiol. 61(1):32-42, 1995) utilizing the transients O-J-I-P. It has been found that low temperature associated to high irradiance potentialize the photoinhibitory effects over the photosynthetic apparatus. The photosynthetic quantum efficiency (FV/FM) was irreversible affected as plants were subjected to high irradiance associated to low temperature, imposing damage symptoms at the 20% level of exposure for 5 min., and of 60 and 80% for the times exposure of 30 and 60 min., respectively. Our data suggested that the reaction center (RC) of photosystem II, in the reduced state, corresponding to the 'P' transient, more apropriated reflect the photoinhibitory damage as compared with the RC of PSII in the oxidized state, corresponding to transient 'O'.

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870_19584.

REGULATION OF PHOTOSYNTHETIC PROCESSES IN SYNECHOCOCCUS DURING ENVIRONMENTAL CHANGE

Grossman A.R., Schwarz R., Dolganov N., Bhaya D.

The Carnegie Institution of Washington, Department of Plant Biology, 260 Panama Street, Stanford, CA 94305.

Keywords:

acclimation, environmental stress, light harvesting complexes, nutrients, phycobiliproteins

To optimize the utilization of photosynthate and avoid damage that can result from the absorption of excess excitation energy, photosynthetic organisms must modify the synthesis and activities of components of the photosynthetic apparatus in response to environmental cues. When cyanobacteria experience nutrient limitation they degrade their phycobilisomes and dramatically reduce the rate of photosynthetic electron flow, primarily as a result of decreased PS II activity. We have developed procedures to identify genes that are differentially expressed upon transfer of cyanobacteria to nutrient limiting growth conditions and have used screens to isolate mutants defective in their ability to respond to nutrient limitation. Complementation of the mutant strains has led to the identification of three genes, nblA, nblR and nblC, important for the acclimation to nutrient limitation. The NblA protein might tag the phycobilisome for degradation or direct protease activity toward phycobilisome polypeptides. The NblC protein might be involved in removing the chromophore from phycobiliproteins prior to their degradation. The NblR protein is a response regulator critical for acclimation to both nutrient limitation and high light. The importance of these genes is not restricted to acclimation to nutrient limitation or high light; they appear to be part of a control pathway that precisely tunes the photosynthetic apparatus to the constantly fluctuating conditions of the environment.

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872_3946.

LIGHT HARVESTING AND PHOTOPROTECTION BY CAROTENOIDS: STRUCTURE-BASED CALCULATIONS FOR THREE DIFFERENT PHOTOSYNTHETIC ANTENNA SYSTEMS

Ritz T., Damjanovic A., Schulten K.

Beckman Institute
University of Illinois
405 N Mathews Ave
Urbana, IL 61801

Keywords:

bacterial photosynthesis, carotenoids, electronic excitation transfer, light harvesting complexes, photoprotection

Carotenoids contribute to light harvesting and protect photosynthetic systems through triplet quenching. Both functions involve excitation transfer between carotenoids and chlorophylls (Chls). This transfer has been investigated computationally on the basis of the x-ray structures for LH-II of Rs. molischianum and Rps. acidophila and for peridinin-chlorophyll-protein (PCP) of A. carterae. The electronic states of Chls and carotenoids have been described by means of semiempirical PPP-SCF-CI calculations. In case of the B850 band in LH-II involving a ring of tightly coupled bacteriochlorophylls (BChls) this description was extended to account for the excitonic character of the respective excitations. The transfer rates between the carotenoid and Chl states have been determined. The results show that efficient singlet excitation transfer involves the optically forbidden S1 state of carotenoids. Transfer through the S1 state cannot occur through the Forster (dipole) mechanism, but instead involves higher multipoles (Coulomb mechanism) and to a smaller extent electron-exchange (Dexter mechanism). In the case of triplet excitation transfer, based solely on the Dexter mechanism, the calculated transfer times are short enough to ensure efficient photoprotection of all the Chls in PCP and of most of the BChls in LH-II. The remaining BChls are not directly protected, but can transfer triplet excitation efficiently to one of the protected BChls.

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873_3756.

OPTICAL PROPERTIES OF BACTERIOCHLOROPHYLLS IN RHODOBACTER CAPSULATUS LH2 COMPLEX ALTERED BY COMBINATORIAL MUTAGENESIS

Hu Q.^*, SturgisJ.N., Robert B., Niederman R.A.<sup>*

*</sup>Dept. of Mol. Biol. & Biochem., Rutgers Univ., Piscataway, NJ, 08855-1059, U.S.A.
SBPM/DBCM, CEA & URA 1290 CNRS, Centre d'etudes de Saclay, 91191, Gif-sur-Yvette, France.

