We have shown the heat-induced photobleaching of phycobilisomes in cyanobacteria. Consitituve expression of HspA in Synechococcus protected phycocyanin from losing its light-harvesting property. The present study aims to reveal the mechanism of the bleaching and how HspA can protect phycocyanin from the bleaching.
Cell extracts were prepared from the reference strain, Synechococcus strain ECT, and the HspA expressing strain ECT16-1 after a heat shock. The former strain showed the phycocyanin bleaching, while the latter one did not. Phycobilisome of the ECT cell was much more prone to heat-denaturation than that of the ECT16-1 cell. Purified phycobilisome was incubated in the presence or absence of HspA at 50^oC in the presence of H₂O₂. This treatment resulted in the protein aggregation and phycocyanin bleaching. However, addition of HspA suppressed the denaturation. Studies on a specific interaction between HspA and a phycobilisome component are in progress.

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The CIRCE element is known to be the target for the HrcA repressor and involved in the temperature-dependent gene regulation for groESL operon and/or dnaK operon in a large variety of microorganisms. We have already reported that the transcription of the groESL1 operon and the groEL2 gene in Synechocystis sp. strain PCC 6803 is derepressed by the hrcA disruption under normal conditions. However, the level of these groE mRNAs in the hrcA mutant increased further upon heat shock or irradiation, suggesting that the transcription is regulated by a mechanism other than the CIRCE/HrcA system. We found that the groE induction in the mutant is enhanced by light and this light acceleration is inhibited by DCMU. A well conserved sequence was found upstream of the groESL1 promoter. In order to identify a novel cis-element, upstream regions were fused to a reporter gene and the groE promoter analysis is in progress.

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In the genome of Synechocystis, there are five genes that encode a putative ADP-ribose pryophosphatase. Each of the recombinant proteins was overexpressed and their enzymatic activities were examined. Sll1054 and Slr0920 hydrolyzed ADP-ribose specifically, while Slr1134 hydrolyzed not only ADP-ribose but also NADH and FAD. By contrast, Slr1690 showed very low activity for ADP-ribose. Slr1690 had four substitutions of conserved amino acids. The replacement of the mutated amino acids with those conserved, increased hydrolytic activity, suggesting that Slr1690 might have been derived from an ADP-ribose pyrophosphatase. Clustering analysis suggested that the ADP-ribose pyrophosphatase family in Synechocystis appears to comprise a bacterial type and four phylogenetically diverse types that diversified via molecular evolution. The growth of insertional mutants, Δsll1054 and Δslr0920, was severely inhibited in the presence of 25 μ ADP-ribose, suggesting that Sll1054 and Slr0920 play a major role in the degradation of ADP-ribose.

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We found out that disruptant of slr1212 encoding a phytochrome-related protein reduced the accumulation of carotenoid after irradiation of 300 to 500 μmol m^-2s^-1 for twelve hours. The accumulation of carotenoid was effectively induced by irradiation of orange (615nm) and violet (395nm) of high light in the wild type and was reduced under both light conditions in slr1212 disruptant. Furthermore, longer irradiation of violet light led to cell bleaching.
In this cyanobacterial genome, response regulator gene slr1213 encoding AraC subfamily is adjacent to slr1212 suggesting that it may function as a transcription factor in the downstream of Slr1212. Inactivation of slr1213 in the slr1212 disruptant restored the carotenoid accumulation. This suggests that Slr1213 functions as a repressor of carotenoid accumulation and Slr1212 represses the activity of Slr1213. Analysis in the composition of cellular carotenoid demonstrated that the accumulation of myxol-DF was reduced in slr1212 disruptant.

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Recently, we have identified pigments of Anabaena sp. PCC7120 and Nostoc punctiforme PCC73102: myxol 2'-fucoside (not rhamnoside), 4-ketomyxol 2'-fucoside, β-carotene, echinenone and canthaxanthin. In this study, we identified pigments of Anabaena variabilis ATCC29413. Myxol (5%, mol% of total carotenoids) and 4-hydroxymyxol (2%) bound no glycosides. Other carotenoids were β-carotene (51%), echinenone (22%) and canthaxanthin (20%). As genomic draft sequence of this strain is available on the Web, the pathway of carotenoid synthesis can be discussed. β-Carotene ketolase (CrtO/W) can introduce keto group to both β-carotene and echinenone to produce echinenone and canthaxanthin, respectively. β-Carotene hydroxylase (CrtR) seems to have unique characteristics, that is, β-end group of deoxymyxol is a good substrate to produce myxol, but that of β-carotene is not a substrate to produce zeaxanthin. Different characteristics of crt-genes might be caused various carotenoids in cyanobacteria. Glycosyl transferase might be inactive, since carotenoid glycosides are absent in this strain.

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Gloeobacter violaceus PCC 7421 is a unicellular cyanobacterium which is thought to be branched off at the earliest stage within the cyanobacterial clade by phylogenetic analysis based on the 16S rRNA sequences. Thus, Gloeobacter might retain traces of the ancestral properties of cyanobacterium. We analyzed carotenoid composition and identified carotenoid biosynthetic genes. The major carotenoid was β-carotene, and oscillol-di(L-fucoside) and echinenone were also identified. However, other carotenoids which exist among various cyanobacteria, were not detected. We searched for the carotenoid biosynthetic genes in the genome sequence of Gloeobacter. Whereas two plant-type desatulase genes (crtP and crtQ) that catalyze desaturation steps sequentially from phytoene to lycopene were not found, a bacterial-type phytoene desatulase (crtI) existed. In vivo functional complementation experiment revealed that CrtI produced lycopene by catalyzing 4-steps desaturation of phytoene. This is the first demonstration that oxygenic photosynthetic organism uses the bacterial-type phytoene desaturase.

