High temperature is one of key stress factors with negative impacts on the growth of plants and the productivity of crops. Previously, we have reported that DREB2A activates expression of many stress-responsive-genes involved in heat stress tolerance and that HsfA3 is the most up-regulated gene in the transgenic Arabidopsis plants overexpressing DREB2A.
In this study, we carried out expression and transactivation analyses of HsfA3 and demonstrated that expression of the HsfA3 gene is directly regulated by DREB2A under the heat stress condition. We also characterized the function of HsfA3 using HsfA3 overexpressors and knockout mutants and found that HsfA3 regulates many heat shock-related genes in response to heat stress. Furthermore, we found that thermotolerance of the HsfA3 overexpressors was increased and that of the mutants was decreased. We will discuss the role of HsfA3 in the transcriptional cascades in response to heat stress.

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To examine mechanisms involved in cold stress response, FOX hunting system was used to isolate Arabidopsis gain-of-function mutants. The isolated overexpressor (DEAR1ox), which shows constitutive cell death on rosette-leaves and accelerated leaf senescence, is caused by over-expression of the DEAR1 (DREB and EAR motif protein 1) gene encoding transcriptional repressor that contains a the DREB domain and EAR motif.
Expression of DEAR1 was induced by cold treatment. Transcriptional repression of the cold inducible genes in the DEAR1ox results in reduction of freezing tolerance. Expression of DEAR1 was also enhanced by pathogen infections. The DEAR1ox showed transcriptional promotion of PR genes and highly accumulation of endogenous SA, indicating that the SA-dependent defense pathway is activated in the DEAR1ox, leading to the HR-like cell death phenotype.
These results indicate that the DEAR1 is a key regulator for cross-talk between biotic (pathogen-resistance) and abiotic (freezing tolerance) stress responses.

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Arabidopsis thaliana possesses three types of hemoglobin (Hb) gene. The ΔAtGLB3 is a T-DNA insertion mutant that lacks the functional gene of trHb, a homologue of bacterial truncated Hb. We compared non-photochemical quenching (NPQ) of photosynthesis between the wild type and ΔAtGLB3. There is no phenotypic difference between the wild type and ΔAtGLB3. We have found that NPQ of ΔAtGLB3 is severely inhibited at 35 °C where that of wild type is tolerant. Interestingly, there was no substantial difference in NPQ between the wild type and ΔAtGLB3 at 25 °C. Since NPQ mechanism can be inhibited by thiol reagents through the modification of functional SH groups of a NPQ enzyme, we consider that NO may inhibit NPQ mechanism in ΔAtGLB3 due to the absence of trHb, an NO scavenging protein. The results strongly suggest that trHb is required to protect NPQ from heat stress.

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Coral bleaching is a recognized phenomenon of the biological impacts of global warming on marine ecosystems. Currently, the exact mechanism involved in the heat induced-coral bleaching phenomenon is still unclear. A recent study has suggested an implication of nitric oxide (NO) in the mechanism leading to the expulsion of symbiotic algae from the coral host. Here we show that the dinoflagellate Symbiodinium microadriaticum produces NO when supplemented with either nitrite or L-arginine as a substrate. This production of NO was confirmed by electrochemical and fluorimetric techniques. When S. microadriaticum were exposed to an acute heat stress (from 27 to 41 °C), an increase in NO production was observed along with a decrease in photosynthetic efficiency, thereby suggesting that excessive NO production upon heat stress could be detrimental to the cells. The implications of these findings are discussed in the light of the coral bleaching phenomenon.

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We reported previously that the genetically modified rice lines expressing OASA1D, a feedback-insensitive anthranilate synthase (AS) α subunit gene of rice, showed remarkable increase of free tryptophan (Trp) in leaves and seeds. For the purpose of practical use of this gene, we tried to develop the direct co-transformation system with linear transgene cassette and selectable maker gene cassette lacking vector backbone sequence. To avoid the integration of any vector backbone sequence in the rice genome, the cassette fragments (pRbcAc::OASA1D-asa1T and p35S::HPT-nosT) were introduced into rice cells by the use of silicon carbide whiskers. This system produced fertile transgenic rice plants with a low copy of transgene, and those contained high level of free Trp in leaves and seeds. In addition, we will show some results of frequency in appearance of selectable maker lacking lines by genetic segregation in the progenies.

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The progress of the molecular biological studies on development and environmental response in marine multicellular algae is hindered by the lack of reverse genetic techniques. To overcome this situation, we developed a transient gene expression system in sea red algae Porphyra yezoensis. For the efficient expression, we found the importance of the making an artificial β-glucuronidase (GUS) coding region (PyGUS) whose codon usage had been adapted to that in P. yezoensis and the employing promoters of endogenous genes. We also found a very weak activity of the cauliflower mosaic virus (CaMV) 35S promoter in P. yezoensis cells. Since the efficiency of promoters on the PyGUS expression resembled the mRNA abundance analyzed by RT-PCR, the transient gene expression system established in the present study is useful for promoter analyses in P. yezoensis.

