The NIBB Seminar

Regulatory Mechanism of Photosynthesis

Organizer: Kintake Sonoike and Norio Murata

April 2 - 3, 1998

Sponsored by National Institute for Basic Biology


The NIBB seminar "Regulatory Mechanism of Photosynthesis" is being organized to promote free exchange of information and to provide a forum for in-depth discussion of research. Now that almost all the structural proteins involved in the photosynthetic electron transport chain have been identified and their genes sequenced, we have the really good static image of photosynthetic apparatus. We are now at the position to try to attack the dynamic aspects of the photosynthesis. The aim of this seminar is to bring together researchers and students working on any aspect of the regulatory mechanism of photosynthesis. Particular attention is given to the regulation, assembly and degradation of the photosynthetic apparatus and auxiliary enzymes that support photosynthesis. Fortunately, leading scientists from Germany, Sweden, Israel, U.S.A. and Russia who are studying such topics are visiting Japan. We are inviting these foreign scientists and nine Japanese scientists as speakers and organizing this seminar on this occasion.


April 2 (Thursday) Afternoon

13:00-13:10 Opening Remarks by K. Sonoike

13:10-13:45 R.G. Herrmann (Ludwig-Maximilians-Universitat)

"Chloroplast thylakoid membranes: a paradigm for biogenetic and evolutionarycomplexity"

13:45-14:20 M. Ikeuchi (University of Tokyo)

"A novel gene, pmgA, specifically regulates photosystem stoichiometryin the cyanobacterium Synechocystis sp. PCC 6803 in response to high light"

14:20-14:55 A. Tanaka (Hokkaido University)

"Interconversion of chlorophyll a and chlorophyll b"

14:55-15:15 Discussion related to above presentation

(may allow short presentations by attendants)

15:15-15:35 Coffee Break

15:35-16:10 T. Kuwabara (University of Tukuba)

"Self-degradation of dithiothreitol-sensitive tetrameric protease(polyphenol oxidase) mediated by active oxygen species"

16:10-16:45 Y. Takahashi (Okayama University)

"Assembly of the photosystem I complex"

16:45-17:20 K. Satoh (Okayama University)

"Carboxyl-terminal processing of the precursor D1 protein in the photosystemII reaction center by a nuclear-encoded endopeptidase"

17:20-17:40 Discussion related to above presentation

(may allow short presentations by attendants)

18:00- Informal Reception

April 3 (Friday) Morning

9:00- 9:35 B. Andersson (Stockholm University)

"An immunophilin-like protein in the thylakoid lumen: Its role forprotein turn-over and protein phosphorylation"

9:35-10:10 S. Shigeoka (Kinki University)

"Scavenging system of active oxygen species in higher plants"

10:10-10:45 K. Asada (Fukuyama University)

"Photoreduction of dioxygen to water in PSI has dual functions"

10:45-11:20 I. Ohad (Hebrew University)

"Mechanism of the reversible light activation of thylakoid proteinphosphorylation"

11:20-11:40 Discussion related to above presentation

(may allow short presentations by attendants)

12:00-13:00 Lunch

April 3 (Friday) Afternoon

13:00-13:35 N. Murata (NIBB)

"Fatty acid desaturases are regulators for the stability of photosyntheticmachinery via the desaturation of fatty acids"

13:35-14:10 H.B. Pakrasi (Washington University)

"Manganese transport in cyanobacteria"

14:10-14:45 S. Shestakov (N. Vavilov Institute of General Genetics)

"Two divergent GAPDH genes play a different role in carbon metabolismof the cyanobacterium Synechocystis 6803"

14:45-15:20 K. Sonoike (University of Tokyo)

"Photoinhibition of Photosystem I and its relation to chilling sensitivityin plants"

15:20-15:40 Discussion related to above presentation

(may allow short presentations by attendants)

15:40-15:50 Closing Remarks

*Each speaker gives 25 minutes presentation, and has 10 minutes for discussion.


Chloroplast thylakoid membranes: a paradigm for biogenetic and evolutionary complexity

Reinhold Herrmann

Botanisches Institut, Ludwig-Maximilians-Universitat, Menzinger Str. 67, D-80638 Munchen, Germany

Thylakoid membranes are unique biomembranes capable of converting solar energy into utilizable high-energy chemical metabolites in the forms of ATP and NADPH. They are lipid bilayers into which different supramolecular protein complexes are intercalated. The intriguing feature of chloroplast thylakoids is their dual genetic origin which implies a coordinated delivery of the components from two sources of protein synthesis in the cytosol and plastid, respectively. The establishments of chloroplasts is embedded into the evolution of the genetic system of the eukaryotic cell which was accompanied by an enormous restructuring of genetic material of the endosymbionts that formed that cell. It included loss, gain, and complex intracellular transfer of genes. The result is an integrated genetic system (rather than semiautonomous organelles) which in its entirety has to be regulated in time, quantitatively, in multicellular organisms also in space.