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Carotenoids in cyanobacteria have diversity from species to species. Last year, we presented the identification of pigments in Anabaena sp. PCC7120; myxol-fucoside, ketomyxol-fucoside, β-carotene, echinenone, and canthaxanthin. In Anabaenas, there are no zeaxanthin, one of major pigments in Synechocystis sp. PCC6803.
In this study, we propose a biosynthetic pathway in A. 7120. Nine genes have already been functionally identified in other bacteria, and close homologues of these genes were found in the genome of A. 7120 by similarity search. We found two homologues of β-carotene ketolase; one is alr3189, a homologue of crtW in Nostoc punctiforme PCC73102 and the other one is all3744, a homologue of crtO in S. 6803. An all3744-deletion mutant lacked echinenone and canthaxanthin, but contained ketomyxol- fucoside. This result indicates that ketolation of β-carotene and myxol-fucoside are catalyzed at least two different ketolases. We are also trying to disrupt other carotenogenesis genes.

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Almost all organisms have lipid modification systems required for anchoring soluble proteins to membranes. Bacterial lipoproteins are a set of proteins modified at their N-terminal cysteine residue with an N-acyl group and S-diacylglyceryl group. This type of lipid modification is ubiquitous in bacteria. Although the lipid modification of lipoproteins has been studied in some bacteria, it has not been studied in cyanobacteria. Because cyanobacteria are unique bacteria containing thylakoid membranes in addition to outer and plasma membranes, lipoproteins in cyanobacteria are expected to have many important roles. In this study, we identified all genes involved in lipid modification of lipoproteins in a cyanobacterium Synechocystis sp. PCC6803 and found that some subunits of the photosystem II complex are lipoproteins. These results suggested that the pathway of the lipid modification of lipoproteins exists in cyanobacteria and lipid modification has important roles in assembly of the subunits to the photosystem II complex.

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Although RNA polymerase purification methods have been reported in a number of cyanobacterial species, they are very complicated and time-consuming.Here we report a simple and rapid purification of RNA polymerase from Synechocystis sp. strain PCC 6803, by using a carboxy terminal His6-tagged RpoC2 (beta' subunit) mutant strain. A vector for chromosomal displacement was made by placing the Cm cassette immediately downstream of the rpoC2-(CAC)6 construct. The generated mutant strain grew as well as the wild type. For purification, cells were disrupted using glass beads, and the resulting high speed supernatant (20 mM Tris-Cl pH 8, 0.5 M NaCl, 5% glycerol) was loaded on a Ni-NTA column. Western analysis revealed that the 20-50 mM eluate contained the subunits of RNA polymerase (beta', beta, gamma, alpha, sigma). We also describe the results of in vitro transcription analysis using the purified enzyme.

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Chloroplast gene expression is largely controlled at post-transcriptional steps, and many RNA binding proteins (RNPs) are known to be involved in these regulation. Moreover, the biological function of some chloroplast RNPs can be regulated by phosphorylation. Although cyanobacteria are thought to be the progenitor of chloroplast, little is known about the function of RNPs in the cells. We analyzed the proteins in the ssDNA binding fraction from Synechocystis sp.PCC6803, since many RNPs are known to have affinity for ssDNA. It was found that the levels of two major proteins (20kDa and 30kDa) and the two protein kinase activities in the fraction from continuously illuminated cells are markedly higher than those from cells in dark. Furthermore, it was assumed that 45kDa and 50kDa ssDNA binding proteins detected in continuously illuminated cells may be phospho-proteins. To understand the biological function of these ssDNA binding proteins in cyanobacteria, further analytical studies are underway.

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The first half of biosynthetic pathway of chlorophylls (Chl) from glutamate to protoporphyrin is shared with heme biosynthesis. Mg-chelatase, a three-subunit enzyme (ChlI, ChlD and ChlH), catalyzes the insertion of Mg²⁺ into protoporphyrin, the first reaction of Mg-branch. Thus, it is suggested that Mg-chelatase plays an important role in the teterapyrrole allocation mechanism. A single recombinant mutant A5101 was isolated from the cyanobacterium Plectonema boryanum in a trial to isolate a chlD-disruptant. Absorption spectrum of dark-grown A5101 cells suggested significant decrease in the Chl and phycobiliproteins contents. The Chl content of A5101 was 12 % of the wild type. While A5101 grew slightly faster than the wild type in darkness, A5101 did not grow under photoautotrophic conditions. Further phenotypic analysis such as the contents of some proteins of photosystems and Chl biosynthesis will be presented and the function of ChlD is also discussed.

