Recently, we succeeded in preparation of oxygen-evolving Photosystem II (PSII) particles from a mesophilic centric diatom, Chaetoceros gracilis. The PSII particles, however, contained a large amount of FCP and their activity was unstable. In this study, PSII complexes were purified from a moderate thermophilic centric diatom, Chaetoceros neogracile which grows optimally at 35 degrees celcius, to prepare a stable PSII complex. After the cells were disrupted by freeze-thawing, thylakoid membranes were isolated and then solubilized with Triton X-100. Crude PSII was prepared from the solubilized thylakoid membranes by centrifuged fractionation. After the crude PSII was solubilized again with Triton X-100, PSII was purified by ion-exchange column chromatography. The purified PSII in which FCP was completely removed showed high oxygen-evolving activity (about 2,000 μmol O₂/mg Chl/h) and contained five extrinsic proteins of PsbO, PsbQ', PsbV, Psb31, PsbU. Heat-stability and pigment compositions of the pure PSII will be reported.

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Oxygen-evolving Photosystem II (crude PSII) prepared from a diatom, Chaetoceros gracilis, contained five extrinsic proteins of PsbO, PsbQ', PsbV, Psb31 and PsbU. To examine binding and functional properties of these extrinsic proteins, we purified the five extrinsic proteins and performed reconstitution experiments of these extrinsic proteins with PSII intrinsic proteins. After release of all of the extrinsic proteins by alkaline Tris-treatment of crude PSII, the five extrinsic proteins were purified by ion-exchange column chromatography. For reconstitution experiments, pure PSII prepared by DEAE column chromatography of Triton-solubilized crude PSII was treated with 4 M urea and 0.2 M NaCl to release all of the extrinsic proteins. This extrinsic protein-depleted PSII was reconstituted with each extrinsic protein either separately or in various combinations. Based on the results obtained, we will propose a model of diatom PSII showing association of the five extrinsic proteins.

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The redox potential of the primary electron acceptor, pheophytin a (Ph), is an important physicochemical parameter for charge separation in Photosystem II, because this potential should be a base point for speculations of the potential of the primary donor P680 inducing a strong power enough for water oxidation, which has not been measured. However, the reported values of Ph redox potential should not be necessarily reliable, though Klimov et al. reported -610 mV vs. SHE and an additional measurement was performed about 30 years ago. Possible problems in this reliability should be due to a titration technique; further they performed measurements at pH 11.0, different from physiological condition, to enhance a reductive power of titrants. In the present study, we have tried to measure the Ph redox potential at pH 6.5 by spectroelectrochemistry, leading us to find the potential higher by ca. 100 mV than the reported values.

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The primary and secondary quinone electron acceptors, Q_A and Q_B, in photosystem II (PSII) both consist of a plastoquinone (PQ) molecule. Whereas Q_B is readily exchanged, Q_A is tightly bound and thought to be unexchangeable without decomposition of protein subunits. In this study, we have performed reconstitution of Q_A with PQ retaining the protein structure of the PSII complex. Q_A is depleted from PSII by treatment with dithionite and benzyl viologen, and then excess PQ was added to the sample. Fourier transform infrared difference spectra upon Q_A photo-reduction showed that more than 70% of Q_A was reconstituted with a proper interaction at the Q_A site. This result was confirmed by reconstitution of ¹³C-labelled PQ, showing downshifts of signals of PQ. Reconstitution of Q_A in PSII will be a useful tool to investigate its structure in the protein and the mechanism for controlling the redox potential.

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Photosynthetic water oxidation is performed in the water oxidizing complex (WOC) in photosystem II (PSII) through a light-driven cycle of S states (S₀-S₄). To clarify the water oxidation mechanism, monitoring water reactions in WOC is essential. In this study, we have studied the water reactions in WOC by detecting DOD bending vibrations using Fourier transform infrared (FTIR) spectroscopy. FTIR difference spectra of the S-state cycle were measured using PSII complexes of T.elongates moderately deuterated with D₂¹⁶O and D₂¹⁸O. By calculating D₂¹⁶O-D₂¹⁸O double difference spectra, DOD bending bands of D₂O molecules in WOC were identified at 1250-1150 cm^-1. The presence of 6-8 peaks in each spectrum indicated that at least two water molecules participate in the reaction in every S-state transitions. The bands at 1240 cm^-1 in the S₂-->S₃ and S₃-->S₀ spectra reflected substrate insertion to WOC from a water cluster in the protein.

