PsbY is a membrane protein of ˜4 kDa found in photosystem II (PS II) complexes from cyanobactria to higher plants. However, it is not shown in the current cryatllographic model, and the function is still not clear. In this work, we deleted psbY gene from Synechocystis 6803 and analyzed the function of PsbY.
No difference was observed in the growth curve and the rate of oxygen evolution between the wild-type and mutant cells. However, the rate of oxygen evolution in the purified mutant PS II complexes was much lower compared to that in the purified wild-type PSII complexes. Electrophoretic profile revealed that most part of PsbU and PsbV, and some part of PsbO and PsbQ were lost in the purified mutant PS II complexes. It was concluded that PsbY is important for the stable binding of extrinsic proteins especially for PsbU and PsbV.

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It has been generally accepted that PsbU in cyanobacterial photosystem II (PS II) is a functional equivalent of PsbP or PsbQ in plant chloroplasts. After the recent discovery of PsbP and PsbQ homologues in cyanobacterial PS II [Thornton et al. (2004) Plant Cell 16, 2164], we investigated the function of PsbU using a psbU-deletion mutant (ΔPsbU) of Synechocystis 6803 focusing on the functional relation to Ca²⁺ and Cl^-. The growth rate was highly suppressed when Cl^- was eliminated, while ΔPsbU grew well when only Ca²⁺ was eliminated. Remarkable suppression in the oxygen-evolving activity in ΔPsbU thylakoids in the absence of Cl^-, which was restored by 60% with NO₃^- and not by SO₄^2-. Based on our results, we concluded that PsbU stabilizes only Ca²⁺, and not Cl^-, through its structural nature. This work was supported by funding from NSF (MCB0215359, HBP), and from Hyogo Prefecture (YK), COE (IT and YK).

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In order to study the roles of lipids in PSII from Thermosynechococcus vulcanus, we used phospholipase A₂ (PLA₂) and lipase to treat PSII core complex and examined their effects on PSII structure and functions. The following results were obtained: 1. PLA₂-treatement decreased the content of PG by 70.6%, leading to a decrease of 40% in oxygen evolution. In contrast, lipase-treatment specifically decreased the content of monogalactosyldiacylglycerol (MGDG) by 71.2% without affecting the contents of other lipids, resulting in a decrease of 16% in oxygen evolution. These indicated that PG was more important than MGDG in maintaining the PSII function. 2. PLA₂-treatment induced neither the dissociation of PSII dimer nor any loss of polypeptides from the PSII core complex. 3. Depletion of PG did not affect the water oxidation site, but caused a damage to the Q_B binding site, leading to an inhibition in the electron transfer from Q_A to Q_B.

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The amount and activity of PSI/II are known to change depending on the light conditions inside leaves of C₃ plant. C₄ cells will also adapt to the inner leaf condition. We measured the fluorescence spectrum of each cell in a leaf and stem of Setaria viridis (NADP-ME type C₄ plant) by the high-spatial-resolution microspectroscopy at room temperature and 77K. The device has a monochrometor attached to the conforcal laser scanning microscope, and has the spatial resolution of 0.3 μm and the wavelength resolution of 1 nm. We can get in vivo spectrum of every single cell and chloroplast. We measured the fluorescence spectrum at various parts in the leaves and figured out PSI/II ratio. In the mesophyll cells, PSI/II ratio is different from that in the bundle sheath cells. We will discuss the relation of the cell environments and the amounts of PSI and PSII.

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Cyanidioschyzon merolae is a thermo- and acidophilic primitive red alga. We purified the Photosystem I (PSI) complexes from C. merolae and analyzed the biochemical and spectroscopic characteristics precisely. The molecular mass of the PSI complexes was estimated as 400 kDa by Blue Native (BN)-polyacrylamide gel electrophoresis (PAGE). It was also confirmed that the PSI complexes associated light-harvesting complexes (LHC) I by the following 2nd dimensional SDS-PAGE. Until now, PsaA, PsaB, PsaC, PsaE, PsaF, and PsaL polypeptides were also determined as subunits. The PSI complexes contained 200 chlorophyll a molecules, which was larger than that of pea, and 36 zeaxanthin per P700. Violaxanthin and antheraxanthin were not detected in the PSI complexes. Only one gene coding LHC was reported in the genome of C. merolae. These results suggest that the PSI complexes from C. merolae has an antenna system different from pea.

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In mammals, electron-transfer flavoprotein:ubiquinone oxidoreductase (ETFQO) accepts electrons from the electron-transfer flavoprotein (ETF) and reduces ubiquinone. ETF is the physiological electron acceptor for at least nine mitochondrial matrix flavoprotein dehydrogenases involved in straight-chain fatty acid β-oxidation, catabolisms of several amino acids, and choline metabolism. Thus mammalian mitochondrial proteins, ETF and ETFQO, plays a key role in multiple metabolic pathways, and are important in supplying mitochondria with respiratory substrates auxiliary to those derived from sucrose. In humans, mutations in either ETF or ETF-QO results in the fatal genetic disease, type II Glutaric acidemia. In contrast to the situation in mammals, nothing is yet known about the function of ETF and ETF-QO in plants so far. Here we will describe a genetic, molecular, and biochemical analysis of an unique Arabidopsis homologue of ETFQO.