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To aim for the production of useful substances in higher plants through metabolic engineering, we have to know the key enzymatic reaction controlling metabolic flux of the substance biosynthesis pathway. We developed analytical method using in vivo isotope dilution from ¹³CO₂ for the identification of rate-limiting step in sugar phosphate and carotenoid metabolism in tobacco leaves. After the feeding ¹³CO₂ into the leaves that have steady-state CO₂ assimilation rate, the ¹³C labeling ratio of intermediate metabolites extracted from the frozen tissue was determined by CE-MS and LC-TOFMS. Under 1,000 μmol m^-2 s^-1 PFD and 600 μmol mol^-1 of ambient CO₂, the labeling ratio of sugar phosphate in Calvin cycle reached maximum level in the 10th minute labeling. The time course of labeling ratio was different between G1P and G6P, indicating that interconversion of G1P and G6P is one of the rate-limiting steps in sugar phosphate metabolism.

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There is high proportion of transcription factors (TFs) in Arabidopsis genome compared to other organisms so that TFs play pivotal role for regulation of various biological processes in Arabidopsis. To identify the functions of transcriptional factors, we have tried to clone all annotated transcriptional factors without stop codon. Until now approx. 1000 transcriptional factors were cloned into entry vector by Gateway technology. This collection is thought as good resource because the clone is easily move to objective destination vectors by LR reaction.
Using this collection, we are establishing novel Arabidopsis transgenic plants over-expressing independent transcription factors which is fused to glucocorticoide receptor for functional analysis. In this system, by application of a steroid hormone, dexamethasone (DEX), introduced transcriptional factor is functionally active. We have performed phenotypical observation of this mutant population after DEX application.
Here we show the detail of these lines and discuss the usefulness for functional genomics.

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We irradiated Arabidopsis thaliana with several kinds of heavy-ion beams to investigate the linear energy transfer (LET)-dependent effects. First, dry seeds were irradiated with C, N, Ne, Ar, or Fe ions at doses ranging from 5 to 400 Gy to compare the sensitivities on flowering and mutation rates among the ion species. The sensitivities to irradiation differed markedly among the ion species. Of the ion species, N (30 keVμm^-1) was the most effective at inducing albino mutants. Second, we examined the effects of LET on mutation induction. The LET of C, N, Ne, and Ar ion beams was controlled at 30-640 keVμm^-1. Regardless of ion species, irradiation with the same LET value resulted in the same mutation rate. Thus, the LET of ion beams appear to be an important factor affecting mutagenesis. We will also report DNA damages induced by heavy-ion irradiation in the conference.

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In Brassica self-incompatibility (SI), self/non-self recognition is controlled by S-haplotype-specific interaction between the pollen-borne ligand, SP11, and its stigmatic receptor-kinase, SRK. Our genetic analysis of a self-compatible mutant revealed the involvement of a cytoplasmic protein kinase, MLPK, in the SI signaling, but its exact physiological function remains unknown.
In this study, we identified two MLPK isoforms, named MLPKf1 and MLPKf2, localizing to the plasma membrane by different molecular mechanisms. While MLPKf1 and MLPKf2 exhibited distinct expression profiles, both were expressed in papilla cells. Although both MLPKf1 and MLPKf2 could independently complement the mlpk/mlpk mutation, their mutant forms that lack the plasma membrane localization motifs failed to complement the mutation. Furthermore, we showed direct interactions between SRK and MLPK in tobacco protoplasts. These results suggest that these MLPK isoforms interact directly with SRK on the plasma membrane to transduce SI signaling

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CEBiP, chitin elicitor binding protein in rice, plays an important role as a cell surface receptor for chitin elicitor signaling1). As CEBiP seemed not to have any intracellular domain, it was suggested that other partner protein(s) are involved for the signaling across the membrane. Blue native PAGE as well as SDS-PAGE/western blotting of membrane proteins with/without the pre-treatment with cross-linking agents also indicated the presence of CEBiP-receptor complex. A receptor-like kinase of rice, OsLysM-RLK9 was chosen as a candidate of such a partner protein, as it has a high sequence homology with CERK1 (Chitin Elicitor receptor Kinase), an indispensable component for chitin elicitor signaling in A. thaliana2), and examined for the possible interaction with CEBiP. Yeast two-hybrid analysis showed the positive interaction between CEBiP and OsLysM-RLK9, indicating the possible receptor complex formation by these two proteins.
1)Kaku et al., PNAS, 103, 11086 ('06), 2)Miya et al., PNAS, in press.

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A simple and non-RI method for the identification and characterization of plant receptors with biotinylated ligands is presented. Biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the characterization of chitin elicitor binding protein, CEBiP. Membrane fractions were treated with GN8-Bio and cross-linked with glutaraldehyde. CEBiP was successfully biotinylated and detected both in the plasma membrane and microsomal membrane fractions by Western blotting with anti-biotin antibody followed by chemiluminescence detection. The binding characteristics of the GN8-Bio to the CEBiP showed a good agreement with the known specificity of CEBiP. The GN8-Bio-tagged CEBiP could also be purified from the membrane using an avidin column. These results indicated the usefulness of biotinylated ligands both for the purification and characterization of putative receptors or binding proteins for the ligands of interest.

