In higher plants, chlorophyll a is converted to chlorophyl b through 7-hydroxymethyl chlorophyll a, and chlorophyll b returns to chlorophyll a again through 7-hydroxymethyl chlorophyll a. This conversion system is called chlorophyll cycle.
With the aim of identifying the enzymes and the regulatory factors that are involved in chlorophyll cycle, we screened EMS-mutagenized Arabidopsis thaliana, and we isolated a mutant that accumulated 7-hydroxymethyl chlorophyll a. We named this mutant hmc1. This mutant showed remarkably slow growth, and accumulated 7-hydroxymethyl chlorophyll a more than 10 times in its early developmental stage compared with those in wild type. We also found that this mutant accumulated more 7-hydroxymethyl chlorophyll a with an increasing leaf age. Currently, analysis of the mutant and fine mapping of hmc1 mutant is being undertaken.

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Recently, MALDI (matrix-assisted laser desorption/ionization)-TOF (time-of-flight) MS (mass spectrometry) is one of principal methods for proteins and amino acids analysis. However, there is a little information on the analysis of plant pigments including chlorophylls (Chls). In this study, we investigate optimum parameters to detect Chl a by MALDI-TOF MS. We selected terthiophene and alpha-cyano-4-hydroxy cinnamic acid (CCA) as a matrix. We found a stable signal of Chl a using terthiophene. When CCA was used, pheophytin a was detected in addition to Chl a, indicating that acid degradation of Chl occurred by matrix used. A simultaneous analysis of Chl a degradation products such as pheophorbide a, chlorophyllide a and pheophytin a was available by these matrixes. We are continuing to study on the analysis of chlorophyll c, bacteriochlorophyll a and other pigments.

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Plant pigments of corals were analyzed by high-performance liquid chromatography (HPLC) to investigate the secondary symbiotic algae of zooxanthella (dinoflagellate). Coral samples were collected from Sesoko, Okinawa. Pigment analysis was carried out using C₈ column and binary elution system with photodiode array detection. HPLC analysis achieved the separation of more than 60 peaks of the pigments from more than 10 species of corals and about 30 pigment species of them were identified. In addition to marker pigments of dinoflagellate, a variety of pigments including chlorophyll d, chlorophyll b, chlorophyll c₁, zeaxanthin, and lutein were detected. These findings suggest that in addition to the pigments from dinoflagellate, some of the pigments are probably due to epiphytic or endophytic algae growing in corals such as cyanobacteria, green algae and diatoms.

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Carotenoids in cyanobacteria have diversity from species to species. Genera Anabaena and Nostoc have unique polar ketocarotenoids, ketomyxol glycosides. In Anabaena 7120, ketomyxol fucoside is obserbed (Takaichi et al., 2005, PCP). Ketolase, CrtW, is the only enzyme that functionally identified among the synthetic pathway from lycopene to ketomyxol glycosides (Mochimaru et al., 2005, FEBS Lett.). We also identified β-carotene hydroxylase, CrtR, in Anabaena 7120.
In this study, we tried to determine fucose synthase by gene disruption. From the disrupted mutant of all4826 (homologue of GDP-fucose synthase from Synechocystis 6803), more polar carotenoid glycosides than those of wild type were obtained. NMR data indicated that the glycoside moiety seemed to be different methylpentose than that of any reported myxol glycosides, such as α-L-fucose, α-L-rhamnose, and α-L-chinovose. It suggests that in Δall4826, fucose is not synthesized and the methylpentose binds to myxol as a substitute.

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Recently, we isolated the purple photosynthetic bacterium, Rhodopseudomonas sp. strain Rits, which is phylogenetically close to Rhodopseudomonas palustris. These bacteria have similar photosynthetic properties that both bacteria synthesize light-harvesting(LH) 4, showing different absorption spectrum from LH2, under low-light conditions, in addition to producing LH1 and 2 under high or normal light conditions. The compositional analysis of chlorophyll pigments from the strain Rits indicated that this bacterium accumulated a significant amount of Bacteriochlorophyll (BChl) a intermediates possessing non/di/tetrahydrogeranylgeranyl groups at the C-17 propionate. However, the localization and function of these intermediates in this organism were still unrevealed. In this study, we analyzed the compositions of BChl a and carotenoids from intact cells of Rits that were grown by irradiation of different light intensity, and decided the localization of these pigments in this bacterium by investigating pigment compositions of RC-LH1, LH2 and LH4 complexes purified from those cells.

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In tetrapyrrole biosynthetic pathway, isoforms of glutamyl-tRNA reductase (HEMA2) and Fe-chelatase (FC1) mainly express in non-photosynthetic organs. Previously, we showed that the expression of HEMA2 and FC1 was induced in photosynthetic organs by wounding or ozone exposure. Here, the effects of oxidative stresses on the expression of HEMA2 and FC1 were assessed using reagents generating reactive oxygen species (ROS). Treatment of methyl viologen or Rose Bengal induced the expression of both genes, suggesting ROS generation is involved in the induction. Heme contents in roots of knockout mutant of each gene were about half of respective wild types. In wild types, heme contents were increased by ozone, while in both mutants, the reduction of the ozone-induction was observed. These results suggest the existence of tetrapyrrole biosynthetic pathway, which is normally function for heme biosynthesis in non-photosynthetic organs, but induced in photosynthetic organs under oxidative conditions to supply heme for defensive hemoproteins.