The study of chloroplast thylakoid biogenesis uncovered that regulation occurs at all levels and with unexpected complexity and subtlety both for matter influx (e.g., greening model) and efflux (maintenance, acclimation, repair etc.). It included the generation of novel regulatory circuitries and biogenetic mechanisms that can, at least in part, be understood from the history of the cell, as well as often different compounds for input and output processes. For instance, the former included promoter changes of the translocated genes, gain and changes of transit peptides etc., the latter kinases, phosphatases, recently discovered rotamases, and proteases.

A novel gene, pmgA, specifically regulates photosystem stoichiometry in the cyanobacterium Synechocystis sp. PCC 6803 in response to high light

Yukako Hihara, Kintake Sonoike*, and Masahiko Ikeuchi

Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan and *Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan

Previously, we identified a novel gene, pmgA, as an essential factor to support photomixotrophic growth of Synechocystis sp. PCC 6803 and reported that pmgA-defective mutant grew better than wild type under photoautotrophic conditions [Hihara and Ikeuchi (1997) Photosynth. Res. 53: 243-252]. To gain insight into the role of pmgA, we investigated the mutant phenotype of pmgA in detail. When low light-grown cells were transferred to high light, pmgA mutants failed to respond in the manner typically associated with Synechocystis to high light. Specifically, the mutants lost their ability to suppress accumulation of chlorophyll and photosystem I and consequently could not modulate photosystem stoichiometry. These phenotypes seem to result in enhanced rates of photosynthesis and enhanced growth during short-term exposure to high light compared with wild type under high light conditions. Both wild type and mutants decreased content of photosystem II and phycocyanin, decreased ratio of phycocyanin to photosystem II and showed elevated activity of CO2 fixation under high light conditions, suggesting that pmgA is specifically involved in accumulation of photosystem I complex for modulation of photosystem stoichiometry. On the other hand, mixed culture experiments demonstrated that loss of pmgA function was selected against during longer-term exposure to high light, suggesting that pmgA is involved in acquisition of resistance to high light stress. Finally, early induction of pmgA expression, detected by the reverse transcriptase-polymerase chain reaction (RT-PCR), upon the shift to high light allowing us to conclude that pmgA is the first gene identified as a specific regulatory factor for high-light acclimation.

Interconversion of chlorophyll a and chlorophyll b

Ayumi Tanaka

Institute of Low Temperature Science, Hokkaido University, Sapporo 060-0819, Japan

Chlorophyll b is a ubiquitous accessory pigment in land plants, chlorophyte algae and prochlorophyte. Chlorophyll b has a formyl group on its B ring, where chlorophyll a has a methyl group. Chlorophyll b is synthesized by the oxidation of the methyl group of chlorophyll a to a formyl group. A study on the regulation of chlorophyll b synthesis is essential to understand how plants adapt to environmental light conditions, because chlorophyll b plays a crucial role in stabilizing light-harvesting complexes. Here we report the isolation of gene for chlorophyll b synthesis and the conversion of chlorophyll b to chlorophyll a.

(1) Isolation of the gene for chlorophyll b synthesis.

A gene involved in chlorophyll b synthesis was isolated by insertional mutagenesis of Chlamydomonas reinhardtii. We isolated 6 chlorophyll b-less mutants by insertional mutagenesis using nitrate reductase gene or argininosuccinate lyase gene as a tag and examined the rearrangement of mutant genomes. We found that a region of the genomes were deleted in all mutants. A cDNA was isolated with the probe for this region. The cDNA is 2885 bp in length and has a single open reading frame. The cDNA has a coding capacity for 463 amino acids, giving a calculated molecular weight of 51341. Its predicted amino acid sequence contains binding domains for [2Fe - 2S] Rieske center and mononuclear iron and shows similarity to the enzymes of methyl monooxygeases, indicating that the gene encodes chlorophyll a oxygenase (CAO). The reaction mechanism of chlorophyll b formation will be discussed.

(2) Conversion of chlorophyll b to chlorophyll a.

We found that chlorophyllide b was converted to chlorophyll a in isolated cucumber etioplasts. We also found that 7-hydroxymethyl chlorophyllide was accumulated when chlorophyllide b was incubated with etioplasts and that 7-hydroxymethyl chlorophyllide was converted to chlorophyll a. These findings indicate that chlorophyll b is converted to chlorophyll a via 7-hydroxymethyl chlorophyll.

These observations indicate the interconversion between chlorophyll a and chlorophyll b. We call this metabolic pathway the Chlorophyll Cycle. chlorophyll a to b ratio would be regulated by chlorophyll cycle. The role of Chlorophyll Cycle in the adaptation of plants to various light intensity and in the chlorophyll breakdown will be discussed.

Self-degradation of dithiothreitol-sensitive tetrameric protease (polyphenol oxidase) mediated by active oxygen species

Tomohiko Kuwabara

Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

In plant chloroplasts, thylakoid proteins are at risk of injury as a consequence of free radicals generated by excess light. Proteolytic systems that specifically degrade damaged proteins must be necessary for maintenance of the architecture of the photosynthetic machinery. However, little is known about the proteases involved in the proteolytic processes.

