Cytoskeleton plays a crucial role in plant development and morphogenesis. Gametophyte cells of the moss Physcomitrella patens are an excellent model to study dynamics of cytoskeleton because gametophytes have simpler structures than sporophyte tissue in other plants. Further, we can make stable transformants with an appropriate construct in a given locus by homologous recombination. In this study, we made reporter constructs by fusing a GFP with P. patens a-tubulin or an actin binding domain of mouse talin, which are expressed under the control of rice actin promoter to visualize microtubules and actin filaments. The Pphb7 gene, a member of HD-Zip gene family, was used as a target locus because its expression is limited to rhizoids and no phenotype was detected in any tissues except for rhizoids. Using the stable lines, we report the dynamics of microtubules and actin filaments in the processes of cell division, cell elongation, and chloroplast movement.

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Rice lip19 that encodes a bZIP protein of 148 amino acids has been isolated as one of low-temperature-induced genes. Hence LIP19 seems to play a regulatory role in gene expression during cold stress, although the details of its function remain to be discovered. Since LIP19 lacks the common ability of bZIP transcription factors to homodimerize and to bind DNA we searched for other putative partners for heterodimerization. A screening for clone(s) whose product interacts with the LIP19 protein using a yeast two-hybrid system disclosed OsOBF1.
Here we confirmed the interaction between LIP19 and OsOBF1 using a pull-down assay. An experiment of domain-swapping between LIP19 and OsOBF1 revealed that a leucine-zipper portion of LIP19 caused the defect in homodimerization. With in situ hybridization, we showed that lip19 and OsOBF1 are expressed at similar sites. Based on the data, we will discuss a role of LIP19 in cold signaling pathway in rice.

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We reported that expression of SAMDC gene, which has a central role in spermidine (Spd) and spermine (Spm) biosynthesis, is increased by low-temperature stress (5 degree C) in young rice plants. In this work we examined whether increase in Spd and Spm is induced by cold or other abiotic stresses. We found that moderate low-temperature (12 degree C) and submergence stresses other than low-temperature stress induce increase in putrescine (Put) in leaves and stems. We also found that ABA induces increase in Put in roots, while ABA does not induce increase in Put in leaves and stems. These evidences indicates that ABA is unlikely to be directly involved in increase in Put induced by moderate low-temperature and submergence stresses in leaves and stems. The results of expression analysis of arginine decarboxylase and ornithine decaboxylase genes, which are responsible for biosynthesis of Put, are also discussed in relation to stress conditions.

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The plant membrane lipids contain a high content of trienoic fatty acids (TAs), which are assumed to play an important role in temperature adaptation. TA synthesis is catalyzed by ω-3 desaturase. In Arabidopsis, two isozymes of the chloroplast-localized ω-3 desaturase (FAD7 and FAD8) have been identified. These two isozymes display individually distinct patterns of expression in response to temperature change. To understand the mechanisms regulating the expression of these isozymes, in this study, posttranslational mechanisms of the regulation were investigated using blue native PAGE, a high-resolution molecular sizing method. The PAGE analysis revealed that both isozymes formed a protein complex. Changes of the oligomeric state of the FAD8 were observed in response to temperature change, while the state of the FAD7 was not altered. We will discuss the temperature effect on the oligomeric state of the FAD7 and FAD8.

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Arabidopsis thaliana can enhance freezing tolerance (FT) by exposure to low, nonfreezing temperatures, a phenomenon known as cold acclimation (CA). CA is driven by expression of CBF/DREB1 but the mechanisms of the enhancement of FT are not fully understood. We herein screened for Arabidopsis mutants that remained FT during "cold deacclimation" (DA). An isolated mutant named freezing tolerant 1 (frt1) exhibited a greater degree of FT than the wild type, not only during DA but also under non-acclimated or CA conditions. RNA gel blot analysis demonstrated that the transcript levels of neither CBF/DREB1 nor its downstream COR genes were enhanced in the frt1 mutant over the wild type. The frt1 mutant accumulated slightly lower levels of proline than the wild type, but accumulated constitutively higher levels of sugars than the wild type, suggesting that enhanced FT of the frt1 mutant is due to its increased levels of sugars.

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Although elucidation of molecular mechanisms of cold acclimation in Arabidopsis plants is rapidly progressing, it is still difficult in some cases to determine responses to low temperatures at cellular levels due to the complexity of plant individuals. To study such responses, we characterized kinetics of cold acclimation with Arabidopsis T87 suspension cultured cells and compared the results with those obtained with seedlings. Acclimation-induced increase in freezing tolerance was detected with the cultured cells only at late lag phase at a narrow time-window (1-2 days) of cold acclimation. Expression of cold-regulated genes (DREB1A, RD29A, and COR15a) increased rapidly after acclimation (6-24 hr) but suddenly decreased thereafter. We also found that an increase in sucrose concentration in cultured medium extended the time-window for cold acclimation. These results suggest that kinetics of cold acclimation are different between seedlings and cultured cells and are influenced by sugars in the cultured medium.