Keywords:

Bchl, circular dichroism, fluorescence, light harvesting complexes, Raman spectroscopy

Two classes of R. capsulatus LH2 (B800-850) mutants obtained by combinatorial mutagenesis of the C-terminal half of the b-apoprotein were examined: a pseudoLH2 (pLH2) class with 14-nm blue shifts in the 850-nm peak; and a pseudoLH1 (pLH1) class lacking an 800-nm peak and with B850 red shifts of up to 30 nm. In all strains, both the H-bonding environment of B850 BChls and their conformation and liganding were intact. In contrast, the intensity of the CD spectra of pLH1 complexes was considerably reduced, consistent with alterations in the relative orientation of B850 pigments and in interactions between them. Elevated fluorescence polarization over the red wing of the B850 band in the pLH2 complexes indicated a reduction of exciton mobility within the ring of BChl molecules. Possible structural alterations governing these spectral properties will be discussed.

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877_28548.

ORIGIN OF THE EXTREME LONGWAVE CHLOROPHYLL FORM OF THE PHOTOSYSTEM I TRIMERIC COMPLEX OF SPIRULINA

Karapetyan N.V.¹, Dorra D.², Holzwarth A.R.², Kruip J.³, Röegner M.<sup>3

1</sup>A.N.Bakh Institute of Biochemistry, 117071 Moscow, Russia;
²Max-Planck-Institut fur Strahlenchemie, 45470 Mulheim;
³Ruhr-Universitat Bochum, 44780 Bochum, Germany

Keywords:

cyanobacteria, energy transduction, photosystem 1, pigment-protein complexes, trimer

PSI trimers of cyanobacterium Spirulina contain the longwave chlorophyll absorbing at 735 nm (Chl735) and emitting at 760 nm (F760). Following data indicate that Chl735 originates from the interaction of Chl molecules bound with different PSI monomeric subunits within a trimer. (i) Deconvolution of the 77K absorption spectrum of trimers shows that each monomer in the PSI trimer contains two molecules of Chl735. (ii) PSI trimers reconstituted from the monomers within the liposomes reveal F760 emitted by Chl735 that appeared as a result of interaction of Chls located on monomeric subunits in the course of the trimerization. (iii) Incubation of detergent-free membranes of Spirulina or reconstituted in liposomes PSI trimers at "high salt" conditions decreases the F760 intensity because of transfer of trimers into monomers and diappearance of Chl735.

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879_15764.

EXCITATION WAVELENGTH DEPENDENT SPECTRAL EVOLUTION IN RHODOBACTER SPHAEROIDES R-26 REACTION CENTERS AT LOW TEMPERATURES: THE Q_X TRANSITION REGION

Lin S., Jackson I., Taguchi A.K.W., Woodbury N.W.

Dept. of Chemistry & Biochemistry, Center for the Study of Early events in Photosynthesis, Arizona State University, Tempe, AZ 85287-1604, U.S.A.

Keywords:

bacterial reaction center, electron transfer, electronic excitation transfer, ultrafast spectroscopy

Femtosecond transient spectra of R-26 reaction center in the Q_X transition region have been measured at 15K. Study has been focused on excitation wavelength and excitation intensity dependence of the formation of charge separated states on the A and B side of the reaction center. Results show that when the excitations were directly into the bacteriopheophytin bands, prompt bleaching appeared at both 532 and 545 nm. The 545 nm band grows continuously on the picosecond time scale forming charge separated state of P⁺H_A^-, while the 532 nm band diminished within the first picosecond presumably transferring the excitation energy to P. Excitations at 590, 750, 760, 800, and 880 nm with low excitation intensity show essentially no difference on the formation of the final charge separated state. Excitation intensity dependence study was carried out with 590 nm excitation. When excitation intensity was increased, a band peaking at 534 nm was formed. The formation time of this 534 nm band is similar to that of the 545 nm band. It is suggested that the long-lived bleaching is originated from the formation of H_B^-. The mechanism for the formation of charge separated state on the B side is most likely due to the double-photon excitation which pumps P to a higher energetic state so that it can overcome the energy barrier to form charge separated state along the inactive branch in the photosynthetic reaction centers.

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880_15073.

DIRECTED MUTATIONS OF orfs IN PURPLE PHOTOSYNTHETIC BACTERIA REVEAL NEW PHOTOSYNTHESIS GENES

Beatty J.T.