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The active site for water oxidation in Photosystem II goes through five oxidation states (S₀-S₄). It consists of a Mn₄Ca-cluster bound to 7 amino acids of the D1 and CP43 polypeptides. To study the role of one of these ligands, the D1-H332Q, we analysed purified PSII from Thermosynechococcus elongatus in which this amino acid residue was substituted by either Gln or Ser. Oxygen-evolution in both mutants under continuous light was 70-80% of that of WT*. However, the S₃ to S₀ transition seemed unaffected. The S₂Q_A^- charge recombination was a multiphasic process. The faster phases were accelerated in both mutants. The S₃Q_B^- charge recombination of H332Q, experimented by thermoluminescence, occurred at a temperature 6⁰C lower than that of the WT*. These results suggest that D1-His332 is involved in some of the S₂ and S₃ thermodynamic properties but not in the S₃Tyr_Z^. to S₀ transition.

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In this study, we found random degradation of a number of subunit components in oxygen-evolving photosystem II particles (crude PSII) isolated from a centric diatom, Chaetoceros gracilis, at room temperature in the dark, which were not observed in PSIIs from other organisms, suggesting the existence of diatom-specific proteases. The degradation was remarkably inhibited by EDTA, PMSF or Cu ion, implying the presence of metalloproteases and serine proteases. The degradation was stimulated with an increase in temperature and optimal at 40-45 degrees celsius but depressed above 50 degrees celsius. To examine the localization of these proteases, the crude PSII was solubilized with 1% Triton X-100 and then applied to DEAE-toyopearl column, which resulted in pure PSII containing no FCP. The significant degradation found in the crude PSII was not observed in the pure PSII. This indicates that proteases in the crude PSII were separated from PSII components during column procedures.

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In certain cyanobacteria, green-light irradiation induces accumulation of a green-absorbing pigment, phycoerythrin, whereas red-light irradiation induces accumulation of a red-absorbing pigment, phycocyanin, in the photosynthetic light-harvesting phycobilisome. We have shown that genes of a cyanobacteriochrome and a transcription factor (ccaS and ccaR) are essential for expression of phycobilisome linker gene in Synechocystis sp. PCC 6803, which does not possess phycoerythrin (Katayama et al 2004, PCP 45, S124). We recently demonstrated the green/red reversible photoconversion of CcaS and its light-regulated phosphotransfer to CcaR (Hirose et al., 2008, PNAS 105, 9528-9533). Here, we generated mutants of ccaS and ccaR in Nostoc punctiforme ATCC 29133 that accumulates phycoerythrin under green light but not phycocyanin under red light (group II chromatic adaptation). Under green or red light, phycoerythrin contents were lower in both mutants than wild type. We will discuss the mechanism how CcaS and CcaR regulate green light-induced phycoerythrin accumulation in Nostoc.

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Phytochromes are well-known photoreceptors mediating photomorphogenetic responses in plants. Plant phytochromes and bacteriophytochromes covalently bind a linear tetrapyrrole as a chromophore, and show red/far-red reversible photoconversion. PixJ1 is a phytochrome-like photoreceptor necessary for positive phototaxis in a unicellular cyanobacterium Synechocystis sp. PCC 6803. We have already reported that PixJ1 protein covalently bound a linear tetrapyrrole and showed a novel blue (λ_Amax= 435 nm)/green (λ_Amax= 535 nm) reversible photoconversion. We have already presented the interconvertible photoreactions of PixJ1 by low temperature light-induced UV-Vis difference spectroscopy analyses. In this study, light extinction wavelength and temperature conditions were improved and provided detailed schemes of photoreactive pathway of PixJ1. We will discuss the possible photoreactions of PixJ1.

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We found two mutants of Lotus japonicus, which were having elongated shoot and had elongated hypocotyls when germinate under red light. Subsequent sequencing analysis revealed to have mutations in the PHYB gene of both lines. Both mutants formed lesser number of nodules compared to the wild type.
Grafting experiments using phyB mutant showed that shoot genotype is responsible for the negative regulation of root nodulation. One possible reason for low nodule number inphyB mutants is due to limiting photosynthates. How about another reason? L. japonicus which was grown under continuous white light for 14 days was moved to high R:FR or low R:FR condition and then nodulation test were carried out. Surprisingly, the number of root nodules of low R:FR plants was dramatically reduced compared with that of high R:FR plants. These results indicate that root nodule formation is one of the R:FR ratio perception reaction.