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Alterations in membrane structure and function occurring in cold acclimation are essential for cell survival under freezing stress since the plasma membrane is the primary site of freezing injury in plants. We previously identified a number of plasma membrane proteins in Arabidopsis that have altered in response to cold acclimation. In the present study, we aimed to elucidate the functional roles of a lipocalin-like protein, one of the cold-response plasma membrane proteins. Effects of the lipocalin-like protein on the freezing tolerance and the occurrence of freeze-induced lesions of Arabidopsis were determined in planta with transgenic plants that overexpress the gene encoding the protein. Freezing tolerance of leaves and protoplasts of the non-acclimated transgenic plants was improved compared with that of wild type plants. Furthermore, there seemed to be a difference in the occurrence of freeze-induced lesions of protoplasts in the transgenic and the wild type plants (Supported by BRAIN).

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It has been reported that the acid rain influenced the growth and metabolism of plants. However, the influence of acid snow on wintering plant has been hardly reported. In this study, the equilibrium-freezing test under acid condition (acid freezing test) was done to study the response of wintering plant to acid freezing stress, which may be caused by acid snow. When leaf segments of cold acclimated winter wheat (Triticum aestivum L. cv. Chihokukomugi) were applied to the acid freezing test, the survival rate after the acid freeze-thaw (pH 2.0) was markedly decreased as compared with that after the freeze-thaw in pure water (pH 5.6), a control experiment. On the other hand, survival rate after supercooling in the acid solution (pH 2.0) was hardly lowered. These results suggested that the acidic condition in a freeze-thaw process of winter wheat promoted the freezing injury.

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Most organisms have developed various strategy to react rapidly to temperature downshift and regulate expression of many genes to acclimate to low temperature. However, information about how low-temperature signal is perceived and transduced is limited. In photosynthetic organisms, such as plants and cyanobacteria, temperature downshift in the light results in an imbalance between the light energy absorbed through photochemistry and the energy utilized through metabolism. Thus, temperature downshift in the light is not only decrease of temperature but also increase of photosystem II excitation pressure, which reflects the relative reduction state of the photosystem. We carried out microarray analyses of responses to low temperature both in the light and in the dark to separate low-temperature effects from effects of excitation pressure. We identified 15 ORFs upregulated by temperature downshift both in the light and in the dark.

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RNA helicases play important roles in various aspects of RNA metabolism, such as ribosome biogenesis, translation initiation and mRNA degradation. In general, RNA helicases are enzymes that unwind base paired RNAs. In the present study, we mutated the crhL gene for a cold-inducible RNA helicase with complete segregation of the native copy of the gene in Synechocystis sp. PCC 6803. The mutant of the RNA helicase (ΔcrhL) exhibited a phenotype of slow growth at low temperatures. DNA microarray and Northern blotting analyses indicated that the ΔcrhL mutation decreased the transcript levels of the groESL1 operon that encode the 10-kDa chaperonin and the 60-kDa chaperonin1 and the groEL2 gene that encode 60-kDa chaparonin2 under cold stress. The ΔcrhL mutation did not affect the stability of groESL1 transcript under cold stress. The involvement of RNA helicase in regulation of the expression of groESL1 and groEL2 genes will be discussed.

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In cyanobacteria, a disruptant of hspA encoding a small heat shock protein homologue, shows decreased cell growth rates at moderately high temperatures, and loss of both basal and acquired thermo-tolerances, which resemble the phenotype of a disruptant of htpG (encoding an Hsp90 homologue). In vitro studies have shown that one of the major functions of small heat shock proteins and Hsp90 is to bind and keep non-native proteins in a refolding-competent state, under denaturing conditions. The aim of the present study is to elucidate whether constitutive expression of HspA can functionally replace HtpG in the cyanobacterium Synechococcus sp. PCC 7942. HspA neither improved the growth of the htpG disruptant at 45^oC nor conferred thermo-tolerance to the mutant. These results suggest that cellular function of HtpG may differ significantly from that of HspA. Experiments are in progress to identify targets of HtpG.

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Phycobilisome is a light-harvesting chromoprotein complex in cyanobacteria. At the last meeting we reported the analysis of phycobilisomes that were isolated from a mutant of Synechococcus sp. PCC 7942 constitutively expressing HspA, a small heat-shock protein. We also examined interactions between HspA and phycobilisome in a purified system. Those results suggest that some components of phycobilisome interact with HspA.
In the present study, we attempted to identify targets of HspA in phycobilisome specifically. A mixture of purified phycobilisome and HspA was incubated for 15 min at 50^oC in the presence of 0.3% H₂O₂. The mixture was fractionated by sucrose gradient ultracentrifugation and each fraction was analyzed by SDS-PAGE. The denaturation of phycobilisome resulted in the mobility shift of HspA, suggesting that HspA interacts with phycocyanin or allophycocyanin. In vitro experiments with purified phycocyanin α or β subunit and HspA are in progress to detect a direct interaction between them.