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Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes. In Arabidopsis, it is suggested that there are 20 genes encoding possible MAPKs, and these MAPKs can be divided into at least four groups (A-D). Group D has 8 members, and forms the largest subgroup. Group D MAPKs contain TDY motifs instead of TEY motifs in their T-loop and larger C-terminal regions relative to other groups. We have been focused on MPK8, one of the group D MAPK, for further analysis. MPK8 functioned as a member of MAPK in yeast mpk1 mutant based on complementation. Yeast two-hybrid analysis revealed that MPK8 interacts with various types of calmodulin. We showed that calmodulin activates MPK8 without calcium in vitro. We also showed that MPK8 is activated by MKK3. These results suggest novel activation mechanisms of MAPK in plant. We will discuss the activation mechanism of MPK8 both calmodulin and MKK3.

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Mechanosensing and response is speculated to involve sensory complexes containing a Ca²⁺-permeable, mechanosensitive channel. A recently-identified plasma membrane protein from Arabidopsis, MCA1, has been suggested to be involved in Ca²⁺ influx and touch sensing. We identified an MCA1 homolog, OsMCA1, in rice. In contrast to Arabidopsis in which two MCA family members are present, OsMCA1 has been shown to be the sole MCA family member in the rice genome. OsMCA1 functionally complemented a yeast mutant defective in the Mid1 gene encoding a putative Ca²⁺-permeable mechanosensitive channel component. To investigate its physiological functions, we developed transgenic plants as well as suspension-cultured cells in which its expression is enhanced or repressed. Ca²⁺ mobilization induced by various stress have also been characterized using apoaequorin-expressing lines. Possible roles of OsMCA1 in stress-induced Ca²⁺ influx will be discussed.

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Modulation of histone N-tail modifications correlates with transcriptional activation and repression in eukaryote. In plant, histone modifications are altered in vernalization process. However, no clear correlations between the status of histone modifications and the stress-inducible genes under abiotic stresses have been reported.
We have studied alterations of the histone modification patterns under abiotic stress conditions in Arabidopsis. We detected temporal changing of enrichment at methylation and acetylation sites in histone N-tail on the drought stress responsive genes, DREB2A, RD20, RD29A, RD29B and RAP2.4, under drought stress condition by Chromatin Immuno Precipitation assay. On the coding regions of these genes, K4me3 enrichments increased with drought stress treatment. We will discuss about the correlation between gene activation and histone status of histone H3 under drought stress conditions.

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Under abiotic stress conditions such as drought and high salinity, ABA levels increase in plants, and ABA regulates the expression of many genes that function in plant stress tolerance. A conserved cis-element designated ABRE (ABA-responsive element), which controls ABA-responsive gene expression, has been identified in promoter regions of ABA-regulated genes. Arabidopsis cDNAs encoding bZIP-type transcription factors referred as ABRE-binding (AREB) proteins were isolated using the yeast one-hybrid screening method. Among these transcription factors, expression of AREB1, AREB2, and ABF3 was upregulated by ABA, dehydration, and high-salinity stresses in Arabidopsis plants. Overexpression of the intact AREB1 is insufficient to lead to expression of downstream genes. Post-transcriptional activation of AREB1 by phosphorylation was necessary for its maximum activation. Using in gel kinase assay, we are analyzing phosphorylation of AREB1 peptide fragments under drought and high-salt stress conditions.

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DREBs are transcription factors that interact with a cis -acting DRE sequence and activate expression of downstream genes involved in abiotic stress response and tolerance in Arabidopsis thaliana. There are four DREB2 homologues in rice, and OsDREB2B is genetically close to other DREB2-type genes in monocots. OsDREB2B transcripts were accumulated by low temperature, high temperature and salt stresses in rice seedlings. OsDREB2B produced two forms of transcripts, and quantitative real-time PCR analyses demonstrated that the functional transcription form of OsDREB2B was significantly induced by stresses compared with the other form. The OsDREB2B protein translated from the functional transcription form mRNA was localized to nucleus and we found that the protein exhibits high transactivation activity by using transient transactivation experiments, suggesting that protein modification is not required for activation of OsDREB2B. We are now also analyzing the target genes of OsDREB2B using transgenic Arabidopsis and rice.

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When plant tissue receives mechanical stress, does occur water movement across cell membranes? If the water movement doesn't occur, the exogenous energy is conserved in the distortion of cells just as mechanical energy potential. On the other hand, when the water movement across cell membranes occurs, a part of the exogeous energy is transformed to water potential energy, and is conserved. This difference causes the fundamental difference in the strategy of plants against exogenous mechanical stress.
If the water movement across cell membranes occurs, participation of some aquaporins which locate on the cell membranes can be assumed. We performed flexural stress relaxation test using mercuric chloride which inhibits water transport through aquaporins. Mercuric chloride delayed the relaxation of flexural stress in living plant tissues, though it did not affect on the viscoelasticity of apoplast at all. This suggested that water movement in grass plants relieves exogenous mechanical stress.

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Wild watermelon (Citrullus lanatus L.) inhabits in the Kalahari desert and possesses well-developed root system to absorb deep underground water resources. Proteomic study showed that Ran GTPase (CLRan) was induced by drought stress in the roots of this plant. Furthermore, Arabidopsis over-expressing CLRan1 showed enhanced root growth, suggesting that CLRan1 may function as a key regulator for root growth. To understand molecular mechanism of root growth stimulation by CLRan1, site-direct mutagenesis approaches were undertaken in this study. Transgenic Arabidopsis over-expressing the constitutive-active form of CLRan1 (G19V) showed enhanced root growth. In contrast, dominant-negative form of CLRan1 (T24N) inhibited root growth in the transgenic plants. These results suggest that CLRan1 may regulate root growth through switching of its activation status.