We previously purified a dithiothreitol-sensitive tetrameric protease (DSTP) from PSII membranes of spinach [1]. Afterward, the protease was shown to be structurally related to polyphenol oxidase (PPO) [2], which has one binuclear Cu site per protein molecule for the reaction center of PPO activity. Recently, immunoaffinity chromatography allowed us to rapidly purify DSTP, minimizing the degradation of the protein during purification [3]. When purified DSTP was subjected to gel-filtration chromatography on Superose 12, two elution peaks appeared at the positions of the tetramer (230 kDa) and the monomer (60 kDa), both with the proteolytic and PPO activities. This result indicates that DSTP and PPO are the same protein. The activity of DSTP degrading the extrinsic 23-kDa protein of PSII was suppressed under anaerobic conditions, suggesting that the proteolytic activity requires molecular oxygen, and thus, it is related to the PPO activity.We thought that the degradation of DSTP represents the essential features of the structure and function of the protein. Therefore, we developed a method to preserve purified DSTP without degradation; the protein was stable in the buffer that contained NaCl, Tween 20, and ethylene glycol, and could be stored for six months without degradation, during which the latent PPO activity was preserved. The degradation of DSTP was induced by the removal of the stabilizing reagents followed by incubation with H2O2 at a moderately high temperature (30-50oC). The degradation of the protein was accompanied by the degradation of H2O2, with the initial rate of 21 mol H2O2 (mol DSTP-derived Cu)-1 s-1. The degradation was inhibited by L-mimosine (specific chelator of binuclear Cu site) as well as other metal-chelators, DL-DOPA (substrate of PPO activity), and scavengers of hydroxyl radicals such as n-propyl gallate and pyrogallol. These results suggest that hydroxyl radicals were generated from H2O2 by the binuclear Cu site. Since the reaction was induced by the addition of H2O2, the Oxy form of the binuclear Cu site should be involved in the reaction. DSTP degraded also in the absence of H2O2, but at a slower rate. This fact suggests that the Oxy form could be generated spontaneously by the incorporation of dissolved oxygen molecule into the binuclear Cu site. Thus, it is likely that the addition of H2O2 enhances the degradation but is not a prerequisite for it. The generation of hydroxyl radicals suggests the occurrence of a Fenton-type reaction, which implies that Cu (II) in the Oxy form was reduced to Cu (I) by some reductant. Since the degradation can occur spontaneously, the presumable reductant is likely to be some amino acid residue(s) in the protein.

[1] Kuwabara, T. and Hashimoto, Y. (1990) Plant Cell Physiol. 31: 581-589.

[2] Kuwabara, T. (1995) FEBS Lett. 371: 195-198.

[3] Kuwabara, T. et al. (1997) Plant Cell Physiol. 38: 179-187.

Assembly of the photosystem I complex

Yuichiro Takahashi

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

The photosystem I (PSI) complex mediates the light-induced electron transfer from reduced plastocyanin or cytochrome c6 to oxidized ferredoxin. This complex consists of five chloroplast-encoded subunits and eight nuclear-encoded subunits as well as more than one hundred molecules of photosynthetic pigments, phylloquinone molecules and three distinctive Fe-S clusters. The biosynthesis of the PSI complex, therefore, depends on the coordinated expression of chloroplast and nuclear genes, the synthesis and integration of the cofactors, and the proper assembly of the subunits to form a functional complex. A number of PSI-deficient mutants have been isolated so far. Molecular biological and biochemical characterization of these mutants revealed that deficiency of expression of one of structural components of the PSI complex results in the loss or decrease of PSI activity and increased turnover of the other PSI subunits. However, mutants specifically deficient in assembly of the PSI complex have not been identified.

A recent study revealed that two chloroplast-encoded proteins are required for accumulation of PSI complex [1]. They are encoded by conserved chloroplast open reading frames, ycf3 and ycf4, and their products were localized in the thylakoid membranes. The Ycf3 is hydrophilic while the Ycf4 contains two putative transmembrane helices. The Ycf3 and Ycf4 accumulated normally in PSI-deficient mutant cells, suggesting that they are not constituent component of the PSI complex. Interestingly, both ycf3- and ycf4-deficient transformants of the green alga, Chlamydomonas reinhardtii, were totally deficient in the PSI complex, although the chloroplast-encoded reaction center proteins, PsaA and PsaB subunits, appear to be expressed normally in these transformant cells. These results, together with greening experiments of the dark-grown y-1 mutant cells, suggested that these two proteins may be involved in the assembly of the PSI complex. Interaction between the Ycf4 and PSI complex will be discussed.

[1] E. Boudreau, Y. Takahashi, C. Lemieux, M. Turmel and J.-D. Rochaix (1997) EMBO J. 16: 6095-6104.

