The heliobacteria are strictly anaerobic photosynthetic bacteria that belong to Gram-positive bacteria. RC of Heliobacteria contain Fe-S type RC. Fe-S type RCs are able to photoreduce NADP⁺ via ferredoxin (Fd), although it has not been proven yet in heliobacteria. We investigated electron transfer process from heliobactrial RC to Fd by NADP⁺ photoreduction and ESR spectroscopy. The absorption spectra of the purified Fd A, B and C from Heliophilum fasciatum and g values of cryogenic ESR spectra were typical of those of the 2[4Fe-4S] cluster type. Fd A decreased an absorbance of 385 nm under air condition, and their half life were 6 h respectively. The purified RC fraction was contained cytochromes. NADP⁺ photoreduction activity with spinach FNR were showed by Fd B and C. However, illuminated ESR spectroscopy in the presence of each Fds and purified RC from H. fasciatum were shown typical of reduced Fe-S cluster.

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The redox-active tyrosine Y_D in photosystem II (PSII) serves as an accessory electron donor to P680. When oxidized, Y_D releases a proton and becomes a neutral radical Y_D^·. Hence, a H-bond network around Y_D plays a crucial role in this reaction. In the last annual meeting, we reported that Y_D has two simultaneous H-bonds. Here, we studied the interactions of water molecules with Y_D using FTIR spectroscopy. Light-induced Y_D/Y_D^· FTIR difference spectra were obtained with the PSII core complexes from Thermosynechococcus elongatus. In the OH stretching region, signals were observed at 3636/3617 and 3594/3584 cm^-1. These peaks downshifted upon H₂¹⁸O substitution, and hence were attributed to two water molecules coupled to Y_D. The recent X-ray structures of PSII revealed that the side chain capable of forming a proper H-bond with Y_D is only D2-His189. Thus, it is likely that one of the two water molecules is directly H-bonded with Y_D.

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A common feature of light and heat stresses to chloroplasts is the stress-induced damage to Photosystem II (PSII) complexes. The reaction centre D1 protein is the primary target of the damage. After the damage, a repair mechanism operates to replace the damaged D1 subunits within PSII by a newly synthesized copy. We investigated the relationship between the light- and heat- induced damage to the D1 protein and unstacking of the thylakoids. For efficient migration of the photodamaged PS II complexes from the grana to stroma thylakoids, and also of FtsH proteases from the stroma thylakoids to the grana, the grana stacking becomes a physical hindrance.At lower temperature (4*), the thylakoid unstacking did not take place by strong illumination, and the cleavage of the D1 protein was not observed. These results suggest that the thylakoid unstacking is necessary for efficient degradation of the photo- or heat-damaged D1 protein in PS II.

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FtsH is a membrane-bound zinc-dependent metalloprotease belonging to the AAA (ATPase associated with various cellular activity) protease family. The cyanobacterium Synechocystis sp. PCC6803 (PCC6083) has four FtsH homologues. These proteases have two transmembrane helices at the N terminus, and an ATP binding domain and a protease domain at the C terminus. The FtsH encoded by slr0228 is involved in selective degradation of the light- or heat- damaged D1 protein. However, the details of the degradation pathway of the damaged D1 by FtsH are unknown.
To isolate FtsH retaining the protease activity, we constructed a mutant in which the slr0228 gene was genetically modified. A his-tag, and a Factor Xa protease recognition region which is necessary to remove the his-tag afterward, were added at the N terminus of the FtsH in the PCC6803 WT cells. In this presentation, we report purification of his-tagged FtsH from PCC6803.

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When spinach thylakoid membranes were subjected to moderate heat stress, the reaction center-binding protein D1 of photosystem II was damaged, and a 23 kDa N-terminal fragment and a 9 kDa C-terminal fragment were produced. According to our previous study, this cleavage is caused by an FtsH protease which is bound to the thylakoid membranes [Yoshioka et al.: J. Biol. Chem. (2006)]. In this work, we tried to improve the purification of the FtsH protease. Several detergents were used to solubilize the FtsH protease from the spinach thylakoids, and the presence of FtsH was monitored by Western blot analysis. We found that 0.1-1.0% of n-dodecyl-β-D-maltoside (DDM) was most effective in the solubilization of FtsH. Protease activity was detected with the DDM-washed supernatant by proteolytic degradation assay as β-casein was a substrate. To further purify the active FtsH protease, the anion-exchange chromatography is now being carried out with the DDM-solubilized supernatant.

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We used a silica mesoporous material (SMM) that has 23 nm pore diameter inner pores of honeycomb-like hexagonal cylindrical structure in its inside as a matrix for the protein function. We introduced the dimmer complex of photosystem II (PS II) reaction centers (756 kDa) that were purified from a thermophilic cyanobacterium Thermosynechococcus vulcanus into SMM that has inner pore. The adsorption to SMM decreased the oxygen evolution activity to be about a half when measured by a Clark-type oxygen electrode. Pigment assembly as well as the protein structure are almost intact inside SMM when tested by the absorption and fluorescence spectrum. The structure and function of PS II reaction center inside the nano-scale pores will be discussed.