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In plants, betaine is synthesized upon abiotic stress via choline oxidation, in which choline monooxygenase (CMO) is a key enzyme. Although it had been thought that betaine synthesis is well regulated to protect plant against abiotic stress, here it is shown that an exogenous supply of precursors such as choline, serine, and glycine in the betaine-accumulating plant Amaranthus tricolor further enhances the accumulation of betaine under salt-stress, but not normal, conditions. Addition of isonicotinic acid hydrazide, an inhibitor of glycine decarboxylase, inhibited the salinity-induced accumulation of betaine. Salt-induced accumulation of Amaranthus tricolor CMO (AmCMO) and betaine were much slower in roots than in leaves. Antisense expression of AmCMO mRNA suppressed the salt-induced accumulation of AmCMO and betaine, but increased the level of choline. The genomic DNA, including the upstream region (1.6 kbp), of AmCMO was isolated.Importance of promoter sequence of CMO and precursor supply were discussed.

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The cyanobacterium Nostoc commune is adapted to the terrestrial environment and highly desiccation-tolerant. It is shown that N.commune accumulates trehalose as compatible solute when N. commune is subjected to desiccation and salt stress. In this study, changes of trehalose content and levels of transcript of the genes for trehalose methabolizing enzymes in N. punctiforme stain M-15 responding to desiccation and biochemical properties of these enzymes were investigated. N. punctiforme accumulated 5 μ mol trehalose per g dry weight when desiccated. The genes for trehalose synthesis ( treYZ ) and trehalose hydrolysis ( treH ) constitute a gene cluster. Levels of transcripts of treY, treZ and treH increased simultaneously after 1 h of dehydration. Trehalase activity decreased in the presence of 50 mM NaCl, and was strongly inhibited by Tris buffer. These results suggest that trehalose level is regulated mainly by change of trehalase activity.

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Polyamines play important roles in cell proliferation and stress adaptation. The polyamine content of cells is regulated by biosynthesis, degradation and transport. Synechocystis sp. PCC 6803 can adapt to various environmental stress. However the enzymes involved in polyamine biosynthesis and transport in Synechocystis has not been completely understood. In this study, we found that the growth of Synechocystis was rescued by addition of exogenous polyamine under osmotic stress condition. Synechocystis sp. PCC6803 has several enzymes for polyamine biosynthesis. We isolated the gene encoding homologous arginine decarboxylase. We generated knockdown mutant for the genes, and observed that the mutants required polyamine for their growth. The arginine decarboxylase activities of the gene products were also decreased compared with that of wild type. These results indicate that polyamines are involved in adaptation for osmotic stress and that the genes studied above may participate in the biosynthesis of polyamines.

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Plants sense gravity and regulate their morphology, known as gravitropism. In the process of gravitropism, changes in the gravity vector (gravistimulation) are transduced into certain intracellular signals. Previous studies showed that gravistimulation induced increases in the cytoplasmic calcium concentration ([Ca²⁺]_c) consisting of an initial peak followed by a second one. However, it is obscure whether the dual peak is evoked by gravistimulation and/or rotation, since gravistimulation is generally accompanied by rotation under 1g condition. We analyzed [Ca²⁺]_c in seedlings of Arabidopsis thaliana expressing aequorin under multiple gravitational acceleration conditions created by parabolic flights. The second [Ca²⁺]_c peak was not induced when the seedlings were turned through 180^o under microgravity condition, whereas the initial one was often observed. The second [Ca²⁺]_c peak was strongly dependent on the gravitational acceleration. These results suggest that the second peak is induced by gravistimulation, whereas the initial one is induced by rotation.

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The cytochrome c₈ isoform, was newly isolated from purple photosynthetic bacterium Rubrivivax gelatinosus mutant lacking HiPIP and high- and low-potential cytochromes c₈. The isocyt c₈ has an approximately 10 kDa molecular mass and the about +280 mV redox midpoint potential. Absorption spectrum of the reduced form showed the α-band at 552 nm. Flash-induced kinetic measurements using the isocyt c₈ and the photosynthetic membrane indicated that the isocyt c₈ has a functional ability of an electron donor to the RC-bound cytochrome subunit. The primary structure of the isocyt c₈, determined by peptide sequencing followed by cloning and nucleotide sequencing, showed 65 % amino acid sequence identity with the known high-potential cytochrome c₈ of R. gelatinosus. The gene arrangement suggested participation of the two high-potential cytochromes c₈ to nitrite reduction.

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The elongation step of translation is sensitive to oxidative stress. We have recently found that elongation factor G (EF-G) is the primary target of inhibition by reactive oxygen species (ROS) within the translational machinery. In the present study, we investigated the redox properties of EF-G (Slr1463) in Synechocystis sp. PCC 6803. The presence of H₂O₂ oxidized two Cys residues in EF-G, suggesting that inactivation of EF-G by ROS is due to the formation of a disulfide bond between two Cys residues. Substitution of Cys105 by Ser rendered Slr1463 insensitive to H₂O₂, suggesting that Cys105 is a target of ROS. The oxidized Cys residues were reduced by thioredoxin, suggesting that thioredoxin reduced the disulfide bond. These results suggest that the redox state of EF-G regulates the activity of translation.