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DNA microarray analysis revealed salt stress enhances the expression of several HSP genes in Synechocystis sp. PCC 6803. Higher enhancement in the case of hspA suggested its role in the protection from salt stress. As a consequence, we studied a deletion mutant of hspA (HK-1) at various salt stresses and examined the expression patterns of some of the HSP genes. To our surprise, HK-1, survived to a higher level after a direct lethal salt treatment, constitutively accumulating higher level of groESL1 and groEL2 transcripts at 30^oC, compared to wild type. However, HK-1 failed to acquire tolerance by moderate salt pretreatment in contrast with wild-type cells. Salt stress influenced groEL2, htpG and dnaK2 transcript accumulation by specific stabilization of these transcripts. Electron micrographs at various salt stresses showed HK-1 failed to undergo changes in ultrastructure that wild-type cells experienced. This further confirms the role of hspA in salt stress management.

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The expression of the isiAB operon, which encodes CP43' and flavodoxin, is induced under heat or oxidative stress conditions as well as iron deficient conditions. The aim of this study is to elucidate their roles for acquisition of stress tolerance with using isiA/isiB and isiB disruptants of Synechocystis sp. PCC 6803. Both mutants were more sensitive to oxidative or heat stress than wild type, suggesting that they play roles in the protection from these stresses. To our surprise, the isiB mutant was more sensitive to methylviologen (5 μM) than the isiA/isiB mutant, while the isiA/isiB mutant was more susceptible to heat stress than the isiB mutant. These results indicate that CP43' and flavodoxin play roles under oxidative and heat stress conditions.

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Drought-tolerant wild watermelon plants accumulate citrulline massively in the leaves under drought. To explore the mechanism of its accumulation, we analyzed glutamate acetyltransferase (GAT) that simultaneously catalyzes the first and fifth steps in citrulline biosynthesis.
The GAT purified from the leaves of watermelon was composed of two subunits. N-terminal amino acid sequences of these subunits were significantly homologous to the 37th to 49th and the 249th to 283rd residues of putative Arabidopsis GAT. The analysis of the corresponding watermelon cDNA suggested the presence of chloroplast transit peptide at its N-terminus. The watermelon GAT was not inhibited by the L-citrulline and L-arginine. Moreover, the enzyme showed optimum temperature of 70 ^οC, suggesting that this enzyme is suitable for the thermogenic condition of the leaf tissue under drought. These results suggest that the watermelon GAT contributes effectively to the massive accumulation of citrulline.

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Choline monooxygenase catalyzes the oxidation of choline to betainealdehyde which is a key enzyme for the betaine synthesis in plants. Purified CMO showed extremely low activity probably due to the labile property of the enzyme. For functional characterization of CMO, we constructed the vectors in which the CDH gene of E. coli bet gene clusters was replaced with CMO or deleted, and used for the transformation of E. coli (DH5a) and fresh water cyanobacterium Synechococcus PCC7942, neither of which could synthesis betaine. We found that E. coli cells in which choline dehydrogenase (CDH) was replaced with spinach CMO accumulate betaine. Changes of Cys181 in spinach CMO to Ser, Thr, and Ala and His287 to Gly, Val, and Ala abolished the accumulation of betaine. Overexpression of spinach CMO in E. coli, Synechococcus, and Arabidopsis conferred resistance to abiotic stress. These results will be presented.

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To elucidate the molecular mechanism of hydrotropism in roots of Arabidopsis thaliana, we have isolated 7 hydrotropic mutants from 4860 T-DNA tagged lines. All of these mutants showed a normal response to gravity. In this study, we report on phenotypic and molecular analysis of a hydrotropic mutant, cs2448.
The roots of cs2448 grow faster than those of wild-type and coiled clockwise on inclined agar plate. However, microscopy showed that there is no obvious morphological difference between cs2448 and its wild-type.
TAIL-PCR showed that the T-DNA was inserted in AKT2 gene. Three T-DNA insertion lines whose T-DNAs were inserted in 3 different sites of AKT2 gene were found to produce an altered hydrotropic response. These results suggested that the partial response of cs2448 to water stimulus was due to the disruption of AKT2 gene, which encodes a potassium channel. Expression of pAKT2::GUS was detected in the stele, primarily in the phloem.

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In plant cells, two distinct types of aquaporins are accumulated in plasma membrane and tonoplast. The finding has drawn our attention to how plants regulate water transport across each membrane. It is assumed that water permeability of tonoplast (Ps2) should be much higher than that of plasma membrane (Ps1), because cytoplasmic water potential could be stabilized by quick water-exchange with vacuole. However, the hypothesis has not been enough proved. Here, we propose a method for measuring Ps1 and Ps2 at the cellular level. Protoplasts were exposed to osmotic shock, and the time course of changes in the cell volume was observed. Swelling or shrinking rate of the protoplasts depends on both Ps1 and Ps2. We developed the numerical model which simulates these processes. Ps2 was directly obtained from swelling/shrinking rate of isolated vacuoles. Ps1 can be determined from the numerical solution of this model combined with the value of Ps2.