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Plants convert light energy to chemical energy by performing photosynthesis in chloroplasts. Chloroplasts are differentiated from proplastids during plant development, but how the differentiation is achieved is only partially understood. Recently, we obtained a clue to elucidate the molecular mechanism in rice. When we ectopically overexpressed OsGLK1 encoding a GARP-type transcriptional regulator in rice, the OsGLK1-overexpressing calli exhibited green color on a 2,4-D-containing medium. The green calli accumulated transcripts for a set of genes involved in photosynthesis or other chloroplast functions. These results showed that OsGLK1 is a key factor regulating chloroplast development. Similarly, overexpression of OsGLK2, a homolog of OsGLK1, generated green calli, indicating not only OsGLK1 but OsGLK2 play crucial role(s) in chloroplast development. Further studies are in progress to characterize the common and different features between the two GLKs by analyzing their gene-expression patterns and candidate genes under the control of individual GLK proteins.

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When rice phytochrome B (phyB) mutants are grown under continuous red light irradiation (Rc), the seedlings display a pale-green phenotype that contain only 20% of chlrophyll as compared with wild type (Nippnbare) seedlings and their chloroplasts are furnished undeveloped thylakoids as well. It suggests that there are phyB-dependent signaling pathways for chloroplast development. To examine the pathways, we tried to isolate factors acting on them through microarray analysis in which comparison of gene expression profiles between phyB and Nipponbare was performed. We hypothesized that reduced expression of the gene for H subunit of Mg chelatase (OsChlH) is the most probable cause for appearance of the pale-green phenotype. To confirm the hypothesis, we exposed Rc to etiolated seedlings grown in the complete darkness for 8 days and observed transitions of OsChlH transcript levels, amounts of chlorophyll precursors and chlorophyll accumulations in the seedlings. Obtained results are consistent with our hypothesis.

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Using a rice microarray, we identified many abiotic stress-responsive genes. Among them, a gene for a bHLH transcription factor down-regulated by drought stress has been studied. The bHLH protein showed a high sequence homology with Arabidopsis PIF, driving us to name the transcription factor OsPIF1. Overexpression of OsPIF1 in transgenic rice plants promoted internode elongation. In contrast, dominant loss-of-function rice mutants with a chimeric repressor resulted in short length of the internode sections. In a transient experiment using rice protoplasts, the transcription of a GUS reporter gene driven by the promoter containing G-box elements was activated by OsPIF1 and we found that the APB motif of OsPIF1 is important for this activation. These data suggest that OsPIF1 functions as an important regulatory factor of plant growth in response to drought stress. Currently, we are analyzing down-stream genes of OsPIF1 by an oligoarray system using OsPIF1 transgenic rice plants.

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Arabidopsis thaliana changes its architecture dependent on environmental condition, temperature, light-intensity, and R/FR-ratio. Under high temperature condition hypocotyls and petioles are elongated like as under low R/FR-ratio condition. We study the molecular mechanism of this morphogenesis at high temperature.
At first, we report the correlation between temperature and light-intensity in morphogenesis. High irradiance diminished morphological changes mentioned above at 28 degrees. This result suggests that temperature and light-intensity signals are cross-talked. Secondary, to identify genes involved in the temperature-signaling pathway we isolated several temperature-insensitive mutants that prevent hypocotyl and petiole elongation at 28 degrees. At one of them, we found that the T-DNA was inserted in Phytochrome Interacting Factor4 (PIF4) gene and no PIF4 mRNA was detected. In wild-type plants, PIF4 mRNA was increased at 28 degrees compared at 22 degrees. Our data suggests that PIF4 function is essential for elongation of hypocotyls and petioles under high temperature.

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In plants grown in a low R/FR ratio, rapid stem and petiole elongation, which enables the plant to receive enough sunlight, is observed. This response is referred to as the shade avoidance response. The end-of-day FR (EOD-FR) treatment also causes similar responses. In addition to the growth response, rapid induction of genes such as AtHB2 and HFR1 is observed. Many auxin-inducible genes are also up-regulated. However, it remains unknown how different parts of the plant communicate in the process of those responses. In the present study, we irradiated petiole and blade separately with FR in Arabidopsis to locate the photo-perceptive site for the response. The results clearly demonstrated that phyB in blades almost exclusively regulated both the elongation and gene expression responses in petioles. Hence, a signal appears to be transduced from the blade to petiole after the light perception. Similar experiments are now in progress for seedlings.