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In plant cells, heme is synthesized in chloroplasts and transported to various organelles. Furthermore, tetrapyrrole intermediates is presumed as plastid-derived signals that regulate the expression of nuclear photosynthetic genes. Previously, we showed the existence of p22HBP/SOUL gene family in Arabidopsis thaliana that is involved in tetrapyrroles transport in animal cells, and actually one of recombinant proteins could bind heme. Here, we analyzed cytosolic tetrapyrrole binding protein (TBP) of Arabidopsis. Among 4 putative cytosolic TBP genes in database, ORF of 1 gene (At1g78450) contained deletion suggesting this is pseudogene. Using purified recombinant proteins of At1g17100 (TBP17100) and At3g37970 (TBP37970), we performed binding analysis to metal porphyrins. Both proteins showed specific binding to heme, while they non-specifically bound to other metal porphyrins. TBP17100 showed specific binding to protoporphyrin IX, whereas TBP37970 possessed only nonspecific binding. These results suggest that TBP17100 and TBP37970 have distinct functions for cytosolic tetrapyrrole transport in plant cells.

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Biosynthetic pathways of chlorophyll (Chl) a and bacteriochlorophyll (BChl) a are common from protoporphyrin IX to chlorophyllide (Chlide) a. Chl a is synthesized from Chlide a by ChlG, while BChl a is synthesized from BChlide a by BchG. Green sulfur bacterium Chlorobium tepidum synthesis three kinds of chlorophylls, BChl a, Chl a, and BChl c. In the genome of C. tepidum, three homologues of chlG/bchG (CT1270, CT1610 and CT1992 ) are found. In this study, we introduced CT1270 and CT1610 to strains of purple bacterium Rhodobacter capsulatus, which were knockouted BChl a biosynthesis genes. We observed that novel pigments were accumulated in the CT1270-introduced cell. We also found that a single pigment component which has a same retention time of Chl a was eluted by HPLC. It was revealed that this pigment has the same molecular mass as phytyl-Chl a by LC-Mass analysis.

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We have tried to measure precisely the redox potential of the primary electron donor of photosystem (PS) I, P700, with a spectroelectrochemical technique. Then, we have succeeded to determine the P700 redox potential, and also have found for the first time the species-dependence of the P700 redox potential. In the present study, we investigate the spectroscopic properties of P700 to elucidate the factors for the species-dependence. The differences in absorbance and circular dichroism (CD) of P700⁺ - P700 of cyanobacteria, red and green algae, and higher plants were different from species to species. The spectroscopic results will be discussed with the species-dependence of the P700 redox potential.

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Cytochrome (cyt) b559 is an essential component of photosystem (PS) II. Although cyt b559 is supposed to play a role for the protection of PS II from damage caused by excess light energy, its function remains unclear. The redox potentials of b-type cyt usually ranges from 0 to +100 mV (vs. SHE), while some researchers have reported using a chemical redox titration technique that cyt b559 exhibits a different redox potential around +400 mV. In the present study, to examine the redox property of cyt b559 precisely, we have tried to measure the redox potential of cyt b559 in D1/D2/cyt b559 reaction center complex isolated from spinach with a spectroelectrochemical technique. The redox potential of cyt b559 in the sample was +89.5 ± 3.0 mV (3 measurements). Furthermore, cyt b559 exhibited a one-electron redox reaction only at the potential in the range from 0 to +400 mV.

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Green sulfur bacteria grow phototrophically using sulfide as well as thiosulfate as electron donor. In this study, we purified thiosulfate-oxidizing multi enzyme from Chlorobium tepidum. Thiosulfate-oxidizing multi enzyme catalyze oxidation of thiosulfate and reduction of cytochrome c. Three fractions (Fraction I, II, III) which are involved in oxidization of thiosulfate were purified by ammonium sulfate fractionation, anion-exchange chromatography and cation-exchange chromatography. Fraction I contains heterodimer of SoxY and SoxZ, Fraction II contains monomer of SoxB, and Fraction III contains heterotrimer of SoxA, SoxX, and CT1020. For electron transfer from thiosulfate to cytochrome c554, all of three fractions were indispensable in reaction mixtures. We estimated the V_max value is 15 µM Cyt c554 reduction/µM each factor/min and the K_m is 0.24 mM.

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State transition is a short-term acclimation to balance excitation ratio between Photosystem I (PSI) and II (PSII) in oxygenic photosynthetic organisms. When PSII was preferentially excited (state 2), a part of light harvesting complex II (LHCII) migrates from PSII to PSI, forming PSI-LHCI/II supercomplex. We have already reported the isolation of this supercomplex containing CP26, CP29, and LhcbM5 from Chlamydomonas cells in state 2. Here we report the effect of ionic strength on association of LHCIIs with PSI-LHCI complex in order to analyze pigment composition of the PSI-LHCI/II supercomplex. We found that LHCIIs are detached from the supercomplex in a high ionic strength, which can be used to dissociate and purify LHCIIs under a mild condition. We will also report pigment contents of LHCIIs bound to PSI complex.

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Photosystem I (PSI) is a large complex composed of a number of subunits and cofactors. We are interested in the function of the chloroplast-encoded Ycf3 and Ycf4 that are essential for PSI assembly. Here mutants were generated in which 2-9 amino acid residues at N-terminus of Ycf4 have been deleted. Deletion of seven residues (N7) drastically affected PSI complex assembly, indicating the N-terminal sequence has an important role in the stability and function of Ycf4. Separation of PSI complex from the thylakoid extracts from N7 revealed the accumulation of a small amount of unusual PSI complex. According to the apparent size, this PSI complex can be a deteriorated complex or assembly intermediate. We also analyzed the chloroplast mutants in which ycf3 and/or ycf4 had been knocked-out. We will discuss a possible involvement of Ycf4 and Ycf3 in an initial assembly stage of PSI complex.