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Although the central part of photosystem II (PSII) is highly conserved from prokaryotic cyanobacteria to eukaryotes, there are some differences in the extrinsic proteins involved in oxygen evolution among different organisms. The crystal structure of PSII from cyanobacteria has been reported; however, no reports have been published on the structure of any eukaryotic PSII. Red alga is one of the eukaryotic algae closely related to cyanobacteria, but its PSII differs from that of cyanobacteria in that the former contains a 20 kDa protein, the fourth extrinsic protein. In order to elucidate the structure of red algal PSII, we have purified and crystallized PSII from a red alga Cyanidium caldarium. We improved the crystallization conditions which yielded crystals that diffracted to a higher resolution than we reported previously. The crystals obtained in the present study will allow us to analyze the structure of red algal PSII.

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In this study, we examined the role of DGDG in photosystem (PS) II using a disruptant of dgdA gene (slr1508) of Synechocystis sp. PCC6803 that is defective in DGDG synthesis. Despite the lack of DGDG, the mutant cells grew photoautotrophically as rapidly as that of the wild-type cells, indicating that DGDG is not essential for photosynthesis in Synechocystis. However, the oxygen-evolving activity of PSII was decreased in the mutant. Analyses of PSII complex purified from the mutant cells indicated that the extrinsic proteins PsbU, PsbV, and PsbO were substantially dissociated from PSII complex. In addition, we found that heat susceptibility but not dark-induced inactivation of oxygen evolution was notably increased in the mutant cells in comparison to the wild-type cells, suggesting that PsbU subunit is dissociated from PSII complex even in vivo. These results demonstrate that DGDG plays important roles in PSII through the binding of extrinsic proteins.

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PsbK and PsbZ are two low-molecular mass subunits of PSII and are conserved from cyanobacteria to higher plants. PsbK has a single trans-membrane helix, whereas PsbZ has two trans-membrane helices. Both subunits are located in the periphery of PSII dimer in adjacent to CP43. Functional analysis of mutants lacking each of the two subunits from various organisms has shown different phenotypes depending on the organisms. In order to study the role of these subunits from a structural point of view, we purified, crystallized PSII from mutants of thermophilic cyanobacteria lacking either PsbK or PsbZ. The crystals from both mutants belonged to the same space group with similar unit cell constants. The space group was also the same as that of wild-type, whereas the unit cell constants of the mutants were significantly different from those of wild-type, indicating an effect on the molecular contact between PSII dimers within the crystal lattice.

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PsbY and PsbZ are the membrane proteins of ~5.0 kDa and ~6.5 kDa in Photosystem II (PSII) complexes. We deleted psbY and psbZ genes from Thermosynechococcus elongatus BP-1.
The oxygen-evolving activity in ΔpsbZ PSII complexes was lower than that in the wild-type PSII complexes. Most of Ycf12 as well as PsbK was lost in ΔpsbZ PSII complexes. These suggest that PsbZ is important for the stable binding of PsbK and Ycf12 with PSII core intrinsic proteins. We analyzed quantity of Mn from those PSII complexes.
No difference was observed in the growth under the several conditions between the wild-type and ΔpsbY. the oxygen-evolving activity of ΔpsbY PSII complexes were 20-30% lower than those of the wild-type. BN-PAGE revealed that the dimeric PSII was no significant reduced in ΔpsbY. Electrophoretic profile and analysis of the crystal structure revealed that PsbY was disappeared and no release of the extrinsic proteins was observed.

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The extrinsic proteins of photosytem II (PSII), PsbO, PsbP and PsbQ, are known to stabilize the oxygen-evolving center (OEC) and regulate the Ca²⁺ and Cl^- requirement. However, the mechanism underlying such functions has yet to be clarified. We investigated the involvement of the extrinsic proteins in the OEC reactions using FTIR spectroscopy. Upon PsbP and PsbQ deletion, clear changes were observed in the amide I bands of the S₂/S₁ FTIR difference spectrum, whereas further PsbO deletion did not change the spectrum. Normal amide I bands recovered by recombination of PsbP, although the recovery was not observed by Δ15PsbP that lacks 15 N-terminal residues. Recombination of ¹³C-labeled PsbP showed the same changes as unlabeled PsbP. These results indicate that the N-terminal residues of PsbP directly interact with OEC and affect its protein conformation. It is suggested that this direct interaction of PsbP induces its function of Ca²⁺/Cl^- retention.

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Psb30 (Ycf12) is a novel hydrophobic subunit of photosystem II (PSII) complex first reported in Thermosynechococcus elongatus (Kashino et al. BBA 2007). In Chlamydomonas, this polypeptide is encoded by the chloroplast gene psb30 (ycf12). We have generated psb30-deficient transformants (Δpsb30) by chloroplast transformation. The transformants grew photosynthetically, indicating that Psb30 is not essential for PSII activity. We are currently testing the growth of these transformants under various stress conditions. We also raised a specific antibody against synthetic oligopeptide of Psb30. It was confirmed that Psb30 accumulates on thylakoid membranes and its level was significantly reduced in various PSII-deficient mutants. In addition, fractionation of thylakoid extracts by sucrose density gradient ultracentrifugation and ion exchange chromatography revealed that Psb30 is co-purified with PSII core complex. We are trying to locate Psb30 in PSII core complex by partial dissociation of the core complex.