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Nicotinamide nucleotides (NAD and NADP) are important co-factors in various metabolic processes in living organisms. Although both NAD and NADP are redox regulators of living cells, these compounds likely play overlapping but different roles in other biological processes. NAD and NADP are synthesized in NAD biosynthetic pathway comprising NADS, NMNAT, and NADK. The levels of NAD and NADP appear to be regulated by these enzymes. In this study, we analyzed transgenic Arabidopsis plants, which have several levels of NAD and NADP to elucidate the regulating mechanisms of these co-factors. Our results showed that the decrease of NADP level inhibited photosynthetic electron transport. Furthermore, fluctuation of NAD and NADP levels affected several metabolite processes, indicating that NAD and NADP are key factors that control plant metabolism and also suggest the possibility that enhancement of co-factor synthesis is a workable strategy for improving performances of plant.

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NADH-GOGAT1 was mainly detected in the vascular bundles of developing organs suggesting an important role in reutilization of Gln, which is transported from source organs. In root, the NADH-GOGAT1 inducibly accumulated in two cell-layers of root surface by NH₄⁺ supply, indicating a role in the primary assimilation of NH₄⁺. In this work, a knock-out mutant by Tos17 insertion for OsNADH-GOGAT1 (nadh-gogat1) was isolated and characterized.
When seedlings of wild-type (WT) and nadh-gogat1 were hydroponically grown with 5 mM NH₄⁺, root length of the nadh-gogat1 was reduced significantly. Although NH₄⁺ uptake was identical, free ammonium ions highly accumulated in the nadh-gogat1 roots associated with the decrease in Glu, Asn and Asp. Since the phenotype appeared only in the roots at the seedling stage, hormonome analysis is currently investigated. In field condition, stem length, the number of panicle and dry weight of the mutant were decreased compared with WT.

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Recent studies have revealed that cytokinin is a signaling molecule involved in the propagation of nitrogen signals throughout the whole plant cells for an integrated network of intracellular and inter-organ signaling of nitrogen availability in addition to nitrate itself. In previous study, we reported that one of the isopentenyltransferase in Arabidopsis, AtIPT3, is a key determinant of nitrogen-dependent cytokinin biosynthesis. In rice, seven genes of isopentenyltransgerase (OsIPT1-5, 7-8) were identified as the genes involved in the cytokinin biosynthesis. The intracellular localization of each gene products is observed in chloroplast, mitochondrion, or cytosol, respectively. We analyzed gene expression of OsIPTs in the nitrogen-treated rice root. As the results, we found that some OsIPTs were preferentially induced by ammonium ion rather than nitrate ion. The induction of OsIPTs were also observed by the addition of external glutamine. We will discuss the mechanism for nitrogen-inducible gene expressions in those OsIPTs.

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In rice, glutamine (Gln) regulates expression of several key genes for nitrogen metabolism including NADH-glutamate synthase1 (NADH-GOGAT1) and NAD-isocitrate dehydrogenase (IDH) genes. However, molecular mechanisms of Gln-signal transduction in plants are unknown. We have focused on a nuclear localizing OsACR9 in rice as a candidate for the Gln sensor since cellular distribution of OsACR9 overlapped with those of NADH-GOGAT1 and IDH in various rice organs. To investigate whether OsACR9 may be involved in Gln-signaling in rice, we searched rice genes expressed in a Gln- and OsACR9-dependent manner by microarray analysis. Comparative-transcriptome profiling in rice roots treated with L-methionine sulfoximine plus NH₄⁺ and/or Gln, provided 388 candidate genes expressed in a Gln-dependent manner. Expression of 59 genes of these candidates was altered in roots of RNAi-mediated OsACR9-knockdown rice. A yeast two-hybrid screening for OsACR9-interacting rice proteins is in progress. Involvement of OsACR9 in Gln-signaling in rice will be discussed.

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We previously reported that expression of the OsMYB-NR2 transcription factor gene was rapidly induced by nitrate supply in rice. To identify the target genes of OsMYB-NR2, we generated transgenic callus overexpressing OsMYB-NR2 and performed microarray analysis using the callus. Based on the result obtained, we selected candidates for the target genes, and examined whether the promoters of these genes could be really transactivared by OsMYB-NR2. Transactivation assays using maize protoplasts revealed that OsMYB-NR2 can transactivate the promoters of genes for tryptophan synthase α subunit involved in tryptophan biosynthesis and for tryptophan decarboxylase catalyzing the first step in the tryptophan secondary metabolism. Contents of tryptophan and serotonin, one of the secondary metabolites of tryptophan, were measured and the physiological function of OsMYB-NR2 was discussed. This work was supported in parts by grants from the Program for promotion of Basic Research Activities for Innovative Biosciences.