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Ribulose 1, 5-bisphosphate carboxylase/oxigenase (RuBisCO) is the key enzyme of photosynthetic carbon assimilation. RuBisCO is composed of eight small subunits (SS) encoded by nuclear gene rbcS and eight large subunits (LS) encoded by chloroplast gene rbcL. In the last year, we reported the transgenic tobacco plants harboring antisense rbcS under the control of dexamethasone (DEX) -inducible promoter (46th annual meeting). In these plants, the amount of endogenous rbcS mRNA can be artificially reduced by induction of antisense rbcS mRNA using DEX treatment. Here, we report the effect of rbcS mRNA reduction on the expression of rbcL gene, the accumulation of RuBisCO and the structure of chloroplast.

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We have previously demonstrated that transgenic tobacco plants expressing FBP/SBPase in chloroplasts exhibit increased photosynthetic capacity and dry matter compared with those of the wild-type plants. To clarify the genes that are associated with increased parameters using a DNA microarray analysis, we produced transgenic Arabidopsis expressing FBP/SBPase in the chloroplasts (ApFS). In ApFS-2 plants with 3.3-fold higher FBPase activity compared to wild-type plants, both fresh weight and photosynthetic CO₂ fixation rate were 1.7-fold higher than those in the wild-type plants. Total RNAs were isolated from hypocotyls, young rosette and basal blade of mature rosette and were used for the DNA microarray analysis. Small, but significant, differences were observed in the expression of many genes. Fifteen transcripts showing more than 1.5-fold expression in ApFS-2 were confirmed by real-time RT-PCR.

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There are three rice starch branching enzymes (BE): BEI, BEIIa, and BEIIb, each having a different function and expression pattern. The individual function and probable interactions of these genes were analyzed by posttranscriptional silencing thraugh RNAi. Seven gene constructs (one each for the three BE genes, and their combinations- BEI+IIa, BEI+IIb, BEIIa+IIb, BEI+IIa+IIb) were made and their T1 seeds were analyzed.
Silencing of BEI or BEIIa gene alone had no visible effect on seed morphology, while silencing of BEIIb resulted into the production of small seeds with whitish endosperm. Although the combined BEI + BEIIa silencing did not affect the seeds, the three silencing combinations BEI+IIb, BEIIa+IIb, and BEI+BEIIa + BEIIb yielded seeds with shriveled whitish endosperm, indicating that the genes interact synergistically to regulate starch biosynthesis in rice endosperm.

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The granule-bound starch synthase I (GBSSI) catalyzes the synthesis of amylose in plastids. We have reported the necleotide sequences of the GBSSI genomic clone and the cDNA clone obtained by screening libraries. In this study we analyzed multiple other clones in order to investigate polymorphism in the GBSSI gene of sweetpotato, hexaploid plant. Five genomic clones and two cDNA clones possessing an intact ORF were analyzed and the nucleotide sequences were classified into five groups. The nucleotide sequence identity of the ORF region was 98.1%-98.9%. The amino acid sequence identity of GBSSI mature protein was 99.1%-99.6% among five groups. This result suggests that sweetpotato has heterogeneous group of GBSSI proteins in plastids. Comparison of the necleotide sequence of introns showed polymorphism in length of all 13 introns (1-89 bp difference). Analysis of the partial length clones is in progress.

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To investigate influence on rice grain-filling metabolism by high temperature, the comprehensive expression analysis using microarray and conventional differential screening was conducted. The kernel grown under high temperature condition (33/28 C) had decreased grain weight and milky-white appearance compared to the control grown under low temperature (25/20 C). Starch analyses revealed that high temperature-grown kernel has lower amylose content and altered amylopectin which is rich in long chains (DP 20-32) and poor in short chains (DP 6-18). Microarray analysis using developing seeds 10d after anthesis revealed that expression of granule-bound starch synthase (GBSSI) and starch branching enzyme (BEIIb) genes was repressed under high temperature, which is in agreement with change of starch composition; decrease of amylose content and elongation of side-chains of amylopectin. These results suggest that storage material in rice kernel is, in part, controlled by gene expression level.

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Chlorophyllide a oxygenase (CAO) converts chlorophyll a to chlorophyll b.The CAO protein levels regulate accumulation of chlorophyll b. We previously demonstrated that the CAO levels were maintained at a low level in plants when a sufficient amount of chlorophyll b was accumulated.In order to reveal this feedback mechanism, we screened for mutants that accumulate a higher level of CAO protein in spite of the presence of chlorophyll b.
In order to monitor the CAO protein levels, we prepared Arabidopsis transgenic lines overexpressing GFP-CAO fusion proteins. The transgenic plant was mutated by EMS and screened with confocal microscopy. We isolated a line that showed a high GFP fluorescence. In the cotyledon of this line, the CAO protein level was higher than that in the parent line, whereas CAO protein was not detected in rossette leaves in both lines. Mapping of the mutation locus is in progress.

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Pheophorbide a oxygenase (PaO) catalyzes oxygenolytic opening of porphyrin macrocycle of pheophorbide a, which is the key step in chlorophyll degradation. We previously reported that PaO was encoded by Accelerated cell death 1 (ACD1) gene. We produced transgenic Arabidopsis plants expressing antisense RNA for ACD1 (As-ACD1) to investigate the cell death processes induced by accumulation of pheophorbide a.
Pheophorbide a was accumulated in As-ACD1 leaves during dark incubation. Dark treated As-ACD1 leaves were bleached upon illumination. It was probably caused by generation of singlet oxygen. We also found that As-ACD1 leaves were not bleached but exhibited cell death when they were incubated in the darkness. This indicate that the accumulation of pheophorbide a induces cell death without generation of singlet oxygen. In order to investigate the light-independent cell death processes, we observed cell ultrastructure and examined the expression of PCD marker genes in As-ACD1 leaves.