Dept. of Microbiology & Immunology, University of British Columbia, 300-6174 University Blvd, Vancouver, BC, Canada V6T 1Z3

Keywords:

assembly, bacterial photosynthesis, biogenesis, gene expression, genetic manipulation

The theme of this presentation is that genome sequences are a rich source of material for experiments on photosynthesis, and that such studies sometimes provide new insights into fundamental processes that were previously thought to be well understood.
Open reading frames (orfs) of unknown function have been discovered by DNA sequencing carried out by several laboratories. In purple bacteria, these orfs are typically located near and sometimes co-transcribed with genes that encode proteins of the reaction center, light-harvesting complexes, or pigment biosynthesis. We have used 'reverse genetics' to systematically disrupt some of these cloned orfs, to evaluate the phenotypes of Rhodobacter capsulatus strains in which the wild type chromosomal orf is replaced by the corresponding disrupted allele. This approach has revealed functions in photosynthetic physiology for such orfs (now known to be genes) as pufX, pucC and lhaA (previously known as orf1696). These results will be summarized and new data, which reveal that additional orfs encode proteins required for optimal photosynthetic growth, will be presented.
It seems that many of these newly discovered genes encode proteins involved in assembly or functional organization of the photosynthetic apparatus.

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882_27651.

STRUCTURAL CHARACTERIZATION OF CAROTENOID PROTEINS ISOLATED FROM SPIRULINA MAXIMA

Kerfeld C.A., Krogmann D.W., Yeates T.O.

UCLA-DOE Laboratory of Structural Biology, Los Angeles, CA

Keywords:

carotenoids, crystallization, cyanobacteria, light harvesting complexes, photoprotection

A 34 kDa orange carotenoid protein (OCP) from the cyanobacterium Arthrospira maxima has been characterized and crystallized. OCP most likely functions in either photo-protection or in light-harvesting. The first 29 amino acids of the purified A. maxima OCP, determined by N-terminal amino acid sequencing, show a high degree of homology with the N-terminus of slr 1963 gene product from Synechocystis PCC 6803. OCP comprises two self repeating segments and contains a putative ATP/GTP binding site and four protein kinase C phosphorylation sites. Crystals of the A. maxima OCP have been grown and native diffraction data have been collected to 2.5Å resolution. A screen for heavy atom derivatives of the crystals is underway. The crystals have a striking optical property. They grow as elongated prisms that, when viewed through a single polarizing filter, are either orange or perfectly clear depending on the orientation of the crystal relative to the direction of polarization. The naturally occurring A. maxima OCP gives rise by proteolysis of the N- and C-termini to a red carotenoid protein of approximately 16 kDa. Microcrystals of this protein have been obtained.

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884_11318.

IS P680⁺ THE DOMINANT CAUSE OF PHOTOINACTIVATION OF PSII IN PLANTS?

Anderson J.M., Park Y.-I., Chow W.S.

Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia

Keywords:

light stress, P680, photoinactivation, photoprotection, photosystem 2, triplet P680

Recently, we proposed a unifying model for PSII photoinactivation in vivo that depends on the generation and/or maintenance of the primary radical pair, P680⁺Pheo^- and the ways in which charge recombination occurs^1,2. Enhanced radical pair concentration may lead to damage to D1 protein in leaves via (i) P680⁺ or (ii) singlet O₂ generated from triplet P680. We propose that P680⁺ is the more likely cause of PSII nonfunctionality in leaves for the following reasons:(1)slower electron transfer from secondary donor to P680⁺ than from Pheo^- to Q_A^-;(2)triplet P680 quenching is increased by two orders of magnitude, in the presence of Q_A^-;(3)³P680 is not observed in leaves, only in isolated PSII membrane fragments or reaction centres;(4)O₂ may not be able to interact with triplet P680 in vivo?
1. Anderson J.M., Park Y.-I. & Chow W.S. (1997) Physiol. Plant. 100: 214-223
2. Anderson J.M. et al. (1998) Photosyn. Res. in print

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886_25501.

EFFECTS OF HIGH TEMPERATURES ON PHOTOSYNTHETIC SYSTEMS

Satoh K., Yamane Y., Emi T., Kashino Y., Koike H.

Dept. Life Sci., Fac. Sci., Himeji Inst. Tech., Harima Science Garden City, Hyogo, 678-1297 Japan

Keywords:

cyanobacteria, fluorescence, heat stress, photosystem 2, phycobiliproteins

High-temperature-induced fluorescence Fo increases and Fm decreases were studied in higher plants and cyanobacteria. In higher plants, the Fo increase was attributed to irreversible detachment of LHCII from the reaction center of Photosystem (PS) II and partly reversible inactivation of PS II (1,2). In cyanobacteria, the Fo increase was found to be due to irreversible release of phycobilisomes from the reaction center complexes of PS II and to partly reversible inactivation of PS II. The Fm decrease was due to inhibition of oxygen evolution. However, Fm levels in the presence of DCMU and NH₂OH were also decreased by high-temperature treatments, showing that the inactivated PS II reaction center complexes become less fluorescent. We will also show that there is another mechanism which causes an increase in the Fo level at high temperatures.
1) Yamane et al. (1997) Photosyn, Res. 52: 57-64.
2) Yamane et al. (1998) Photosyn. Res. In press.