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We have isolated and characterized of novel genes encoding ACT domain containing proteins, ACT1 and ACT2, from Arabidopsis. ACT1 and ACT2 contain an ACT domain, an amino acid-binding domain. The domain is a regulatory domain mainly found in proteins involved in amino acid and purine metabolism. RT-PCR analysis indicated that the expression pattern of ACT1 and ACT2 genes was deferent in various organs from Arabidopsis. ACT1 mRNA was accumulated mainly in rosette leaves. In contrast, ACT2 mRNA was accumulated mainly in roots. To understand a role of ACT1 and ACT2, we are examining the characterization of Arabidopsis mutants ( act1 and act2 ) defective in ACT1 and ACT2 gene products.

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Plants mainly absorb nitrogen as nitrate and assimilate nitrate into amino acids. These reactions consume large amounts of energy, and, in illuminated leaves, most of the energy can be efficiently supplied by photosynthesis. However, plants often assimilate nitrate in non-photosynthetic organs and the distribution of nitrate assimilation among the organs differs depending on species, environments and growth stages. The aim of this study is to elucidate ecophysiological meanings of the distribution of nitrate assimilation through estimating the amounts of nitrate assimilation in each organ. Using hydroponically-grown Zinnia elegans, we followed the diurnal changes in the activity of nitrate reductase, the rate-limiting enzyme, and calculated the total amount of nitrate assimilated. Then, from the nitrate assimilated and the changes in nitrogen concentration, we estimated amounts of nitrogen translocated. Based on these data, we discuss the independency of nitrogen acquisition in each organ, and the advantage of nitrogen assimilation in non-photosynthetic organs.

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Leaves represent the largest pool of nitrogen in vegetative grasses. As leaves expand, large quantities of nitrogen flux into them; with more than 60% of this deriving from internal remobilization. As leaves age, influx decreases and efflux becomes the major factor determining leaf N content. N flux within the plant should therefore be non-uniform through time. Leaves connect together at a small organ through which all leaf N must pass, known as the tiller axis. To test our hypothesis of non-uniform N concentrations in the tiller axis, we measured the N concentration in ryegrass, barley and wheat, finding strong evidence of a steady oscillation, which correlates with leaf growth and developmental patterns, and is hypothesized to be a potential control mechanism. This oscillation appears to be genotype specific, predominantly driven by protein turnover, and sensitive to environmental conditions, such as exogenous N supply and temperature.

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Nitrogen utilization of rice is determined by several factors, such as uptake, assimilation and other various steps. Morphology of rice root dramatically changes according to the supply of different ammonium concentration. Chromosome segment substitution lines (CSSLs) developed between Koshihikari and Kasalath were employed for mapping QTLs controlling root length. A QTL stimulating root length under low NH₄⁺ condition was detected on chromosome 8. A CSSL, SL-225, which has a Kasalath segment on a long arm of chromosome 8, and Koshihikari were hydroponically grown under various concentration of NH₄⁺ (5-500 μM). Root length of the SL-225 was significantly longer than Koshihikari under all concentration tested, and the elongated root reduced the uptake of NH₄⁺ at 50 μM NH₄⁺. Backcrossed progenies derived from SL-225 were used for the high resolution linkage analysis. The QTL was narrowed down at approximately 40 kb-region on chromosome 8.

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¹⁵N, a stable isotope, has long been used in studies about kinetics of nitrogen fixation and transport of the fixed nitrogen. However, it is difficult to analyze instant responses to an environmental (ex. temperature, light conditions) change with this method because it requires invasive assay. In this study, we have developed a method to produce ¹³N-labeled radioactive nitrogen gas (¹³N₂) with high purity. We fed the ¹³N₂ mixed with defined concentrations of O₂ and N₂ to the underground part of nodulated soybean plants and obtained movies of fixation of nitrogen gas using PETIS (positron-emitting tracer imaging system) successfully. The rates of fixation within 10 min were estimated quantitatively.