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The translational machinery is particularly sensitive to oxidative stress. Using an in vitro translation system from a cyanobacterium, we have demonstrated that elongation factor G (EF-G) is the primary target of oxidation by reactive oxygen species within the translational machinery. In the present study, we examined the effects of the overexpression of EF-G on the tolerance of cyanobacteria to oxidative stress. Overexpression of EF-G enhanced protein synthesis under oxidative stress and, moreover, mitigated the photoinhibition of photosystem II (PSII). It appears that increased levels of EF-G might minimize oxidative damage to protein synthesis and accelerate the repair of photodamaged PSII.

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Genes encoding subunits of photosystem I (PSI genes) in the cyanobacterium Synechocystis sp. PCC 6803 are actively transcribed under low-light conditions, whereas their transcription is coordinately and rapidly down-regulated upon the shift to high-light conditions. To identify the molecular mechanism of this phenomenon, we searched for common light-responsive elements in the promoter region of PSI genes. We found that these promoters possessed a common light-responsive element located just upstream of the basal promoter region. These AT-rich upstream sequences enhanced the basal promoter activity under low-light conditions and their activity was transiently suppressed upon the shift to high-light conditions. Thus, these AT-rich upstream elements are responsible for the coordinated high-light response of PSI genes dispersed throughout Synechocystis genome. Identification of regulatory factors interacting with the upstream elements will be reported.

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Many cyanobacterial species possess two copies of genes encoding putative transcriptional regulators having an AbrB-type DNA binding domain at their C-termini. In this study, we made the disrupted mutants of sll0359 and sll0822 encoding AbrB-type regulators in Synechocystis sp. PCC 6803. Both mutants showed slower growth rate and lower content of photosynthetic pigments than wild-type strain. DNA microarray analysis of sll0822 mutant revealed decreased expression of several genes involved in nitrogen uptake or photosynthesis in the mutant under normal growth condition. Northern blot analysis showed that induction of nitrogen uptake genes such as nrtA and urtA was abolished in the mutant upon nitrogen starvation. Now we are examining the amount and oligomerization state of Sll0822 in wild-type cells under various nitrogen conditions by using its specific antibody.

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Capillary electrophoresis mass spectrometry (CE/MS) was applied for the comprehensive survey of changes in amounts of metabolites upon the shift from photoautotrophic- to photomixotrophic conditions in Synechocystis sp. PCC 6803. When glucose was added in the light, the drastic change in metabolic processes, i.e., up-regulation of the oxidative pentose phosphate pathway and glycolysis together with down-regulation of the Calvin cycle, was observed in the wild-type cells. These responses seem prerequisite for the coordination between anabolic and catabolic reactions under photomixotrophic conditions. CE/MS analysis of pmgA-disrupted mutant which cannot grow under photomixotrophic conditions suggested that CO₂ fixation rate was higher in the mutant than in the wild-type cells upon the addition of glucose. Concomitantly, 1) decrease in ATP and NADPH contents, 2) failure in activation of the oxidative pentose phosphate pathway and of respiratory electron transport and 3) aberrant accumulation of isocitrate were observed in the mutant under photomixotrophic conditions.

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Some cyanobacteria accumulate sucrose as the compatible solute during salt stress. In Synechococcus elongatus PCC 6301/PCC 7942 and Thermosynechococcus elongatus BP-1, sucrose-phosphate synthase (SPS) and sucrose 6-phosphatase (SPS) genes are fused together and may function as the single polypeptide.
Effect of various salts to induce sucrose synthesis during the liquid culture of S. elongatus PCC 7942 was examined. Sucrose accumulation was hardly observed in low-salt conditions, but after the addition of 0.2 M NaCl, the intracellular sucrose concentration attained to approximately 160 mM within 24 hours. NaNO₃ showed comparable effects as NaCl, while KCl and KNO₃ were less effective.
The SPS-SPP gene in S. elongatus PCC 7942 was disrupted by targeted mutagenesis, resulting in the complete loss of the ability to synthesize sucrose. The growth rates of the mutant in the presence of 0.3 M NaCl were substantially reduced, indicating the significant role of the sugar under salt stress.

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Members of small heat shock protein (sHsp) family range in monomer size from 12−43 kDa, are characterized by a conserved alpha-crystallin domain. In order to elucidate the role of sHsp under oxidative stress generated by methylviologen or hydrogen peroxide, we analyzed phenotype of a cyanobacterial mutant which constitutively expresses sHsp or whose gene encoding sHsp is disrupted. Small Hsp stabilized phycocyanin and photosystem complexes in the cyanobacteria Synechococcus sp.PCC 7942 and Synechocystis sp. PCC 6803.