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A glucose-tolerant strain of Synechocystis sp. PCC6803 grows both photoautotrophically and photoheterotrophically consuming glucose as the source of reduced organic carbon. Additionally, light is necessary for the maintenance of photoheterotrophic growth. However, there is little knowledge about the role of the light irradiation to be activating photoheterotrophic growth. The objective of this study is to elucidate the effect of the light on the glucose utilization during the photoheterotrophic growth. Extensive analysis of the activities of the glycolytic enzymes revealed that glycolytic enzymes require light irradiation to maintain the activity. The changes in expression profiles at the protein level in cells incubated in heterotrophic growth conditions in the presence of intermittent light were investigated. Synthesis of at least 30 proteins was induced by light irradiation in the presence of glucose. These results suggest that enzyme activity and protein expression are controlled by light irradiation that enables to maintain the photoheterotorophic growth.

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The genome of Synechocystis sp. PCC 6803 cells encodes four group-2 sigma factors including sigE, and in this study, we have found that a mutant deficient in SigE fails to proliferate under the heterotrophic growth condition. Microarray and Northern analyses demonstrated that the transcript levels of oxidative pentose phosphate (OPP) pathway, glycolysis, and glycogen catabolism were reduced in the sigE mutant under the normal growth condition, and activities of two significant enzymes for the OPP pathway, glucose-6-phosphate dehydrogenase and 6-phophogluconate dehydrogenase, were actually decreased by the sigE disruption. Previously, Muro-Pastor demonstrated that SigE was induced by nitrogen deprivation (Muro-Pastor et al., 2001). We showed here that the sugar catabolic genes were actually activated by nitrogen depletion dependent on SigE.

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Synechocystis sp. PCC6803 is a Gram-negative bacterium and has a cell surface structure composed of peptidoglycan, outer membrane, and surface layer (S-layer) from the inner side. We have shown that abundant hemolysin-like protein (HLP) binds to the S-layer of the glucose-tolerant strain (GT). Physiological function of HLP is unknown, although purified HLP is shown to be a trimer that binds Ca²⁺. In the present study, the wild type (WT) and the GT strains were compared in terms of Western blotting and immunoelectron microscopy with polyclonal antibodies to HLP. The results revealed that no HLP was present in WT. Furthermore, it was suggested that HLP binds Ca²⁺ in the S-layer. The existence of abundant Ca²⁺ on the cell surface may have some physiological and/or ecological meanings in GT.

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Many of N₂-fixing cyanobacterial strains have two types of [NiFe]-hydrogenase: an uptake hydrogenase (Hup) and a bidirectional hydrogenase (Hox). The biosynthetic pathways leading to active [NiFe]-hydrogenase formation are complex processes that require the collaboration of Hyp (Hydrogenase pleiotropy). HypF is involved in the CO and CN ligand syntheses of [NiFe]-cluster from carbamoyl phosphate and also has a central role in assembly of the cluster. To see if disruption of hypF gene is beneficial to photobiological H₂ production by N₂-fixing cyanobacteria, we have constructed two hypF disruption mutants from Anabaena sp. PCC 7120 wild-type and the ΔhupL mutant: ΔhypF and ΔhypF/ΔhupL. These mutants showed high H₂ production activity at a rate 5 to 8 times that of wild-type under nitrogen-fixing conditions. The disruption of hypF seems to be useful as an alternative means for improvement of hydrogen production in nitrogenase based system.

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When cucumber seeds germinate in a horizontal position, seedlings form a peg on the lower side of the transition zone. We have suggested that gravistimulation activates auxin efflux and suppresses peg formation on the upper side of the transition zone. To understand this mechanism, in this study we isolated 5 cucumber cDNAs of PIN auxin efflux carrier gene in addition to CsPIN1 cDNA previously isolated and investigated their mRNA accumulation by Northern hybridization and in situ hybridization. Our results showed that tissue specificities of CsPINs expression could be classified into three categories; CsPIN1-4 are expressed in vascular tissues, CsPIN5 is expressed in epidermis and cortex of roots, and CsPIN6 is expressed in root columella cells. Further investigations of CsPIN1 and CsPIN3-6 that are also expressed in the transition zone will reveal the mechanism of auxin transport to be involved in gravity-regulated peg formation.

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Cucumber seedlings grown in a horizontal position develop a peg on the lower side of transition zone between the root and the hypocotyl. We have shown that auxin induces peg formation and that following gravistimulation auxin and mRNA of auxin-responsible genes accumulate asymmetrically across the transition zone. We showed that CsARF2 and CsIAA1 mRNAs accumulate in the same tissue at the stage of peg initiation. We therefore proposed that CsARF2 may activate auxin-responsible genes responsible for peg formation and that co-expression of CsARF2 and CsIAAs is important for transcriptional regulation of the auxin response. In this study, we examined transcriptional activity of CsARFs and CsIAAs by tobacco BY-2 protoplast transient assay. We found that CsARF2 activated the transcription of auxin-responsible DR5 promoter in the assay. We discuss the mechanism of the transcriptional regulation of auxin responsible genes by CsARFs and CsIAAs, which may play a role in peg formation.

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We have isolated a number of shoot gravitropism (sgr) mutants of Arabidopsis to elucidate the molecular mechanism of shoot gravitropism. We have previously shown that the endodermal cells, which contain sedimented amyloplasts, are essential for shoot gravitropism. The sgr6 recessive mutant exhibited reduced gravitropic response in the inflorescence stem. Amyloplasts were normally sedimented to the direction of gravity in the endodermal cell of sgr6. In addition, sgr6 exhibited normal phototropism. The SGR6 gene encodes a protein with 1703 amino acids containing two HEAT repeat motifs. Endodermis-specific expression of the SGR6 using the SCR promoter restored shoot gravitropism of sgr6-1. These results suggest that SGR6, which is a novel big protein with unknown function, is involved in the gravi-perception and the signal transduction process which take place in the encodermal cells.