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Photosystem (PS) II consists of D1, D2, CP43, CP47, and other many small subunits, and is responsible for the water oxidation reaction. It is known that PS II fluoresces at 685 and 695 nm at 77K, and CP43 and CP47 are attributed to their origins, respectively. We constructed the CP43-deletion mutant of Synechocystis sp. PCC 6803 by replacement the gene (psbC) with the antibiotics resistant gene. This strain could not grow autotrophically but grow under the presence of 5 mM glucose with the following two light conditions, i.e. photo-mixotrophic growth with a continuous light and light-activated heterotrophic growth with illumination of light for 15 min per day. This strain showed two fluorescence maxima at 686 and 725 nm at 77K, and the former might come from phycobiliproteins. The time-resolved fluorescence spectra gave essentially identical results. Based on these observations, fluorescence properties will be discussed in details.

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Objective of our research is to enhance the ability of CO₂-fixation of plants by improving their photosynthetic performance. In C3-plants, CO₂-fixation rate at low CO₂ is limited by factors relating to Rubisco-carboxylase reaction, at high CO₂ by factors relating to RuBP-regeneration. In this work, for the identification of regulatory elements of CO₂-fixation in plants we try a screening of Arabidopsis mutants that show abnormalities in photosynthesis at low or high CO₂. In the screening of mutants we adopted a quantum yield of PSII (Φ(PSII)) as an index of photosynthesis rate for a high through put analysis. Now, we have three mutants which showed low Φ(PSII) with independence of CO₂, compared to wild type (2 mutants); high Φ(PSII) at low CO₂, compared to wild type (1 mutant). We identified that these mutations were located in the chromosome 1, 3, and 4, respectively.

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Plant mitochondria have multiple energy-dissipating pathways. In the present study, we examined a regulation mechanism for the respiratory gene expression under high-light stress and the differences among the respiratory pathways in Arabidopsis. Gene expression of AOX1a, NDA1, NDB2, NDC1, COX6b, and CI76 were induced after the high-light treatment. NDB2 co-expressed with AOX1a strongly, but some light-induced genes showed distinct expression patterns. Manipulation of the photosynthesis or respiration using several chemicals revealed that there are distinct regulation manners for each respiratory gene expression. When high-light was treated in conjunction with high CO₂, the several respiratory genes were further induced, suggesting that the changes in metabolic pathways by elevated photosynthesis are involved in the respiratory gene expression. From these results, we propose the regulation model of respiratory gene expression under the high-light stress.

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The chloroplast NAD(P)H dehydrogenase (NDH) complex reduces plastoquinones and generate H⁺ gradient using NAD(P)H in photosystem I cyclic electron flow and chlororespiration. The NDH complex is found in higher plants and cyanobacteria, and a number of NDH-related genes were characterized, while whole machiney of NDH is not identified yet. Last few years, we have succeeded to identify novel genes related to NDH activity, NDH-Dependent cyclic electron Flow (NDF), using a co-expression analysis of Arabidopsis. Among NDF genes, NDF2 had a weak homologue gene, NDF5.
Here we report that NDF5 is related to NDH activity as well as NDF2. Unique presence of NDF2 and NDF5 in higher plants suggests that these genes are acquired by higher plants evolutionally. Functional roles of these proteins in NDH activity are investigated in the comparison with known NDH subunit using Blue Native PAGE analysis.

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Chloroplastic NAD(P)H dehydrogenase (NDH) genes were identified as a homologous gene cluster of a respiratory enzyme, complex I, when the whole chloroplast genome sequence was determined. So far, 14 ndh genes were identified in higher plants. While 11 of ndh genes are coded in chloroplastic genome and the others are in nuclear genome, no electron-accepter subunits have been identified yet. The comparison with eukaryotic complex I, which consists of more than 40 subunits, suggests that several missing subunits exist in chloroplastic NDH. Our recent approach using in silico screening method based on co-expression analysis has predicted several missing subunits of NDH. In this report, we characterized a novel gene NDF6 which is essential for NDH accumulation.

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In higher plants, the chloroplastic NAD(P)H dehydrogenase (NDH) complex is involved in PSI cyclic electron transport. Eleven genes encoding the subunits of the NDH complex have been discovered in chloroplast genome based on their sequence similarity to the subunit genes of the mitochondrial complex I (NADH dehydrogenase).Recently three novel subunits encoded in nuclear genome have been isolated by the biochemical method. To understand full structure of the chloroplastic NDH complex, we have screened 7 novel candidate ndf (NDH-Dependent cyclic electron Flow) genes essential for NDH activity through co-expression analysis and phylogenetic methods. In this report we show the results of Blue Native-PAGE analysis of NDH complex in Arabidopsis ndf mutants, which lack NDH activity, and C₄ plants, which show high expression of NDH genes as compared with C₃ plants. From these analyses, we discuss the interaction of the novel ndf subunits and the known NDH subunits.

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Glutathione is a ubiquitous tripeptide that is synthesized via two reactions dependent on photosynthetic ATP production. To study whether glutathione is associated with photosynthesis regulation, we investigated effects of decreased glutathione levels on photosynthesis using an Arabidopsis mutant, cad2-1, which has 15-30% of wild-type levels of glutathione. The cad2-1 mutant showed low photosynthetic electron transports and high non-photochemical quenching (NPQ). These results are considered to suggest that low levels of glutathione suppress photosynthesis. The mutant had 75% Rubisco content of the wild-type. It has been found that the another Calvin cycle enzyme, aldorase, is regulated by glutathione. Taking these into account, it is considered that low levels of glutathione suppress the Calvin cycle. We will discuss relationship between glutathione levels and the Calvin cycle based on aldorase activity and CO₂ assimilation in cad2-1 under various CO₂ concentrations.