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In vitro activity of a methyltransferase, BchU, which catalyzes methylation at the C-20 position of a chlorin moiety in biosynthetic pathway of bacteriochlorophyll c, was investigated. The bchU gene derived from a photosynthetic green sulfur bacterium, Chlorobium tepidum, was over-expressed in Escherichia coli as a His-tagged protein (His₆-BchU), and the His₆-BchU was purified. The enzymatic activities of this His₆-BchU were measured using a variety of synthetic pigments as substrates and S-adenosylmethionine as a donor of methyl group. These results suggest that in the biosynthetic pathway, native BchU catalyzes the methylation of bacteriochlorophyllide d homologs possessing a 1-hydroxyethyl group at the C-3 position and functions after the hydration of the C-3 vinyl group by BchF and V. This finding was supported by analysis of the crystal structure of BchU. We will discuss the role of BchU in the bacteriochlorophyll c biosynthetic pathway.

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In Rhodobacter capsulatus, 3-vinyl hydroxyethyl hydratase, which is encoded in bchF, is involved in conversion of C-3 vinyl group to hydroxyethyl group. Green sulfur bacterium Chlorobium tepidum synthesizes three kinds of chlorophylls, namely, BChl a, Chl a, and an antenna pigment of BChl c. Whole genome of C. tepidum has been deciphered and two paralogs of bchF (CT1421 and CT1776) are found. It is proposed that the C-3 carbon of BChl c is a chiral center that can exist in R- or S- stereochemistry, and CT1421 relates R-type, while, CT1776 concerns S-type BChl c. In this study, we introduced CT1421 and CT1776 to cell of mutant of R. capsulatus in which bchF was inactivated. We observed the enzymatic function of bchF in R. capsulatus was complemented by either genes from C. tepidum. We also introduced these genes as well as bchF from R. capsulatus into cyanobacteria Synechocystis sp. PCC6803.

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Most photosynthetic organisms including cyanobacteria have two types of protochlorophyllide (Pchlide) reductases; light-dependent Pchlide reductase (LPOR) and nitrogenase-like dark-operative Pchlide reductase (DPOR), which are structurally unrelated. While LPOR was demonstrated to be essential for growth under high light conditions in cyanobacteria, it is still unknown as to the physiological significance of DPOR co-existing with LPOR among wide variety of photosynthetic organisms. We compared growth of a DPOR-lacking mutant (chlL-disruptant) with wild type under various light conditions. Significant growth delay (ca. 8 h) was observed when the dark-grown mutant was transferred to high light conditions (>200 μmol m^-2 s^-1). During photoautotrophic growth under dark-light cycle (16h-dark/8h-high light), the growth delay of the mutant was obvious in the first light stage though the chlorophyll content was the same as wild type. These results suggested that DPOR is needed for initial adaptation process to high light in cyanobacteria.

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In carotenogenesis, lycopene cyclases catalyze lycopene to β-carotene via γ-carotene. At present, three distinct functional enzymes are known. (1) Bacteria (CrtY), two cyanobacteria (CrtL), and plants (CrtL, CrtL-b) have homologus enzymes. (2) Some bacteria (Mycobacterium, Myxococcus, etc.) have a hetero-dimer of CrtYc and CrtYd, and archaea and fungi have a fused CrtYc and CrtYd monomer. (3) Green sulfur bacterium (Chlorobium tepidum) has CruA, whose homologs have been found from some cyanobacteria.
In this study, functions of cruA-like genes were studied. The disrupted mutant of Anabaena sp. PCC 7120 had no effects on carotenoid composition, while that of Synechocystis sp. PCC 6803 could not be segregated. When the genes from Synechocystis 6803 and Gloeobacter violaceus were expressed in Echerichia coli, which could produce lcopene, β-carotene was not produced. Therefore, cruA-like genes in cyanobacteria have not the functions of lycopene cyclase, and a novel fourth enzyme must be present in cyanobacteria.

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We have identified all the carotenoids in Anabaena sp. PCC 7120: myxol fucoside, ketomyxol fucoside, β-carotene, echinenone and canthaxanthin (Takaichi et al. (2005) PCP 46, 497). Further, we have functionally determined two distinct β-carotene ketolases: CrtO (all3477) for echinenone and CrtW (alr3189) for ketomyxol synthesis (Mochimaru et al. (2005) FEBS Lett. 579, 6111).
In this study, we searched cartenogenesis enzymes by sequence homology. The enzymes, whose functions have already been confirmed in other bacteria, were chosen for the query sequences. Since a disrupted mutant of alr4009, produced (keto)-deoxymyxol fucoside instead of (keto)-myxol fucoside, its function was confirmed to be β-carotene hydrogenase (CrtR). Although the functions of CrtD and CruA were OH-γ-carotene 3,4-desaturase and lycopene cyclase, the disrupted mutants of all5123 and alr0920 had no effects on carotenoids, respectively. Therefore, these enzymes were not for carotenogenesis in Anabaena 7120. There are some differences among species, and further studies are needed.

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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 (dimethylsulfoxide) respiration system. DmsS has no putative membrane-spanning hydrophobic amino acid regions, although other sensor histidine kinase generally contains transmembrane region. To analyze membrane-binding region of DmsS, dmsS-lacZ fusion genes were constructed. Each strain had the LacZ activity, suggesting that each LacZ fusion protein was located in the cytoplasm. The LacZ fusion protein with no hydrophobic region and other proteins with the hydrophobic region were recovered in the cytoplasmic and membrane fractions, respectively, suggesting that the fusion proteins bound to the membrane from the cytoplasmic side. Although DMSO induces the expression of DmsA protein under anaerobic conditions, no change of dmsS promoter activity was observed by DMSO, suggesting that DMSO itself is not a signal for the transcription of dmsS.