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893_3308.

THE RICE IMPORTIN a1: LIGHT-REGULATED EXPRESSION AND MEDIATION OF NUCLEAR PROTEIN IMPORT IN VITRO BY RECOMBINANT PROTEIN

Jiang C.J., Matsuki R., Shoji K., Inagaki N., Yamamoto N.

Photosynthesis Lab., Plant Physiology Dept., National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, 305-8602, Japan

Keywords:

protein import, protein targeting, protein translocation, proton transport, importin

Nuclear imR>
Nuclear import of most nuclear proteins is initiated by recognition of nuclear-localization signal (NLS) by importin a. A importin a homologue was isolated from rice plant and was tentatively named rice importin a1. Recombinant rice importin a1 protein bound specifically to plant NLSs and, in conjunction with other vertebrate nuclear transport factors, mediated import of NLS-proteins into the nucleus in digitonin-permeabilized HeLa cells. These data provide strong and direct evidence suggesting that the rice importin a1 functions as a component of the NLS-receptor in plant cells. RNA blot analysis revealed that transcription of the rice importin a1 is down-regulated by light in the leaves. Possible involvement of the rice importin a1 in photomorphogenesis-related gene regulation will be discussed.

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897_13678.

CLONING AND EXPRESSION OF GENES ENCODING CYTOCHROME C-550 AND A 12 kDa PROTEIN, TWO EXTRINSIC PROTEIN OF PS II, FROM A RED ALGA CYANIDIUM CALDARIUM

Ohta H.¹, Okumura A.¹, Katoh T.¹, Shen J.-R.², Kamo M.³, Enami I.<sup>1

1</sup>Dept. Biol., Fac. Sci., Sci. Univ. of Tokyo, Tokyo 162-8601,
²RIKEN Harima Inst., Hyogo 679-5143,
³Res. Inst. for Biosci., Sci. Univ. of Tokyo, Chiba 278-8510, Japan

Keywords:

O2 evolution, OEC proteins

Cytochrome (cyt) c-550 and a 12 kDa protein are members of the four extrinsic proteins of the oxygen-evolving PS II complex from a red alga, Cyanidium caldarium [Enami et al. (1995) B.B.A. 1232, 208-216]. In this study, we cloned genes encoding these proteins from the red alga and expressed them in E. coli. The cyt c-550 gene encodes 171 aa, of which, the first 35 aa were served as a leader sequence required for transport into lumen of the thylakoids. The mature apoprotein of cyt c-550 contains 136 aa with a calculated molecular mass of 14799 Da. The cDNA encodes a putative polypeptide of 154 aa with a calculated molecular mass of 16714 Da. The 12 kDa protein involves two characteristic transit peptides for envelope and thylakoid transfer signals. Cleavage of these transit peptides results in a mature protein of 93 aa with a calculated molecular mass of 10513 Da. These sequence features indicate that cyt c-550 is encoded in chloroplast DNA and the 12 kDa protein is encoded in nuclear DNA. Recombinant proteins were expressed in E. coli using the T7 polymerase pCAL-n-EK expression system. Reconstitution experiments with these expressed proteins were examined.

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899_27933.

QUANTITATIVE ANALYSIS OF INTRATHYLAKOID pH AND XANTHOPHYLL CYCLE EFFECTS ON PSII Chl a FLUORESCENCE LIFETIMES AND INTENSITY

Gilmore A.M.¹, Shinkarev V.P.², Govindjee², Hazlett T.L.<sup>2

1</sup>ANU/RSBS, Canberra, ACT 0200 Australia
²UIUC, Urbana, IL 61801 USA

Keywords:

coupling/uncoupling, energy dissipation, fluorescence lifetimes, pH-gradients, xanthophyll cycle

The xanthophyll cycle and intrathylakoid pH contribute to a vital photoprotective capacity by dissipating excess absorbed light energy. Based on time-resolved and pulse-amplitude modulation (PAM) fluorescence data, we model the influence of the intrathylakoid pH and the xanthophyll cycle on the Chl a fluorescence lifetime distributions and intensity. The model assumes (1) a specific binding site for zeax- (Z) (or antheraxanthin, A) in the PSII minor antennae (CP24, 26 or 29); (2) binding site activation by low intrathylakoid pH (pK_a=4.5); (3) one Z or A molecule binds to the active site, and (4) binding 'switches' a PSII unit to a decreased fluorescence lifetime and intensity by forming an exciton trap with a rapid heat dissipation rate constant. The model explains both the fraction of the 0.5 ns lifetime distribution as a function of the [Z] (and [A]) and intrathylakoid pH and the dependence of the intensity on the 0.5 ns fraction. Quantitative model analysis yields an association constant per PSII for Z (or A) ranging from 1.0 to 2.6 in several higher plant species.
We thank NSF DBI 96-02240.

