In Arabidopsis thaliana, there are about 2,000 transcription factors. These transcription factors are expected to make complicated regulation networks. On the other hand, we found about 12% of all genes undergone alternative splicing in Arabidopsis. It is possible that alternative splicing events occurred in pre-mRNAs encoding transcription factors make transcriptional networks more complicated. We analyzed alternative splicing events on Arabidopsis transcription factors, and studied the effect of alternative splicing events on transcriptional networks. We found about 100 (5% on all) genes undergone alternative splicing out of 1,968 transcription factor genes. The fraction of the alternative splicing events was small. But in several transcription factor families, bHLH, C2C2-type Zn-finger and CCAAT-binding factor, we found comparatively large number of alternative splicing events. We also found that the alternative splicing events often affected non-DNA-binding regions and UTRs. It was indicated that alternative splicing events had important role for fine-tuning of transcription factor functions.

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Plants respond and adapt to drought, cold and high-salinity stresses in order to survive.Many stress-regulated genes have been identified by the expression profiling studies using the DNA microarrays. However, we think that novel antisense RNAs, non-coding RNAs, small RNAs and alternative splicing mechanisms have a function in the plant responses to the stresses.
Recently, whole-genome tiling array has become a powerful tool for the analysis of the whole transcriptome, including analyses of sense-antisense transcripts, non-coding RNAs, small RNAs and alternative splicing. Now we are also applying Arabidopsis Affymetrix whole-genome tiling arrays (6.4 million 25-mer oligos) to study the whole transcriptome under drought, cold, and high-salinity stress conditions. The tiling array experiments for the 10-hr drought treatment indicated that the novel drought-responsive transcriptional units and antisense RNAs exist in the Arabidopsis genome and that the alternative splicing events occur in response to the drought stress.

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There are two kinds of interactions between genes and metabolites. One is regular interactions, and the other is conditional ones. In this study, we extract the characteristic interactions that depend on specific stress conditions.
Based on a state-space model that evaluates the transitions of the time-dependent interactions from a probabilistic view point, the time points at which interactions are changed are detected. Then, genes and metabolites are extracted that relates to the transition time points. Conclusively, conditional interactions are compared among the stress conditions. We suggest a statistical approach to analyze these processes systematically.
We apply the proposed method to time series data of Arabidopsis thaliana stress responses and extract genes and metabolites that express functions that depend on specific stress conditions.
By means of the extraction of genes and metabolites related to some specific stress responses, the regulation for the adaptation to various environmental conditions will be able to be verified.

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Gene expression profiles of Arabidopsis under various conditions and in various organs are available in public databases. Analysis of co-expressed genes using such microarray data enables us to predict gene function comprehensively. In our previous study on integration of transcriptome and metabolome data of sulfur-starved Arabidopsis, we were able to identify the genes encoding the enzymes involved in particular metabolic pathways. In a similar way, we can predict the genes encoding transcriptional factors involved in particular metabolic pathways. The genes which co-express in all experimental conditions are considered to be regulated by a single regulatory mechanism, while the genes which co-express under particular conditions are considered to be regulated by several different mechanisms. Such a comprehensive analysis of all available microarray data enables us to predict regulatory networks. Collection of metabolite profiles under various conditions and integrated analysis with gene expression profiles is useful for precise prediction of gene function.

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Integration of transcriptomics and metabolomics enabled us to predict the co-regulated gene and metabolite in Arabidopsis under sulfur deficiency (1). In particular, it was useful to identify the gene group in a same metabolic pathway, e.g. glucosinolate (GLS) biosynthesis. To identify other gene function, our integrated analysis was applied to identification of leucine (Leu) biosynthetic genes.
In Leu and methionine-derived GLS biosynthesis, a similar side-chain elongation reaction has been estimated. Candidate genes of Leu biosynthesis enzyme have appeared on the metabolic database (Aracyc), but their function have not been precisely tested. In this study, the candidate genes of GLS side-chain elongation enzymes and transcription factors were successfully predicted based on co-expression pattern with known GLS biosynthesis genes. The other genes, in contrast, did not show apparent co-expression with known GLS biosynthesis genes, suggesting that they are involved in Leu biosynthesis but not in GLS biosynthesis.
1. Hirai et al.,JBC (2005)

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Interpretation of DNA microarray data sets requires tools that enable us to obtain appropriate coexpression clusters. Here, we propose tools of network analysis to systematically analyze gene coexpression using many DNA microarray data sets of Arabidopsis instead of cluster analysis.
Computational analysis was performed using correlation coefficient data file between gene expressions that was calculated from 771 DNA microarrays. Genes which show high correlation coefficient and are highly specific to targeted genes over adequate threshold were selected as coexpressed candidate genes. The present tools were applied to genes related to secondary metabolism in Arabidopsis to validate adequate threshold setting.
In the majority of the applied genes related to secondary metabolism, coexpressed candidate genes were successfully expected by setting adequate threshold of correlation coefficient and specificity to each target gene.

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Cellulose, a major component of the cell walls, is the most abundant biopolymer in plants and serves many uses as industrial materials. However, the cellulose synthesis is so complicated that the mechanisms are poorly understood. In the present study, we analyzed a comprehensive gene coexpression profiles of the cell wall formation-related genes based on the publicly available microarray data sets from RIKEN and the Max Planck Institute.
The cellulose synthase genes (CesA) involved in primary and secondary cell wall formation were classified into two separate groups. In these groups, genes that have been previously shown to participate in cell wall formation, such as COBRA and chitinase-like protein gene, were correlated with CesA. Additionally, CesA also correlated with genes encoding enzymes and transcription factors that have been uncharacterized so far. These genes may participate in and play distinct roles in the formation of primary and secondary cell wall.