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The green alga Chlamydomonas reinhardtii contains nine distinctive LHCIs; seven out of nine accumulate at a stoichiometoric amount with PSI subunits and the remaining two are present at a lower level. Since LHCIs associate stably with PSI complex, they are purified as PSI-LHCI supercomplex in the presence of a nonionic detergent. In order to characterize the structure/function relationship of PSI-LHCI supercomplex, we have developed an efficient method for the supercomplex purification. Thylakoid membranes were solubilized with dodecyl-maltoside, fractionated on sucrose density gradient, and subsequently purified by anion exchange column chromatography. We optimized sucrose density gradient for ultracentrifugation and NaCl gradient in elution buffer of DEAE chromatography for the satisfactory purification of a larger amount of PSI-LHCI supercomplex. The resulted preparation retains most constituent polypeptides except two peripheral subunits and shows a high PSI activity. The present purification method can be applied for PSI-LHCI supercomplex on a large scale.

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The green alga Chlamydomonas reinhardtii has 14 photosystem I (PSI) genes, but PsaN and PsaO have not been detected in the purified PSI preparation. PsaN appears to be an extrinsic protein on the lumenal surface while PsaO with two putative transmembrane helices appears to be intrinsic. Their location in PSI complex is not known because these two subunits are lost in the PSI preparations used for crystal structure analyses. Here we raised antibodies against synthetic oligopeptides corresponding to PsaN and PsaO, respectively. We found that both PsaN and PsaO are stably present in the isolated wild type thylakoids but are easily lost during treatments of the thylakoids with chaotropic agents or mild detergents. It was clearly shown that no PsaN and PsaO accumulate in PSI-deficient mutant, strongly suggesting that they are PSI subunits in Chlamydomonas. We will also discuss the results obtained from otherChlamydomonas PSI mutants.

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Ferredoxin-NADP-oxidoreductase (FNR) catalyzes NADPH production by photosystem I in the presence of ferredoxin. A part of FNR in the chloroplast is stably associated with the purified thylakoids and is fractionated with cytochrome b₆f complex in higher plants, suggesting its possible involvement in the cyclic electron transport. Here we have estimated the amount of FNR in the thylakoids purified from wild type and various photosynthetic mutants of Chlamydomonas. The thylakoids purified from wild type contained 40% of total FNR and showed NADP photoreduction activity in the presence of ferredoxin. In contrast, the thylakoids isolated from photosystem I mutant lacked FNR. These results suggest that the association of FNR with the thylakoids is stabilized by the presence of photosystem I complex. However, the absence of cytochrome b6f and photosytem II also affected the stability of FNR on the thylakoids. We will discuss a possible function of the bound FNR.

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We have introduced functional membrane protein complex into a silica mesoporous material (SMM) that has the nano-scale pores of honeycomb-like hexagonal cylindrical structure in its inside. The photosynthetic reaction center (RC) complex (129 kDa) and light-harvesting LH2-complex (137 kDa) that were purified from a thermophilic purple photosynthetic bacterium Thermochromatium tepidum were introduced into SMM already shown to be (ref.).
In this study, we introduced the trimer complex of photosystem I (PS I) reaction center (1068 kDa) purified from a thermophilic photosynthetic cyanobacterium Thermosynechococcus (T.) elongatus into SMM that has pore of 23.5 nm diameter. The photochemical activity and pigment assembly as well as the protein structure are almost intact inside SMM. Adsorption to SMM were increased the heat stability of P700 photochemical activity and protein structure.
The PS1-SMM conjugate can be a new material for the solar energy conversion.
Ref.I.Oda,Y.Shibata.T.kajino,Y.Fukushima,S.Iwai,S.Ithoh. J.Phys.Chem.B2006,110,1114-1120

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The cyanobacterium Acaryochloris marina has Chld as a major pigment and contains a small amount of Chla. Whether the special pair chlorophyll (P) of PSII in A. marina is formed by Chld or Chla remains controversial. In this study, we have identified chlorophyll species of P in A. marina using FTIR spectroscopy. Light-induced FTIR difference spectrum upon P oxidation was measured for purified PSII core complexes of A. marina and compared with the spectra of P680 in Synechocystis 6803 and spinach, which consists of Chla. The chlorin ring vibrations of A. marina showed clear differences from those of Synechocystis and spinach, indicating that P of A. marina is formed by Chld. The feature of keto C=O bands of P⁺ was similar to that of P680⁺, suggesting that the charge distribution on P⁺ is also similar. It was thus proposed that P of A. marina is a homo-dimer of Chld molecules.

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When spinach thylakoids were subjected to heat stress, the D1 protein of photosystemII was cleaved, and a 23kDa N-terminal fragment was produced. The cleavage, however, was not observed when the thylakoids were treated with 2M-KSCN, indicating that the protease responsible for the heat-induced cleavage of the D1 protein was solubilized by the KSCN-treatment. In the supernatant of KSCN-solubilized thylakoids, protease activity was detected by gelatin activity gel electrophoresis. The protease activity was stimulated by the addition of Zn, and inhibited by EDTA. Western blot analysis, using antibody against FtsH protease showed that the supernatant of KSCN-washed thylakoids contains FtsH proteases. In addition, we identified the homologue to Arabidopsis FtsH2 and 8 by MALDI-TOF mass analysis of the thylakoids. Reconstitution of the FtsH protease with the thylakoids was also carried out. All these results suggest that the FtsH protease is responsible for the proteolysis of the D1 protein under heat stress.