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900_26364.

CHARACTERIZATION OF THE AMINO ACID SUBSTITUTION F363R IN THE HYDROPHILIC LOOP E DOMAIN OF CP47

Clarke S.M., Eaton-Rye J.J.

Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand

Keywords:

cyanobacteria, OEC proteins, photosystem 2, CP47

Amino acid substitutions of Asn-361, Phe-362 and Phe-363 have been introduced into the chlorophyll a-binding protein CP47. The mutants N361K and F362R were found to grow photoautotrophically and evolve oxygen at rates similar to the control, however for F363R the doubling time was extended from 12 hours to 26 hours and this strain was found to be readily photoinactivated. In chloride-limiting conditions the photoautotrophic growth was 48 hours for F362R with both the control strain and N361K having a doubling time of 20 hours. The mutant strain F363R was unable to sustain photoautotrophic growth in chloride-limiting conditions. The phenotype of the mutants was further investigated following the selective removal of the extrinsic proteins of the water-oxidizing complex. It was found that F363R was unable to grow photoautotrophically in the absence of either the PSII-O or PSII-V (cyt c-550) proteins, but F362R was able to support photoautotrophic growth in the absence of PSII-O. However, F362R exhibited a photoautotrophic doubling time that was twice that observed for either N361K or the control in the absence of PSII-V. The PSII-U (12 kDa) protein was also found to be important for the stability of the F363R mutant strain. These results demonstrate that Phe-363 is important in PSII assembly and/or stability and indicate that the absence of PSII-V is more detrimental than the absence of PSII-O.

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910_22761.

CemA HOMOLOGUE IN CYANOBACTERIA (PxcA) INVOLVED IN PROTON EXCHANGE

Sonoda M., Katoh H., Vermaas W., Ogawa T.

Bioscience Center, Nagoya University, Nagoya 464-8601, Japan, Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, AZ 85287-1601, USA

Keywords:

anions, bicarbonate, chloroplast, chloroplast genes, CO2 concentrating mechanisms, CO2 uptake, cyanobacteria, electron electron transport, molecular biology, mutants, photosystem 1, photosystem 2, proton transport

PxcA is a cyanobacterial gene homologous to CemA in chloroplast genomes and is involved in light-induced proton exchange. PxcA was located in the cytoplasmic membrane and was similar in size to CemA of Marchantia and Chlamydomonas but was larger than CemA of higher plants. Inactivation of PxcA abolished light-induced proton extrusion. In the presence of 2,5-dimethyl-p-benzoquinone, the net proton extrusion by Synechocystis PCC 6803 ceased after a minute of illumination and a postillumination influx of protons was observed. This suggested that the PexA-dependent proton extrusion is accompanied by the influx of protons. Measurement of proton extrusion in a photosystem(PS)I-deletion mutant and a PSII-deletion mutant indicated that PSII is a major factor in light driven proton extrusion. At pH 6.5, PxcA^- mutant did not take up CO₂ and NO₃^- at 100 µM Na⁺ and showed very low activity of CO₂ uptake even at 15 mM Na⁺. PxcA-dependent proton exchange may have a role in charge and pH homeostasis during uptake of CO₂, HCO₃^- and NO₃^-.

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911_23966.

DIBUCAINE COMPETES WITH THE DpH INDICATOR 9-AMINOACRIDINE FOR UPTAKE IN THYLAKOID MEMBRANES WITHOUT UNCOUPLING

Yamasaki H.^1,2, Gilmore A.M.<sup>1

1</sup>PBE, RSBS, ANU, ACT 0200, Australia
²Fac. Sci., Univ. Ryukyus, Nishihara, Okinawa 903-0213, Japan

Keywords:

ascorbate, coupling/uncoupling, energy dissipation, pH-gradients, xanthophyll cycle