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Differed from roots and stems, there is no sufficient knowledge about the nature or mechanisms of tropism of leaves. We showed that the rosette leaves of Arabidopsis thaliana exhibit negative gravitropism in dark. On the other hand, leaves did not stand up under white and red light, suggesting that red light controls the motion of leaves.
To investigate interaction between light and gravitropism of leaves,Arabidopsis seedlings were put in normal and upside-down position under white light, dark and red light for 9 hours. The angles between first and second leaves were measured before and after the treatments. As a result, all seedlings showed the leaf gravitropism, while the behavior of leaves varied among light conditions. Motion of leaves was, judged from the above data, divided into gravity-dependent and gravity-independent movement that is under control of red light. Analyses of the leaf-tropism mutants are also in progress.

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Heat stress during early ripening period cause abnormal maturing of seeds such as milky white kernels in rice. The aim of this study is to elucidate its mechanism. We harvested immature rice seeds with or without high temperature stress, and examined seed quality and activities of α-amylase and ADPglucose hydrolyzing enzyme NPPase that are relatively heat-stable compared with the other enzymes related to starch metabolism. Both activities were shown to increase in cultivars which exhibited poor quality of seed compared with the others. Proteome analysis showed that the expression of proteins related to glycolytic pathway, reactive oxygen species elimination, and chaperonin are affected by high temperature stress. Furthermore, we analyzed oxidation status of proteins in the immature seeds with biotinhydorazide that binds to carbonyl group of proteins, indicating that proteins oxidized under high temperature stress significantly increased in heat-susceptible cultivars.

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Molecular chaperones DnaK and DnaJ are widely known to act as partner proteins during proper folding of various proteins. In cyanobacteria Synechococcus sp. PCC7942, these chaperones were found to exist as multi-gene family with 3 dnaK (dnaK1, dnaK2, dnaK3) and 4 dnaJ (dnaJ1, dnaJ2, dnaJ3, dnaJ4) homologs. We have previously reported that dnaJ1, dnaJ2 and dnaJ3 are essential for nomal growth, with the latter exist as partner of the dnaK3 and co-localize of the thylakoid membrane.
In order to elucidate other specific function of DnaJ3, we carried out genetic analysis. Using PCR-mutagenesis to introduce point mutation in non-conserved C-terminal region, we were able to isolate a temperature sensitive mutant resulting from mutation F193L. Moreover, two of the suppressor mutations mapped severally in the Rbp2 (syc2097) or PNPase (syc1666) . We are currently analyzing the possible mechanism involved in the suppression of DnaJ3 mutation.

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The cyanobacterial Rbp proteins are RNA-binding protein with a single RNA recognition motif (RRM). The unicellular cyanobacteria Synechococcus sp. strains PCC6301 and PCC7942 have three homologous genes, rbp1, rbp2 and rbp3, encoding Rbp1 (107 aa), Rbp2 (99 aa) and Rbp3 (142 aa). In our previous research, we reported that the expression of rbp1 was cold-inducible and the rbp1 disruptant grew poorly at low temperatures, whereas that of rbp2 was cold-depressed and the rbp2 disruptant grew normally even at low temperatures. In order to characterize three rbp promoter activities under the different stress conditions, chimeric genes containing each rbp upstream sequence fused to the bacterial luciferase (luxAB) as a reporter were transformed in Synechococcus PCC7942. We report the results of the promoter responses of three rbp genes under the different stress conditions.

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In northern part of Japan, rice crop production is occasionally damaged severely by low temperature in summer due to the disturbance in pollen formation. Rice is most chilling sensitive at the onset of microspore release. We identified 3 novel genes whose expression levels were remarkably affected by chilling in rice anther at the microspore release stage. Through the application of chilling stress in rice anther, the expression of OPDAR1 (12-oxo-phytodienoic acid reductase 1) and Radc1 (Rice anther down-regulated by chilling 1) was repressed and the expression of SAMDC1 (S-adenosylmethionine decarboxylase 1) was induced. Expression patterns of these genes were histochemically examined in anthers from transgenic rice plants bearing individual gene promoter::gus fusions. We will discuss the role of jasmonate, polyamine and these genes under cool temperature stress in pollen development stage.

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To investigate changes in polyamine level in rice seedlings treated under various environmental stresses and plant hormones, polyamines are extracted and subjected to HPLC analysis. The HPLC analysis revealed that level of a major polyamine, putrescine, is significantly increased in rice seedlings in response to chilling and submergence stresses as well as ABA. It is well known that arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) are involved in putrescine biosynthesis in plants. To test either of ADC or ODC is responsible for the increase in putrescine level in rice seedlings treated under the stresses and ABA, we have isolated ADC and ODC genes from rice by RT-PCR. The gene-specific probes were prepared from 3'-UTR of the ADC and ODC genes for northern analysis. Results of northern analysis with the gene-specific probes are discussed in relation to the increase in putrescine level in rice seedlings treated under chilling and submergence stresses.