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Moderate heat stress (40^οC, 30 min) on spinach thylakoids induced cleavage and aggregation of the D1 protein in Photosystem II. An N-terminal 23-kDa and a C-terminal 9-kDa fragments of the D1 protein, as well as aggregates of the D1 protein were detected by Western blot analysis with specific antibodies. FtsH proteases, which are responsible for cleavage of the heat-damaged D1 protein, were abundant in the stroma thylakoids, but less abundant in the grana or the Photosystem II membranes. Interestingly, FtsH was present even in the Photosystem II core. In the heat-stressed thylakoids, the D1 proteins was dephosphorylated and cleaved, while a large amount of aggregates of phosphorylated D1 protein was detected in the grana and Photosystem II membranes. We suggest that the heat-damaged D1 protein was degraded when dephosphorylated and recognized by FtsH proteases, while the damaged and phosphorylated D1 protein aggregated with the D2 protein and CP43.

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Crystal structural model of photosystem II (PS II) revealed the presence of an unknown low molecular weight transmembrane polypeptide in PS II core complexes from Thermosynechococcus elongatus. We analyzed precisely the polypeptide composition of PS II complexes purified from T. elongatus and found one Ycf protein as a PS II component. To confirm the association of this protein in other cyanobacterial PS II complexes, and to investigate the function of it in PS II, we deleted the corresponding gene in Synechocystis 6803.
The mutant cells grew and evolved oxygen at almost the same rate as wild type. However, the oxygen-evolving activity in the purified mutant PS II complexes diminished compared to that in the wild-type PSII complexes. Analysis of polypeptide profile revealed a partial release of extrinsic proteins in the purified mutant PS II complexes. These results indicate that this protein is a structural component in PS II complexes.

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Digalactosyldiacylglycerol (DGDG) is one of the lipid molecules present in thylakoid membranes. DGDG exists not only as a lipid bilayer-forming molecule but also as a component of various complexes in thylakoid membranes. It has been reported that fourteen lipid molecules were bound to photosystem II complex (PSII) of Thermosynechococcus elongatus, and four of them were DGDG. Although the findings suggest that DGDG plays important roles in PSII, its physiological role has not been clarified.
Search with our Gclust server (http://gclust.c.u-tokyo.ac.jp/) predicted that a novel DGDG synthase, which is different from that of higher plants, is conserved among several cyanobacteria and Cyanidioschyzon merolae. Disruption of slr1508 gene in Synechocystis sp. PCC 6803 resulted in a loss of ability to synthesize DGDG. With this mutant, we found that DGDG is not an essential component of PSII, but is required for stabilization of PSII.

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The crystal structure of PSII dimer from Thermosynechococcus vulcanus with its psbTc gene inactivated by insertion mutation of an antibiotic cassette in a site in the C-terminal region, was analyzed at 3.8 Å resolution. In the crystal structure of the mutant PSII, the transmembrane helix of PsbTc remains existed, whereas the stromal loop of PsbTc disappeared. In addition, the PsbM subunit, which is lost in a mutant PSII of T. elongatus lacking psbTc, was still present. While the content of PSII monomer was increased in the psbTc-disrupted mutant than that in the wild type, the dimeric form was still the major one in the mutant. These results indicated that the psbTc-disrupted mutant of T. vulcanus lacks only the stromal loop of PsbTc, which did not affect the binding of PsbM but partially destabilized the dimeric form of PSII, leading to the increase of PSII monomer.

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Cyanobacteria possess a hight activity of cyclic electron transport. The several pathways have been proposed, but it is not established. NAD(P)H-Quinone Oxidoreductase (NQR) is one of the protein which shows NAD(P)H oxidation activity. In Synechocystis sp. PCC 6803, this gene is registered as a drug resistance gene (drgA). However, it's function is not fully elucidated yet.
NQR shows an NAD(P)H oxidation activity and hence it may participate in cyclic electron transport. To investigate the possibility, we have constructed drgA (slr1719) inactivation mutant and characterized it.
Under high-light intensity, the growth rate of the MT was a half of the WT. For measurement of NAD(P)H oxidation activity, we found an acceptor specific to NQR and inhibitors as well. A possible function of NQR estrimated by measuring redox changes of P700 in cell-free system will also be reported.

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Carbon partitioning of photosynthetic products is thought to be regulated by capacity of sucrose synthesis. To clarify the contribution of cytosolic fructose-1,6-bisphosphatase to the photosynthetic carbon metabolism, we generated transgenic tobacco plants expressing cyanobacterial FBP/SBPase in cytosol (TcFS) and then analyzed photosynthetic characteristics under high CO₂ conditions.
In TcFS plants, lateral shoot, leaf number and fresh weight were increased compared with those in the wild-type plants under high CO₂ (1200 ppm) conditions. TcFS showed increased photosynthetic activity compared with that in the wild type. The level of hexose of wild type accumulated in upper leaves, while levels of sucrose and starch of TcFS plants accumulated in lower leaves and lateral shoots. These findings suggest that the cytosolic FBPase contributes to efficient conversion of hexose into sucrose, and that the change of the carbon partitioning affects photosynthetic capacity and morphogenesis.