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2-Cys peroxidredoxin (2-Cys Prx) reduces reactive oxygen species (ROS) such as hydrogen peroxide through the S-S/-SH catalytic cycle which requires the active cysteine to be regenerated by reductant. To date, no electron donor reducing the cyanobacterial 2-Cys Prx has been identified. In this work, a peroxiredoxin reductase (PrxR) was co-purified with 2-Cys Prx from a thermophilic cyanobacterium Thermosynechococcus elongatus cells as an NADPH dehydrogenase induced by oxidative stress. This result indicated that the PrxR interacts with the 2-Cys Prx. An in vitro assay with purified recombinant 2-Cys Prx and PrxR revealed that both proteins were essential for the efficient electron transport from NADPH to hydrogen peroxide. These results suggested that the PrxR transfers reducing power from NADPH to 2-Cys Prx, which reduces ROS in cyanobacteria grown under oxidative stress conditions.

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Hsp90 is an essential and a ubiquitous chaperone protein in the cytosol of eukaryotic cells. The phenotype of mutants of HtpG which is a prokaryotic homolog of Hsp90 had not been observed in Escherichia coli and Bacillus subtilis. However we showed that the htpG disruptant strain from the cyanobacterium Synechococcus sp. PCC 7942 was sensitive to heat shock, indicating an essential role of HtpG. In the present study, we show that HtpG interacts directly with DnaJ, a prokaryotic homolog of Hsp40, by biochemical experiments such as co-immunoprecipitation studies and genetic analysis of yeast two-hybrid assays. Hsp40 is one of the cochaperones of Hsp70. Cyanobacteria have DnaKs which are prokaryotic homologs of Hsp70. It is known that Hsp90 cooperates with Hsp70/Hsp40 in eukaryotic cells, thus HtpG may collaborate with the DnaK chaperone system in Cyanobacteria.

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The 90-kDa heat shock proteins (Hsp90s) are a widespread family of molecular chaperones found in bacteria and all eukaryotes. Eukaryotes require a cytoplasmic Hsp90 for viability under all conditions tested. Like other major chaperones such as Hsp70 and Hsp60, Hsp90 has a weak ATPase activity. Structural analysis of Hsp90 and HtpG, a prokaryotic member of Hsp90, has revealed dynamic, ATPase-coupled conformational changes. However, the ATPase-coupled chaperone mechanism is largely unknown. We showed that HtpG is essential for the thermal stress management in cyanobacteria in contrast to Escherichia coli and Bacillus subtilis. We also reported at the last meeting that HtpG interacts with a linker polypeptide of the phycobilisome from the cyanobacterium Synechococcus sp. PCC 7942. In this presentation, I will show the effect of nucleotides and nucleotide analogs on the suppression of heat inactivated linker polypeptides by HtpG.

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Effects of light and glucose on the expression of soluble proteins were analyzed by means of two-dimentional electrophoresis in Synechocystis sp. PCC6803 which exibits not only photoautotrophic growth but also light-activated heterotrophic growth (LAHG) under dark with glucose as a carbon source. Various proteins were induced by light in the presence of glucose, while glucose addition in the dark was not effective for induction of many proteins, such as Fructose-1,6-bisphosphate aldolase (FBA). Therefore, both light and glucose were required for the full induction of protein synthesis.
One of the response regulators, sll1330, was demonstrated to be involved in regulation of FBA expression by light and glucose. FBA was synthesized only slightly under LAHG conditions in the disruptant of this gene. The disruptant of sll1330 grew scarcely in heterotrophic growth under intermittent light but did normally in autotrophic condition. These results suggest that sll1330 is the key regulator of FBA expression.

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For improvement and better sustainability of photobiological hydrogen production based on nitrogenase and photosynthesis by cyanobacteria, we have created several hydrogenase and homocitrate synthase mutants. The mutant of Nostoc sp. PCC 7120 disrupted in uptake hydrogenase gene (ΔhupL) showed improved hydrogen production activity over the wild-type. Nostoc sp. PCC 7422 has high nitrogenase activity, and the ΔhupL cells were able to accumulate hydrogen to about 30% (v/v) in 3 to 8 days, with the efficiency of light energy conversion into hydrogen of as high as 3.7% vs visible light. In these mutants, the high hydrogen production activity stages were relatively short-lived, lasting for 10-20 hours. The mutant (ΔnifV1) disrupted in one of the homocitrate synthase genes with PCC 7120 ΔhupL as the parent showed improved sustainability of production. Addition of low concentrations of CO₂ and N₂ to Ar gas sometimes improved the sustainability.

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We previously reported that cell flocculation is induced under conditions of light plus low temperature to avoid photoinhibition in Thermosynechococcus vulcanus RKN and that the flocculation is released by treatment with cellulase. However, the substance responsible for the aggregation could not be evaluated properly, since it was not completely digested even after excess treatment of cells with cellulase.
Here, we established the protocol for quantitation of cellulose by combination of the cellulase digestion with pretreatment of cells with sonication in ethanol. With this method, we evaluated cellulose accumulation in the process of the low temperature-induced cell flocculation. While cells began to aggregate at 24 h and mostly completed in 72 h, the cellulose accumulation preceded the aggregation and was maintained up to 72 h. Under the regular growth conditions, very little accumulation of cellulose was detected. These results suggest that cell flocculation is caused by cellulose accumulation.