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Analysis of gene co-expression network can facilitate characterization and prediction of gene functions. We analyzed a mutual relationship between genes involved in amino acid biosynthesis using co-expression network constructed from publicly available correlation coefficient data of Arabidopsis (ATTED-II). The genes were clustered into three co-expressed groups. Group 1 contained the genes involved in valine, leucine, and isoleucine biosynthesis. Group 2 contained genes involved in arginine biosynthesis. Group 3 contained genes involved in glutamine, glutamate, serine, and glycine biosynthesis. We analyzed the co-expression patterns of the genes in each group with genes that were not directly involved in amino acid biosynthetic pathways. The result demonstrated the presence of the genes that could related the expression of amino acid biosynthesis genes. We are currently making overexpressors of these candidate genes in Arabidopsis T87 culture cells, and effects on the transcript and metabolite accumulation will be discussed.

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We investigated the regulatory mechanisms of isoprenoid biosynthesis, especially with reference to cis-prenyltransferase (cPT) which is involved in rubber synthesis. By analyzing co-expression of isoprenoid biosynthesis genes using the correlation coefficients calculated from 771 AtGenExpress data, some gene networks are inferred. 1) Genes in methylerythritol phosphate (MEP) pathway were closely connected, whereas those of mevalonate (MVA) pathway were only clustered when using the categorized array data from tissues. 2) Phosphomevalonate kinase gene was constantly isolated from the cluster of mevalonate pathway genes owing to its constitutive high expression. 3) Using the tissue-categorized array data each cPT gene was not connected with other cPT genes or the isoprenoid biosynthesis genes, but connected with some transcription factor genes.
For candidate transcription factors for cPT, generation of transgenic lines with overexpression or RNAi construct is in progress.

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Sulfur is an essential nutrient for growth of plants. Recently, a cis-acting element which responds to sulfur deficiency was identified in Arabidopsis (Maruyama-Nakashita et al. 2005). Our aim is to search transcription factors regulating sulfur assimilation.
Two public databases, ATTED (Arabidopsis thaliana trans-factor and cis-element prediction database; http://www.atted.bio.titech.ac.jp/) and AtGenExpress JPN (http://pfg.psc.riken.jp/AtGenExpress/index.html) were used to find transcription factors of which expression correlate with those of sulfur assimilation related genes and are induced under sulfur deficiency. Here, we investigated a Myb gene as a candidate. We over-expressed a full-length cDNA fragment of the gene in Arabidopsis suspension-cultured cell lines. Transcriptome analysis showed reduction of expression of a high affinity sulfate transporter gene. Furthermore, metabolite analyses using capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) showed reduction of contents of sulfur-related metabolites. The results suggest that the Myb transcription factor down-regulates sulfur assimilation.

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Over-expression of the PAP1 gene, which encodes an MYB-like transcription factor in Arabidopsis thaliana, resulted in up-regulation of a set of genes that were assigned or putatively assigned for flavonoid biosynthesis and accumulation of cyanidin derivatives in leaves and roots (Tohge et al., Plant J. 42:218-235. 2005). In this study, we over-expressed the PAP1 gene under 35S CaMV promoter in Arabidopsis suspension-cultured cells and examined transcriptome and anthocyanin analyses of transgenic cell lines. Analysis with liquid chromatography-coupled mass spectrometry indicated that PAP1 over-expressing transgenic cell lines produced seven species of cyanin-derived anthocyanins, while no anthocyanins were detected in wild type cell lines. Mass fragmentation pattern analysis of these anthocyanin derivatives indicated that the major species among them had three glycosides and two acyl-moieties to the cyanin aglycone. The transcriptome analysis of the cells using DNA microarray chips indicated induction of anthocyanin biosynthesis genes.

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An approach to understand the function of transcription factors (TFs) that coordinately regulate expression of genes involved in metabolic pathways is important for metabolic engineering in plant. As a basis for the functional analysis of TFs, we have investigated the potential TF genes encoded in the Arabidopsis genome and collected sequence data for 1860 genes encoding known and putative TFs that belong to 56 gene families. For the further functional analysis, ERF, DOF, STK, CO, BRX, NIN and MYB families were selected, and their ORF cDNA were subcloned into the Gateway entry vector. Then, we transformed Arabidopsis cultured cells (line T87) with cDNAs of the selected TF genes driven by a CaMV35S promoter. We examined transcriptional profiles in the TF gene-overexpressed T87 calli using an Arabidopsis DNA microarray. From the results, metabolic pathways controlled by the TF genes are discussed.

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Chlamydomonas cells utilize inorganic carbon very efficiently and suppress photorespiration by the operation of CO₂-concentrating mechanism (CCM) when adapted to limiting CO₂. Only limited number of CCM mutants are available, and their photosynthetic characteristics are still not fully investigated. To help understand the CCM, photosynthetic characteristics of the mutants, ca1, which over-accumulates DIC in the cells but cannot utilize, and pmp1, which cannot accumulate DIC, are compared with wild type and RPR1, a mutant with reduced rate of photorespiration. NPQ around CO₂-compensaton point was about 0.6 in 5% CO₂-grwon cells of all strains studied. On the other hand, in air (300 ppm CO₂)-adapted cells, NPQ was much higher in ca1 than that of wild type and RPR1 (about 1.5), and was as low in pmp1 as that in 5% CO₂-grown cells. In addition, a partial operation of DIC transport was suggested only at very low DIC concentrations in air-adapted cells.