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Plant responds and adapts to environmental stress at gene expression level as well as physiological level. Genetic and molecular analysis indicates that several signal transduction pathways to control stress-responsive gene expression. We have analyzed gene expression profiles of Arabidopsis during rehydration from dehydration stress using a 7k RAFL-cDNA microarray and identified many rehydration-inducible genes (Plant J, Oono et al. 2003). We think recovery process from abiotic stress also has several pathways like stress response process. In the present study, we present the results of expression profile of low-temperature stress and recovery from cold stress using the 7k RAFL-cDNA microarray and an Agilent 22k oligo-array. We identified many genes that are regulated during recovery process from low-temperature stress. We compared gene expression profiles during recovery process from low-temperature stress with those from drought stress. We will discuss specificity and common processes in the recovery process from cold stress and drought stress.

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Arginine decarboxylase (ADC) catalyzes the first step of polyamine biosynthesis to produce putrescine (Put) from arginine. One of 2 Arabidopsis ADC genes, AtADC2 was induced in response to drought and salt stresses causing the accumulation of free Put. To analyze the roles of stress-inducible AtADC2 and endogenous Put in stress response, we characterized the adc2-1 mutant under drought and salt stress. In the adc2-1 mutant, free Put content was reduced to about 25% of that in the control plants and did not increase under salt stress. This result suggests that AtADC2 is a key gene for the production of Put under not only stress conditions, but also normal conditions. Furthermore, the adc2-1 mutant was more sensitive to drought and salt stresses than the control plants. These results indicate that Put plays an important role in drought and salt stress response in Arabidopsis.

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We selected pst (photoautotrohic salt tolerance) mutant lines of Arabidopsis for revealing the mechanism of salt tolerance (Plant Cell, 11, 1195-1206, 1999). The cDNA macroarray and oligo-microarray were performed for pst2 and wild-type lines grown under a non-stress condition. Higher expression of a transcription factor with bHLH motif in pst2 plants was confirmed by real-time RT-PCR. There was another specie of mRNA produced by alternative splicing under the salt stress. The RACE has clarified that transcription initiation site is single. The salt-induced mRNA retained an intron on C-terminal side among two introns, and stop codon and its subsequent initiation codon in the retained region generated the two peptide molecules which looked like cleaved forms of the original protein. Protein species produced in plants have been analyzed by immuno-blotting, and the function of bHLH transcription factor has been examined with its transgenic plants.

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Plants generate active oxygen species (AOS) by environmental stress such as salinity. The generated AOS involves increased activity of antioxidant enzymes. However, catalase activity was often decreased by stress. Previous studies reported that catalase binds to salicylic acid (SA), and its activity is inhibited. We studied that relation between SA and catalase, and induction or inhibition of benzoic acid 2-hydroxylase (BA2H, cytochromeP-450) which catalyzes SA biosynthesis, in salt stress.
In rice treated by NaCl, catalase activity decreased and SA content increased. We suggested that SA accumulated by salt stress was decreased catalase activity. NaCl treatment increased BA2H activity. It was, therefore, considered that the activation of BA2H by salt stress increased SA content. We investigated the inhibition of BA2H by various compounds (gibberellin biosynthesis inhibitors, herbicides and substrate analogues). Gibberellin biosynthesis inhibitors were the most effective. It is suggested that direct P-450 inhibitor is also effective inhibitor of BA2H.

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A cDNA that encoded mangrin, a novel protein which may play an important role for the salt tolerance of a mangrove plant, Bruguiera sexangula, was previously isolated using a functional screening method. Enhanced growth rates were observed in the transformants that expressed mangrin in both Escherichia coli and tobacco-suspension-cultured cells. Interestingly, the amount of mangrin mRNA in the B. sexangula cultured cells increased within 3 h following the addition of over 50 mM NaCl. However, there are no information about the mangrin promoter region. Therefore, mangrin promoter region was amplified by PCR and the sequence was determined. The sequence analysis indicates that there are four putative W-boxes in the promoter region. The promoter region was introduced into tobacco cultured cells (BY2) and tobacco plant, respectively, and the promoter activity was investigated using beta-glucuronidase gene as a reporter gene.

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Betaine is an important osmoprotectant in many plants, but its transport activity has only been demonstrated using a proline transporter (LeProT) from tomato, a betaine non accumulating plant. Weisolated two full length and one partial transporter genes from betaine accumulating mangrove Avicennia marinar. Their homologies to betaine transporters from bacteria and betaine/GABA transporters from mammalian cells were low, but high to proline transporters from Arabidopsis and tomato. Two full length transporters could efficiently take up betaine and proline with similar affinities and maximum velocities. The uptakes of betaine and proline were significantly inhibited by mono- and di-methylglycine, but only partially inhibited by betaine aldehyde, choline, and GABA. Sodium- and potassium-chloride markedly enhanced betaine uptake rates with optimum concentrations at 0.5 M whereas sucrose showed only modest activation.

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Amaranthus tricolor is an unique C₄ plant which produces three-colors in leaves, green apices, yellow middle, red basal regions before the onset of flowering. We previously reported that the yellow and red regions possessed greatly reduced levels of Chl, less than 10 % of the green regions although the net photosynthetic CO₂ fixation rates of yellow and red regions were about 40 % of the green regions (Plant Cell Physiol. 40, 668-674 (1999)). Here, we examined the expression of choline monooxygenase genes from Amaranthus tricolor. Upon salt stress, the levels of choline monooxygenase and betaine increased in both green regions as well as red and yellow regions. Changes of the levels of glycinebetaine and choline monooxygenase upon the changes of salinity were investigated. We also examined the efects of choline precursors on the accumulation levels of glycinebetaine. These results will be presented.