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Important functions of carotenoids in photosynthetic organisms are protection against photooxidative damage and light-harvesting. Nevertheless, the correlation between the chemical structure of carotenoids and their function has not yet been clarified. To assess functional features of cyclic carotenoids, we have constructed mutants of the purple photosynthetic bactererium, Rubrivivax gelatinosus (IL144), that accumulated cyclic carotenoids. To construct a Β-carotene-accumulating mutant, lycopene cyclase gene (crtY) from Pantoena ananatis (Erwinia uredovora) was introduced into the lycopene-accululating mutant. Then, crtW and/or crtZ from Paracoccus sp. N81106 (Agrobacterium aurantiacum) were introduced to this mutant to obtain three mutants synthesizing canthaxanthin, zeaxanthin and astaxanthin.
These cyclic carotenoids were found in cytoplasm, membranes and pigment-proteins. It was suggested that the cyclic carotenoids could function as antenna pigments for growth of the purple bacterium. Resistance of the mutants against singlet oxygen was as high as the wild-type Rvi. gelatinosus that accumulates spheroidenone in the presence of O₂.

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It has been reported that in bacteria, such as Azotobacter vinelandii, several factors are involved in the assembling of iron-sulfur cluster. The genes encoding these factors constitute iscSUA-hscBA-fdx cluster on the genome. Green sulfur bacterium Chlorobium tepidum, an anaerobic photosynthetic bacterium, has an iron-sulfur type reaction center and nitrogenase for nitrogen-fixation. In the whole genome of C. tepidum, homologues of iscU (CT1994) and iscS (CT1995) are found. In A. vinelandii, another gene nifU is involved in assembling of nitrogenase, homologue of nifU C-terminal domain (CT1786) is also found in the genome of C. tepidum. To investigate the mechanism of iron-sulfur cluster assembly in C. tepidum, we constructed recombinant IscU (CT1994), IscS (CT1995), truncate form of IscU and NifU homolog (CT1786) and over-expressed in E. coli. Absorption spectra and cryogenic ESR spectra suggested that the purified IscU binds [2Fe-2S] cluster.

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The photosynthetic reaction center complex (RC) purified from a thermophilic purple photosynthetic bacterium Thermochromatium tepidum was introduced into a folded-sheet silica mesoporous material (FSM). Adsorption of RC to FSM was determined optically and by the adsorption isotherms of N2. The FSM compounds with internal pore diameters of 7.9 and 2.7 nm adsorbed RC at 0.29 and 0.02 mg/mgFSM, respectively. Photochemical activity of RC in FSM was retained, even after heating for 10 min at temperatures higher than 60 degrees C. On the other hand, RC in solution was fully inactivated after heating at 60 degrees C. Photoreduction of DCPIP was observed in FSM. The hydrophobic silica nano-pores can, thus, provide a new environment for the membrane proteins to reveal their functions. The FSM-protein conjugate will be useful for the construction of a new probe and reaction systems.

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Ferrochelatase assay or heme content was frequently determined by the pyridine-hemochrome method, but this was disadvantageous because of low coefficient of pyridine-hemochrome and toxicity of pyridine. In plants, heme is biosynthesized in chloroplast and distributed to various organelles. To elucidate heme trafficking, a safe and sensitive heme assay is necessary. Here, we developed an sensitive assay using reconstitution with apo-horseradish peroxidase (HRP), and determination of peroxidase activity by measuring chemiluminescence. Active peroxidase was reconstituted within 30 min, and stable chemiluminescence was obtained 30 min after reaction. This method was linear with heme concentration and was extremely sensitive with detection limit as little as 10 pM. Various porphyrins and metalloporphyrins did not affect the chemiluminescence. The luminol oxidizing effect of Fe²⁺ was less than 3 magnitude of heme. Application of this method, such as heme efflux from tetrapyrrole-binding protein, the ferrochelatase assay, and heme content in organelles are being analyzed.

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In tetrapyrrole biosynthetic pathway, isoforms of glutamyl-tRNA reductase (HEMA) and Fe-chelatase (FC) mainly express in non-photosynthetic organs, such as roots and hypocotyls, suggesting they function for heme biosynthesis in these organs. However, the expression of HEMA2 and FC1 was induced in photosynthetic organs by cycloheximide, suggesting that the expression of HEMA2 and FC1 in photosynthetic organs was usually repressed but induced under some stress conditions. To clarify this hypothesis, Arabidopsis HEMA2 or FC1 promoter- GUS construct was subjected to stress conditions. The GUS activity specifically increased in wounded area and the whole body of ozone fumigated leaves, showing that the expression of HEMA2 and FC1 is induced under oxidative stress conditions. cDNA array analysis of wounded leaves revealed that HEMA2, FC1 were rapidly induced within 15 min, and co-induced with heme-proteins related to defense response. The physiological functions of HEMA2 and FC1 under oxidative stress conditions will be discussed.