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Marine diatoms are thought to contribute at least 25% of the global carbon fixation. Diatoms have been shown to possess the inorganic carbon-concentrating mechanism (CCM) that facilitates an ample flux of CO₂ to photosynthesis under CO₂-limited environment. The Diatom CCM is regulated primarily by [CO₂] but not other Ci species in the bulk medium and elevated [CO₂] has strong repressive effect on the high-affinity photosynthesis. However, detailed molecular mechanisms of CO₂ sensing and CCM regulation in marine diatoms are not known. We show evidences that cAMP functions as a major signaling molecule for the changes in the ambient [CO₂] and light in the marine diatom Phaeodactylum tricornutum. Increment of cAMP level in the cytosol appears to operate to repress chloroplastic carbonic anhydrase gene, ptca1, which is known to be a CO₂-responsive gene, under elevated [CO₂] condition via functions of a cAMP responsive element in the promoter region of ptca1.

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The β-type carbonic anhydrase (PtCA1) localizes as clustered particles at the surface of the girdle lamellae in the marine diatom Phaeodactylum tricornutum. The PtCA1 gene (ptca1) contains 138 bp of 5'-sequence (pre138), which encodes an N-terminal presequence of 46 amino acids (Pre46AA). The Pre46AA comprises of an ER-signal, which is followed by a chloroplast transit region. However, the chloroplast transit region is yet to be addressed in detail. A series of truncations was carried out on the 3'-terminus of pre138. These truncated ptca1 were ligated with the GFP gene (egfp). As a result, a short motif, FNAN, around the ER cleavage site of Pre46AA was shown to be critical as a chloroplast transit. GFP analysis of N- or C-terminus truncations of the mature PtCA1 suggested that 1-20 region from C-terminus might participate in the particle formation of PtCA1.

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The haptophyte alga Emiliania huxleyi possesses two carbon fixation pathways to produce organic and inorganic compounds in photosynthesis and calcification, respectively. Using a radiotracer technique, the photosynthetic primary products were determined after the incubation of cells with 2 mM NaH¹⁴CO₃ for 10-120 s under the light and the labeled compounds were identified by thin-layer chromatography. The time course and the pulse-chase experiment showed that phosphate esters, mostly the Calvin-Benson (C₃) cycle intermediate, and Asp were synthesized first and metabolized to lipids and amino acids such as Ala and Glu. These data showed the initial carbon fixation was driven by both the Calvin-Benson cycle and β-carboxylation reaction and then the products were immediately metabolized via TCA cycle and its associated pathway.

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The lamina joint (LJ) and pulvinus of grasses are slightly pale green as compared with the leaf blade, leaf sheath, and stem. In Arundinella hirta, the blade, sheath and stem show Kranz anatomy and the C₄ pattern expression of photosynthetic enzymes. We investigated the cell structure and photosynthetic enzyme expression in the LJ and pulvinus. These tissues substantially lacked stomata and Kranz anatomy, but the parenchyma cells included a few granal chloroplasts. The chloroplasts of pulvinus contained large starch grains. Western blots demonstrated that the LJ and pulvinus also accumulate PEPC, NADP-ME, Rubisco, and PPDK, but the NADP-ME differed in the molecular weight from NADP-ME of the blade, sheath, and stem. Immunogold electron microscopy revealed that high-level expression of Rubisco and low-level expression of PEPC and PPDK occur within the same cell. These data suggest that a single-cell-type C₄-like metabolism may operate in the LJ and pulvinus.

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To search for new member of C4 photosynthesis, we have isolated several C4 plant-abundantly expressed genes by transcriptome analysis between Flaveria trinervia (C4) and F. pringlei (C3). Among them we found out a novel gene, encoding a CP12 domain-containing protein, named FtCP12L. Its biochemical function was totally unknown in C4 plants. Here we will show our trial to identify the binding partner of the FtCP12L. The recombinant His-tagged FtCP12L protein was incubated with crude protein extract from F. trinervia leaves under the oxidative condition. Using the Ni-chelating column, recombinant protein was affinity purified and co-eluted proteins were analyzed with the SDS-PAGE. We found two ca. 43-kDa- and ca. 65-kDa-protein bands as candidates and the former was recognized with the antibody for phosphoribulokinase. These results indicate that the FtCP12L forms a novel protein complex, including phosphoribulokinase and the 64-kDa-protein in C4 Flaveria.

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Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a key enzyme in photosynthetic CO₂ fixation. In higher plants, RuBisCO is a multimeric enzyme composed of large and small subunits encoded by the chloroplast rbcL gene and nuclear rbcS genes. RuBisCO is biosynthesized via complex processes, hence overall mechanism is still unknown. To understand the molecular mechanism for these processes, we have designed a positive method for screening RuBisCO biosynthesis mutants using methionine sulfoximine (MSX) which is an inhibitor of glutamine synthetase. Wild-type plants treated with MSX could not survive because photorespiratory NH₃ accumulated massively depending on RuBisCO oxygenase activity. In contrast, rca mutants which lacked the activation of RuBisCO could survive under the same condition. After screened of 11000 EMS mutagenized seedlings, we obtained eight mutants which show either decreased RuBisCO amounts or low activities. These results indicate that this system is useful for screening of RuBisCO biosynthesis mutants.