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Legumes are able to establish a symbiosis with rhizobium bacteria. Recognition of the rhizobial Nod Factors (NFs) is crucial to initiate symbiotic process in legume plants. Structural backbone of NFs is N-acylated chitin oligomer. Chitin oligomers are known as elicitors which induce defense responses in many plant species. NFs and chitin elicitors have been shown to be recognized by closely-related LysM receptor kinases. We focus on the mechanisms controlling these opposite responses elicited by the similar molecules.
Microarray analyses using a model legume Lotus japonicus indicated that the genes involved in defense responses such as phytoalexin synthesis were drastically up-regulated by NFs as well as chitin treatment. NF-induced, but not chitin-induced, defense gene expression was abolished in nfr1 mutant. In-gel kinase assay revealed that SIPK and WIPK-like activities were induced in NF-treated roots.

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Legumes establish symbiosis with rhizobia in root nodules. Recently, various symbiotic genes have been identified from model legumes to reveal molecular mechanisms of the symbiosis. We have isolated a symbiotic mutant, alb1 of Lotus japonicus Gifu B-129. When alb1 was infected with Mesorhizobium loti under nitrogen-starved conditions, it showed a serious growth inhibition phenotype compared with wild type. alb1 could form immature nodules, but they failed to develop mature nodules. In the wild type, numerous infected cells were developed in the central zone of nodules. However, no infected cells were observed in alb1. In alb1, infection threads were arrested in epidermal cells, and M. loti was not released to nodule cells. By map-based cloning followed by complementation of alb1 phenotype, it was revealed that ALB1 encoded a LRR-receptor like kinase.

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Leguminous plants form root nodules for symbiosis with soil bateria Rhizobia to fix dinitrogen in the atmosphere. To utilize symbiotic nitrogen fixation, it is indispensable to understand the molecular mechanism of nodulation process. Nitrogen fixation activity of rhizobium is controlled by the host plant under symbiotic condition. Fixation minus (Fix^-) mutants are good materials to identify the plant factors involved in development and maintenance of symbiotic nitrogen fixation.
In this study, we had analyzed a novel Fixation minus (Fix^-) mutant of Lotus japonicus Ljsym102. This mutant formed pale pink nodules with low nitrogen fixation activity and exhibited early senescence symptons in the nodule tissues. These preliminary observations showed Ljsym102 established a symbiotic association with Rhizobia but had defect to express effective nitrogen fixation activity. We report further characterization of Ljsym102 mutant and map-based cloning of its causal gene.

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Most of rhizobia are able to fix N₂ only when they inhabit in the compatible host legume nodule cells and differentiate into bacteroids. This indicates that N₂ fixing activity of rhizobia is strictly controlled by interactions with host legume plants. However, it is unclear how the host plants regulate nitrogenase activity of rhizobia in the nodules.
A Lotus japonicus Fix^- mutant, fen1, forms small and pale pink nodules. Acetylene reduction activity of the fen1 nodules is very low, though rhizobia infected in the nodule cells appeared to differentiate into bacteroids. Therefore, Fen1 gene is presumed to be essential for controlling nitrogenase activity of rhizobia. We identified the Fen1 gene by map-based cloning and complementation test. Here we report a possible function of FEN1 protein in the development of N₂ fixing activity, based on the results of functional analysis of recombinant protein in Saccharomyces.

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We present here the effects of sodium chloride (NaCl) and brassinosteroids (BRs) on nodulation of Lotus japoicus. When Lotus seedlings were inoculated with rhizobia on a medium containing 100-150 mM NaCl, the number of nodules was slightly reduced compared with that on the same medium free of NaCl. Growth enhancement of the seedlings due to symbiotic fixation was rarely observed in this condition. Moreover, white-colored nodules were increased, in which leghemoglobins are supposed not to function properly. Interestingly, rhizobia harbored in white-colored nodules were much larger in number than those in red-colored ones. In the presence of more than 175 mM NaCl, nodulation hardly occurred. By contrast, neither applications of the most bioactive BR, brassinolide (BL) nor a specific inhibitor of BR biosynthesis, brassinazole influenced nodulation in the absence of NaCl. However, BL treatment at 10^-8 M or more obviously enhanced the inhibition of nodule formation caused by NaCl.

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The root nodules of leguminous plants will be the most suitable model system to understand the mechanism of endosymbiosis. However, host plant molecules that induce bacteroid differentiation are still unknown. Morphology of bacteroids from Medicago truncatula and Lotus japonicus are quite different from each other. The differences may be attributed to the different strategy of the host plant to establish symbiosis. The NCR genes of M. truncatula, that encode several hundred of cysteine-rich and potential-antimicrobial peptide, are absolutely expressed in nodules and can be associated with bacteroid differentiation. We postulate that intracellular rhizobia in the nodules of M. truncatula are challenged with a battery of the NCR peptides that induce the terminal bacteroid differentiation. Testing this hypothesis relies on localizing the peptides in nodules and analyzing their in vitro activity on Sinorhizobium meliloti, the microsymbiont of M. truncatula, and their in planta activity in L. japonicus nodules.