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Salicornia europaea, a member of the family Chenopodiaceae, is a highly salt-tolerant halophyte, which is distributed in the salt marsh. To analyze the mechanisms of salt tolerance at cellular level, callus-initiating conditions were investigated using the sterilized leaves. Murashige-Skoog medium containing 1x10^-7 M of 2,4-dichlorophenoxyacetic acid and 1x10^-6 M of 6-benzylaminopurine is the most efficient medium for the callus initiation (under 16 h of light and 8 h of darkness condition). Interestingly red and greening calluses were generated from the leaves, respectively. Both calluses could grow in the presence of over 300 mM NaCl. In this study, effect of salt stress on the growths and ion contents in these calluses were investigated. Furthermore, genes responsible for the salt tolerance were also investigated.

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Plant has mechanisms to adapt to sulfur deficiency and grow apparently normal. To elucidate a whole adaptive response to sulfur deficiency, we analyze transcriptome and metabolome of Arabidopsis which are grown normally under sulfur depleted condition. In this presentation, we will show time course data of transcriptome and metabolome of a series of plants, ranging from just after transfer to -S condition to after equilibrium of changes in plant's internal status.
Arabidopsis was grown for three weeks on sulfur-sufficient control medium, transferred to control or -S medium and harvested at 3, 6, 12, 24, 48 and 168 hour-after-transfer. These plants were grown normal after transfer. Transcriptome and non-targeted metabolome of leaves and roots were analyzed using microarray and Fourier transform ioncyclotron MS. Targeted metabolic profiling was also conducted using HPLC and capillary electrophoresis. In this presentation we will show putative adaptive processes to sulfur deficiency.

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Cystathionine Γ-synthase (CGS) is the key enzyme of Met biosynthesis in higher plants. Expression of the CGS gene is negatively feedback-regulated at the level of mRNA stability in response to S-adenosylmethionine (SAM), the direct metabolite of Met. A short stretch of amino acid sequence (MTO1 region) encoded by the first exon of CGS itself is involved in this regulation. Since the CGS exon 1 acts in cis, we have proposed that this regulation occurs during translation. Accumulation of partial translation products was observed when an RNA carrying GST-tagged CGS exon 1 was translated in vitro in the presence of SAM, suggesting that translation arrest occurs during the regulation. In addition, polysome profiling analysis showed that decay intermediates of the CGS exon 1-mediated mRNA degradation were detected in ribosome fractions, but not in ribosome-free fractions, implying that a ribosome stalls on a decay intermediate of CGS mRNA in vivo.

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Cystathionine γ-synthase (CGS) catalyzes the first committed step of methionine biosynthesis. Expression of the CGS gene is feedback-regulated at the level of mRNA stability by S-adenosylmethionine (SAM) in Arabidopsis. There is a 41 amino acid region of exon 1 that is highly conserved among plant species (conserved region). Since the exon 1 polypeptide acts in cis, we have proposed that the regulation occurs during translation. We used a wheat germ extract translation system to analyze the decay intermediate of CGS mRNA. When in vitro-transcribed RNA carrying CGS exon 1 was translated in the presence of SAM, a 5'-truncated RNA that was about 300 bases shorter than the full-length RNA was detected. Analyses of a series of deletions within CGS exon 1 revealed that the conserved region is necessary and sufficient for production of the 5'-truncated RNA.

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Boron (B) is an essential micronutrient for plants, but B can become toxic at elevated concentrations. In B rich soils, crop yields and qualities are decreased. Hence, the toxic effects of high B are important problem on agronomic policy. However, the molecular mechanisms of B toxicity are still poorly understood.
The aim of this study is to identify genes involved in B tolerance and to understand the mechanisms of B toxicity. For the purpose, yeast was transformed with an Arabidopsis cDNA library and transformants were selected on toxic 80 mM B concentrations. In this screening, we isolated several yeast that showed enhanced B tolerance. Sequence analysis showed that one of the cDNA clones from the yeast encodes poly(A) binding protein PAB2. PAB2 has been reported to involved in translation initiation and stability of mRNA. Possible role of PAB2 in B tolerance will be discussed.

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An oxygen sensor may play an important role in Anabaena sp. PCC 7120. FixL of rhizobia and DOS of Escherichia coli have a heme PAS domain as an oxygen sensor. In our latest study, a homologous heme PAS domain of COS (Alr2428) in Anabaena 7120 showed oxygen-responsive nature in vitro. Additionally, this protein contains histidine kinase domain at the C-terminal side of the heme PAS domain and response regulator and DNA-binding domains at the very N-terminus. Here, we prepared a recombinant protein with heme PAS and histidine kinase domains and analyzed the self-phosphorylation activity in the reduced or oxidized form. Next, we prepared another recombinant protein with response regulator and DNA-binding domains and analyzed the DNA-binding activity in the presence or absence of the acetyl phosphate. From these, possible oxygen-sensing mechanism of this protein will be discussed.