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In a cyanobacterium Synechocystis PCC 6803, an FtsH protease (FtsH2, slr0228) is involved in the proteolysis of the D1 protein damaged by light stress and heat stress. Thus, the FtsH protease is essential for cell growth under these stress conditions.
To see if the synthesis of the protease is stimulated under the stress conditions, We carried out Western blot analysis to estimate the amount of FtsH in the wild-type cells subjected to moderate heat stress (42^oC, 2h), light stress (1000uEm^-2s^-1, 2h), and low temperature stress (4^oC, 18h) conditions. The antibody against DS9 protein corresponding to an FtsH protease identified in tobacco was used for the immunoblotting. We found that under these stress conditions, the level of the FtsH protease in the cells increased by 5-40 % compared with the cells grown under normal conditions.

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Chloroplasts have many defense systems against excess light. Some of these systems involve the reactions with mitochondria. Although excess reducing equivalents generated in chloroplasts can be exported to mitochondria, detailed mechanisms in intact leaves are not clear. In our prevent study, it was suggested that the mitochondrial alternative oxidase (AOX) has essential roles in dissipation of excess reducing equivalents (Yoshida et al. 2006). To conclude this probability, we analyze the respiratory properties in the leaves under the excess light. We used Arabidopsis thaliana mutants which lack the major defense systems in chloroplasts, the cyclic electron transport around PSI and D1 protein turnover. When exposed in high light, the leaves in the mutants showed photoinhibition more than wild type and higher rates of KCN-resistant respiration. These results suggest that AOX would function as a sink of excess reducing equivalents and contribute the mitigation of photoinhibition.

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Plant mitochondria have alternative oxidase (AOX). AOX catalyzes the O₂-depedent oxidation of ubiquinol, producing ubiquinone and water. AOX is not coupled to proton translocation and thus represents non-energy-conserving branch of the electron transport chain. It is suggested that AOX plays roles in preventing production of reactive oxygen species as well as in maintenance of the TCA cycle activity.
Arabidopsis thaliana has AOX1a-1d and AOX2, whose expression patterns differ among organs or stress treatments. Although it was demonstrated that AOX gene was induced and AOX protein was activated by stress treatments in suspension cells, it is not clear whether the expression and activation patterns of AOX correlate with its in vivo activity. In this study, we investigate the effects of expression and activation level of AOX on its in vivo activity, respiration and growth in A. thaliana plants under low-temperature or high-light stresses.

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It has been established that a green alga, Chlamydomonas reinhardtii, induces carbon-concentrating mechanism (CCM) and accumulates inorganic carbon under CO₂ limiting condition. However, the driving mechanism of CO₂-uptake systems remains unclear so far. We characterized the effects of several kinds of photosynthetic inhibitors on CO₂-uptake activity in wild type cells. Both O₂ evolving activity (H₂O to NaHCO₃) and CO₂-uptake activity were inhibited to 50% of original levels in the presence of 0.2 μM DCMU and the inhibitory effect of DCMU on both activities showed good fit to Michaelis-Menten equation, suggesting that light-induced CO₂-uptake is directly driven by photosynthetic electron transport. Now we characterize the property of CO₂-uptake in some photosynthetic and respiratory mutants to address the sites of photosynthetic electron flow that contributes considerably to the light-dependent CO₂-uptake of Chlamydomonas.

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Chlamydomonas raudensis is a psychrotrophic green alga that was isolated from perennially ice-covered lake, Lake Bonny (Antarctica). The lake is covered with a 3-5m ice-cap that transmits only selective wavelengths of light and avoids fluctuation of light intensity. This green alga has been reported to lack the ability of photoacclimation probably due to the extremely stable light environment of the natural habitat. We compared the ability of state transitions in this Antarctic phytoplankton with a model organism Chlamydomonas reinhardtii. The fluorescence quenching profiles and the 77K fluorescence spectra indicate that the two Chlamydomonas species have significantly distinct manner of the energy redistribution between PS1 and PS2. The relationship between the light environment and the photoacclimation mode observed in phytoplankton will be discussed.

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Reef-building corals harbor various bacteria whose biological significance for corals remains obscure. Here we report a novel cyanobacterium that has been found within the skeleton of reef-building corals. We have successfully isolated a filamentous cyanobacterium that inhabits within the skeleton of Goniastrea aspera, a massive reef-building coral. Characteristics of the filamentous cyanobacterium are: (1) 1μm in diameter, (2) nonheterocyst-forming, and (3) Chl a as the sole chlorophyll pigment. 16S rDNA analysis has affiliated the filamentous cyanobacterium with Halomicronema sp., a genera that has recently been identified in benthic microbial mats in a hypersaline pond. The biological interaction between Halomicronema sp. and the host coral is discussed in terms of a new symbiotic relationship.

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Japanese beech (Fagus crenata) is deciduous broad-leaved tree species and differently responses to high-light between the Pacific Ocean type (PAO) and the Japan Sea type (JAS). When PAO and JAS saplings were grown under high-light (H) and low-light (L) condition, only JAS-H turned its leaf color to pale green. To elucidate these phenomena, we analyzed about the two photosystems. JAS-H decreased D1-protein level, resulting in decreasing in Fv/Fm and ETR, while PAO-H highly maintained. P-700 photooxidation kinetics showed that the intersystem electron pool size in JAS-H was 1.4-times higher than that in PAO-H. Furthermore, re-reduction kinetics of P-700⁺ showed that the PS I cyclic electron transport in PAO-H was 1.2-times faster than that in JAS-H. These results demonstrated that PAO shows acclimatization mechanisms against high-light, whereas JAS is vulnerable to continuous high-light due to increases of the intersystem electron pool size and suppression of electron transport activity.