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RuBisCO and phosphoribulokinase (PRK) are enzymes unique to Calvin cycle and we are interested in their evolution. In order to study the evolution of PRK, we have cloned three PRK homologues, glr2296, glr4426 and gll2122 (PRK-I, II and III, respectively) from the early diverging cyanobacterium, Gloeobacter violaceous. Analysis of PRK activities of purified proteins expressed in Escherichia coli reveals that PRK-I is the bona fide PRK possessing activity similar to other cyanobacterial PRKs, while PRKII does not possess any PRK activity. Interestingly, PRK-III possesses significant, albeit lower, PRK activity. In phylogenetic tree, PRK-III is placed in a clade with PRK homologues from methanogenic archaea. This protein could be an ancestral form of PRK which could have an archaeal origin. We discuss the properties of these two PRK homologues with respect to their amino acid sequences. The knowledge of true activity of PRK-III could suggest the probable course of PRK evolution.

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Chlamydomonas reinhardtii induces CO₂-concentrating mechanism (CCM) to acclimate to CO₂-limiting stress (LC). Although previous studies have identified LC-inducible proteins during the CCM induction, detail subcellular localization of the proteins have not been examined. In this study, we focus on identifying the localization of LCIC protein, which showed rapid and strong induction during the CCM induction. The predicted amino acid sequence of LCIC shared 55.9% identities with that of LCIB, which also identified as a LC-induced protein. We examined the subcellular localization of LCIC by generating the strains harboring GFP-tagged LCIC and also by immunoelectron microscopy using anti-LCIC antibody. The possible role of the LCIC protein will be discussed.

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The carbonic anhydrase (PtCA1) in the marine diatom Phaeodactylum tricornutum is regulated in response to changes in the ambient [CO₂]. Our previous studies have shown that critical CO₂-response region of the (Pptca1) promoter, comprises a cAMP-response element (CRE1) and a p300-binding site (p300bs) within -70 to -10bp relative to the transcription start site. In the present study, we attempted to model a fundamental structure of promoter in response to CO₂. CRE1 or p300bs was fused directly to minimal promoter of Pptca1 (mPptca1), and its transcriptional activities were evaluated. As results, CRE1 could confer mPptca1 with clear CO₂ response whereas p300bs could not, indicating that CRE1 is one of critical elements for CO₂ response. Meanwhile, three anti parallel repeat sequences located overlapping to CRE1 and p300bs and upstream region of CRE1 were recently found to be related in response to CO₂. Topological relations of these putative cis-elements will be discussed.

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The Β-type carbonic anhydrase (PtCA1) localizes as clustered particles at the surface of the girdle lamellae in the chloroplast of the marine diatom Phaeodactylum tricornutum. PtCA1 has been suggested to play important role in CCM but details of PtCA1 particle isn't known. PtCA1 cluster is formed only in the chloroplast, therefore this cluster is probably requires some aid of unknown stromal factors. In this study, we attempted to detect stromal factor. Rubisco was first thought to be the factor and thus localized using GFP laveling method. However, Rubisco localized at the stroma didn't form particles. LciB, a protein which is thought to be essential for CCM in the green algae localizes as cluster around the pyrenoid of C. reinhardtii. P. tricornutum has a homologue of LciB gene (PtLciB), which was cloned and fused with the egfp. Localization of PtLciB and results from immunoprecipitation assay by PtCA1 antibody will be discussed.

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Marine diatoms can grow in various salt concentrations. However, the mechanisms of acclimation to low salt environment in marine photoautotrophs have not been reported. In this study, low-salt-response was investigated at transcription levels using the marine diatom Phaeodactylum tricornutum. Cells were transferred from standard (0.5 M [Na⁺]) to low salt (0.1 M [Na⁺]) and growth profile was monitored. RNA was obtained from each acclimation stages, pre-acclimation, low-salt-shock and acclimation stages, and then a semi-exhaustive transcriptome analysis was performed using cDNA-AFLP (cDNA-amplyfied fragment length polymorphism) technique. At present, 34 cDNA-fragments were identified as low-salt-response genes. Expression profiles of these fragments were categorized into 3 types; low-salt-induced, low-salt-shock-induced, and low-salt-repressed. The numbers of fragments of these types were 12, 11 and 12, respectively. ABC transporter SMC protein was found to be low-salt-induced type and Na⁺/H⁺ antiporter was found to be low-salt-shock-induced type. Casein Kinase II β subunit was found in low-salt-repressed type.

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The two-component system consisting of DmsS/DmsR is involved in the inductive expression of dmsCBA operon of Rhodobacter sphaeroides f. sp. denitrificans by DMSO (dimethyl sulfoxide) under anaerobic conditions. Sensor histidine kinase DmsS has no putative membrane-spanning hydrophobic amino acid regions, although other sensor proteins usually contain transmembrane region. Analysis of the transmembrane regions of DmsS by LacZ fusion showed it did not stick out to the periplasm.
In this study, we constructed and assayed the expression of dmsS-phoA
fusion genes. All of the strains showed no PhoA activity, suggesting that DmsS was located in the cytoplasm. These results indicated that the sensing domain of DmsS was located in the cytoplasm and DMSO in the cell is a transcriptional signal of dms operon.