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PAS domain superfamily, which binds flavin, is known as sensors of blue light or redox changes. The FAD-binding PAS domain of Aer protein is known as redox sensor in Escherichia coli to regulate aerotaxis. We found in the cyanobacterial genomes that one of the PAS domains of Slr1759 protein is significantly homologous to the FAD-binding domain of Aer. Slr1759 protein is a multidomain protein which contains two PAS, GAF, His kinase and response regulator domains. We overexpressed the segment containing the PAS domain as His-tagged fusion protein in E. coli. The purified protein showed absorption spectrum typical for the oxidized flavin but quite distinct from that of Aer. We will discuss possible role of the flavin-binding PAS domain together with the disruption phenotype in Synechocystis.

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Cyanobacreria generally possesses many genes that encode phytochrome-like protein. We performed following experiment to examine the regulatory role of phytochrome-like protein on light-responsive gene expressions. We incubated mutant cells in which genes sll0821, sll1124, sll1473, slr0473, slr1212, slr1393, slr1805, slr1969 encoding phytochrome-like protein have been destroyed under the darkness for 12-hour and then illuminated by white light for one hour. We selected light-responsive genes whose expression level was increased more than twice or reduced less than half by transition from dark to light, but the rate of induction or reduction was decreased less than half in the mutants by DNA microarray analysis. In consequence, It was clarified that the induction of the expression of cpcG2 by illumination was almost eliminated in sll1473 mutant. In addition, characteristic effects were observed in other mutants. We will also report our ongoing study about the regulation light-responsive cpcG2 expression.

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Though Ca²⁺-permeable stretch-activated channels are postulated to play crucial roles in various signal transduction pathways in plants, their molecular identity is totally unknown. We here identified genes from rice (OsMID1) and tobacco (NtMID1A/B) that functionally complemented a yeast mutant defective in the MID1 gene encoding a putative Ca²⁺-permeable SA channel component. To investigate functions of these genes, we developed transgenic plants as well as suspension-cultured cells in which their expression are enhanced or repressed. Possible interaction of the plant MID1 family with other types of Ca²⁺ channels will also be discussed.

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Phospholipase D (PLD) is the enzyme that hydrolyze phosphatidylcholine and generate phosphatidic acid and choline.
Arabidopsis thaliana encodes 12 PLD genes, which are categorized into plant-type PLDs and animal-type PLDs by their domain structures. Plant-type PLDs are known to play roles in the signal transduction of environmental stresses. On the other hand, AtPLDζ1, one of two animal type PLDs in Arabidopsis, has been proposed to play an important role in the root-hair cell morphogenesis. In this study, We analyzed the expressions-pattern of AtPLDζ2, the other animal-type PLD.
A 3-Kbp promoter region of AtPLDζ2 was fused to GUS gene and introduced into Arabidopsis. The transgenic plants showed the GUS activity mainly in floral organs, indicating that AtPLDζ1 and AtPLDζ2 have different expression patterns.

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Peroxynitrite (ONOO^-) is a reactive nitrogen species (RNS) that can be produced through the reaction between the reactive oxygen species (ROS) superoxide and the RNS nitric oxide (NO). ONOO^- has been considered to be the most toxic RNS in mammalian systems because of its capability to nitrate tyrosine residue. Here we report that as similar to tyrosine, plant phenolic compounds also can be nitrated by an ONOO^--independent mechanism. The phenolic compound p-coumaric acid (p-CA) was nitrated in the presence of horseradish peroxidase (HRP), NaNO₂ and H₂O₂. The nitrating reaction was inhibited by KCN, an inhibitor for HRP. The antioxidant ascorbate suppressed p-CA nitration and its suppression time strongly depended on ascorbate concentration. The results suggest nitrogen dioxide radical is involved in the HRP-dependent p-CA nitration.

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Bacteria colonize the exterior and interior habitat of leaves, or phyllosphere, nourished by sugars and other products of plant metabolism. To establish a population on a leaf, a bacterium must be able to overcome several abiotic and biotic stresses including plant-derived potentially toxic hydrogen peroxide (H₂O₂). Here we report that H₂O₂ tolerance of Rhodococcus sp. strain APG1, in relation to nitric oxide (NO) production by cells cultured on a variety of C sources. Relative to cells grown on other sources of C, sucrose-grown cells were found to reach lower growth yields and show higher tolerance to H₂O₂ that correlated with increased formation of NO. The results suggest that sucrose may enhance H₂O₂ tolerance of Rhodococcus APG1 by increasing cellular NO producing capacity. We propose a regulatory role for NOS in promoting tolerance of Rhodococcus APG1 to oxidative stress in the phyllosphere.

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Atmospheric NOx, which includes nitric oxide and nitrogen dioxide, is usually regarded to act as N source or to exert detrimental effects on plants. No reports on its signaling effect, however, have so far been reported. In this study we cultured plants (Nicotiana plumbaginifolia) under the natural light for several months in the air "polluted" with ¹⁵N-labelled NOx (100 to 200 ppb) and in the "clean" air with no or very low level of NOx (<5 ppb). We have discovered that the biomass yield, element contents, total free amino acids and crude proteins were almost doubled in the plants cultured in the polluted air than the ones cultured in the clean air. Isotopic mass spectrometry showed that the nitrogen derived from NOx made only a negligible contribution in the total nitrogen of plants. Therefore, the present findings imply that urban NOx "pollution" is a plant vitalization signal.