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Rice (Oryza sativa L.) plants were transformed with the rice rbcS cDNA under the control of its own promoter by Agrobacterium-mediated method. The primary transformants were screened for the ratio of Rubisco-N to leaf-N. In the progeny of the selected lines, the mRNA levels of rbcS and rbcL were increased from 2.1- to 2.8-fold and 1.2- to 1.9-fold, respectively. Rubisco content was significantly increased by 30% on a leaf area basis. The ratio of Rubisco-N to leaf-N was also increased by 10 to 20%. However, light-saturated photosynthesis was not enhanced even when the rate was measured at low [CO₂] where Rubisco becomes limiting for photosynthesis. We conclude that introduction of additional sense rbcS leads to overexpression of rbcS and that this overexpression slightly up-regulates the gene expression of rbcL at a transcript level and enhances the amount of Rubisco holoenzyme. However, overproduction of Rubisco protein does not improve photosynthesis.

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Carbonic anhydrase of Phaeodactylum tricornutum (PtCA1) is encoded by the nuclear genome and its expression is regulated at the transcriptional level by changes in the ambient [CO₂] and light. Critical region required for [CO₂] and light-responsive transcription of ptca1 were located between -70 and -30 bp relative to the transcription start site. This critical region contains four cis-elements, that is, two cAMP response elements (CRE1:Pptca1-70/-63,CRE2:Pptca1-21/-14), a p300-binding site (Pptca1-52/-46) and a Skn-1 binding site (Pptca1-35/-28). It is suggested that the Skn-1 binding site is involved in stimulation of PtCA1 expression in response to [low CO₂] and light. In this study, Yeast one-hybrid assay was done to identify transcription factors, which bind to Skn-1 binding site. As a result, 155 positive clones were obtained. Inserted sequences in these positive clones were determined.

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The mechanism of CO₂-response in fresh water algae have been studied extensively, whereas little information is available in mechanistic bases of CO₂-response in marine micro-algae. In order to understand CO₂-response in marine diatoms, CO₂ responsive genes were screened by cDNA-AFLP(cDNA- amplified fragment length polymorphism) comparing cDNA speacies from cells grown in 0.5% CO₂ and air using the marine diatom Phaeodactylum tricornutum. CO₂-responses of 20 cDNA fragments were so far confirmed. Of these 13 genes, including gene for triosephosphate isomerase, were found to repressed in air that is in agreement with the case of cyanobacterial CO₂-response. On the other hand, the remaining 7 genes including genes for ferredoxin nitrite reductase were found to be induced in air that is opposite to the case of cyanobacterial CO₂-response, indicating that CO₂-response of nitrogen metabolism in marine diatoms is different from that in cyanobactera.

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Marine diatoms are thought to possess a CO₂-sencing mechanism, but this mechanism is yet to be elucidated in detail. Activity of the promoter region of a carbonic anhydrase gene in the marine diatom, Phaeodactylum tricornutum (Pptca1) was analyzed, and it has been demonstrated that the critical CO₂-response sequence was located downstream 70bp relative to the transcription-start site. This core-regulatory region comprises two putative cAMP-response elements, CRE1, 2 and a putative p300-binding site and was demopnstrated that Pptca1 was under control of cAMP. A series of manipulated construct of Pptca1 was ligated to uidA reporter gene and introduced into P. tricornutum. Activities of manipulated promoters were measured as GUS levels in lysates grown in 5% CO₂ or air. As a result, a new candidate for CO₂ -responseive cis-element was identified. Relationship between cis-elements and cAMP, and interaction of the cis-elements, will be discussed.

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Some cyanobacterial species including Cyanobacterium sp. MBIC 10216 synthesize distinct α-polyglucans semi-amylopectin instead of glycogen. Semi-amylopectin is characterized as the content of long α-1,4-glucan chains being much more abundant as compared to glycogen, and its apparent molecular size close to that of rice amylopectin. To elucidate the genetic background of semi-amylopectin synthesis, the genomic sequence of the strain MBIC 10216 is being determined. Among the genes responsible for the biosynthesis of storage polysaccharides, we have found three distinct genes whose products possess all the essential amino acid residues of starch/glycogen branching enzymes (BE) belonging to GH13 family. The distinct BE activities were demonstrated with the recombinant proteins, although they remain undetectable in the crude extracts of the cyanobacterium. Catalytic specificities of the three BE isoforms were studied with synthetic α-1,4-glucans as the substrate. Chain length distribution of the branched glucans with α-1,6-linkage was markedly different between these isoforms.

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Four C4 photosynthetic enzymes (PEPC, PPDK, NADP-MDH, NADP-ME) were overexpressed in rice leaves. The overexpression of PEPC alone slightly suppressed photosynthesis, while co-expression of PPDK, MDH, and ME in addition to PEPC restored photosynthesis, although it caused slight stunting. Comparison of three different double (PEPC+PPDK, PEPC+ME, MDH+ME) and one triple (PEPC+PPDK+ME) transformants indicated that the overexpression of PEPC acted to suppress photosynthesis, and the co-expression of PEPC and ME caused stunting. PPDK and MDH are active only in the daytime while PEPC and ME are active day and night. It is thus likely that the co-expression of PEPC and ME results in consumption of once fixed carbon at night and under weak light illumination. PEPC becomes more active at night than in the daytime in rice leaves. The stunting of the quadruple transformant observed in this study might be unique to rice.