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Green sulfur bacteria grow phototrophically using sulfide as well as thiosulfate as electron donor. We purified three proteins (Fraction I, II and III) which are involved in oxidation of thiosulfate from Chlorobaculum (formally Chlorobium) tepidum cell extract. Fraction I contains heterodimer of SoxY and SoxZ, Fraction II contains SoxB, and Fraction III contains heterotrimer of SoxA, SoxX and CT1020. For electron transfer from thiosulfate to cytochrome c-554 which is an immediate electron donor to photosynthetic reaction center, all of three fractions were indispensable in reaction mixtures. We purified fourth component (Fraction IV) from C. tepidum cell extract. Fraction IV contains SoxF2 and its absorption spectra indicated SoxF2 is a flavoprotein. When SoxF2 was added to the mixture of Fraction I-III, the reduction rate of cytochrome c-554 was accelerated about twice. In this study, we will report some of biochemical properties of SoxF2 involved in thiosulfate oxidation in C. tepidum.

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Green sulfur bacteria grow phototrophically using sulfide as well as thiosulfate as electron donor. We purified three proteins which are involved in oxidation of thiosulfate from Chlorobaculum (formally Chlorobium) tepidum cells. For electron transfer from thiosulfate to cytochrome c-554, SoxY, SoxZ, SoxAX-CT1020 were indispensable in the reaction mixtures. As for CT1020, which is a subunit of heterotrimer of SoxAX-CT1020, the presence of this component is limited to the species of green sulfur bacteria which have an ability of thiosulfate oxidation. Other thiosulfate oxidizing bacteria have heterodimer SoxAX without CT1020. To investigate whether CT1020 is essential for the thiosulfate oxidation in C. tepidum, we expressed SoxA, SoxX and CT1020 in Escherichia coli respectively. The reaction mixture contained three recombinant proteins showed almost the same thiosulfate oxidation activity as that of purified C.tepidum cells, however, if one of three was omitted from the reaction mixture, no activity was observed.

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The green sulfur bacteria are strictly anaerobic photototrophic bacteria. Their reaction center (RC) complex is supposed to form a homodimeric structure and is classified into the type 1 RC which contains Fe-S clusters as terminal electron acceptors. The electron transfer reactions have been studied so far with various biochemical and spectroscopic analyses. However, physical properties of cofactors involved in the reaction have not completely been clarified yet. Especially, it is still controversial whether functional quinone molecule is present within the RC.
Molecular genetic methods are thus expected to be the most suitable approach to resolve the issues. Since Chlorobium tepidum is amenable to a genetic manipulation and the information of its genome sequences is available, we have executed recombinant experiments to obtain mutated RC core proteins. We would like to discuss the best methodology to explore the electron transfer pathway and its mechanism within the RC.

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We previously reported that the disruptants of slr0846 express lower levels of psaAB, and exhibit lower content of photosystem I and chlorophyll than wild-type. Slr0846 shows homology to IscR, which is an Fe-S cluster-binding redox responsive transcription regulator in Escherichia coli, but all critical cysteine residues required for the Fe-S cluster are not conserved. In this study, we examined multiple transcription start points of psaAB in slr0846 mutants by using the primer extension method. As a result, the slr0846 disruptants showed decrease in the psaAB genes transcription and over-expressing mutants showed increase in the transcription irrespective of the transcription start points. Since degradation of psaAB mRNA in the mutants is comparable to wild-type, it is suggested that Slr0846 is involved in the psaAB transcription. We also present data of pseudo-revertants isolated from the slr0846 disruptants.

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We had found phototropic response in filamentous cyanobacterium Rivularia sp. IAM M-261. Further analysis is in progress, but application of genetic analysis to this strain has been unsuccessful.
In order to obtain transformable species, we looked for other cyanobacteria showing phototropism. As a result, we found that following cyanobacteria exhibit phototropic response, Scytonema sp. IAM M-291, Tolypothrix distorta IAM M-98, Calothrix thermalis PCC7715, Calothrix desertica PCC7102 and one Calothrix species isolated from field. Most of these are terrestrial species and phototropism is expected to be advantageous to live on land. Phototropic response of PCC 7715 was induced by blue light as well as M-261.

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Lichens are symbionts of fungi with algae or cyanobacteria. We analyzed photosynthetic system of cyanobacteria inside lichens by the measurement of fluorescence spectrum of chlorophyll with a confocal laser microscope that is combined with a high-resolution spectrometer. The system allows the measurement with a spatial resolution of 200x200 nm for x-y plane and 1000 nm for z-axis, with the wavelength resolution of 1 nm. We measured the spectrum and image of each cyanobacterial cell, calculated their PS I/II ratio , and studied the distribution of cyanobacteria inside the lichens. We selected four species of lichens that contain cyanobacteria. We confirmed that cyanobacteria in Nephroma and Collema complanatum retain their filamentous cell shapes. However, we could not identify the clear filamentous structure in Peltigera polydactyla and Peltigera praetextata. The features of their photosynthetic systems compared with the cultured cells of cyanobacterium isolated from xxxx.