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Cyanobacteria have a high nitrogen-to-carbon ratio (~0.2) and invest a lot of energy in nitrogen assimilation. Regulation of gene expression in response to changes in nitrogen availability therefore comprises an important part of environmental adaptation of cyanobacteria; Many genes involved in acquisition and assimilation of nitrogen are regulated by NtcA, a global nitrogen regulator. To identify new nitrogen-regulated genes and to gain a deeper insight into global nitrogen regulation in Synechococcus elongates, we used the high-density DNA microarray of S. elongates PCC 6301. Transfer of ammonium-grown cells to nitrate-containing medium induced many genes in addition to the known nitrogen-regulated genes such as nirA operon, cyanase operon, and the glutamine synthetase and ammonium transporter genes. By contrast, many of the CO₂ assimilation genes were found to be downregulated. We are currently analyzing the contribution of NtcA to the global change in gene expression.

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In Physcomitrella patens, we previously identified and characterized five NRT2 genes and three Nar2 genes that are supposedly involved in active nitrate transport. In this study, we identified three new NRT2 genes (PpNRT2;6, PpNRT2;7 and PpNRT2;8) by screening the genome database. The deduced proteins are similar to PpNRT2;5 in lacking the hydrophilic amino acid segment conserved in the N-terminal region of PpNRT2;1-PpNRT2;4. Nitrogen regulation of PpNRT2;6 and PpNRT2;7 was similar to that of PpNRT2;1-PpNRT2;4 and distinct from that of PpNRT2;5, which strictly requires nitrate or nitrite for expression and is not susceptive to negative feedback caused by nitrogen assimilation. In contrast to PpNRT2;5, PpNRT2;8 was negatively regulated by nitrate and nitrite. In nitrate-replete protonemata, the PpNRT2;3 transcript was the most abundant among the PpNRT2 transcripts, suggesting the principal role of PpNRT2;3 in nitrate uptake from medium.

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The filamentous heterocystous cyanobacterium Anabaena variabilis harbors three genetically distinct nitrogenases. The conventional Mo-nitrogenase, encoded by nif1, expresses in the absence of a fixed nitrogen source when Mo is available. The V-nitrogenase encoded by vnf accumulates under Mo deficient condition in the presence of V, whereas, nif2 coding for another Mo-nitrogenase is expressed in vegetative cells only, under anaerobic condition. We report here the expression of the nif1 and vnf in heterocysts. Using reporter genes gfp and bfp, we have constructed 3 kinds of transcriptional fusion nif1-gfp-Ω, vnf-bfp-C.K3 and nif1-gfp-Ω-vnf-bfp-C.K3 in the shuttle vector pRL25C. The resulting vectors were transformed into the host cell with the help of conjugative plasmid RP4 by triparental mating method. The in situ expression of nif1 and vnf was detected by observing the over-expression of GFP and BFP under the regulation of a variety of environmental factors.

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Recently, many articles have shown that in plants as well as animals nitric oxide (NO) plays a key role of many important physiological processes (disease resistance, stomata movement, floral transition and so on). However, little is known about molecular mechanisms of NO signaling in plants. We produced transgenic Arabidopsis plants (cDrNOS) constitutively overexpressing nitric oxide synthase (NOS) from Deinococcus radiodurans. In the last annual meeting of JSPP (the 46th) we showed the dwarfphenotype of the cDrNOS plants. Expression levels of about 25,000 genes in rosetta leaves of the cDrNOS plants were analyzed using a microarray analysis. We also made a transgenic plants (iDrNOS) transiently overexpressing DrNOS gene after ethanol treatment. We will show the expression pattern of genes, which indicated remarkably different expression levels between WT and the cDrNOS plant on the microarray analysis, in the iDrNOS plants after ethanol treatment and discuss the functions of these genes.

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The potential of sweet potato to benefit from biological nitrogen fixation has been reported. In this study, culture-independent method based on the direct PCR amplification of nitrogenase gene (nifH) was applied to obtain information on the endophytic nitrogen-fixing bacteria in sweet potato. Genomic DNA and RNA from the leaf, petiole, stem and tuber samples of field-grown sweet potato were extracted. The fragments of nifH gene were amplified with two sets of degenerate primers by nested-PCR. The DNA sequence with high similarity to the nifH gene from Bradyrhizobium japonicum and Paenibacillus azotofixans were found in petiole, Anabaena variabilis, Herbaspirillum seropedicae, A. 7120 and Nostoc commune were found in stem, and Rhizobium leguminosarum and B. japonicum were found in tuber. RT-nested-PCR analysis revealed that the clones homologous to Bradyrhizobium sp. and uncultured N₂-fixing bacterium were expressed in stem, and clones homologous to uncultured N₂-fixing bacterium was expressed in tuber.

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Symbiotic nitrogen fixation of leguminous plant is established through complex signal exchange between rhizobacterium. Genome analysis of a model legume Lotus japonicus is in progress. Using the genome sequence information, several key regulatory genes of Nod factor perception and initial signal transduction were identified. We took a comprehensive approach to analyze the gene expression profile of the nodulation process by SAGE method (Asamizu et al. 2005). A cDNA array study on this process was reported (Kouchi et al. 2004). These transcriptome analyses led us to identify 20 transcription factors whose expression is up regulated in the nodulation process. In this study, we took several approaches to functionally characterize them. Nodules over-expressing the TFs were constructed utilizing hairy root transformation system and downstream genes were examined by oligo array experiments. A TILLING mutant showing considerable delay in nodule formation compared to wild type was obtained. The latest status will be presented.