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The effect of growth under elevated CO₂ on the capacity of photosynthesis with a single leaf and a whole plant was investigated with a radish cultivar of White Cherish. The plants were grown under ambient (ca. 400 &mumol mol^-1) or elevated CO₂ (ca. 750 &mumol mol^-1). The rates of photosynthesis per leaf area with a whole plant and a single leaf of various aged plants (15 to 26 days after planting) were measured under ambient and elevated CO₂. The rates of photosynthesis were increased by 20-28% by elevated CO₂. There was no effect of growth under elevated CO₂ on the rate of photosynthesis clearly indicating no downward acclimation of photosynthesis to elevated CO₂. Elevated CO₂ increased dry weight accumulation by more than 27%. The effect of elevated CO₂ on other growth characteristics will be also shown.

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The structural gene coding for ADP-glucose pyrophosphorylase (AGPase) in the cyanobacterium, Synechococcus elongatus PCC 7942 was disrupted by the insertion of blasticidin S resistant gene. The growth rate of the AGPase mutant in liquid culture medium was considerably lower compared to that of the wild type. The enzymatic activity of AGPase in the crude extract of the mutant was below undetectable level. Glycogen synthase activity was also decreased to 1/5 of that in the wild type. Accumulation of glycogen was completely abolished in the AGPase mutant. In the presence of 0.2 M NaCl, sucrose was synthesized in S. elongatus PCC 7942. The rate of sucrose accumulation was slower in the AGPase mutant and the sucrose content attained in the mutant was 1/2 of that in the wild type. The physiological characteristics of the AGPase mutant were examined in comparison to those of the glycogen synthase mutant that we isolated previously.

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We reported previously in 2004, the analysis of four starch synthase I (SSI)-deficient mutant lines having different activity levels. In this study, we identified the physiological characteristics of wild type and SSI mutant for understanding the physiological function in SSI in rice developing endosperm. The SS activity of the soluble fraction in rice developing endosperm obtained by anion exchange chromatography exhibited two peaks, designated as SSI and SSIIIa. It is known that SS in higher plants are enhanced by 0.5M citrate and glucan primers as well as ADP-glucose are needed for the reaction. Partial purified SSI by the anion exchange is enhanced by citrate and primer non-dependence, while SSIIIa showed primer-dependence, consistent with the reports with maize endosperm. SSI seemed to function from the very early (7 Days after flowering) through the late stage (25 DAF) of endosperm development.

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The present study established that there are two distinct polymeric forms of isoamylase1 (ISA1) in rice endosperm: presumably a homo-pentamer of ISA1 and a ISA1-2 hetero-hexamer composed of five ISA1 and one ISA2. The molecular size of homo- and hetero-multimers were approximately 420 to 480 kDa and 510 to 550 kDa, respectively. The ISA1-2 multimer exhibited higher affinities for various branched polyglucans, especially for phytolglycogen, which had a Km value that was approximately 12 times lower relative to that with the ISA1 complex. The ISA1 complex was active even when incubated at 50 degree for 10 min, while the homo-multimer was completely inactivated at 40 degree for 10 min. These results suggest that the ISA1-2 multimer plays a predominant role in the amylopectin biosynthesis in rice endosperm although the ISA1 multimer can complement the function of the ISA1-2 multimer at least to some extent.

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Though it is known that β-polyglucan is a storage product in Haptophyceae (Prymnesiophyceae), it has not been analyzed yet. We studied its structure and content in Pleurochrysis haptonemofera, Haptophyceae. β-polyglucan extracted from its cell extract was composed of β-(1,3)- and β-(1,6)- linkage by NMR analysis. The molecular weight of the β-polyglucan was 34,000 Da (D.P. 206) in peak value with gel permeration chromatography. The enzymatic determination method was established for determination of β-glucan content. Enzymatic determination revealed that β-polyglucan was localized in a soluble fraction and contained 20-50 pg per cell. The culture level of β-polyglucan kept constant until the log phase, increased at the stationary phase and decreased in the dark without relating to growth phase.

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The Positron Emitting Tracer Imaging System (PETIS) and caron-11-labeled carbon dioxide (¹¹CO₂) can image carbon movement during photoassimilation and export in a plant leaf, and ¹¹C kinetics make it possible to estimate physiological function parameters in those processes. With an exposure ¹¹CO₂ -gas to a leaf, PETIS experiments were performed under four light conditions on a single leaf. In order to estimate the rate constants of photoassimilation parameters, time activity curves of ¹¹CO₂ gas input and leaf response were fitted to an appropriate compartmental tracer kinetic model, which applies influx and efflux for photoassimilation and photosynthate export rate constants respectively. These results are reasonable response against light environment and important to discuss photosynthesis in plant physiology and agriculture. Kinetic parameters are fitted enough with pixel-by-pixel analysis, so that our method suggest that a parametric imaging of photosynthesis, which is indicative of a plant molecular imaging.