Dibucaine (DC) has been interpreted to have unique uncoupling effects on the DpH and xanthophyll cycle-dependent energy dissipation. However, the uncoupling mechanism was not understood. Detailed analyses of the inhibitory effects of DC in thylakoids suggest that the apparent uncoupling is really due to competitive uptake between DC, 9-aminoacridine (9AA) and other lipophilic amines. Low concentrations (below 2 QM) of 9AA and DC exhibit similar fluorescence quenching due to DpH-dependent uptake into thylakoids. Both amines also exhibit mutual competitive uptake inhibition. The fluorescence yields of both in aqueous solution are strongly quenched by sodium ascorbate. Both 9AA and DC similarly inhibit deepoxidation, electron transport and PSII energy dissipation. Although, 9AA is 2 to 5 times more potent than DC in all cases, there are few fundamental differences in the effects of these molecules. We conclude that DC does not possess a unique uncoupling effect as previously suggested.

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915_28262.

PROTEIN TARGETING TO THE CHLOROPLAST OUTER MEMBRANE

Li H.-M., Chen L.-J., Tu S.-L., Su Y.-S., Tsay L.-Y.

Institute of Molecular Biology
Academia Sinica
Taipei 11529
TAIWAN

Keywords:

chloroplast, protein import

Nuclear-encoded chloroplastic proteins can be roughly divided into two groups. Proteins in the first group are synthesized as higher molecular weight precursors with N-terminal extensions called transit peptides. This group of proteins includes all proteins destined for the interior of chloroplasts and two proteins destined for the outer envelope membrane. The second group of proteins consists of the rest of outer membrane proteins identified so far. These outer membrane proteins are synthesized at their mature size in the cytosol without a cleavable transit peptide. From the second group of proteins, we have chose a protein, OEP14, to study the mechanism of protein import to the outer membrane. The outer membrane targeting/insertion signal of OEP14 has been localized to the N-terminal first 30 amino acids of the protein. This signal shares some features with the ER-targeting signal peptides but is nevertheless specific for the chloroplastic outer membrane. The binding and insertion of OEP14 to the outer membrane require a trypsin-sensitive and an NEM-sensitive component, respectively.

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916_13706.

NEWLY IDENTIFIED GENES IN TOBACCO PLASTID GENOME

Tsudzuki T.¹, Tsudzuki J.², Nakashima K.², Wakasugi T.³, Hirose T., Sugita M., Sugiura M.

¹Data Processing Center, Aichi-Gakuin Univ., Nisshin 470-0195; ²School of Life Studies, Sugiyamajogakuen Univ., 464-8662; ³Dept. of Biology, Toyama Univ., Toyama 930-0887; Center for Gene Research, Nagoya Univ., Nagoya 464-8602, JAPAN

Keywords:

chloroplast genes, higher plants, RNA editing, sequence analysis

Twenty four new genes have been identified in tobacco chloroplast genome since the first publication of complete nucleotide sequence and gene map in 1986. Those comprise one small RNA gene (sprA) and 23 protein-coding genes (psaC,I,J, psbI,J,K,L,M,N,T, petG,L, ndhG,H,I,J,K, rpl23,36, rpoC2, matK, accD and clpP).
Our group found some sequence errors after the publication and we have examined errors reported and suggested by other groups by our re-sequencing. The numbering system for the sequence was changed according to these corrections. The genome size is now 155,939 bp long, consisting of LSC of 86,686 bp, SSC of 18,571 bp and IR of 25,341 bp each.
Details for newly identified genes and corrections will be presented along with RNA editing sites found on the genome.

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917_30102.

LOCALIZED POLY-b-HYDROXYBUTYRATE ACCUMULATION IN CYANOBACTERIA

Miyake M., Kataoka K., KhatipovE.A., Shirai M., Kurane R., Asada Y.

National Institute of Bioscience and Human-Technology, Higashi 1-1, Tsukuba, Ibaraki 305, and Division of Biotechnology, Ibaraki University, Ami, Inashiki, Ibaraki 300-03, Japan

Keywords:

algae, bioreactors, biosynthesis, biotechnology, bleaching, cyanobacteria, environmental stress, membrane protein, membrane structure, microalgae, thylakoid membranes, ultrastructure, poly-b-hydroxybutyrate

In cyanobacteria, poly-b-hydroxybutyrate (PHB) is considered as a carbon storage. Recently, we reported the posttranslational regulation of PHA synthase depending on C/N ratio (1). In this congress, we will report a localized accumulation of PHB granules at the photosynthetic membranes.
PHB granules isolated from some cyanobacteria that are originally capable of PHB accumulation were associated with the thylakoid membranes, and the PHA synthases were located at ynthases were located at the membrane fractions. PHB granules from a photosynthetic bacterium and a genetic engineered cyanobacterium which expressed PHA synthesis genes of Alcaligenes eutrophus were not associated with the photosynthetic membranes, and PHA synthases were soluble. PHB granules in cyanobacteria may be accumulated at the thylakoid membranes.
(1) Miyake, M., et al. (1997) J. Bacteriol. 179, 5009-5013.