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Defect of the endosperm development is observed in inter-specific crosses of many plant species. Abnormal endosperm development causes seed abortion, therefore, this phenomenon is known to be a reproductive barrier after fertilization. Several reports suggested that defect of endosperm development is caused by the functional difference between parental genome. One of the possible molecular mechanisms underlying this phenomenon can be genomic imprinting, an epigenetic mechanism resulting in mono-allelic gene expression by parent-of-origin dependent manner. Recent advances of the study of genomic imprinting in Arabidopsis are applied to understand the reproductive barrier of hybrid endosperm in rice.
To begin with, we analyzed how imprinted genes are involved in the development of hybrid endosperm. We conducted inter-specific cross between cultivated and wild rice and observed developmental defect in the hybrid endosperm. We also investigated the expression of imprinted gene. Possible involvement of genomic imprinting in the hybrid endosperm will be presented.

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The seed-shattering gene, qSH1 encodes a BEL-type homeobox gene highly similar to Arabidopsis REPLUMLESS (RPL) gene and is required for the formation of abscission layer in rice. Since BEL and KNOX type homeobox genes can form a heterodimmer and control the downstream genes in Arabidopsis, we looked for the ortholog of BP gene, the hetrodimer partner of KNOX-type homeobox for RPL and found that it's the OSH15 in rice. We next crossed the d6 (osh15) mutant plant with a nearly-isogenic line of qSH1 and analyzed their progeny. The results revealed that the OSH15 played a role for the seed-hattering. A complementation test further proved that OSH15 is another seed-shattering gene of rice. Histological analysis suggested that OSH15 might be involved in the degradation process of abscission layer in rice rather than the formation. Now we are performing a microarray analysis to identify the gene involved in this process.

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We have identified 45 RLKs expressed in pollen and the pollen tubes of Arabidopsis thaliana through a comparison of several tissues' gene expression profiles. No dramatic phenotypes were observed in vivo in each single gene T-DNA insertion line, so double disruptions in sister genes seemed to be necessary. Nine sets of double gene disruptant lines were made for further analyses. We found one line, which hardly harbored seeds although its plant shape and size looked normal. Reciprocal crosses demonstrated its male defects. In vitro, 88% of pollen tube tips burst just after starting germination, thereby stopping its growth. In vivo, the pollen tube growth was stopped from papilla-stigmatic to style tissues. Phenotypes such as skewed segregation rate in self-fertilization and impaired pollen tube growth in disruptants were complemented with native promoter-driven YFP/CFP fusion proteins. We will discuss roles of RLK regulated pollen tube growth in the fertilization.

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Double fertilization is an important mechanism for angiosperm reproduction. However, little is known both morphologically and genetically due to the position of the embryo sac. Since the embryo sac is embedded inside of the ovule tissue deeply, it had been extremely hard to observe it directly. To overcome this difficulty, we have developed confocal laser microscope system which allows us to observe gametophytic cells inside the ovule as a sequential 3D movie. We have searched fluorescence protein makers which enable us to capture the real time imaging of the double fertilization. Here, we show the real time imaging of the double fertilization process for the first time. This result leads to new findings that sperm cells migrate toward the place for the fertilization within 1min. after pollen tube discharge. We observed that the fertilization of the central cell occurs first, followed by the egg cell fertilization.

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Many plants measure changes in day-length to ensure that flowering occurs under optimum conditions. In Arabidopsis, the most prominent molecular mechanisms in the photoperiodic flowering pathway are the clock-regulated gene expression of CONSTANS (CO) and the stabilization and activation of CO protein by light. We show that the timing of the blue-light dependent formation of FLAVIN-BINDING, KELCH REPEAT AND F-BOX 1 (FKF1) and GIGANTEA (GI) protein complex is crucial for regulating the timing of CO gene expression. The expression of FKF1 and GI is clock regulated, and the peak expression of both genes coincides in long days (LD) but not in short days (SD). Hence, the timing of the FKF1-GI complex formation is regulated by the coincidence of both external and internal cues. We propose the importance of external and internal coincidence mechanisms on CO transcription regulated by the FKF1-GI complex.

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Circadian rhythms are generated by endogenous circadian clock system. In the past decade, molecular genetic analyses using Arabidopsis thaliana provided novel insights into the plant circadian clock system. A large number of circadian clock genes (CCGs) are involved in a complex gene network of the plant clock system. With systems biology analyses, the clock system in Arabidopsis was modeled as three transcriptional feedback loops (loop I, II and III) that consist of a single Myb genes (LHY/CCA1) and pseudo-response regulator genes (PRRs). However, a molecular phylogeny of these genes in angiosperm has not been determined. In order to clarify the phylogeny, we analyzed intron-exon structures, phylogenetic trees and chromosome syntenies among CCGs of three model plants (Arabidopsis, Rice and Poplar). We revealed that some CCGs were duplicated via whole genome duplication events and that, after these events, some duplicated CCGs were deleted from the genomes.

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In the cyanobacterium Synechococcus elongatus, all promoter activities are under the control of the circadian clock in continuous light conditions. Previously, Kucho et al. used PCR-fragment-based DNA microarray to examine genome-wide transcription profiles in Synechocystis and identified 9% of all genes as clock-controlled genes. However, the genome-wide expression in synechococcus in which amplitude and precision of circadian transcription rhythm are much higher, has not been analyzed as far.
We employed high-density oligonucleotide arrays (GeneChip) to examine genome-wide Synechococcus gene expression. Our analysis identified at least 30% of 2,538 possible ORFs as clock-controlled genes. Most of the genes were classified into two groups, one peaking at subjective dawn and the other peaking at subjective dusk. Such circadian control was nullified in kaiABC-null strains. These results are consistent with our previously suggested model (Tomita et al. 2005). Based on these results, we will discuss the genetic network among clock-related genes.