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Recently, we demonstrated that the central oscillator of cyanobacterial circadian clock was KaiC phosphorylation cycle. Moreover, we could reconstitute circadian oscillation of KaiC phosphorylation in vitro with three Kai proteins and ATP. Hence, the mechanism of the generation of the KaiC phosphorylation rhythm can be elucidated by biochemical analysis of three Kai proteins. In cyanobacterial cells, the accumulation levels of KaiC and KaiB show circadian rhythm, while level of KaiA is constant. When the amount of KaiA was altered, the period and amplitude of the in vitro KaiC phosphorylation rhythm were both changed. On the other hand, the period and the amplitude did not depend on the amount of KaiB, though indispensable for the generation of the KaiC phosphorylation rhythm. These results imply the stability of KaiC phosphorylation rhythm in vivo and the role of KaiA and KaiB in the generation of the KaiC phosphorylation rhythm.

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Circadian rhythms are endogenous oscillations of physiological activities with a period of about 24 hours, which allow organisms to efficiently adapt to the day/night alteration of their environment. Cyanobacteria are the simplest organisms that exhibit circadian rhythms. Three genes, kaiA, kaiB and kaiC are essential components of circadian clock in the cyanobacterium Synechococcus elongatus PCC 7942. Phosphorylation dynamics of KaiC is modulated by KaiA and KaiB, and phosphorylation state of KaiC oscillates in the cells with a 24-hour period. Two amino acid residues on KaiC to which posphoryl groups attach were identified, showing that the phosphorylation residues are necessary for in-vivo oscillation. We have reconstituted the self-sustainable oscillation of KaiC phosphorylation in vitro with three recombinant Kai proteins and ATP. We are investigating the molecular mechanism of in-vitro KaiC phosphorylation cycle.

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The heterocyst is a specialized cell for nitrogen fixation in the filamentous cyanobacteria, and its development is triggered by limitation of combined nitrogen in the medium. During heterocyst development, patterns of gene expression change dramatically. We identified seven genes encoding transcriptional regulators that were upregulated by nitrogen deprivation in Anabaena sp. PCC 7120, using an Anabaena oligonucleotide microarray. Among them, the nrrA gene, which encodes a response regulator of the OmpR family, has shown the most prominent induction after nitrogen deprivation. Expression of nrrA increased all through the filaments within 3 h of nitrogen deprivation and became higher in proheterocysts than in vegetative cells after 12 h. In the nrrA deletion mutant, heterocyst development was delayed and the induction of hetR, a master gene in regulation of heterocyst development, was suppressed up to 24 h nitrogen deprivation. It is concluded that nrrA influences heterocyst development.

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The first step of N₂-fixation is catalyzed by the enzyme complex, nitrogenase. As nitrogenase is highly sensitive to O₂, aerobic N₂-fixation of cyanophytes usually occurs during the night or in the specialized cells for N₂-fixation(heterocysts). However, N₂-fixation of non-heterocystous diazotrophs of genus Trichodesmium is light-dependent. Inter-cellular localization of N₂-fixing system in Trichodesmium has been studied but the results are still in controversy; some reports suggested the presence of N₂-fixing cells (diazocytes). In-situ immuno-cytochemical analysis of Trichodesmium sp. NIBB1067 revealed that (1) nitrogenase was expressed in almost all cells grown under N₂-fixing conditions, (2) there was no correlation between the abundance of diazocyte-like cells and the potential activity of nitrogenase, and (3) diazocyte-like cells were also found in cells grown with combined nitrogen. The factors that may caused the inconsistent results and the possible mechanism(s) to protect N₂-fixing system from O₂ in Trichodesmium will be discussed.

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Nitrogenase produces hydrogen as an inevitable byproduct of the reaction. In cynobacterial nitrogenase-based photobiological hydrogen production, the presence of hydrogenase seems to decrease the productivity by reabsorbing the hydrogen. Many cyanobacteria have both uptake (Hup) and bidirectional hydrogenase (Hox). Nostoc sp. PCC 7422 has both hup and hox genes, but its Hox activity is extremely low. We have constructed ΔhupL mutant and found that the hydrogen production (120 μmoles H₂/mg Chl/hr) of the mutant was 3 times that of the wild-type. Because no significant change was observed in nitrogenase between these two, the above difference is attributed to inactivation of reabsorption in the mutant. The mutant cells accumulated hydrogen to about 30% (v/v). The maximum level was affected by gas phase, cell density, cell culture etc. After hydrogen accumulation, the culture started to produce hydrogen although at a lower rate when the gas phase was renewed to Ar.

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Some cyanobacteria produce hydrogen as an inevitable byproduct of nitrogen fixation. We have created three uptake hydrogenase mutants from Anabaena PCC7120 and shown that the ΔhupL and ΔhupL/ΔhoxH produced H₂ at a rate 4-7 times that of wild-type. However, in the presence of N₂, the H₂ productivity is lowered because about 75% of the electron flux through nitrogenase is allocated to NH₃ formation. Biochemical studies of nitrogenase from Klebsiella pneumoniae indicate that the catalytic MoFe₇S₉ cluster in nitrogenase binds homocitrate and that nitrogenase of homocitrate synthase gene nifV mutant catalyzes the reduction of protons more preferably than of N₂ compared with that of the wild-type. In Anabaena PCC7120 genome two homocitrate synthase genes, nifV1 and nifV2, were identified. We have created three mutants (ΔnifV1, ΔnifV2, ΔnifV1/ΔnifV2) on the ΔhupL and examined their H₂ productivities. The expression patterns of nifV1 and nifV2 were also analyzed by Northern blots and GFP reporters.