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918_30760.

TOPOLOGICAL ANALYSIS OF PS II REACTION CENTER USING MONOCLONAL ANTIBODIES

Tomo T., Ogawa K.^*, Inoue Y., Shen J.-R.

RIKEN Harima Inst., Hyogo 679-5143, Japan, ^*RIKEN Animal Res. Cen., Saitama 351-0198, Japan

Keywords:

antibody, D1 protein, D1/D2-cyt-b-559, photosystem 2, Qb

In order to analyze the topology of PSII reaction center (RC) proteins, we constructed monoclonal antibodies (Mab) using PSII RC preparation as an antigen. Four types of Mabs with high reactivity against D1, D2 and cytochrome b-559 a subunit, respectively, were obtained. Among these, the anti-D1 Mab had a high reactivity against denatured thylakoids, BBY, PSII core and RC preparations. This Mab also recognized the D1 protein in native PSII core and RC, but not in native thylakoids, BBY membranes and a partially purified PSII particle retaining the Q_B-binding site. Using synthesized peptide fragments, the epitope of the anti-D1 Mab was determined to be 235E-240G of D1, which is located in the close vicinity of the Q_B-binding site. Since thylakoids and PSII preparations not recognized by the anti-D1 Mab all retained the Q_B-site, whereas the PSII core and RC did not, we conclude that the epitope regio around the Q_B-binding site originally had a compact structure not accessible by the Mab; upon destruction of the Q_B-site, this region became exposed to the outer surface, resulting in the easy access by the Mab.

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919_2273.

HEAT-INDUCED DEPHOSPHORYLATION OF PHOTOSYSTEM II CORE PROTEINS

Rokka A.^1,2, Vener A.V.², Aro E.-M.¹, Andersson B.<sup>2

1</sup>Department of Biology, University of Turku, FIN-20014, Turku, Finland
²Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-10691 Stockholm, Sweden

Keywords:

D1 protein, D2 protein, heat stress, photosystem 2

Studies on the temperature dependence of thylakoid protein dephosphorylation revealed a specific heat-induced acceleration of dephosphorylation of photosystem II (PSII) core phosphoproteins. A rise in the temperature up to 42^oC induced nearly a 20 fold increase in the dephosphorylation rate of the D1 and D2 reaction centre proteins in isolated spinach thylakoids, while a 3 to 4 fold enhancement of LHCII proteins dephosphorylation was observed. In vivo studies on spinach, by using a phosphothreonine antibody, confirmed an almost complete dephosphorylation of the D1 and D2 proteins during 10 min of the 'heat-shock'. Detailed characterisation of the dephosphorylation reactions in vitro disclosed two major factors for heat-induced D1/D2 dephosphorylation: firstly, fast lateral migration of PSII cores from appressed to non-appressed thylakoid regions and secondly, dissociation of the thylakoid cyclophilin-like protein, TLP40, from the inner thylakoid membrane surface. The implications of heat-induced dephosphorylation of PSII proteins will be discussed in biochemical and physiological terms.

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923_3958.

QUENCHING OF ENERGY TRANSFER IN CHLOROSOMES FROM CHLOROFLEXUS BY THE ADDITION OF SYNTHETIC QUINONES

Tokita S., Frigaard N.-U., Hirota M., Shimada K., Matsuura K.

Dept. of Biology, Tokyo Metropolitan Univ., Minamiosawa, Hachioji, Tokyo 192-0397, Japan

Keywords:

Bchl, chlorosomes, fluorescence, quenching, quinones

Chlorobiumquinone has been suggested to have a role in the redox-dependent regulation of energy transfer in Chlorobium chlorosomes. To study the quenching mechanism, a number of synthetic quinones were tested as quenchers of BChl c fluorescence and BChl c to BChl a energy transfer in chlorosomes from Chloroflexus aurantiacus. Among the naphthoquinones which were found to have fluorescence quenching activity in the oxidized form, 5-hydroxy-1,4-naphthoquinone had higher quenching activity than 2-methyl-1,4-naphthoquinone. Phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone) had much less quenching activity. Among the anthraquinones, 1,4-dihydroxy-anthraquinone or 1,5-dihydroxy-anthraquinone had much higher quenching activity than 2-methylanthraquinone. The higher quenching activity in the less hydrophobic quinones is consistent with the suggestion that chlorobiumquinone (which is a 1'-oxonaphthoquinone) has much higher quenching effect than menaquinone in chlorosomes.