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A marine unicellular alga Emiliania huxleyi(Haptophyta) is the most abundant coccolithophorid. The alga gives the great impact on the global carbon cycle by blooms and the biological pump. Such activities should be supported by its photosynthetic activity. In this study we focused on the metabolism of glycolate, the product of Rubisco oxygenase reaction, since our previous study showed that the algal photosynthesis is sensitive to oxygen.
A homologous sequence of glycolate dehydrogenase (GDH) was found by sequence analysis of the E. huxleyi EST database. Northern blot analysis revealed that GDH mRNA level increased under light condition, but not dark condition. Main metabolites of ¹⁴C-glycolate were Glu and Gln. From the results, we concluded that GDH functions as a glycolate oxidizing enzyme to produce glyoxylate and then the compound is metabolized to Glu and Gln in E. huxleyi via a unique pathway that is different from the known glycolate pathway.

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Plastidic phosphate translocators are classified into four subfamilies; triose phosphate/phosphate translocator (TPT), phosphoenolpyruvate/phosphate translocator (PPT), glucose-6-phosphate/phosphate translocator (GPT) and pentose phosphate/phosphate translocator. Four kinds of cDNAs (McTPT1, McPPT1, McGPT1 and McGPT2) belonging to three of these subfamilies were isolated from a facultative Crassulacean acid metabolism (CAM) plant, Mesembryanthemum crystallinum. The transcript levels of these genes are differentially regulated during C₃-CAM transition of M. crystallinum by salt stress.
To compare the structures of these translocator genes, we are trying to isolate their genomic clones. So far, we have sequenced transcribed regions of McGPT1 and McGPT2, and found that they are separated by four introns at the same positions as GPT genes in Arabidopsis and rice. Moreover, we have isolated the 0.9 kb and 2.6 kb upstream regions of McTPT1 and McGPT2, respectively. The putative promoter structures of these genes will be compared with other CAM-specific and nonspecific genes from M. crystallinum.

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It has been established that a green alga, Chlamydomonas reinhardtii, induces a carbon-concentrating mechanism (CCM) and accumulates inorganic carbon (Ci) under CO₂-limiting conditions. However, CO₂ transporters remain to be identified. We previously demonstrated that a CCM regulatory mutant lcr1, which is deficient in a Myb transcription factor, LCR1, was unable to induce three low-CO₂ inducible genes, Lci1, Lci6, and Cah1, and showed a decreased affinity against Ci.¹⁾ Since Lci1 encodes a membrane protein, we introduced a chimeric Lci1 gene, which was fused with an upstream region of Nia1 gene, into the lcr1 mutant to induce the expression of Lci1 by switching nitrogen source in the media from ammonium to nitrate in high-CO₂ condition. We will discuss the function of LCI1 based on the CO₂-uptake activity and affinity against inorganic carbon of the transformants carrying chimeric Lci1.
1) Yoshioka et al. Plant Cell (2004)

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Green sulfur bacterium Chlorobium tepidum, an anaerobic photosynthetic bacterium, has an iron-sulfur type reaction center and nitrogenase. In the whole genome of C. tepidum, homologues of iron-sulfur (Fe-S) cluster assembly factors, iscU (or nifU) and iscS (or nifS), are present and they form an operon (CT1994 and CT1995). In a previous study, we reported that purified CT1994 bound a [2Fe-2S] cluster on C-terminal domain. We also reported that another Fe-S cluster was able to reconstitute on N-terminal domain in the presence of iron, sulfide, and reducing agent under an anaerobic conditions. In the present study, we purified CT1995 and found that the purified protein showed desulfurase activity. The K_m value is 4.0 µM, and the V_max value is 8.3 mol sulfide/mol CT1994/min, using L-cysteine as substrate. We also found that reconstitution of Fe-S cluster on N-terminal domain of CT1994 occurred in the presence of CT1995 and L-cysteine instead of sulfide.

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In Rhodobacter sphaeroides f. sp. denitrificans, the DmsS/DmsR two-component signal transduction system regulates expression of dmsCBA operon encoding DMSO (dimethyl sulfoxide) respiration system. Although other sensor histidine kinase generally contains transmembrane region, LacZ analysis showed that the fusion proteins bound to the membrane from the cytoplasmic side. In the dmsA-deficient mutant which is not capable of DMSO respiration the amount of the dmsC-lacZ activity increased about 3-fold in the presence of DMS (dimethyl sulfide) and increased about 10-fold in the presence of DMSO. It is thought that DMS converted and a little DMSO generated in the media. These results indicated that DMS itself is not a signal and DMSO itself is a signal for the transcription of dmsCBA.

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Oxidative stress inhibits the repair of photosystem II by suppressing the synthesis de novo of proteins in the elongation step of translation. We examined effects of oxidative stress on elongation factor G (EF-G), which is known to be particularly sensitive to oxidative stress, in Synechocystis sp. PCC 6803. EF-G (Slr1463) protein was oxidized by H₂O₂ and subsequently reduced by different concentrations of dithiothreitol (DTT). Analysis of reduced Cys residues with a SH-modifying reagent revealed that the reduction of Cys residues occurred stepwise. Addition of thioredoxin promoted the reduction of oxidized EF-G, suggesting that EF-G interacts with thioredoxin. Substitution of Cys105 by Ser resulted in the insensitivity of EF-G to oxidation by H₂O₂ and to thioredoxin, indicating that Cys105 is involved in the formation of disulfide bond and is also a target of thioredoxin.