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Freezing temperatures are major factors limiting the geographical locations suitable for growing crops, and periodically account for significant losses in plant productivity. In order to identify rice stress-inducible genes, recently transcriptome analysis was conducted and a number of genes were identified to have high expression level in response to various abiotic stresses, including cold stress. To identify a novel cis-acting element that function in cold stress-response in rice, we performed promoter analysis using promoter regions of five cold stress-inducible genes identified by rice microarray analysis. These promoter regions were fused to the GUS reporter gene and introduced into rice. We analysed these transgenic plants and identified a novel cis-acting element for cold-stress-response gene expression in rice. We found this element in the all five promoter regions. To assess the frequency of occurence of this sequence a homology search within the promoter regions of rice cold-stress inducible genes was conducted.

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Rice is a typical chilling sensitive plant. Cell suspension cultures of rice also suffer injuries at chilling temperatures. However, we found out that exogenous application of ABA at 25C induces freezing tolerance in rice cell cultures as well as in rice seedlings. We found more than 80 CBB-stainable proteins were newly expressed or increased in ABA treated cells using 2D electrophoresis. One of them was RAB24, thiol antioxidant. We cloned a homologue of RAB24 from bromegrass cells which increased freezing tolerance more drastically by exogenous ABA. We introduced the bromegrass RAB24 into rice plants using agrobacterium transformation. We determined physiological and morphological traits of the transgenic lines including some stress tolerance

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As temperature decreases, cold-hardy plants acquire freezing tolerance gradually. Freezing tolerance can be induced in bromegrass (Bromus inermis Leyss) suspension cultures by exposure to low temperature or by exogenous application of abscisic acid (ABA). To understand the mechanisms of freezing tolerance in bromegrass, we analyzed the genes responsive to low temperature and ABA using rice cDNA microarray. Previously, we cloned a chitinase gene induced by cold and analyzed activities of the gene products expressed in E. coli.
We performed Northern blot analysis of this gene using total RNA extracted from bromegrass cells under various stresses. We found that the expression pattern differed considerably from those of other chitinase genes. To determine functions of the cold-induced chitinase, we constructed transgenic plants in an attempt to identify the activities and contribution of this gene to freezing tolerance.

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Cold shock domain (CSD) protein is widely distributed in bacteria, plants, and animals. The highly conserved CSD serves as a nucleic acid-binding domain for the functions in translational and/or transcriptional regulation. The cold shock (domain) proteins (Csp) from E. coli have a RNA chaperone activity. We have identified a cDNA encoding a CSD protein (WCSP1) from winter wheat. The cold-induced WCSP1 protein showed a nucleic acid binding activity and also complemented a cold sensitive phenotype of the E.coli csp mutant. To further investigate the function of WCSP1 in vitro, a molecular beacon system was utilized to demonstrate in vitro nucleic acid melting (unwinding) activity. The wild-type WCSP1 showed a nucleic acid melting activity, while the WCSP1 protein with point mutations in RNA-binding motifs were unable to do it. The data supported a function of WCSP1 in destabilizing RNA secondary structures during cold acclimation in wheat.

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Winter wheat plants can develop freezing tolerance after a period of exposure to low but nonfreezing temperature. To analyze this process we have undertaken a large-scale analysis of the wheat transcriptome. For cold treatment the wheat plants (cv. Valuevskaya, highly freezing tolerant) were exposed to 2/0.5^oC for 7 weeks. Total RNA was isolated from control and cold-acclimated leaves. The RNA samples were labeled with Cy3- or Cy5-dCTP and hybridized to 22k wheat oligo microarrays. In the comparison between control and cold-acclimated leaves, 2,435 genes (15.5% of the 15,679 genes) were significantly regulated by long-term cold acclimation. In addition to the using of winter wheat cv. Valuevskaya, we also used spring wheat cv. Haruyutaka (very low freezing tolerant) for comparing gene expression patterns of both cultivars. As a result, 1,571 genes (9.0% of the 17,503 genes) including 900 genes showing cold-dependent expression were differentially expressed between the two cultivars.

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Plants respond and adapt to abiotic stress to survive under stress conditions. Many genes that respond to drought and cold stress at transcriptional level have been described, but there are few reports on genes involved in the recovery process from abiotic stress. We have analyzed gene expression profiles of Arabidopsis in the process of dehydration stress and rehydration from dehydration stress using a 7k RAFL-cDNA microarray (Oono et al. 2003, Plant J). In this study, we analyzed gene expression profiles in the process of cold acclimation and deacclimation using the 7k RAFL-cDNA microarray and an Agilent 22k oligonucleotide array (submitted) and are studying functional analysis of several genes.
We analysed relationship between gene expression profiles and metabolic profiles in cold acclimation and deacclimation. We discuss roles of key genes involved in the biosynthesis of carbohydrates such as raffinose and maltose, and proline that have important roles in cold acclimation.

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Although cold acclimation process has been studied extensively using Arabidopsis plants, determination of responses to cold at cellular level may be difficult due to the complexity of plant individuals. To study such responses, we characterized cold acclimation process of Arabidopsis T87 suspension cultured cells. Freezing tolerance increased only in cells at the late lag phase after 2 days of cold acclimation. Expression of cold-regulated genes reached maximum at 1 day and subsequently decreased rapidly. Changes in sugar content and osmotic concentration were not associated with freezing tolerance. From these results, we hypothesized that an increase in freezing tolerance both in whole plants and cultured cells from 0 to 2 days is primarily due to the cellular responses to cold and the maintenance of the tolerance in plant individuals after 2 days requires the whole plant responses. To verify this hypothesis, microarray and 2D-PAGE analysis are in progress.