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The comparison between gene expression profiles revealed that there were 45 RLKs expressed in pollen and/or pollen tube of Arabidopsis thaliana. Single gene T-DNA insertion lines did not show any strong phenotypes and double disruptions in sister genes seemed to be required for the further analyses. We made 10 sets of double gene disruptant lines, which covered 20 genes.
We found one line, whose pollen germination rate in vitro reduced to almost 0% using the wild type as 100% control. Although the segregation rate in self-fertilization looked normal, the seed numbers, especially in first 10 siliques, were reduced to half of wild type in their median. There might be interfered pollen germination and elongation in smaller number of pollen attached on the papilla. We also found a line, which showed unexpected segregation rate and gave us no double disruptants. Reciprocal crosses suggested that pollen from the line won't fertilize.

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One of the previously isolated genes from Arabidopsis thaliana, designated AtM532, encodes a protein homologous to yeast Rpf2, a pre-rRNA processing factor responsible for the processing of 27Spre-rRNA to 25SrRNA and 5.8SrRNA in ribosome biogenesis. AtM532 gene is highly expressed in meristems and nuclear/nucleolar localization of that product was confirmed by expressing the GFP::AtM532 fusion protein in onion epidermal cells. The AtM532 gene exists as a single copy in the genome and the homozygous for the T-DNA insertion causes embryonic lethal phenotype. Transgenic plants expressing anti-sense AtM532 that show reduced levels of AtM532 protein accumulation exhibited abnormal growth with high accumulation levels of 27SPre-rRNA. Moreover, interaction of AtM532 protein with a ribosome-associated protein was confirmed by the yeast two-hybrid method. These results suggest that AtM532 is a pre-rRNA processing factor.

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Plastids and mitochondria contain their own DNAs. The majority of angiosperm species display maternal inheritance of the organellar DNAs, whereas some plant species are known to exhibit biparental inheritance of organellar DNAs. Male gametophyte (pollen) consists of vegetative and generative cells (the progenitor of sperm cells). The amount of organellar DNA increases in generative cells of species that display the biparental inheritance but decreases in the case of species that display the maternal inheritance. Therefore, it is suggested that disappearance of organellar DNAs in generative cells is one of the mechanisms causing maternal inheritance. To understand the molecular mechanisms of organellar inheritance in higher plants, we used Arabidopsis thaliana and Medicago truncatula, which exhibit maternal and biparental inheritance, respectively, to screen mutants in which the amount of organellar DNAs in generative cells was altered. We will report on microscopic observation, genetic analysis and the progress of the screening.

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To investigate the flowering process of woody plants, we are analyzing genes expressed at flowering stage of Eucalyptus. First we isolated RNAs from flower buds of Eucalyptus, and then constructed a cDNA library using the RNAs. MADS-box genes were identified as transcription factors that function in flower formation in many plant species. The EST analysis showed that at least 14 MADS-box genes were expressed during flower formation in Eucalyptus. In this study, we focused on the floral homeotic gene AGAMOUS (AG) orthologs to understand the mechanism of stamen formation. Three genes that have homology with AG gene were expressed in Eucalyptus, and were named as EAGL1, EAGL2, and EAGL3 respectively. EAGL1 and EAGL2 genes were strongly expressed in flower buds, whereas expression of EAGL3 was at low level. Now we are constructing genes that repress expression of the agamous like genes using RNAi method.

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Linolenic acid (LA) suppresses flowering in Arabidopsis thaliana. Compared to 22 °C, a low temperature (15 °C) increases LA content and retards flowering in wild-type plants (Col). Since triple mutations in FAD3, FAD7 and FAD8 encoding enzyme synthesizing LA reduced the floral retardation by low temperatures, is considered to be attributed to LA. Considering flowering in 35S-AP1 plants overexpressing the flowering determination AP1 is early, and delayed by low temperatures or a cross with 35S-FAD3 plants overexpressing FAD3, LA directly influences the flowering determination by AP1. Here we investigated the relationship between the flowering determination and LA content in ap1 mutants. A decrease in content of LA followed AP1 expression when Col was grown at 22 °C. Among ap1 mutants was a mutant having less flowering-delay response to low temperatures than Col. These suggests that has a domain directly interacting LA, and this interaction controls AP1 function.

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Rice is a short-day (SD) plant and promotes flowering by the expression of Hd3a in SD. FT-L1 and FT-L3 are highly homologous to Hd3a.
To examine the expression patterns of Hd3a -like genes, we investigated their mRNA levels every 10 days until flowering. FT-L1 was expressed constitutively, and Hd3a and FT-L3 were highly expressed at 30 days before flowering in SD. We analyzed the function of Hd3a -like genes by RNAi. In FT-L3 RNAi plants flowering time was similar to that of the wild type (WT). However Hd3a RNAi plants flowered 30 days later than WT and interestingly FT-L1 and FT-L3 were highly expressed at 30 days before flowering. These results indicate that FT-L1 or FT-L3 may replace Hd3a to promote flowering in SD in the absence of Hd3a.

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Day-length is one of the major environmental factors used by plants to determine optimal time to flower. We study the variation of photoperiodic flowering pathway among the various rice cultivars. To investigate natural variation in rice (Oryza sativa L.), we employed the world rice core collection that was generated by NIAS gene bank. The core collection contains the maximum possible genetic diversity of rice with minimum of cultivars. In rice, previous reports showed that Hd3a is considered as a floral activator gene. We therefore investigate the expression level of Hd3a and days to flowering among the core collection. The results showed that strong correlation was observed between levels of Hd3a expression and flowering time. In addition, we investigate night-break effect on Hd3a expression among the core collection. We found that almost all cultivars possess the NB sensitivity. This result suggests that NB effect is a universal phenomenon among rice.