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Genomic DNA of prokaryotes and plastids exists as nucleoids, which associated proteins play functional roles in the global regulation of such essential DNA functions as replication, recombination, transcription, and protection of DNA against environmental stresses. HU is major nucleoid protein in Anabaena sp. PCC 7120 and conserved in not only prokaryotes but also in plastids of red algae and cryptophyte algae. HU is encoded by the hanA gene in Anabaena sp. PCC 7120, however hanA is mostly uncharacterized about transcriptional regulation. In this works, hanA is expressed mainly in the log phase cells and induced under the low temperature or low nitrate conditions. To make deletion mutants of the hanA genes, we attempted to replace hanA by a homolog of Cyanidioschyzon merolae. We obtained mutant strains that were unable to form heterocysts under low nitrate condition. Role of HU in heterocyst differentiation is discussed.

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Plant nuclear genomes encode many cyanobacterial-like genes. The number of these genes is predicted to be ~3,000 in Arabidopsis, but most of these genes have not been identified. We are attempting mass identification of highly conserved genes among 8 cyanobacteria, Cyanidioschyzon, and Arabidopsis but not conserved among nonphotosynthetic organisms. Consequently, The 56 proteins were selected. We termed these as Chloroplast Proteins of Endosymbiont Origin (CPRENDO). The SALK T-DNA insertion lines for the 56 CPRENDO (CPR) genes were systematically characterized. The cpr27 mutant showed cotyledon-specific albino phenotype. Interestingly, some cells of albino cotyledon contain mature chloroplasts as well as undeveloped plastids, suggesting that CPR27 is critical for plastid development in cotyledons. Although no visible color phenotypes were observed, analysis by the saturation pulse method revealed that CPR27 also affects chloroplast functions in foliage leaves.

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We analyzed disruptants of Synechocystis genes that are homologous to CPR genes (encoding CPRENDOs or chloroplast proteins of endosymbiont origin) in Arabidopsis thaliana. Among the 38 disruptants, 5 were not completely segregated, suggesting that these are essential genes. Various phenotypes, such as growth retardation, light sensitivity, changes in pigment composition, changes in fluorescence induction and PAM pattern, were detected in 22 disruptants. These results demonstrate that these genes are related to photosynthetic growth of the cyanobacterium, which explains why CPRENDOs have been maintained as chloroplast proteins in plants.

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Based on the whole genome sequence of Mesorhizobium loti strain MAFF303099, we carried out large-scale analysis of protein-protein interactions by yeast two-hybrid (YTH) system. As the target for analysis, we selected M. loti genes whose up-regulation during symbiosis and microaerobiosis was revealed by the macroarray analysis and genes exist in symbiosis island. Furthermore, we also focused the interaction of the gene products conserved in other rhizobium species (Sinorhizobium meliloti and Bradyrhizobium japonicum), especially in the protein with unknown or regulatory function. At present, about 1000 bait clones have already been screened and over 1500 independent interactions consist of 1200 proteins were identified. In deduced large networks of M. loti proteins, we found various interactions of two component signal transducer and of symbiosis related gene with unknown function genes. Along with the YTH screening, the interaction database and its display system in RhizoBase are being prepared.

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Bacteroid differentiation is one of the most important events in symbiotic nitrogen fixation between Rhizobium-legume symbiosis. However, there is little information available to understand bacteroid differentiation of Rhizobium. Here we show that differentiation of bacteroids in indeterminate nodules involves genome amplification and cell elongation. Mature bacteroids in indeterminate nodules are functional but non-viable cells. In contrast, bacteroids in determinate nodules are comparable to free-living bacteria in their DNA content, cell size and viability. By using recombinat Rhizobium strains that are able to nodulate both determinate and indeterminate legumes, it is revealed that bacteroid differentiation is controlled by the host plant not by the rhizobial genetic background. These results suggest that leguminous plants employ at least two different strategies to control bacteroid differentiation of their micro-symbionts.

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A genome-scale cDNA-AFLP screen identified a number of Lotus japonicus genes transcriptionally activated during AM symbiosis. Among the rapid and symbiosis-specific genes, two serine-protease genes, SbtM and SbtS were identified. Analysis of the genomic sequence of L. japonicus revealed the presence of a gene family for SbtM located in at least two clusters each of which contains one pseudogene. Quantitative real-time PCR revealed that the transcript levels of SbtM genes and SbtS are increased during AM and differentially regulated in symbiosis processes. Analyses of roots transformed with SbtM and SbtS promoter-GUS fusions revealed that SbtM and SbtS are specifically induced around fungus-infected regions.We refine the promoter analysis with the aim to identify cis-element acting in early stages of AM development. Functional genetic studies were also performed using RNAi silencing and TILLING of SbtM genes.

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Legumes can utilize atmospheric dinitrogen by highly coordinated symbiosis with rhizobium. Recently the legume genes involved in early nodulation process have been clarified with molecular genetic approach using the model legumes Lotus japonicus and Medicago trancatula. However, the mechanisms for later stages, infection process and nodule organogenesis, are still unknown. We report cloning and characterization of the LjSYM101 gene essential for the infection thread formation and subsequent nodule organogenesis.
Ljsym101 is a recessive and monogenic mutant with the infection process arrested in the middle part of the root hairs and nodulation blocked at the stage of bumps.
Linkage analysis allowed us to position the Ljsym101 locus within ~87kb region of the chromosome 5. We identified the causative gene by the sequence analysis and confirm it using 5 alleles of Ljsym101. LjSYM101 was predicted to code 1485aa. The expression was detected in uninfected roots and enhanced by inoculation with rhizobium.