Carboxyl-terminal processing of the precursor D1 protein in the photosystem II reaction center by a nuclear-encoded endopeptidase

Kimiyuki Satoh, Yumiko Yamamoto and Noritoshi Inagaki*

Department of Biology, Okayama University, Okayama 700-8530 and *National Institute for Basic Biology, Okazaki 444-8585, Japan

The D1 subunit of the photosystem II (PSII) reaction center is synthesized on membrane-bound ribosomes as a precursor which is immediately converted into the mature form by excising its carboxyl-terminal (C-terminal) extension consisting of 8-16 amino acids. The C-terminal cleavage of the precursor D1 protein (pD1) is catalyzed by a nuclear-encoded endopeptidase in eukaryotic organisms which functions in the lumenal space of thylakoids. The proteolytic processing of pD1 is a typical example of nuclear-regulation of the chloroplast gene expression at the post-translational level. This process is indispensable to the integration of manganese-cluster in the water-splitting machinery in the PSII. However, the physiological role of the presence of C-terminal extension in the precursor protein, as well as the molecular mechanism of integration of the manganese-cluster coupled with the C-terminal cleavage of pD1, is totally unknown.

The protease involved in the C-terminal processing of pD1 has been extracted and purified in near homogeneity from spinach (Fujita et al., 1996) and more recently in its pure state from Scenedesmus obliquus (Trost et al., 1997). The gene for the protease (ctpA) has been identified based on information regarding the partial amino acid sequence for spinach and Scenedesmus proteins, as well as on the genetic complementation analysis for the PSII-deficient mutant in Synechocystis sp. PCC 6803 (Shestakov et al., 1994). In collaboration with Dr. Pakrasi, we have analyzed the active center of the protease by site-directed mutagenesis of ctpA gene in Synechocystis and concluded that the enzyme is classified as a unique, new type of serine protease, which uses the catalytic hydroxyl/amine dyads as its active center.

We have established a system for over-expressing the spinach enzyme in E. coli and the enzyme was purified by utilizing His-tag at the N-terminus. We analyzed the mechanism of C-terminal cleavage, in order to understand the molecular processes of integration in vivo of the water-splitting machinery in the PSII, using the over-expressed spinach enzyme and the pD1 integrated into the thylakoid membrane of Scenedesmus obliquus LF-1 mutant which is deficient in the processing activity, together with substituted synthetic oligopeptides corresponding to the C-terminal region of the precursor protein, as the substrate. The result indicated that there are marked differences in the affinity of substrate to the enzyme and the pH dependency of the catalytic reaction, between the substrate integrated into the thylakoid membrane and that in solution, suggesting the involvement of specific interaction(s) at the membrane surface, although in both cases, in vivo and in vitro, specific amino acid side chains around the cleavage site, for example Ala-345, play crucial roles in the recognition.

An immunophilin-like protein in the thylakoid lumen: Its role for protein turn-over and protein phosphorylation

Hrvoje Fulgosi2, Alexander V. Vener1, Lothar Altschmied2, Reinhold G. Herrmann2 and Bertil Andersson1

1Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden.
2Botanisches Institute der Ludwig-Maximilians-Universitat, Menzinger Strasse 67, D-8000 Munchen, Germany.

The first high molecular weight immunophilin-like plant protein of the cyclophilin type has been identified in chloroplasts as a 40 kDa component co-purifying with a thylakoid protein phosphatase activity (Fulgosi et al. 1998 EMBO J. in press). This protein was shown to be located in the thylakoid lumen, originate from a single copy nuclear gene and possess several structural domains including a cyclophilin-like C-terminal segment of 20 kDa, a predicted N-terminal leucine zipper as well as a putative phosphatase-binding domain. The immunophilin is made as a precursor of 49.2 kDa including a bipartite lumenal targeting transit peptide of 104 residues. The mature protein can attach to the inner membrane surface with a binding strength intermediate to plastocyanin and the 23 kDa extrinsic protein (PsbP). Furthermore it has a heterogeneous location along the membrane system being confined predominantly to the non-appressed thylakoid regions, the site of protein integration. Biochemical measurements gave direct experimental support to the structural prediction that the protein possesses a peptidyl-prolyl cis-trans isomerase (PPIase) protein folding activity typical for immunophilins but is not inhibited by cyclosporin A. Despite a pronounced enzymatic activity the cyclophilin domain of the protein has a greatest divergency among of other known cyclophilins with only about 25% of identity to human cyclophilin A. Furthermore, an intriguing multifunctionality was implied from enzymatic measurements demonstrating that this protein, designated TLP40 (Thylakoid Lumen PPIase), possesses a regulatory effect on thylakoid phosphoprotein dephosphorylation. The TLP40, representing a type of proteins previously not thought to be associated with chloroplast thylakoids, is suggested to be part of a transmembrane signal transduction mechanism that appears to link turnover, folding and phosphorylation of photosynthetic proteins. The TLP40 protein is present in etiolated tissue which gives further support to a role in thylakoid biogenesis. The presence of a protein with functional and structural features such as TLP40 in the thylakoid lumen suggests that this often overlooked chloroplast compartment as well as the light-mediated protein phosphorylation process of the photosynthetic membranes are significantly more complex than presently anticipated. A role in D1 protein turn-over will be discussed.