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Bj488, an up-regulated gene in Bradyrhizobium japonicum bacteroid from nodules at 28 days after infection, was investigated. It displayed a conserved gene homologue to COX17, sharing a potential metal binding motif H(M)X₁₀MX₂₁HXM. COX17 is one of the metallochaperones which conserved in the yeast and human proteins, consistent with a role as a shuttle protein for delivering copper to mitochondria. Metallochaperones are necessary in copper ion delivery and insertion for the assembly of cytochrome c oxidase. The Bj488 mutant resulted in defective symbiotic nitrogen fixation and showed the Fix- phenotype. The significant decrease of Cytochrome c oxidase activity was observed in a Bj488 bacteroids under microaerobic condition, whereas the activity was comparable in cells grown aerobically in free-living cell. It is suggested that Bj488 protein is proposed to have an important role in copper ion delivery to cytochrome c oxidase, which might be induced at low oxygen concentrations.

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A parametric intellectual framework^* for the analysis of transcriptome data is demonstrated to yield coincident results when applied to data acquired using various microarray platforms. Microarrays are widely employed to acquire transcriptome information, and several platforms of chips are currently in use. However, discrepancies among studies are frequently reported, casting doubt on the reliability. The inconsistency among observations can be largely attributed to differences among the analytical frameworks employed for data analysis. The existing frameworks are based on different philosophies often based on ad hoc styles, and yield different results. Here, a parametric framework based on a statistic model that bases on thermodynamic model of transcriptome formation. The framework is tested in data acquired using several slide-glass-type chips and GeneChip. The expressional changes observed and genes selected are coincident between platforms, achieving superior universality of data compared to other frameworks.

^*assumptions, theories and ideas on which we can think

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ATTED-II (http://atted.jp) and COXPRESdb (http://coxpresdb.hgc.jp) are databases we constructed for gene coexpression in Arabidopsis and in mammals, respectively. These databases can be used to design a wide variety of experiments, including the prioritization of genes for functional identification or for studies of regulatory relationships. Although Pearson's correlation coefficient (PCC) is widely used as a coexpression measure, we applied mutual rank (MR) of PCC for ATTED-II (ver. 5.0 and later) and COXPRESdb (ver. 3.0 and later). We will present the following characteristics of MR. (i) High potential to predict gene function. (ii) Independence against genes and data sources. (iii) Potential to draw understandable gene network.

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Knowledge on the functions of Arabidopsis genes has been rapidly accumulated, and it is a challenge to reveal the expression system of genes with useful functions. To perform a global approach for obtaining the information on such system, we selected 187 Arabidopsis genes, constructed the T87 cultured cells in which the genes are over-expressed and/or down-regulated by RNA interference, and then executed an analysis based on 214 DNA microarray chips using the cells. In the analysis, the expression value of each gene was standardized. Based on the standardized values, we searched genes that show the expression specific in the cell for an over-expressed or down-regulated gene of interest and then associated the specifically-expressed genes with the gene of interest as candidate regulatory relationships. Using the information of such associations, we depicted a gene regulatory network in a T87 cultured cell.

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It is very laborious for each wet researcher to classify proteins that share similar biological functions registered in public databases in silico. To get hints to elucidate the biological function of annotated proteins, we have developed and opened a genome-wide database, termed SALAD database, based on similarity clustering and distribution diagrams of evolutionarily conserved peptide motifs found in seven plant species, rice, sorghum, Arabidopsis thaliana, Physcomitrella patens, green alga, red alga, and yeast. Using this database we can compare any annotations of your interest and discuss on their evolutional relationships efficiently and quickly. In addition, to order to compare specific gene expression of microarray data for paralogous (or related) genes, we integrated the LM microarray data on rice pollen development into our clustering in the SALAD database (termed SALAD on ARRAYs). This service is now available for public microarray-data sets. We will introduce these topics on SALAD database.