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⁶Max Planck Institute of Molecular Plant Physiology, University of Tokyo
A Lotus japonicus symbiotic mutant sen1 forms ineffective nodules under symbiotic condition, indicating that Sen1 is responsible for establishment of nitrogen-fixing symbiosis. To clarify the role of SEN1, Sen1 was identified by map-based cloning and expression profile was investigated. By linkage analysis, the Sen1 locus was delimited to a region of about 200kb on the chromosome 4. In one of 15 genes predicted in this region, a mutation was found by sequencing analysis. In further 3 more sen1 alleles, mutations were found in the same gene. Transformation of an entire gene into the sen1 could complement the mutant phenotype. Thus, we concluded that this gene is the Sen1. The Sen1 encodes a polypeptide of 266 amino acids, which contain 5 transmembrane regions. Expression of the gene was detected exclusively in nodules and was limited in the nodule infection cells.

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On infection with Mesorhizobium loti, the nod- mutant of Lotus japonicus, tinod can induce calcium spiking and root hair deformation, but cannot form infection threads or start cortical cell division. The mutant was also blocked in induction of early nodulins, like ENOD40 and NIN by M. loti. We have positionally cloned the causal gene, TINod, encoding a novel member of plant-specific transcription factor GRAS family. Expression of TINod was restricted only to roots, especially in their stele, and was suppressed rapidly upon inoculation with M. loti, suggesting that TINod is functioning on the downstream of calcium spiking, as a surveillance factor of rhizobial infection to initiate transcription of several nodulation-related genes. Most interestingly, TINod was first, as the plant GRAS members, shown to function analogously with animal STATs (Signal Transducers and Activators of Transcription) in its SH2 domain, which have been known to be conserved in both families.

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To investigate the tolerance mechanisms to high boron, we identified Arabidopsis thaliana genes that confer boric acid tolerance to yeast. Among the identified genes, AtRBP47c' was analyzed in this study. AtRBP47c' encodes a splicing enhancer which has three RRM (RNA recognition motif). Recently, RRM is reported to interact not only with RNAs but also with proteins. Hence, we searched a protein which interact with AtRBP47c' among yeast splicing factors. By using a two-hybrid assay, we identified MUD2 as a potential AtRBP47c'-interacting protein. Over-expression of AtRBP47c' could not increase boric acid tolerance in Δmud2 mutant, indicating that MUD2 is necessary for AtRBP47c' to confer boric acid tolerance to yeast. Furthermore, over-expression of AtRBP47c' ameliorated splicing inhibition of several genes under high boric acid condition. These results suggest that AtRBP47c' confers boric acid tolerance to yeast by enhancement of splicing efficiency through interaction with MUD2.

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To study the regulations of gene expressions by sulfur deficiency, we previously reported analyses of mutants with altered GFP fluorescence compared to parental transgenic Arabidopsis thaliana line NOB7 expressing green fluorescent protein (GFP) under control of a sulfur-responsive chimeric promoter. In the present study, EMS-mutagenized M2 seeds were grown for ten days and transferred to plates containing 10 mM O-acetyl-L-Ser (OAS), a precursor of Cys. OAS application causes general up-regulations of sulfur-responsive gene expressions. Plants showing altered GFP fluorescence compared to NOB7 plants were selected as putative mutants. Several mutant lines were further analyzed through mappings of the causal genes, quantitations of transcript accumulations of sulfur-responsive genes and analyses of metabolites. One of the mutant lines exhibited reduced GFP fluorescence both under sulfur sufficient or deficient conditions than NOB7. In this mutant, mutations were found in At4g21670, a gene shown to be involved in ABA signaling and development.

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Chromatin assembly factor-1 (CAF-1) is involved in nucleosome assembly following DNA replication and nucleotide excision repair. In Arabidopsis thaliana, the 3 CAF-1 subunits p150, p60 and p48 are encoded by FAS1, FAS2 and MSI1, respectively. Since nucleosome assembly is expected to be reduced or delayed in mutants lacking CAF-1 activity, we asked whether genomic stability is altered in fas1 and fas2 mutants. Depletion of either subunit enhanced the frequency of somatic homologous recombination (HR) in planta 40-fold. The frequency of T-DNA integration was also elevated. A delay in loading histones onto newly replicated or repaired DNA might make these DNA stretches more accessible, both to repair enzymes and to foreign DNA. Furthermore, fas mutants exhibited increased levels of DNA double-strand breaks, a prolonged S-phase, and elevated levels of mRNAs coding for proteins involved in HR. Also these factors could contribute to up-regulation of HR frequency in fas mutants.

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The most major prevalent lesions produced by ultraviolet (UV) are cyclobutane pyrimidine dimmer and (6-4) photoproducts. These photoproducts impair DNA replication and transcription processes and induce mutation. In higher plants, these photoproducts are thought to be restored with photolyase and nucleotide excision repair (NER). Moreover, it has been thought that these lesions which are not restored by photolyase and NER could be restored by homologous recombination (HR).
In the present study, we have analyzed intra-chromosomal homologous recombination frequency in AtRad1 or AtRad2 knock-out mutant of Arabidopsis. With the result, AtRad1 KO (uvh1) suppress intra-chromosomal homologous recombination, on the other hand AtRad2 KO (uvh3) enhance intra-chromosomal homologous recombination. We have crossed photolyase deficient mutant of Arabidopsis (uvr2-1) with uvh1 or uvh3 to produce double KO mutants, uvr2-1/ uvh1 or uvr2-1/ uvh3, respectively. We are currently analyzing ICHR frequency, UV-sensitivity and accumulation of UV-photoproducts in these double KO mutants.