Scavenging system of active oxygen species in higher plants

Shigeru Shigeoka

Department of Food and Nutrition, Faculty of Agriculture, Kinki University, Nakamachi, Nara 631, Japan

In plant tissues the chloroplasts are potentially the most powerful source of oxidants and sites within the cell most at risk from photooxidative damage. When plant leaves are exposed to excess photons which can not be utilized in photosynthesis, excess photons generate reactive molecules such as active oxygen species, organic radicals, and triplet excited pigments, which oxidize the target molecules in chloroplasts resulting in photoinhibition. The production rate of O2- and H2O2 is estimated to be about 160 and 80 mM s-1 in chloroplasts under normal conditions. Because 10 mM H2O2 is sufficient to inhibit the photosynthetic assimilation of CO2 by 50%, photosynthesis is significantly impaired within a fraction of a second if H2O2 is not promptly scavenged. The primary target molecules are thiol-modulated enzymes of the PCR cycle in the stroma and the D1 protein in PSII reaction center and reaction center complex of PSI in the thylakoids. For protection of the target molecules, immediate scavenging of reactive molecules at the site where they are photoproduced is essential. Chloroplasts of higher plants develop the ascorbate-glutathione cycle including ascorbate peroxidase (AsAP) isozymes which exist as stromal soluble (sAsAP) and thylakoid-bound (tAsAP) forms. AsAP isozymes have also been detected in cytosol (cAsAP) and microbody (mAsAP). We have cloned and characterized cDNAs encoding AsAP isozymes from spinach leaves [FEBS Lett. 367: 28-32,1995; FEBS Lett. 384: 289-293, 1996; Plant Cell Physiol. 39: 23-34, 1998; Arch. Biochem. Biophys. in press]. In addition, we have found that spinach chloroplastic AsAP isozymes are generated from an identical gene (Apx II) by alternative mRNA splicing of 3'-terminal two exons [Biochem. J. 328: 895-900, 1997].

Inflow of CO2 through stomata into chloroplasts and the subsequent fixation and reduction must be balanced with the formation of chemical energy in photosynthesis. Many stresses cause the closure of stomata and the limitation of transpiration to inhibit the photosynthetic capacity. As soon as the CO2 concentration decreases in chloroplasts, there is a lower availability of NADP to accept electrons from PSI, thus initiating dioxygen reduction with the concomitant generation of O2- and H2O2. We have found that tobacco leaves suffering from drought at high light intensity lose the chloroplastic AsAP activities as well as the photosynthetic activity. To examine the possibility that resistance to oxidative stress of plants may be diminished by the instability of AsAP isoforms themselves under oxidative conditions and to enhance the ability to withstand photooxidative stress in higher plants, we introduced the chimeric H2O2-scavenging enzyme into tobacco chloroplasts. Transgenic plants that expressed the catalase (kat E) from E. coli in chloroplasts showed increased tolerance to photooxidative damage caused by drought at high light intensity or by the paraquat treatment as measured by visible leaf injury. In transgenic lines, photosynthetic activities monitored by CO2 fixation and chlorophyll fluorescence were much less influenced by stress treatments. The chloroplastic AsAP isoforms were completely inactivated with the thiol-modulated enzymes still active in the transgenic lines, suggesting that the ascorbate-glutathione cycle can function only in the early steps of photoinhibition due to its sensitivity under oxidative stress.

Photoreduction of dioxygen to water in PSI has dual functions

Kozi Asada

Department of Biotechnology, Fukuyama University, Fukuyama 729-0251, Japan

Univalent photoreduction of dioxygen in PSI produces superoxide radical in thylakoids and its prompt reduction to water at the site of its generation is indispensable to protection from the inactivation of active oxygen-sensitive enzymes of CO2-fixation cycle in the stroma and probably of PSI itself. Rapid reduction of superoxide radical and hydrogen peroxide to water prior to their diffusion to the stroma is guaranteed by attachment of CuZn-SOD and binding of ascorbate peroxidase to the stromal thylakoids, and also by the reduction of monodehydroascorbate radical (MDA) reductase for regeneration of ascorbate with the photoreduced ferredoxin in PSI, being the reducing complex to water microcompartmented at the site where superoxide is photogenerated. Actually the Mn-SOD-enriched in chloroplasts but chloroplastic CuZn-SOD-inactivated transformant of tobacco was sensitive to strong light and its PSI was inactivated, indicating that only the properly compartmented SOD to the PSI complex is effective in the disproportionation of superoxide to suppress photoinhibition.Under photon-excess environments electron acceptors are not available for PS I, which causes the overreduction of the intersystem electron carriers and photoinhibition in PSII and PSI. Under such conditions the photoreduction of dioxygen to water in PSI allows the linear electron flow and protects from the photoinhibition. We have found that the FAD enzyme MDA reductase which catalyzes the reduction of MDA by NAD(P)H, also attaches to the thylakoids and catalyzes the photoreduction of dioxygen to superoxide in PSI. This unexpected finding indicates that MDA reductase enhances the linear electron flow even in the absence of electron acceptors of PSI. Thus, the photoreduction of dioxygen to water, as observed in intact leaves, protects from photoinhibition as far as the reactive species of oxygen are "safely" reduced to water by the microcompartmented scavenging complex in PSI.