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DNA microarray has been used widely to analyze functions of genes or drugs. The data shows genome-wide-transcriptome state of samples. Data-repository databases, such as Gene Expression Omnibus (GEO), provide a large number of experiment data. By comparing various gene expression profiles, we will be able to find biological connections between experiments, such as phytohormone and its inhibitors. However, microarray data is noisy snapshot. It is very difficult to compare biological similarity of gene expression across different laboratories, tissues. It is required to exclude noise to detect biological similarity between experiments.
To compare various publicly available microarray data, we applied network analysis using Co-module method in Arabidopsis. As a result of model data analysis, our approaches were successful to detect biological relationships between experiments from different laboratories. In addition, negative or partial connections were also detected. We constructed a novel Web-based database as an analysis tool of gene expression profiles.

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Prediction of metabolites in the peaks detected by liquid chromatography (LC) - mass spectrometry (MS) requires human insights to re-construct probable molecules from various information, such as 1) mass values on predicted ionization states, and origin of the peaks (¹³C isotopic, multivalent, or auto-fragmented peaks), 2) partial structure of metabolites deciphered by tandem MS/MS analysis and UV absorption, and 3) results of database and literature searching. Therefore, enormous time and labor have to be spent to predict all the peaks detected even in one analysis. This is one of major factors that restricts comprehensiveness of metabolites in the further data analyses. To accelerate understanding of matabolome obtained by LC-MS, we have constructed the database KOMICS (http://webs2.kazusa.or.jp/komics/) to store and reuse the annotation results. Here we report software PowerFT to accelerate annotations of the peaks detected by LC-Fourier transform ion cyclotron resonance (FT-ICR)-MS and the development of KOMICS.

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RIKEN Arabidopsis Genome Encyclopedia (http://rarge.psc.riken.jp/) - commonly known as RARGE - is an online database that provides basic information on the Arabidopsis genome, such as that related to cDNA sequences and transposon insertion mutants. Recently, 2 datasets regarding the traits of plants with disrupted genes were included on the RARGE site. These datasets provide a substantial amount of new information. Kuromori et al performed a comprehensive observational study on transposon insertion mutants, and we developed an online database including this data in order to enable searching and browsing. Further, Myouga et al analyzed transposon or T-DNA insertion mutants with disrupted nuclear-encoded chloroplast proteins from a different perspective, and we developed a Web site for the database containing their data as well. Although both these datasets are available on individual Web sites, we integrated the 2 databases into RARGE in order to make referencing easier.

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Establishment of transgenic plants is a time-consuming process, and it also runs the risk of releasing an antibiotic-resistant gene of a selectable marker into the environment. Here we describe a new technology that overcomes these difficulties in Arabidopsis thaliana and named it as FAST (Fluorescence-Accumulating-Seed Technology). This method uses a selectable marker composed of oleosin-GFP, which enables us to instantly identify the heterozygous transformed 'dry seeds' among the T1 population and then to identify the homozygous seeds among the T2 population with a false-discovery rate of less than 1% under a fluorescence microscope. The selectable marker protein is expressed just in seeds that are used for selection. The FAST method does not require sterilization, clean-bench protocols, the handling of large numbers of plants, or large spaces to grow plants. The FAST method is well suited for maintaining and monitoring the safety of genetically engineered crops, too.

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Gene modification by gene targeting (GT) via homologous recombination is a powerful tool not only for the analysis of the function of the gene of interest but also for the molecular breeding of crops. GT is thought to be clean transformation technology because it can modify the only targeted gene predictably, although transgenes are integrated at random loci in conventional transformation system. In this study, we succeeded in the modification of endogenous rice OASA2 gene, that encodes anthranilate synthase, a key enzyme in tryptophan (Trp) biosynthesis, by T-DNA mediated GT. Southern blot analysis in T₀ and T₁ generation showed that true GT, in which wild-type OASA2 gene was modified as expected, was occurred in this plant. Moreover, the content of free Trp in the leaf of GT plant homozygous of modified OASA2 gene was much higher than that in wild-type.

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The induction of double-strand DNA breaks (DSBs) greatly increases rates of homologous recombination-mediated gene targeting or of gene mutation by error-prone non-homologous end joining. Both repair mechanisms would be important basis for molecular breeding to modify the targeted gene on the genome. However, these approaches depend on the capability to create a DSB on the specific genomic sequence of interest.
Zinc finger nucleases (ZFNs) have been recently developed to introduce a site-specific DSB. ZFNs function as dimers with each monomer composed of a non-specific cleavage domain from the Fok I endonuclease fused to a zinc-finger array engineered to bind a target DNA sequence of interest.
To demonstrate the ZFN approach to higher plants, especially rice, we developed systems for designing of ZFN, confirming digestion in in vitro and in vivo. We are on going to establish the system for induction of genome editing on the rice genome.

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