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934_14801.

PHOTOSYSTEM II ELECTRON TRANSPORT IN TRANSGENIC TOBACCO

Baldewijns L., Valcke R.

Limburgs Universitair Centrum, Dept SBG, Universitaire Campus, B-3590 Diepenbeek, Belgium

Keywords:

electron transport, hormones, photosystem 2, quinones, transgenic plants

The structure and function of the photosynthetic apparatus is under the regulatory control of endogenous phytohormones. Cytokinins and ethylene for instance affect the gene transcription and translation. In this study, transgenic plants wich contain a supplementary ipt-gene resulting in a higher endogenous content of cytokinins on the one hand and transgenic plants containing a sam1-gene which affects the SAM-metabolism on the other hand are used. The photosystem II electron transport capacity was measured on isolated thylakoid membranes using polarographic and spectrophotometric techniques. Using combinations of different electron donors (H₂O, DPC) and electron acceptors (several quinone derivatives, K₃Fe(CN)₆), the capacity of the whole FSII-complex or parts of it was measured. Results indicate clearly that the electron transport capacity of the FSII-complex is affected by the endogenous level of the cytokinins and by the level of expression of the sam1-gene resulting in a changed methionine metabolism (change in ethylene production). Moreover, the results depend on the type of quinone used as electron acceptor indicating different structural arrangement of the PSII-complex.

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939_9731.

ACTIVITIES AND KINETICS OF ELECTRON TRANSFERCTRON TRANSFER IN A REACTION CENTER COMPLEX FROM THE GREEN SULFUR BACTERIUM CHLOROBIUM TEPIDUM

Seo D., Kusumoto N., Sakurai H., Brettel K.^*, Sétif P.^*

Dept. of Biology, Sch. of Education, Waseda Univ., Nishiwaseda, Shinjuku, Tokyo 169-8050, JAPAN,
^*DBCM, CE de Saclay, Gif-sur-Yvette 91191, FRANCE

Keywords:

cytochromes, ferredoxins, green sulfur bacteria, reaction centers, spectroscopy

A purified RC complex from C. tepidum was composed of 5 kinds of polypeptides: 65 kDa (RC core), 41 kDa (FMO protein), 32 kDa (Fe-S protein), 23 kDa (Cyt C), 18 kDa (function unknown). In the presence of ferredoxin (Fd) from this bacterium and from spinach, the RC complex photoreduced NADP⁺ through FNR from spinach at 313 and 81 mmoles/mg BChl a/hr, respectively.
Multi flash-induced optical kinetic studies at room temperature of purified RC from C. tepidum indicated: The charge recombination rate between the terminal Fe-S center and (P840/Cyt c551)⁺ was considerably slowerer (t_1/2 > 400 ms) than that between the terminal Fe-S center and P700⁺ (30-40 ms) in PSI. The flash-generated P840⁺ was rereduced by Cyt c with t_1/2 of 50-120 msec, and we could not observe any t_1/2 = 1-10 msec decaying components. After the fourth flash, the charge recombination rate was already rapid (t_1/2 = about 20 ns) indicating that there are only three electron acceptors which can stably hold electrons in msec time range.

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948_8628.

RUBISCO: KINETIC CONSTANTS OF PARTIAL REACTIONS AND THEIR RELATION TO V_MAX AND K_M

Viil J.

Institute of Experimental Biology, Harku, Harjumaa, EE3051 Estonia

Keywords:

CO2 uptake, enzymes, modelling, regulatory processes, Rubisco

As calculated from a mathematical model of Rubisco, the same values of V_max and K_M may be obtained with different values of kinetic parameters of partial reactions of carboxylation: rate constants of binding of RuBP and CO₂, rate constants of relieving the products, and the concentration of competent carboxylation centers. An attempt is made to elucidate under which conditions their relations are the most unequivocal. V_max is determined mainly by the rate constant of the release of the product and the concentration of operating carboxylation centers. Rate constants of binding of substrates start to influence V_max only if they decline by an order of magnitude or more from their ordinary values. Relations between K_M for RuBP and CO₂ and the parameters of partial reactions are more complicated.

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954_12670.

EFFECTS OF GROWTH CONDITIONS AND PARAQUAT TREATMENT ON ANTIOXIDATIVE ENZYMES IN THE GREEN ALGA CHLAMYDOMONAS REINHARDTII

Fujiwara T., Watabe Y., Sakurai H.

Dept. of Biology, Sch. of Education, Waseda Univ., Nishiwaseda, Shi