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In cyanobacterium Synechococcus elongatus PCC 7942, KaiC is an essential protein for circadian rhythm generation. Here, we screened KaiC-association protein and isolated DnaA, which is well known as DNA replication initiation factor in bacteria. Disruption of dnaA gene in wild-type cell shortened the period of gene expression in Synechococcus elongatus PCC 7942, and mutants with inactivated dnaA were viable. Disruption of dnaA in some KaiC point mutants changed circadian gene expression rhythm besides their original phenotype. We also found that DnaA and KaiC formed complex especially at night and DNA binding domain of DnaA was important for DnaA function. Thus, we proposed that DnaA is a modifier of circadian rhythm by associating and modulating the KaiC.

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KaiC phosphorylation oscillation is the pacemaker of the cyanobacterial circadian clock. We recently demonstrated a self-sustainable robust circadian oscillation of KaiC phosphorylation by reconstituting KaiA, KaiB and KaiC proteins with ATP in vitro. A sequential program of phosphorylation of KaiC on two sites, S431 and T432, has been characterized and found that the phosphorylation state of each of these two residues regulates the phosphorylation /dephosphorylation of the other. The temperature-compensated ATPase activity of KaiC oscillates in a circadian manner in vitro. The activities of KaiC mutant variants without KaiA and KaiB were directly proportional to their cycle frequencies, indicating that the ATPase activity defines the circadian oscillation period. The mechanism of circadian clock is intrinsic to KaiC itself and at least three enzymatic activities, ATPase, kinase, and phosphatase, of KaiC mutually influence each other. Recent biochemical analysis of KaiC will be presented.

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The Arabidopsis thaliana mutant, cfa1, was originally isolated as a mutant that showed altered chlorophyll fluorescence induction kinetics. Furthermore, cfa1 showed somewhat slower growth compared with WT plants especially under high light condition. When we used pulse amplified modulation (PAM) fluometry under weak light (40 μmol/m²/s) and 0% CO₂ with different O₂ concentrations, cfa1 showed lower photochemical quenching (qP) as well as lower non-photochemical quenching (NPQ) than WT only under 2-5% O₂ conditions. The results indicate that the electron transport chain was more reduced and the thermal dissipation was suppressed in cfa1 compared with WT. Since cfa1 and WT did not show significant difference in photosynthetic parameters under 0% O₂, we concluded that CFA1 functioned in enhancing the affinity to O₂ in O₂-dependent reaction such as photorespiration or water-water cycle.

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We describe a methodology to visualize three dimensional
distributions of photosystems in the thylakoid membrane in
chloroplasts under nearly in-vivo conditions. Chlorophyll
fluorescence spectra from the chloroplasts in leaves of
Zea mays were analyzed at room temperature by a newly
developed spectroimaging microscope employing a two-photon
excitation at 800 nm. We have first determined two
wavelength regions, by which one can exclusively probe the
fluorescence from photosystem 1 and 2, each of which
includes associated light-harvesting complexes. The map
of photosystem 2 fluorescence often show fine structures
in which fluorescence intensity exhibit local peaks and
valleys with a characteristic length as short as 0.3
μm. The local peaks plausibly correspond to the granal
stacked part of the membrane. The distribution of PS1
fluorescence was certainly different from that that of
PSII. Implications of these results for the detailed
picture of thylakoid membrane in the chloroplasts will be
discussed.

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Chlorella, a green alga, has a chloroplast that contains thylakoid membrane with photosynthetic pigments both under light and dark conditions. We have studied their fine structures and spectra of subchloroplast regions by fluorescence spectromicroscope, in an effort to understand correlation between ultrastructural morphology and physiological functions of chloroplasts in general. Chlorella chloroplast seems to be an interesting control sample compared with plant chloroplast, from the viewpoint of spectromicroscopic investigation. Chlorella chloroplast is free from differentiation. It is a single chloroplast in the small cell, where light can penetrate easily. It is thus possible to quantitatively estimate the concentration of the photosynthetic pigments, by which reabsorption effects on subcellular fluorescence spectra can be well appreciated. Electron microscopic studies in the past have shown that it is free from granal stacking of thylakoid membrane. It is thus regarded as a nice control sample in comparison with plant chloroplast having granal stacking.

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Light-harvesting complex II (LHCII) of photosystem II (PSII) adjusts light absorption cross section of photosystem I (PSI) and PSII under changing light environments, a process called state transitions. This short-term acclimation mechanism consists of three processes: phosphorylation of LHCIIs, unstacking of thylakoid membranes, and migration of LHCIIs from PSII to PSI. However, detailed mechanism for each process still remains unclear. Here, to investigate the role of unstacking of thylakoid membranes in state transitions, we analyzed 1) fluorescence quenching, 2) phosphorylation of LHCIIs, and 3) observation of thylakoid membranes with electron microscope during dark period using several mutants. The results indicated Δycf9 mutant, which was deficient in a formation of PSI-LHCI/II supercomplex, still showed the decrease in PSII fluorescence, phosphorylation of LHCIIs as well as unstacking of thylakoid membranes. Therefore, we suggest unstacking of thylakoid membranes has an important role for decreasing the excitation energy transfer between PSIIs in state transitions.