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We have unexpectedly discovered that about one-third of the nitrogen derived from ¹⁵N-labeled nitrogen dioxide (NO₂) taken up by Arabidopsis thaliana plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen (UN). In this study, the generality of the formation of the UN across species, nitrogen sources and cultivation environments for plants has been studied. All of 12 plant species studied were found to form the UN. Tobacco plants fed with ¹⁵N-nitrate appeared to form the UN. And the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. Our present findings imply that there is a novel nitrogen metabolism for the formation of the UN in plants. We propose that UN-bearing compounds may provide a chemical basis for the biological action of reactive nitrogen species (RNS).

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All plants examined to date possess non-symbiotic hemoglobin that is genetically and structurally distinct from its symbiotic counterpart (leghemoglobin). Although the ubiquitous distribution of this hemoprotein in the plant kingdom strongly suggests its potential importance, the physiological function remains unknown. To examine the possible participation in inorganic nitrogen/reactive nitrogen metabolism, we studied on an Arabidopsis non-symbiotic hemoglobin, AtGLB1, and found:
(1) AtGLB1 mRNA increased in response to nitrate and nitrite.
(2) Recombinant AtGLB1 (rAtGLB1) exhibited peroxidase activity.
(3) rAtGLB1 was nitrated on its tyrosine residues when nitrite was used as the electron donor in peroxidase reaction, indicating the formation of nitrogen dioxide from nitrite.
These results suggest that AtGLB1 might be involved in nitrite metabolism. Nitrite, a metabolic intermediate in nitrate assimilation, is regarded a reactive nitrogen species whose accumulation is poisonous to plants. Possible roles of AtGLB1 will be discussed in view of defense against nitrite toxicity.

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A QTL associated with panicle weight of rice was located on chromosome 2 (Obara et al., 2001 JEB 52, 1209-1217). In this case, the Kasalath alleles contributed to increase in the panicle weight. A nearly isogenic line, C-22, is consisted of the substituted small segment from Kasalath chromosome in the target QTL of Koshihikari's genetic background. The C-22 showed significant increases in tiller number at the early vegetative stage, total panicle weight, and panicle number when compared to Koshihikari. Nine substituted lines, in which the 50 cM segment from Kasalath chromosome of C-22 was recombinated, were selected to determine the ratio of developing tiller formation in the 2th axil. Linkage analysis showed that QTL for panicle number was detected between DNA markers, 2-S152 and 2-S173. This region was approximately 10 cM. For further delimitation of QTL region, linkage analysis with recombinant plants between 2-S152 and 2-S173 is now in progress.

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Glutamate glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to 2-oxoglutalate and glycine. GGAT has essential roles for the transamination to photorespiratory cycles in peroxisome. We had identified photorespiratory GGAT gene by analyzing the knockout line of alanine 2-oxoglutarate aminotransferase (AOAT) -like gene (Igarashi et al. Plant J 33, 975- 2003).
To better understand the function of GGAT1 gene on the amino acid metabolisms, we obtained many GGAT1 overexpression lines (OE) that had various levels of GGAT1 mRNA. The levels of free serine were increased drastically in transgenic Arabidopsis plants (maximum level of the OE was 20-times higher than that of the non-transgenic plants). There is a clearly correlation between the level of serine contents, the level of GGAT1 mRNA expressions and GGAT activities. These results suggested that photorespiratory transamination would participate in regulation of the amino acid metabolisms.

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Reduction of nitrate and oxalate content in spinach has become the major concern in the breeding program in terms of their toxicity to the human health. Although various experiments have already been performed to determine the nitrate and oxalate contents in spinach, no definitive factors that affect their contents have been elucidated. In the present study, we have determined genetic factors that affect nitrate and oxalate contents in spinach using 200 varieties by growing them in fall, winter and spring from 2002 to 2003 under the same cultural condition (N=10kg/10a) in vinyl houses. In both nitrate and oxalate, a wide range of variations was detected not only among the varieties but also the growing season. Interestingly, amount of nitrate negatively correlated with that of oxalate. Further results will be discussed.

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Nitrate uptake is the first step of the nitrate assimilation pathway. In Physcomitrella patens, transcript levels of the five NRT2 genes, encoding nitrate transporters, were low in ammonium-grown cells and drastically increased in several hours after transfer of the cells to nitrate-containing medium or nitrogen-free medium, indicating that all the NRT2 genes are inducible. Expression of the NRT2 genes was induced in a shorter time under high CO₂ conditions than under low CO₂ conditions and was not observed in the dark, suggesting close relation of NRT2 expression to carbon assimilation. In higher plants, ammonium assimilation products repress expression of the NRT2 genes, but the actual signaling molecule remains to be elucidated. In P. patens, the repressive effect of ammonium was abolished by an inhibiter of glutamine synthetase but not by an inhibiter of glutamate synthase, indicating that glutamine acts as the regulator of NRT2 expression.