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In a previous report, synaptotagmin-like protein, a membrane repair-related protein, which quickly increased during cold acclimation was identified in the Arabidopsis plasma membrane fraction (Kawamura and Uemura 2003). Rapid membrane repair is a general phenomenon in animal cells although no report has been published on this mechanism in plant cells. The mechanical disruption of plasma membrane results in the incursion of extracellular calcium into cytoplasm, and consequently, endomembranes fuse into the damaged plasma membrane through the exocytosis in which SNAREs and synaptotagmin family are involved. When the plasma membrane is mechanically disrupted by the ice formation and/or by the freeze-induced dehydration, it is possible that the plant cells rapidly repair the disruption dependently on the extracellular calcium. In our results, the freezing tolerances of Arabidopsis protoplasts isolated from control and cold-acclimated leaves remarkably decreased when survival tests were carried out in the buffer without calcium. (Supported by JSPS Research Fellowships)

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We have already reported that the acidification to pH 2.0 with sulfuric acid solution in the freeze-thaw process promoted the freezing injury of leaves of cold-acclimated winter wheat (Triticum aestivum L. cv. Chihokukomugi). In this study, we examined a modified freezing test to estimate the influence of freeze-thaw cycles in the acid meltwater in early winter or early spring on the viability of wintering plants. When freeze-thaw cycles were repeated four times, survival rates of leaves treated with sulfuric acid solution gradually decreased as freeze-thaw was repeated. Further, we estimated the influence of acid condition in the freezing, thawing or post-thawing process on the viability of leaves of winter wheat. Survival rates of leaves decreased by existence of sulfuric acid solution in the thawing process. These results suggested that acid condition in the thawing process might become a factor to promote freezing injury of winter wheat.

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Systemic acquired resistance (SAR) is one of plant defense systems. To investigate the influences of environmental stress on SAR induction, we analyzed the effects of abscisic acid (ABA) on SAR. In this study, we used 1,2-benzisothiazole-3(2H)-one 1,1-dioxide (BIT) and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) for SAR induction, which activate upstream and downstream of SA in SAR signaling pathway, respectively. ABA treatment suppressed SAR induced either by BIT or BTH in Arabidopsis. The environmental stress such as salinity, drought and ABA suppressed the expression of PR-1 gene. On the other hand, the expression of some of ABA-biosynthesis or ABA-responsive genes were down-regulated by treatment with BIT or BTH. These data suggest that there should be some crosstalks between the ABA-mediated signaling and SAR signaling.

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The production of reactive oxygen species at the cell surface, called the oxidative burst, is the earliest events detected during incompatible interactions between plants and pathogens. Treatment of potato tubers with the pathogen-derived elicitor caused a rapid and transient accumulation of H₂O₂, followed by a massive oxidative burst 6 to 9 h after the treatment. StrbohA and StrbohB are thought to contribute to the bursts. Because NADPH-dependent O₂^--generating activity was located predominantly in plasma membrane fraction, Strboh has been suggested to localize at the plasma membrane. Here we demonstrate that StrbohA and StrbohB were localized in plasma membrane fractions separated by sucrose density gradient centrifugation using their specific antibodies. Additionally, NADPH-dependent O₂^--generating activities, which were DPI-sensitive and NaN₃-insensitive, were also distributed in plasma membrane fractions. These data suggest that StrbohA and StrbohB are localized at plasma membrane and regulate the oxidative burst in the defense responses.

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To clarify the processes involved in plant immunity, we isolated and characterized a single recessive Arabidopsis mutant, cad1 (constitutively activated cell death 1), which shows a phenotype that mimics the lesions seen in the hypersensitive response (HR). This mutant shows spontaneously activated expression of pathogenesis-related (PR) genes, leading to a 32-fold increase in salicylic acid (SA), indicating that activated SA signaling leads to the HR in cad1.
Inoculation of cad1 mutant plants with Pseudomonas syringae pv tomato DC3000 shows that cad1 mutants show a defense activation phenotype. Cloning of CAD1 reveals that this gene encodes a protein containing a domain with significant homology to the MACPF (Membrane attack complex and perforin) domain of complement components and perforin that are involved in innate immunity in animals. Furthermore, genetic analysis showed that the CAD1 protein negatively controls the SA-mediated pathway of programmed cell death in plant immunity.

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Microarray analysis of the chitin oligosaccharide elicitor-treated cultured rice cells revealed the enhanced expression of a receptor kinase gene, of which extracellular domain showed a high homology with a PR protein. The expression of this gene was induced by chitin oligosaccharides depended on their size and structure. Cycloheximide itself also induced the expression of the gene in the absence of the elicitor. Protein kinase/phosphatase inhibitors and DPI did not affect the induction of the gene expression. The encoded receptor kinase protein was detected in the plasma membrane by western blotting. Intracellular domain of the receptor kinase expressed in E. coli as a GST-fusion protein showed a strong auto-phosphorylation activity but those proteins with mutations for putative active site amino acid residues showed no or weak autophosphorylation. Knock-down cell lines by RNAi as well as over-expressor are being developed.