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The maize INDETERMINATE1gene, ID1, regulates the transition to flowering in maize. ID1 encodes a putative transcription factor which has four different types of zinc finger motifs. ID1 is the founding member of a family of a plant-specific zinc-finger protein that is defined by a highly conserved amino sequence (ID domain) including zinc fingers.
Searches of sequence data showed that there are sixteen, fifteen and thirteen of ID family genes in Arabidopsis,rice and maize, respectively.
Comparison of ID domain of these family proteins revealed that ID1 is distinct from other ID family proteins by having a longer spacer between the first and the second zinc finger.
In order to define the function of ID1, we analyzed the transcriptional activity of ID1 using yeast system and also examined whether the additional sequence between the first and the second zinc fingers of ID1 has effect on transcriptional activity.

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The Lemna genus includes the two different species that show photoperiodic flowering responses of a long-day type (L.gibba G3) and a short-day type(L.paucicostata 6746).We have isolated a number of Lemna homologues of circadian clock-related-genes and photoperiodic flowering genes in Arabidopsis and rice. Clock-related homologues for CCA1/LHY, PRRs, GI and ELF3 showed rhythmic expression profiles similar to the counterparts of Arabidopsis. Then we tried to establish methods for functional analysis of those homologues. We firstly developed a bioluminescent reporter system to monitor circadian rhythms of Lemna plants using a particle bomberdment method. Repoters with Arabidopsis CCA1- and TOC1 promoters showed circadian rhythms in Lemna plants. By introducing overexperssion/RNAi constructs of Lemna homologues with repoters in Lemna plants, we tried functional analysis of Lemna homologues. We will present their effects on the circadian system and discuss the comparison with those in Arabidopsis.

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The core circadian clock in cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC. This clock widely affects transcriptional regulation. In order to reveal the timing system for transcription, we have tried a systematic and high-resolution analysis for temporal profiles of gene expression. We have chosen a strategy using a genome-wide bioluminescent-reporter system for living cells under various conditions that include environmental changes. The 0.6-1.2 kbp upstream regions of predicted ORFs of Synechococcus elongatus PCC 7942 were used for the reporters as [promoters]. As a first step, we intensively analyzed reporter strains for 124 ORFs in a genome region (5% of the whole genome). The environments with various light and temperature conditions were analyzed for them. We also compared bioluminescence traces between wildtype- and a circadian clock-less mutant background cells. Cloning/monitoring strategies and temporal characterization of promoter activities will be discussed.

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Cyanobacteria are the simplest organisms known to exhibit circadian rhythms. Three clock proteins, KaiA, KaiB, and KaiC are essential elements of the circadian clock of cyanobacteria. We demonstrated that the oscillation of KaiC phosphorylation is the primary oscillation of cyanobacterial clock. Thus, kaiC mutants display a wide range of circadian phenotypes. To understand the biochemical functions of KaiC protein, we obtained 900 mutants with PCR-error based mutagenesis followed by a high throughput screening for rhythmic phenotypes with bioluminescence and analyzed their phenotype. While mutations mapped to various locations of KaiC sequence, two clusters of period-altering mutations were found. We analyzed a correlation between the mutation sites and the effects of temperature and light on the period length or phase of the bioluminescence rhythm. We will also discuss on relationships between phenotype of in vivo circadian rhythms and properties of KaiC phosphorylation rhythm of the mutants.

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An auxin-insensitive, dominant mutant, massugu2 (msg2), is caused by a mutation in one of 29 Aux/IAA genes, IAA19. Chimera genes between MSG2 promoter (pMSG2) and axr2/iaa7 or slr/iaa14 were introduced into Arabidopsis for studying specific function of each protein of the gene family. Studies of the transgenic plants show both promoter- and protein-dependent phenotypes (Muto et al., 2005, PCP 46, s208). Interestingly, plants expressing pMSG2:axr2 or pMSG2:slr lack one or both cotyledons in 30% of the population. This aberrant development of cotyledon has never been observed in msg2-1, axr2-1 or slr-1. Because pMSG2:GUS is expressed in cotyledon primordia of heart-stage embryo, ectopic expression of axr2 or slr may cause aberrant development of cotyledon. This observation implicates a mechanism which suppresses the dominant msg2-1 activity in cotyledon primordia. To investigate this hypothesis, further mutation was introduced into msg2-1 seeds and the progeny will be screened for seedlings with defective cotyledonary development.

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DNA methylation is known to involve in the regulation of gene expression. In animals, it is known that the hypomethylation of DNA leads to the aberrant embryogenesis with abnormal expression of embryogenesis-related genes. On the other hand, in plants, there is limited information about the relation between DNA methylation and embryogenesis.
In this study, we investigated the relation between DNA methylation and expression of embryogenesis-related genes (e.g. LEC1, ABI3 and FUS3) in Arabidopsis. The expression of embryogenesis-related genes were analyzed in various tissues (cotyledon, apical explant, rosette leaf, root, flower bud, flower and somatic embryo) of mutants deficient in DNA-methylation-related genes (e.g. ddm1 and cmt3). Some genes showed different expression profiles in the mutants as compared to the wild type. This result suggests that DNA methylation might involve in the regulation of expression of embryogenesis-related genes.

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