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Polygonaceous plants contain high concentrations of oxalate. Excess uptake of this substance is toxic to animals. In plants, oxalate plays an important role as a source of hydrogen peroxide, which may act as defense molecules. Furthermore, some plants secrete oxalate to the rhizosphere, which could detoxify metallic ions.
Oxalate is known to be synthesized through several pathways. However, detailed studies concerning biochemical and molecular analysis of the pathways have not been presented. In this investigation, we analyzed the biochemical regulation of oxalate accumulation in polygonaceous plants. Measurement of metabolites by CE-MS indicated that oxalate levels were quite different among plant species. Enzyme assays demonstrated that the lactate dehydrogenase showed highest activity among other enzymes. The metabolic significance of such pathway in oxalate synthesis in polygonaceous plants will be presented.

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Unicellular calcifying algae (coccolithophorids) produce biomineralized scales, called coccoliths, that are mainly composed of calcite carbonate crystals. In Emiliania huxleyi, the coccolith production process proceeds internally and is genetically manipulated organic matrix-mediated calcification. We found that the rate of coccolith production of E. huxleyi cells was stimulated by low temperature. Inhibition by cycloheximide suggested that the involvement of the de novo synthesis of proteins in the regulation of coccolith production. In order to identify such proteins, a tracer experiment with ³⁵S-methionine/cysteine was performed under both the calcification-stimulating and non- stimulating conditions. SDS-PAGE analysis revealed that the amount of ³⁵S-labeled proteins with molecular masses of 32-kDa and 42-kDa increased significantly accompanied with the strong increase in coccolith production at low temperature. Amino acid sequence analysis of the proteins is undergoing.

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We have cloned a gene (Lsi1) responsible for Si uptake in rice, a silicon-accumulating plant. In the present study, functional characterization of this gene was conducted. Lsi1 is mainly expressed in the roots. The expression was constitutive, but regulated by Si supply; the expression was decreased by one fourth by continuous Si supply. Lsi1 is expressed in the main roots and lateral roots, but not in root hair. Further investigations show that the transport protein is localized only on the exterior side of plasma membrane of both exodermis and endodermis, where Casparian strips exist. When this gene is suppressed, the Si uptake is also reduced correspondingly. Furthermore, when the cRNA encoding Lsi1 was injected into Xenopus laevis oocytes, an increased transport activity for silicic acid was observed. All these evidence show that Lsi1 is a transporter for silicon in rice roots.

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Rice is known as a Si-accumulating plant, but the mechanism responsible for high Si uptake is poorly understood. We have isolated a rice gene (Lsi1) which is associated with Si uptake. In the present study, we report a novel rice gene (Lsi2) for Si uptake. Rough mapping showed that (Lsi2) was located on chromosome 3. Fining mapping resulted in identification of (Lsi2) candidate region of 106 kb, which is predicated to contain 17 candidate genes. Sequence analysis revealed that a single nucleotide substitution occurs at one candidate gene in the mutant. The protein encoded by this gene shows a high similarity to a putative anion transporter. To confirm the function of this gene, complementation test was conducted by introducing a genomic DNA containing the (Lsi2) region into the mutant. Result shows that the Si uptake in the transgenic plants was recovered, indicating that (Lsi2) gene is responsible for Si uptake.

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Silicon is a beneficial element for plant growth and all plants contain a significant amount of Si in their body. However, the molecular mechanism underlying silicon uptake is poorly understood. Recently, we cloned two genes (Lsi1 and Lsi2) which are responsible for Si uptake in rice mutants defective in Si uptake. In the present study, the functional analysis of Lsi2 was conducted. Lsi2 was constitively expressed in the roots and the expression was decreased by Si supply. Experiment with GFP fusion shows that the protein is localized at the plasma membrane of onion epidermal cells. In situ hybridization and immunostaining revealed that Lsi2 was localized at both exodermis and endodermis where Casparian strips are located. Furthermore, in contrast to Lsi1, Lsi2 was localized in inner side of those cells. Taken together, our results indicate that Lsi2 is a transporter responsible for efflux of Si from the cells.

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Iron (Fe) and zinc (Zn) are essential nutrients for plants. However, little is known how Fe and Zn are translocated in rice plants especially to the ears after absorption from roots. We traced ⁵²Fe and ⁶²Zn in rice plants (cv.Nipponbare) of 3 weeks after flowering by the 'PETIS method'. Image of the radioactivity appeared simultaneously in the ear and the flag leaf within 4 hours after supplying ⁵²Fe³⁺-DMA to the roots, suggesting that ⁵²Fe was not translocated to the ear via the flag leaf in short time. Image of the ear of rice appeared within 3.5 hours after supplying ⁶²Zn²⁺ and very little was translocated to the flag leaf during this experiment, suggesting that different from Fe, Zn was preferentially translocated to the ear at maturing stage. Real time translocation of ¹¹C-methionine and ¹³N-NH₄⁺ were also performed in rice plants.

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