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Most cyanobacteria are capable of nitrate assimilation and possess nitrate reductase (NR). In Synechococcus sp. strain PCC7942, three NR-related genetic loci, narA, narB, and narC, have been characterized. The narB locus contains the NR structural gene, narB, and the narA and narC loci contain genes involved in biosynthesis of the molybdenum cofactor, an essential prosthetic growth of NR. In addition to these we have identified another NR-related locus carrying a novel gene, narM, from a Synechococcus mutant that cannot grow in nitrate-containing medium. narM, encoding a small hydrophilic protein, has its homolog only in the cyanobacterial strains that are capable of nitrate assimilation. Insertional inactivation of the narM gene resulted in specific loss of the cellular NR activity, but did not effect the expression of narB and the molybdenum cofactor biosynthesis genes. These results suggested that a novel component is required for the functional expression of NR in cyanobacteria.

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Cyanobacterial PII protein is modified by phosphorylation/dephosphorylation in response to the cellular nitrogen status. Formerly, we found high-level expression of the ammonium assimilation genes in a PII-deficient mutant of Synechocystis sp. PCC 6803 and proposed the presence of a hitherto unknown mechanism that specifically regulates the ammonium assimilation genes. In this study, to further investigate this mechanism, we examined the ammonium assimilation activity of the PII-deficient mutant. It was found that the PII-deficient mutant grows much slower than the wild-type strain on ammonium under the conditions of high light and glucose supplementation. The rate of ammonium assimilation increased under these conditions in the wild-type strain but not in the PII-deficient mutant. These results suggested that the ammonium assimilation activity is upregulated by high light and glucose in the wild-type Synechocystis strain in a PII-dependent manner.

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The terrestrial cyanobacterium Nostoc commune is highly desiccation-tolerant and has a cosmopolitan distribution. The desiccation tolerance of photosynthesis and the role of extracellular polysaccharides for that tolerance in N. commune were studied. The activity of oxygen evolution was not detected from a desiccated sample, and was rapidly recovered with rehydration. The air-dried N. commune was treated with dry oven at 80C and approximately 10% of water was removed from the air-dried N. commune. The oxygen evolving activity was still remained approximately 60% after the desiccation treatment. N. commune colonies were homogenized and filtered with a coarse paper filter to separate the cells from extracellular polysaccharides. This treatment did not ruin the oxygen evolving activity, but the activity was severely damaged after desiccation in the cells. This result suggests that extracellular polysaccharides have an important role for the desiccation tolerance in N. commune.

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Previous studies in our laboratory indicated that disruption of uptake hydrogenase gene (hup) in Anabaena sp. PCC 7120 greatly improved photobiological hydrogen production (4-7 times that of wild-type) and that disruption of bi-directional hydrogenase gene did not improved the production. To see if disruption of hydrogenase genes increases hydrogen productivity in other cyanobacteria also, we have chosen Nostoc sp. PCC 7422 because it was one of the highest hydrogen producing strains among 15 N₂-fixing heterocystous cyanobacteria and its Hox activity was low. We have determined the nucleotide sequence of hupSL by cloning and sequencing, and disrupted the hupL gene by homologous recombination. The Δ hupL produced H₂ more than 10 times that of wild-type and at a rate of 50-70 μmoles H₂ (mg Chl) ^-1 hr^-1. These results indicate that disruption of hup gene is effective for improving hydrogen productivity in nitrogenase-based hydrogen production.

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In oxygen-evolving photosynthetic organisms, photosystem stoichiometry (PSI/PSII) is modulated in order to optimize electron transport in acclimation to various environmental changes. Under high light condition, PSI/PSII is reduced by decreasing PSI amount to avoid excess electron transport. In the cyanobacterium Synechocystis sp. PCC 6803, pmgA was characterized as a factor responsible for this regulation. This mutant lost the ability to decrease PSI amount and consequently could not modulate PSI/PSII. In the present study, we identified sll1961 and glnA as novel factors for moderate PSI/PSII. These mutants showed impaired regulation of PSI/PSII under high light. The growth of Δsll1961, ΔglnA and ΔpmgA under medium light condition were severely suppressed by addition of glucose to growth medium, which no phenotype was observed under low light. glnA encodes glutamate-ammonia ligase related to the metabolism of nitrogen. These data suggest that PSI/PSII may be regulated by the balance between carbon and nitrogen (C/N ratio).

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We reported at the former JSPP meeting that mutation in some genes not involved in photosynthesis could influence the chlorophyll fluorescence kinetics. In our previous report, random mutagenesis was adopted to generate mutants. This method had several demerits: low probability in disruption of small ORFs or difficulty in selecting mutants that grow slowly. To overcome these demerits, we are constructing a genome-wide set of mutants using transposon-inserted DNA at known position. Among 23 mutants constracted so far, 4 mutants showed major different fluorescence kinetics and 5 mutants showed minor different kinetics compared with wild type. Further construction of mutants is in progress aiming at functional classification of genes according to the fluorescence kinetics.

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