Mechanisms of the reversible light activation of thylakoid protein phosphorylation*

Itzhak Ohad

The Minerva, Avron, Even-Ari Center for Photosynthesis Research, The Hebrew University, Givat Ram, Jerusalem 91904, Israel

Redox controlled phosphorylation of thylakoid membrane proteins represents a unique system for the regulation of light energy utilization by eukaryotic photosynthetic organisms as well as for controlling the maintenance and turnover of the PSII proteins. Current views suggest that both, the plastoquinone pool and the cytochrome bf complex control the activation of the thylakoid protein kinase (TLPK) [1]. Using a new method for the activation of the thylakoid protein kinase by a transient acidification in the darkness of the thylakoid suspension [2] and based on results obtained by flash photolysis combined with optical and EPR spectroscopy we have established that the TLPK activation process occurs when a plastoquinol molecule occupies the oxidizing Qo-site of the cytochrome bf complex, having its high potential path components in a reduced state [2, 3]. A linear correlation between kinase activation and accessibility of the Qo-site to plastoquinol was established. The kinase activity persists as long as one plastoquinol molecule per reduced cytochrome bf is still available. Withdrawal of one electron from this plastoquinol, following a single-turnover flash inducing oxidation of P700 leads to deactivation of the kinase parallel with a decrease in the gz EPR signal of the reduced Rieske Fe-S center. These results give direct evidence for a functional cytochrome bf-kinase interaction, analogous to a signal transduction system where the cytochrome bf is the receptor and the ligand is the plastoquinol at the Qo-site. Based on the data obtained thus far one can conclude that TLPK should contain a matrix exposed domain harboring the enzyme active site and able to interact with the matrix exposed substrates as well as a domain reaching within the thylakoid membrane to interact with the PQ pool or the cytochrome bf Qo site respectively [3].

Using perfusion chromatography we have been able to obtain TLPK preparations that phosphorylate LHCII as well as the CP43 protein in isolated PSII cores [4]. Illumination increased the extent and phosphorylation rate of isolated LHCII but not of the LHCII apoprotein or of histone. The light affects the substrate by exposing phosphorylation sites to the enzyme activity. The light activated state of the substrate persists in darkness for at least 15-20 minutes and is due to a conformational change exposing the N-terminal LHCII segment containing the phosphorylation site, preferentially in the trimer LHCII population. Light exposure of the cores during the phosphorylation process results in the phosphorylation of the D1 and possibly the D2 proteins as well [5]. Direct evidence for the light activation of the substrate in situ could not be obtained previously for the simple reason that during illumination it is not possible to distinguish between the activation of the kinase by the reduction of plastoquinol and cytochrome bf and the light induced activation of the substrates. The long lasting activated state of LHCII in vitro observed in this work and reported previously [1] may shed new light on the mechanism of the slow kinase deactivation process following transfer of chloroplasts or isolated thylakoids from the light to darkness.

[1] Gal, A., Zer, H. and Ohad, I. (1997) Physiol Plant. 100: 869-885.

[2] Vener, A.V., van Kan, P.J.M., Gal, A., Andersson, B. and Ohad, I. (1995) J. Biol. Chem. 270: 25225-25232.

[3] Vener, A.V., van Kan, P.J.M., Rich, P.R., Ohad, I. and Andersson, B. (1997) Proc. Natl. Acad. Sci. USA 94: 1585-1590.

[4] Gal, A., Zer, H., Robol-Broza, M., Fulgosi, H., Herrmann, RG, Ohad, I. and Andersson, B. (1995) in: Photosynthesis: from Light to Biosphere (P. Mathis ed.) Kluwer Academic Publishers, Dordrecht, 3, 341-344

[5] Zer H, Vink M, Herrmann RG, Andersson B, and Ohad I (in preparation)

*[Supported by HFSP]

Fatty acid desaturases are regulators for the stability of photosynthetic machinery via the desaturation of fatty acids

Norio Murata

National Institute for Basic Biology, Okazaki 444, Japan

Fatty acid desaturases are enzymes which introduce unsaturated bonds into fatty acids of membrane glycerolipids and thereby provide the membranes with suitable fluidity [1]. Tolerance to low temperature is highly related to the unsaturation of membrane glycerolipids [2]. The low-temperature-dependent desaturation of membrane glycerolipids by the desaturases is regarded as the strategy of organisms to adjust their tolerance to changes in ambient temperature.

A recent study of the gene knockout of fatty acid desaturases has demonstrated the direct involvement of the unsaturation of membrane lipids in tolerance to low temperature [3]. Synechocystis sp. PCC 6803 contains four desaturases and they can be inactivated by insertional mutation in a step-wise manner. After deletion of polyunsaturated fatty acids in membrane glycerolipids, this organism does not grow at 20oC, at which the wild-type strain grows very well. Further study has demonstrated that this effect is caused by enhanced recovery of the photosynthetic machinery from the low-temperature photoinhibition and that the unsaturation of membrane lipids accelerated the processing of the precursor to the D1 protein in the photosystem II complex.