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We reported the purification and partial structure of the glycan elicitor from Alternaria alternata 102 at the last annual meeting. As the further structural analysis, the detail structure was analyzed by reducing sugar analysis, MALDI-TOF/MS and NMR spectroscopy. These results suggested that the elicitor was non-cyclic β-1,3-1,6-oligoglucan and the polymerization of the elicitor was in the range from 8 to 17.
Glucan elicitor could induce class IV chitinases, but not SA and MeJA treatments in BY-2 cells. To better understand an elicitor response signaling, we analyzed glucan elicitor responsible genes using a cDNA microarray, which contained about 16,000 EST. About 300 genes were induced 1h after the treatment with laminarin (β-1,3-glucan) or sugar fraction form A. alternata culture medium. In comparison to data sets of 2,4-D, ABA, BA, MeJA and SA treatments, 14 putative transcription factor genes were identified as an early and glucan elicitor specific inducible genes.

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Interaction of suspension-cultured rice cells with rice blast fungus, Magnaporthe grisea, was investigated. Suspension-cultured rice cells were incubated with spores of compatible or incompatible race of M. grisea in the same medium and the production of H₂O₂, expression of defense-related genes and induction of cell death were analyzed. So far tested, we could not see any significant differences in gene expression profile and induction of cell death. H₂O₂ was transiently produced and the peak was 2-3 h after inoculation with spores of compatible and incompatible races of M. grisea. Incompatible races induced higher level of H₂O₂ than compatible races, whereas the addition of spores washed by centrifugation induced the similar level of H₂O₂ irrespective of compatibility. The supernatant fraction of spore-suspension had H₂O₂-degrading activity, and the activity disappeared by heating at 100C for 5min, indicating that proteinaceous factors are responsible for the degrading action.

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Jasmonic acid (JA) is known to be an important signaling molecule in defense response against wounding. JA is synthesized in the octadecanoid pathway, which employs several enzymes including allene oxide synthase (AOS), allene oxide cyclase (AOC), and 12-oxo-phytodienoic acid reductase (OPR). We generated transgenic rice plants in which the expression of the genes encoding AOS, AOC, and OPR (OsAOS, OsAOC, OsOPR1 and OsOPR3 ) is suppressed by RNAi method. JA content in leaves of the OsAOS-suppressed transformant was reduced approximately 62% of the wild-type level. JA content was undetectable in leaves of the OsAOC-suppressed transformant. JA content in leaves of the OsOPR1-and OsOPR3-co-suppressed transformant was reduced approximately 3% of the wild-type level. These materials will provide a useful tool to elucidate the functions of the intermediates (e.g., 12-oxo-phytodienoic acid) of the octadecanoid pathway in rice defense responses.

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Immunoelectron microscopy and a quantitative analysis of immunogold labeling of a glutamine synthetase (GS; EC 6. 3. 1. 2) revealed that, in mesophyll cells of mature leaves of Azolla filiculoides, almost all GS was present in chloroplasts. By contrast, in hair cells, abundant labeling of GS was observed both in chloroplasts and in the cytoplasm. In hair cells of cyanobiont-free plants, the labeling of GS of both chloroplasts and cytoplasm was very weak compared to that of cyanobiont-containing plants. The findings suggest that hair cells play an important role in the assimilation of ammonia released by the cyanobiont.

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Plants respond to insect attack and pathogen by activating the expression of genes involved in disease resistance. In rice, jasmonic acid (JA) is assumed to play an important role as a signaling molecule in defense responses. The synthesis of linolenic acid (LA), the precursor of JA, is catalyzed by two plastidial ω-3 fatty acid desaturases which are encoded by two genes, FAD7-1 and FAD7-2. To determine the role of JA, we generated transgenic rice in which the expression of both FAD7-1 and FAD7-2 is suppressed by RNAi method. LA content in leaves of the FAD-suppressed transformant (F7Ri) was reduced to approximately 13% of the wild-type level. Wound-induced JA accumulation of the F7Ri was reduced to approximately 10% of the wild-type level. These results indicate that F7Ri line is a useful tool for studying the role of JA in rice.

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To establish an efficient, high-throughput screening/evaluation method for mutants for defense signaling in A. thaliana, we first studied the ROS generation induced by chitin oligosaccharide elicitor in Arabidopsis. By using 96-well microtiter plates and young seedlings, we could analyze the ROS generation quite efficiently on a single plant base and also recover the plants for further cultivation and analyses. Chitin oligosaccharide elicitors induced ROS generation in a size and dose dependent manner as similar to those observed in cultured rice cells. Interestingly, ROS generation was mostly confined in the roots of the seedlings, whereas the expression of typical defense genes were observed in both roots and leaves of the chitin-treated seedlings. Pharamacological studies indicated the involvement of protein phosphorylation and NADPH-oxidase in the ROS generation. The advantage of the experimental system for dissecting plant defence signaling and screening of new mutants for chitin responsiveness will be discussed.

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In legumes, ENOD (early nodulin) genes are expressed in early stage of nodulation. ENOD40 is one of the ENOD genes which is also found in nonlegume plants. Although extensive research has been carried out, its function is still largely unknown. We recently found an antisense ENOD40 (asENOD40) in Lotus japonicus. We investigated the expression of asENOD40 by Real Time RT-PCR, and revealed that the expression of asENOD40 is far weaker than sense RNA (sENOD40), although it is up-regulated in nodule. Furthermore, in situ hybridization analysis showed that asENOD40 RNA exists in the infected region of the nodule, compare with sENOD40 expression near the vascular bundles of nodule. We hypothesize that asENOD40 can have a distinct role for nodulation other than its possible role for suppression of sENOD40.