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In Arabidopsis thaliana, PRR5,PRR7 and PRR9 are the components of the central oscillator of circadian clock. prr5/7/9 triple mutants lose the sensitivity of light in hypocotyl elongation (Nakamichi et al., 2005). But their leaves restore sensitivity to light. Under light-dark condition, they show smaller leaf blades and longer petioles, like a shade-avoidance phenotype, than those grown under continuous lighting (Niimura et al., submitted). In addition, shade-avoidance marker genes, AtHB-2 and PIL1 are highly expressed during dark period in the leaves of prr5/7/9, although the expression of the genes in prr5/7/9 are very low as those in WT during light period. These result suggest the existence of the unique mechanism to repress shade avoidance response in leaves, and that it might be related with PRR5,PRR7 and PRR9.

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PSEUDO RESPONSE REGULATOR9 (PRR9), PRR7, and PRR5 are involved in circadian clock mechanism that generates circadian rhythms. PRR9, PRR7, and PRR5 proteins negatively regulate accumulation of mRNA for CARCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), while the molecular functions of PRR proteins were unknown.
In this report, we analyzed PRR proteins using T87 Arabidopsis cultured cells. The dual-luciferase reporter assay was developed, and the assay allowed us to calculate promoter activity of CCA1 gene in vivo. Transiently expressed PRR9-CFP, PRR7-CFP, and PRR5-CFP proteins localized in the nuclei of cells, and PRR proteins can repress transcriptional activity of CCA1 promoter. The DEX-GR system revealed the essential role of nuclear localization to PRR function. PRR proteins conjugated to intrinsic transcriptional activator VP16 could activate CCA1 promoter, suggesting that PPR proteins can bind to DNA directly. These results suggested that PRR are nuclear localized proteins and repress CCA1 promoter directly.

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In Arabidopsis thaliana, it is currently thought that the core circadian clock is composed of the CCA1/LHY and TOC1 gene-products, which together generate a fundamental circadian rhythm by forming a negative/positive transcriptional feedback loop. This intrinsic Arabidopsis clock plays roles in a variety of biological processes, which are common in higher plants. So far, nevertheless, no mutant lacking all these clock genes, CCA1, LHY, and TOC1, has yet been characterized. Here, we characterized for the first time such a cca1 lhy toc1 triple loss-of-function mutant with reference to the circadian clock-associated phenotypes, including, for instance, the photoperiodic control of flowering time and the light-regulated hypocotyl elongation. To gain insights into the molecular picture of plant clock, based on the results of this and other studies, a unified view as to the sophisticated genetic linkages among these clock-associated core genes will be proposed.

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Plant development is responsive to environmental cues, and many of the regulatory proteins that mediate these responses are only found in plants. In Arabidopsis, GIGANTEA (GI) plays key roles in the control of circadian rhythms, flowering determination and light signaling through phytochrome signaling. We show here that Drought induced 19 (Di19), a zinc-finger protein, interacts with GI in vitro and in planta. Di19 protein localizes to the nucleus. di19 mutation suppresses late flowering of phyA mutants and enhances the elongation of hypocotyl under red light conditions when combined with mutations in genes encoding related zinc-finger proteins genes. The loss of function of Di19 affected the period of circadian rhythms in GIGANTEA expression. Together with these data, we propose the importance of interaction between GI and Di19 in phytochrome-mediated regulation of the control of circadian rhythm, light signaling and flowering in Arabidopsis.

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A circadian clock resetting protein Pex in the cyanobacterium Synechococcus elongatus PCC 7942 accumulates dark condition and regulates the phase and period of the rhythm. Pex binds to the promoter of the clock essential gene kaiA, whose protein KaiA accelerates the phosphorylation of KaiC protein and shortens the circadian cycle. Then, Pex represses the expression of the clock gene. An alpha helix and a positively charged loop between two beta sheets in Pex is essential to bind the promoter in vitro and the in vivo period regulation. Here, we examined molecular weight of Pex in vivo by gel-filtration method and determined as 25 kD in size, suggesting Pex dimer because its monomer shows 13.1 kDa in SDS polyacrylamide gel. Then, we also show the importance of the dimer formation by using bioluminescence rhythm monitoring method and several reporter strains expressing an amino acid replaced Pex protein.

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In higher plant, storage proteins are synthesized on rough ER as precursors and then are transported to protein storage vacuoles to be processed into mature forms. We recently reported an Arabidopsis mutant, maigo2 (mag2), which accumulated large amount of novel structures in the ER (1). The structures contain an electron-dense core which is composed of the precursor forms of 2S albumin. MAG2 protein might function as a complex with ER-localized SNAREs, involving the exit of storage protein precursors from the ER. In order to clarify mechanism of MAG2-dependent transport, we identified factors that form a complex with MAG2. Here, we report characterization of one of the factors, designated MAG2-complex component 1 (MACC1). Our results suggest that MACC1 plays a significant role in the transport of storage proteins in Arabidopsis.
(1) Li et al., Plant Cell 18: 3535-3547 (2006)

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