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PsbP is an extrinsic subunit of photosystem II (PSII) specifically found in land plants and some green algae. To understand the physiological function of PsbP, tobacco plant (ΔPsbP tobacco) in which PsbP was severely down-regulated by RNAi has been analyzed. In ΔPsbP tobacco, maximal quantum yield of PSII (Fv/Fm) was severely decreased, whereas accumulation of PSII subunits was comparable to that in wild-type [Ifuku et al (2005) Plant Physiol. 139: 1175-1182]. In this study, we examined supercomplex organization of thylakoid membrane proteins in ΔPsbP tobacco by Blue-Native PAGE. Phosphorylation status of LHCII and major PSII subunits was also analyzed. The results showed that the amount of PSII-LHCII supercomplex was decreased and PSII was mainly accumulated as dimer in ΔPsbP leaves. Furthermore, LHCII and major PSII subunits were almost dephosphorylated in ΔPsbP leaves. The function of PsbP will be discussed in terms of the dynamic life cycle of PSII.

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Photosystem II (PSII) is composed of membrane intrinsic and extrinsic protein subunits. While most intrinsic subunits are encoded by the chloroplast genome, extrinsic subunits, known as oxygen-evolving complex (OEC), are nuclear-encoded protein in higher plants. In addition to authentic OEC proteins in PSII, higher plants have number of OEC homologs in thylakoid lumen, indicating that genes for OEC proteins were susceptible to diversification during endosymbiosis and subsequent gene transfer to the host nucleus. However, molecular function of OEC homologs has not been elucidated. In this study, we investigated mRNA expression profiles of Arabidopsis OEC homologs using the microarray data from AtgenExpress. Our bioinformatic analysis suggests that some OEC homologs would be involved in the regulation of PSII function, and others in NAD(P)H dehydrogenase complex. The validity of our in silico analysis has been proven in vivo by using corresponding Arabidopsis mutants (Ishihara et al. [2007] Plant Physiol. 145:668-679).

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Extrinsic proteins PsbO, P, and Q are bound to PhotosystemII(PSII)at the lumenal surface of thylakoid of higher plants, and maintain oxygen evolution activity. The PsbO protein is known as the manganese stabilizing protein, which is released from PSII by light or heat stresses. Moderate heat stress(40 degrees for 30 min)induces damage to the reaction center-binding D1 protein of PSII, and our recent data suggest that reactive oxygen species are involved in this event. As it is possible that reactive oxygen species generated around PSII induce damage to the extrinsic proteins, we investigated heat-induced release of PsbO from PSII under aerobic and anaerobic conditions using PSII-enriched membranes from spinach. We found that the release of the PsbO protein was suppressed under anaerobic conditions. The addition of Na-ascorbate suppressed the release of PsbO under aerobic conditions. These results suggest that reactive oxygen species are involved in the release of extrinsic proteins.

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Some bryophytes are known to have high desiccation tolerance. In order to clarify the mechanism of the tolerance, we have investigated changes in various photosynthetic activities caused by dehydration using bryophytes grown under different water environments. We found that PSII fluorescence is quenched and the PSII reaction center activity is lost by dehydration, which seems to be important to protect PSII from high light under dry conditions.
It is known that bryophytes have the xanthophyll cycle which protects the cells by inducing nonphotochemical quenching (NPQ) of PSII fluorescence. However, even when the xanthophyll cycle was stopped by dithiothreitol, desiccation-induced NPQ was observed. HPLC analyses suggested that the desiccation-induced NPQ had no relation to the xanthophyll cycle.
Desiccation-tolerant moss, B. argenteum, became less tolerant when it was cultured in a liquid medium, and the responses for desiccation were modified. Acclimation processes to desiccation in cultured B. argenteum will be discussed.

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Irradiance beneath the sea ice also changes diurnally. We investigated the photosynthetic response of ice algae in Saroma-ko lagoon to the change of irradiance. The photosynthetic properties were assessed by PAM.
The maximum electron transport rate (ETRmax) increased from sunrise until around noon and decreased toward sundown. The maximum efficiency of photosynthesis changed inversely. Under 350 μmol photons/m²/s, which was enough to saturate the electron transport in the subjective ice algae, the level of non-photochemical quenching showed the same variation pattern as ETRmax. This means that ice algae adjust their capacity to dissipate excess light energy according to the diurnal change of irradiance.
These results indicate that ice algae regulate photosynthetic system totally to achieve efficient photosynthesis according to the diurnal change of irradiance. This regulation system may be essential for the productive photosynthesis of ice algae to utilize the weak light avoiding damages to photosystems.

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The Positron Emitting Tracer Imaging System (PETIS) and caron-11-labeled carbon dioxide (¹¹CO₂) can image carbon kinetics and parametric images of photosynthesis. With an exposure ¹¹CO₂ -gas to a leaf, PETIS experiments were performed against several temperature conditions in one individual plant. In order to make photosynthetic parametric images quantitatively, all time activity curves each pixels were fitted to a tracer kinetic model, the photosynthetic compartment model. These results are reasonable response against temperature conditions, and important to discuss photosynthesis in plant physiology and agriculture. Kinetic parameters are fitted enough with pixel-by-pixel analysis, so that our method suggest that a parametric imaging of photosynthesis, which is indicative of a plant molecular imaging.

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Soybean have lower nitrogen use efficiency than rice. Lower photosynthetic rate per unit nitrogen of the leaves and high respiratory rate would be one of the reason for lower nitrogen use efficiency in soybean. However, it is not clear about the detail of metabolic mechanism that caused mentioned above physiological phenomenon in the leaves. In this study, photosythesis and metabolite were measured in leaves of rice and soybean in order to get the basic knowledge of the difference of metabolic mechanism.
The quantum yeild of PSII and non-photochemical quenching (qP) was lower in soybean than in rice and Fv'/Fm' in soybean was similar to that in rice. Leaf photosynthetic rate per unit amount of nitrogen, stomatal conductance, and leaf intercellular CO2 concentration in soybean was lower than that in rice. We also measured the primary metabolites with CE/MS and comparison of metabolism between soybean and rice would be futher discussed.