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Three dnaK genes and four dnaJ genes have been identified in the genome of cyanobacterium Synechococcus elongatus PCC 7942. Among them, DnaK2 and DnaJ2 are induced by several stresses. Our yeast two-hybrid analysis revealed the specific interaction between DnaJ2 and RNase E, which is an essential endoribonuclease and involved in the regulation of psbAII in cyanobacteria. We examined the effect of DnaK2 and DnaJ2 on RNase E activity, monitored by the digestion of psbAII transctipt in vitro. Addition of DnaK2 and DnaJ2 obviously inhibited the RNase E activity in the ATP-dependent manner. These results suggest that DnaK2 and DnaJ2 are involved in the RNA degradation through interacting with RNase E.

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The genome of cyanobacterium Synechococcus elongatus PCC 7942 contains three dnaK and four dnaJ homologs. Among them, DnaJ3 is essential for normal growth and localized onto the thylakoid membrane as is DnaK3. In order to elucidate specific functions of DnaJ3, we carried out genetic analyses. Using PCR-mutagenesis to introduce point mutations into the C-terminal region of dnaJ3, a temperature sensitive mutant carrying a missense mutation F193L (t598c) was isolated. Moreover, we obtained several suppressor mutants from F193L strain and mapped their mutations in syfpcc7942_2440 (pnp) and syfpcc7942_1999 (rbp2) genes. When compared to the wild type strain, dnaJ3 mRNA of the F193L strain was more stable. Here we discuss the involvement between of the dnaJ3 mRNA stability with the temperature sensitivity of growth.

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The Synechococcus elongatus PCC 7942 strain carrying a missense mutation in the peptide-binding domain of DnaK3, one of the essential dnaK gene products, exhibited temperature-sensitive growth. We also isolated suppressor mutants from this strain. One of the suppressors was mapped in the ribosomal protein gene rpl24 (synpcc7942_2221), which encodes the 50S ribosomal protein L24. Subcellular localization of three DnaK proteins was determined, and the results indicated that a amount of DnaK3 was dislocated from membrane-bound polysomes at non-permissive temperature in the dnaK3 temperature-sensitive mutant, whereas, in the suppressor mutant, the normal pattern of DnaK3 was restored. Possible roles of the DnaK3 protein, involving the translational control in the thylakoid membrane-bound polysomes, will be discussed.

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In Synechocystis sp. PCC 6803, decrease of photosystem I (PSI) content is observed under high-light conditions. Since the pmgA-disrupted mutant, which has defect in this regulation, shows severe growth inhibition under high light, the regulation achieved by PmgA is indispensable for growth under high-light conditions. However, the function of PmgA is totally unknown, except that PmgA shows weak homology to RsbW, an anti-sigma factor in Bacillus subtilis. We found that two promoters of psaAB encoding reaction center subunits of PSI shows higher activity under high-light conditions in the pmgA mutant than in WT cells, although the light-responsive mechanism of the two promoters is totally different. This result suggests PmgA functions as an anti-sigma factor. Now, by DNA microarray analysis, we are trying to identify genes whose expression is under the control of PmgA.

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Protochlorophyllide (Pchlide) reduction in chlorophyll biosynthesis is catalyzed by two structurally unrelated enzymes; dark-operative Pchlide oxidoreductase (DPOR) and light-dependent Pchlide oxidoreductase (LPOR). It is suggested that DPOR is the older enzyme, and LPOR evolved after oxygenic photosynthesis had been established. Molecular phylogenetic analyses suggest that Gloeobacter violaceus PCC 7421 is the earliest diverged cyanobacterium, which is supported by some unique characteristics such as lack of thylakoids and very low content of chlorophyll. G. violaceus may have primitive features of chlorophyll biosynthetic pathway. Here we report enzymatic properties of LPOR from G. violaceus. The LPOR gene glr2486 was expressed in E. coli and Glr2486 protein was purified. Glr2486 showed Pchlide reduction activity dependent on light and NADPH. Km for Pchlide was 0.7 μM, which was the lowest value in the known cyanobacterial LPORs. This result suggests that LPOR of G. violaceus is an efficient enzyme in terms of affinity for Pchlide.

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Small non-coding RNAs (ncRNAs) have been identified and characterized in various organisms including cyanobacteria. To gain insight into the roles of ncRNAs in environmental adaptation, we searched for low-CO₂ responsive ncRNAs in Synechococcus elongatus PCC 7942, using DNA microarrays that have oligonucleotide spots for both strands of ORFs and intergenic regions. Ten intergenic regions, whose expression levels are high and whose expression patterns are different from the adjacent ORFs, were selected as candidates of the regions harboring ncRNA genes. Low-CO₂ inducible small transcripts were detected by Northan blot analysis in three of these intergenic regions, suggesting the occurrence of ncRNAs involved in adaptation to low-CO₂ conditions. We are currently determining the transcription start point and the size of each of the putative ncRNAs to design antisense RNA to interfere with their expression.