Gene-engineered enhancement of tolerance to low temperature has been achieved by introducing one of the desaturase genes (the desA gene for the D12 desaturase) from Synechocystis into a low-temperature-sensitive strain Synechococcus sp. PCC 7942. The wild-type cells of Synechococcus contain only 9 desaturase, and fatty acids of their membrane lipids are either saturated or monounsaturated. The genetic introduction of the desA gene to Synechococcus enables this organism to synthesize diunsaturated fatty acids in the membrane glycerolipids, and to tolerate low temperature [4]. Further study has indicated that the enhancement of the tolerance is caused by protection of the photosystem II complex against low-temperature photoinhibition by acceleration of the recovery of the photosynthetic machinery from the photoinhibited state.

[1] N. Murata and H. Wada (1995) Biochem. J. 308: 1-8.

[2] I. Nishida and N. Murata (1996) Annu. Rev. Plant Physiol. Plant Mol. Biol., 47: 541-568.

[3] Y. Tasaka, Z. Gombos, Y. Nishiyama, P. Mohanty, T. Ohba, K. Ohki and N. Murata (1996) EMBO J., 15: 6416-6425.

[4] Z. Gombos, E. Kanervo, N. Tsvetkova, T. Sakamoto, E.-M. Aro and N. Murata (1997) Plant Physiol. 115: 551-559.

Manganese transport in cyanobacteria

Himadri B. Pakrasi

Department of Biology, Washington University, Box 1137, 1 Brookings Drive, St. Louis MO 63130-4899, USA

Manganese is required in trace quantities for the growth of all organisms. Only a handful number of enzymes are known to require Mn for their activities. These include redox enzymes such as Photosystem II (PSII), Mn-superoxide dismutase and Mn-catalase, as well as metabolic enzymes such as pyruvate carboxylase and PEP carboxykinase. PSII is a multisubunit pigment-protein complex in the thylakoid membranes of oxygenic photosynthetic organisms. Four manganese atoms form a Mn-cluster in the lumenal region of PSII, and play an important catalytic role in the oxidation of water. As a part of an ongoing study on the PSII complex in the cyanobacterium Synechocystis 6803, we have identified the first Mn-transporter complex in any organism, although transport of Mn into various prokaryotic as well as eukaryotic cells was studied in detail more than two decades ago. During recent years, a class of proteins have been described in microbes to man that are involved in the transport of a diverse array of macromolecules across various biological membranes. Together they form what is now known as a ABC superfamily or the family of traffic ATPases. In bacterial inner membrane systems, a typical ABC transporter protein has (a) one or two integral membrane proteins, (b) one or two peripheral membrane proteins exposed to the cytoplasm and (c) a periplasmic soluble protein. Among these, the cytoplasmic protein has two domains, called the Walker motifs A and B respectively, that are found in a number of ATPases. Because of the presence of these motifs, the term 'ABC' (ATP Binding Cassette) transporter has been coined. In our newly identified Mn-transporter complex, the MntA protein contains the Walker motifs, MntB is an integral membrane protein, while MntC is presumably a periplasmic Mn-concentrating protein. To investigate further Mn-transport in Synechocystis 6803, we have generated targeted inactivations in each of the genes in the mntCBA gene cluster. Similar to the BP13 mutant, the growth of these mutant strains were significantly slower in a Mn-deficient medium and were restored to near normal levels upon addition of micromolar concentrations of Mn2+, indicating the presence of a second transport system for manganese in this organism. 54Mn2+ uptake experiments indicated that the MntABC transporter is induced under manganese starvation conditions, whereas the second transporter system is induced in the presence of micromolar levels of manganese. Both of these systems were nonfunctional at low temperatures and could transport trace levels of 54Mn2+, reflecting high affinity active transport. The initial rates of Mn2+ uptake for cells grown with or without manganese exhibited biphasic saturation kinetics suggesting that Mn2+ can also be accumulated by a low affinity system in these bacteria. Accumulation of manganese by this system was competitively inhibited by Cd2+, Co2+ and Zn2+. In contrast, the second high affinity system was highly specific for manganese and was not inhibited by any tested metal ion. We have also demonstrated that in this organism, photosynthetic electron transport is necessary for optimal rates of Mn uptake.

[1] Bartsevich, V.V. and Pakrasi, H.B. (1995) Molecular identification of an ABC-transporter complex for manganese: analysis of a cyanobacterial mutant strain impaired in the photosynthetic oxygen evolution process. EMBO J. 14: 1845-1853.

[2] Bartsevich, V.V. and Pakrasi, H.B. (1996) Manganese transport in the cyanobacterium Synechocystis sp. PCC 6803. J. Biol. Chem., 271: 26057-26061.