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In photosynthesis of C3 plants, CO₂ molecules diffuse depending on [CO₂] gradient between the ambient and mesophyll cell chloroplasts. When plants are subjected to drought stress, stomatal conductance (g_s) generally decreases in response to abscisic acid (ABA) produced from the roots. However, it is little information about effects of ABA on internal conductance to CO₂ diffusion from the stomatal cavities to the active sites of Rubisco (g_i). Synthetic ABA (0.125-5 μM) was fed from the petioles of tobacco (Nicotiana tabacum Xanthi.), and changes in net photosynthesis rate on an area basis (A) against [CO₂] in the stomatal cavities (C_i) were obtained. Simultaneously, [CO₂] in the chloroplasts (C_c) was calculated from the combined measurements with chlorophyll fluorescence intensity. g_i and initial slopes of A-C_i responses significantly reduced after the ABA applications although the initial slope of A-C_c did not change, suggesting that g_i as well as g_s is regulated by ABA.

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Absorption of excess-light energy causes generation of reactive oxygen species that leads to irreversible damages of photosynthetic apparatus. Thus, the effective elimination of excess-light energy is essential for the prosperity of land plants. Excess-light situation does not simply occur in parallel with the intensity of light but is enhanced by environmental conditions such as low temperature and water deficit under low light. Plants possess multiple mechanisms against excess-light and the method is varied for the stress conditions. In evergreen conifer, Taxus cuspidata, the nuclear-encoded thylakoid membrane protein early light-induced proteins (Elip) are drastically induced in response to low temperature. It is recently reported that one-helix thylakoid membrane protein (Ohp) and two-helix proteins (Sep) in Arabidopsis were accumulated in response to excess-light. In this study, we will report that the accumulation levels of Ohp and Sep in the needles of Taxus cuspidata in relation to seasonal changes.

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The reaction center-binding protein D1 of Photosystem II (PSII) is damaged by high temperature or excessive visible light. The damaged D1 protein is degraded and removed from PSII or form aggregates with other PSII subunits.
When the thylakoids membranes from WT of a cyanobacterium Synechocystis sp.PCC6803 were illuminated, degradation and aggregation of the D1 protein were observed. Whereas degradation of the D1 protein was not detected in the thylakoids from the lacking of FtsH (slr0228) mutant, and D1 aggregation became prominent in these thylakoids. When the thylakoids with an increased level of FtsH obtained from low temperature-treated WT cells were illuminated with strong light, the D1 aggregates were less significant compared with the WT thylakoids. From these results, it is suggested that the FtsH proteases play a dual role as a protease for D1 degradation and a molecular chaperone to prevent accumulation of the D1 aggregates.

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Vitamin E (α-tocopherol) is an antioxidant that quenches singlet oxygen and free radicals. However, the mechanism by which vitamin E protects photosystemII (PSII) against photooxidative stress remains unclear. In this study, we examined the role of vitamin E in the photoinhibition of PSII in a mutant of Synechocystis sp. PCC 6803 that lacks the synthesis of vitamin E.
The PSII activity of the mutant decreased more rapidly than that of wild type under strong light at 1,500 μE m^-2 s^-1, indicating that photoinhibition was promoted by the lack of vitamin E. In contrast, there was no difference in the progress of photodamage to PSII between the two types of cell in the presence of chloramphenicol. It appears that vitamin E protects PSII from photoinhibition by alleviating the inhibition of the repair of photodamaged PSII.

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We previously reported comprehensive analysis of nitrogen-responsive gene expression in the shoots of rice and identified two MYB proteins (MYB-NR1 and MYB-NR2) as candidates for nitrogen-responsive transcription factors in rice. Here we show our recent progress in the analysis of nitrogen-responsive gene expression in rice. We found that a nitrogen-supply up-regulated 732 genes and down-regulated 190 genes in the roots of rice. Our analysis also revealed that MYB-NR1 and MYB-NR2 are induced by the nitrogen-supply in both shoots and roots. Further characterization of MYB-NR1 and MYB-NR2 indicated a strong expression of these genes in germinating seeds and leaf blades. By the use of maize protoplast transient assays, we also show nuclear localization of MYB-NR1 and MYB-NR2 fused to GFP. We are currently attempting to analyze transgenic rice plants overexpressing MYB-NR1 or MYB-NR2. This work was supported in parts by grants from the Program for promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

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Nitrate is an important nitrogen nutrient source and is also a signaling molecule that modulates gene expression, metabolisms, growth and development in higher plants. It has been well known that the genes involved in nitrate assimilation are induced in response to nitrate application. Recent studies based on microarray experiments have also shown that nitrate induces changes in expression of a broad range of genes. As a first step for understanding the mechanism of nitrate signal transduction, we made several reporter lines and analyzed their nitrate response. The results showed that the flanking sequences of these genes mediate nitrate-responsive gene expression, suggesting the presence of cis-element(s). We are now carrying out the deletion analysis of these reporter lines in order to delineate the DNA region which is important for nitrate response.
This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

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