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Cyanobacteria can be used as solar energy converter into hydrogen. We have chosen nitrogenase for the enzyme of hydrogen production because it catalyzes essentially unidirectional production of hydrogen. Our previous studies show that inactivation of uptake hydrogenase (Hup) is effective in enhancing hydrogen production activity. However, a high production activity stage lasted for only about 10 hours. Homocitrate is bound to the catalytic FeMo-cofactor of nitrogenase and is synthesized by NifV. Nitrogenase from Klebsiella pneumoniae nifV mutants showed that it reduced dinitrogen poorly, but acetylene and proton at rates comparable to the wild-type enzyme in the presence of citrate. Nostoc PCC7120 has two nifV genes, and we have created 6 nifV mutants by disruption of either one or both genes on the wild-type and ΔhupL background. The duration of hydrogen high production was longer in the mutant ΔnifV1/ΔhupL than the wild-type. Differential role of two nifV genes is discussed.

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Photosynthesis of a leaf is regulated not only by own light environment, but also the light environment of the whole plant (Weaver et al. 2001). Thus, plants would have some mechanisms, which recognize and compare the environments among the leaves. To examine effects of environments around a part of leaves on photosynthesis in themselves and the other leaves in a plant, we treated a couple of primary leaves of bean with 1000, 400 and 150 ppm CO₂ concentration. After treatments, we measured changes in photosynthesis of the primary leaves (PL) and the first trifoliate (FT).
The photosynthetic rate at 360 ppm CO₂ of 1000 ppm-treated PL and FT decreased faster than that of 400 ppm during the treatment period, and 150 ppm-treated PL decreased slower. Thus, CO₂ environment around PL affects the photosynthetic rate of PL and FT.

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Evergreen species can store chlorophyll (Chl) even in winter; therefore they can dissipate light energy put into Chls depending on seasons. To examine how they dissipate excess energy at low temperature condition, we examined energy flow by means of steady-state and time-resolved fluorescence. Time-resolved fluorescence spectra (TRFS) of Taxus cuspidate needles were measured with a picosecond time-correlated single-photon counting system. TRFS of summer and autumn needles showed relaxation kinetics similar to that of Arabidopsis thaliana, while those of winter and spring showed different features. In TRFS of winter needles, contribution of photosystem (PS) II fluorescence was quite small, and PSII fluorescence lifetimes were twice shorter than that of A. thaliana, indicating quenching of excitation energy and low accumulation of PSII. In spring, the PSI fluorescence showed excitation wavelength dependence. Both PSI and PSII were modified to dissipate excess energy. We will also discuss about the seasonal change of thylakoid stacking.

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As one of plant photo-protection systems, it is known that excess reducing equivalents could be exported from chloroplasts and be oxidized by mitochondrial respiratory chain. Alternative oxidase (AOX), the unique respiratory pathway in plant, could dissipate these reducing equivalents efficiently, because the electron flow via AOX is not restricted by the cellular ATP/ADP ratio. In fact, we previously reported that accumulation of reducing equivalents in the chloroplasts caused the synchronized up-regulations of the transport activity of the reducing equivalents and AOX capacity. These results indicate that AOX has an important role for photo-protection, and raised the new question about the messenger for AOX up-regulation. We found that the yellow variegated 2 (var2) mutants induced AOX without the accumulation of the reducing equivalents. The green sector in var2 leaves was suffered from the oxidative stress, implying that the oxidative stress in chloroplasts would transduce some signals for AOX expression.

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Higher plants have multiple mechanisms to cope with environmental changes. In photosystem II, absorption of excessive light energy induces thermal dissipation, which can be detected as nonphotochemical quenching (NPQ). To characterize the molecular mechanism of the adaptation to fluctuating environments, Arabidopsis thaliana plants were treated with continuous low-temperature or fluctuating-temperature conditions. The fluctuation of temperature modified the light-intensity-dependence of NPQ. Based on this information, mutants defective in the regulation of photosynthetic electron flow under fluctuating temperature were screened using chlorophyll fluorescence imaging, resulting in identification of candidates. We also characterized the effect of continuous light or fluctuating-light conditions and found the similar strategy of plants of modifying light-intensity-dependence of NPQ.

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The accumulated evidence reveals that the increases in citrate synthesis and exudation enhance the resistance of plants to Aluminum (Al). The carbon skeleton used for synthesis of citrate is supplied from photosynthesis. To investigate whether the increase in citrate syntheses in the leaves of plants would improve their resistance to Al, citrate synthase (CS) was overexpressed in tobacco leaves under the control of tomato rbcS-3C promoter and the overexpressed CS was localized in the cytoplasm. Overexpression of CS resulted in 1-5 fold increase in citrate synthase activity in transgenic tobacco leaves compared with wild-type plants. The staining experiments for roots showed that the transgenic tobacco lines were much more tolerance to Al than wild-type plants after exposure to 100-300 μmol Al.

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Photosynthetic organisms are able to enhance the thermal stability of photosystem II (PSII) when they grow at moderately high temperatures. We studied molecular mechanisms responsible for the acclimation of PSII to high temperature in thylakoid membranes of suspension-cultured cells of soybean. The thermal stability of PSII in isolated thylakoid membranes was enhanced when the growth temperature was shifted from 25^oC to 35^oC. Disruption of the vesicular structure of thylakoid membrane with 0.05% Triton X-100 abolished the enhanced thermal stability. The components released by Triton X-100 from thylakoid membranes from cells grown at 35^oC were able to increase the thermal stability of Triton-treated membranes. Boiling or filtration of the released components abolished the ability to increase thermal stability, suggesting that the active components should be proteins. Thus, proteins that are associated with thylakoid membranes might be involved in the enhancement of the thermal stability of PSII.

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