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A model legume Lotus japonicus has been utilized for transgenic studies. The 35S promoter has been widely used in plant transformation systems, but it is suggested that its activity is relatively low in L. japonicus. To search for a new promoter, we have isolated polyubiquitin gene (LjUBQ) from L. japonicus, containing an intron immediate upstream of the initiation codon. Because it was known that an intron enhanced the polyubiquitin gene expression in Arabidopsis thaliana, chimeric genes containing 5'-upstream region including the intron of LjUBQ and the β-glucronidase (GUS) coding region were constructed. In the case of Agrobacterium rhizogenes-mediated transformation, the GUS activity under the LjUBQ promoter was two-fold greater than those under the 35S promoter. In Agrobacterium tumefaiens-mediated transformation, LjUBQ promoter showed higher activity in shoot and root, in comparison with the 35S promoter. These results indicate that the LjUBQ promoter is suitable for constitutive gene expression.

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Leguminous plants have the capacity to perform a nitrogen-fixing endosymbiosis with rhizobia. The plant symbiotic program is triggered by perception of highly specific signals, Nod factors, secreted by rhizobia. Invasion of rhizobia followed by differentiation of a nitrogen-fixing nodule involve complex interaction mechanism of both organisms. In the model legume Lotus japonicus, cDNA macroarray analysis of the nodule formation process has been reported (Kouchi et al. 2004). For a comprehensive approach to analyze the expression profile of the nodulation process, we performed Serial Analysis of Gene Expression (SAGE) of uninfected root and nodulating root abundant in nodule primordia. We have a special interest in transcription factors because they may represent key regulators of the nodulation program. The cDNA array and SAGE analyses have led us to identify 22 transcripts harboring sequence similarity to known transcription factors. Utilizing the hairy root transformation system, we initiated functional analysis of these genes.

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The nodulation mutant crinkle (crk) was identified from the model legume Lotus japonicus, which shows abortion of infection thread development between the junction of epidermis and cortex. The mutant shows defects in development of trichome, root hair, as well as pollen tube. We have recently identified that the male sterility is caused by the improper maturation of microspore, possibly caused by abortion in tapetum vacuolation. We regard crk is important for polar growth in general, and intend to clone the gene by map-based strategy. Initially crk locus was mapped in the middle of the linkage group 5, between TM0151 and TM0024. From these markers, TAC/BAC contigs were generated, and by the F2 population of 838 individuals, we narrowed down the candidate region into roughly 500kb.

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Tm-1 is a semi-dominant trait in tomato that inhibits the multiplication of Tomato mosaic virus (ToMV) within a single cell without inducing the hypersensitive cell death. Tm-1 has been widely introduced in commercial tomatoes, however, it remains unidentified because of its difficulty in genetic mapping. Recently, an in vitro translation-replication system for ToMV RNA has been established using the lysate of evacuolated protoplasts from uninfected tobacco BY-2 cells. In the BY-2 lysate supplemented with the Tm-1 lysate, ToMV RNA replication was specifically inhibited without any detectable effect on the accumulation of ToMV replication proteins. This result suggests that the Tm-1 factor inhibits the function of ToMV replication proteins, but does not affect the production or the stability of these proteins. The property of the Tm-1 activity on the liquid chromatography will also be reported.

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Bacterial blight ( Xanthomonas oryzae ) is one of the most destructive pathogens to rice in south-west area of Japan and South East Asia. The rice cultivar "Nipponbare" shows the high field resistance against bacterial blight disease. We inoculated bacterial blight pathogens to "Nipponbare" mutants which were induced by the insertion of the endogenous retrotransposon Tos17 , and have screened 20 susceptible mutants among 3,000 mutant lines in two years. By genomic southern hybridization analysis and investigation of genetic segregation among progenies, two mutant lines were found to have tightly close relationship to the Tos17 insertion. We are analyzing the affected genes and the function of the genes to elucidate the mechanism of the field resistance against bacterial blight disease in rice.

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LSD1 (Lesion Simulating Disease 1) is a negative regulator of oxidative stress-induced cell death involved in disease resistance. To clarify the function of LSD1 as a regulatory protein, we are focusing on the protein-protein interaction partners identified by yeast two-hybrid screening. One of those, AtbZIP10 specifically interacts with LSD1. Although AtbZIP10 has a potential NLS, these are co-localized in cytoplasm. However any change of AtbZIP10 distribution was not observed in lsd1. Using split YFP analyses, we could confirm the LSD1-AtbZIP10 interaction in planta. The abolishment of AtbZIP10 partially suppressed the lsd1-induced runaway cell death (lsd1-rcd). On the other hand, AtbZIP10 overexpression induced more lsd1-rcd. Similarly AtbZIP10 overexpressor demonstrated more resistance to both compatible and incompatible Peronospora Parasatica (P.p.), whereas enhanced susceptibility to incompatible P.p. was observed in atbzip10. Taken together, AtbZIP10 would regulate the target genes involved in the programmed cell death at the hypersensitive response in plant disease resistance.

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