[Supported by fundings from US NIH, USDA-NRICGP and the International Human Frontier Program]

Two divergent GAPDH genes play a different role in carbon metabolism of the cyanobacterium Synechocystis 6803

O. Koksharova (1,2), M. Schubert (2), R. Cerff (2), S. Shestakov (1)

(1) N. Vavilov Institute of General Genetics, Moscow 117809, Russia
(2) Institute of Genetics, University of Braunschweig, D-38106 Braunschweig, Germany

The cyanobacterium Synechocystis sp. PCC 6803 harbors two separate highly divergent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, gap1 and gap2, which are closely related at the sequence level to the plant nuclear genes encoding cytosolic and chloroplast GAPDH, respectively. Genes gap1 and gap2 were cloned and subsequently inactivated by insertional mutagenesis to identify their metabolic functions. Completely segregated gap1-mutants have lost the capacity to grow heterotrophically on glucose while growth on organic acids as well as under CO2 and high light was not impaired. Homozygous gap2-mutants can grow only on glucose but not under photosynthetic conditions. Measurements of the anabolic activities (reduction of 1,3-bisphosphoglycerate) in extracts from the wild type and mutant cells showed that Gap2 is a major enzyme with dual cosubstrate specificity for NAD and NADP, while Gap1 displays a minor NAD- specific GAPDH activity. However, if measured in the catabolic direction (oxidation of glyceraldehyde-3-phosphate) Gap2 activity is very low and increases after gel filtration of extracts over Sephadex G25. Our results suggest that enzymes Gap1 and Gap2, although coexpressed in cyanobacterial wild type cells, play distinct key role in catabolic and anabolic carbon flow. Gap2 operates in the Calvin- Benson photosynthetic cycle and in non-photosynthetic gluconeogenesis but Gap1 seems to be essential only for glycolytic glucose breakdown, conditions under which catabolic Gap2 activity is repressed by a specific low molecular weight inhibitor. A possible role of this inhibition in regulation of carbon metabolism in vivo will be discussed.

[This work was supported by a grants from Deutsche Forschungsgemeinschaft and the Russian Foundation for Basic Research.]

Photoinhibition of Photosystem I and its relation to chilling sensitivity in plants

Kintake Sonoike

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Hongo 7-3-1, Tokyo 113-0033, Japan

The decrease of photosynthetic activity is observed when plants receive too much light (photoinhibition). The main target of photoinhibition has long been believed as photosystem II (PSII) and it is well known that photoinhibition is accelerated at chilling temperatures. On the other hand, chilling itself causes the damage to plants even in the dark, and it is now evident that the site of damage to photosynthesis by chilling stress varies with different light conditions [1]. Upon the chilling/weak-light treatment, it was found that PSI is selectively photoinhibited in chilling sensitive plants such as cucumber, tomato or common bean [2]. Here we compare the effects of chilling temperatures on the extent of photoinhibition between PSI and PSII, and also report the role of reactive oxygen species in the photoinhibition of PSI which is different from that in the photoinhibition of PSII.

(1) Different effects of chilling temperatures on PSI and PSII photoinhibition.

To understand the role of chilling temperatures in the two types of photoinhibition, we compared the temperature dependency of photoinhibition of PSI and PSII upon light-chilling treatment of cucumber leaves under various photon flux densities. By the moderate light of 200 umol m-2 s-1, PSI activity decreased sharply below 10oC, while quantum yield of PSII decreased almost linearly as temperature decreased. The extent of the photoinhibition of PSII was correlated with the relative redox state of QA which is determined by both photon flux density and temperature, while that of PSI was mainly determined by the temperature. Thus, the main site of damage under high light condition is PSII while that under low-light/chilling condition is PSI.

(2) The role of reactive oxygen species in the photoinhibition of PSI.

The photoinhibitory process in PSI involves the inactivation of the iron-sulfur centers and the degradation of PsaB protein, a reaction center subunit. In isolated thylakoid membranes, selective photoinhibition of PSI is observed even at room temperature, suggesting that chilling sensitive component is lost during isolation of thylakoid membranes. The inhibition of PSI activity and the degradation are observed only under aerobic conditions and suppressed by the presence of n-propyl gallate, a scavenger of reactive oxygen species. To confirm the involvement of reactive oxygen species in the inhibition process, we examined the effect of hydrogen peroxide on the PSI activity using spinach thylakoid membranes. When 10-30 mM hydrogen peroxide was added in the darkness, no decrease of PSI activity nor degradation of reaction center subunit was observed. By the week illumination of 80 umol m-2 s-1, the PSI activity decreased to less than half of the original level, and this residual activity was diminished by the addition of hydrogen peroxide. This clearly indicates that hydroxyl radical formed from hydrogen peroxide at the photoreduced iron-sulfur centers of PSI is the cause of photoinhibition. This contrasts sharply with the case of PSII where the involvement of singlet oxygen plays an important role.

[1] Sonoike, K. (1998) J. Plant Res. 111: 121-129.

[2] Sonoike, K. (1996) Plant Cell Physiol. 37: 239-247

[Supported by fundings from the Ministry of Education, Science, Sports and Culture, Japan and the International Human Frontier Science Program]