Enteromorpha compressa is a euryhaline organism, capable of growing with a wide range of salinity. When we transferred E.compressa to a hyper saline medium, DMSP was significantly accumulated. DMSP, which is reported to be accumulated in limited species of marine algae and higher plants, is the only major compatible solute in E.compressa. The aim of this study is to investigate the metabolic regulation of DMSP biosynthesis in E.compressa for the environmental adaptation.
We fed the ³⁵S- or ¹⁴C- labeled precursor of DMSP to E.compressa, and examined the labeled metabolites. The activity of DMSP biosynthesis from [³⁵S]-SO₄^2- and [³⁵S]-cysteine were increased, on the other hand, the activity from [¹⁴C]-methionine remained constant. The obtained results suggest that the biosynthesis of DMSP in salt-treated E.compressa is regulated by the supply of methionine.

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Salicornia europaea, a member of the family Chenopodiaceae, is a highly salt-tolerant halophyte, which is distributed in the salt marsh. To analyze the mechanisms of salt tolerance at cellular level, suspension-cultured cell was established. The suspension-cultured cell can grow in Murashige-Skoog medium containing 1x10 ^-7 M of 2,4-dichlorophenoxyacetic acid, and 1x10 ^-6 M of 6-benzylaminopurine. In addition, this suspension-cultured cell could grow in the presence of 600 mM NaCl. To find the key genes for the salt-tolerance, functional screening was performed using Escherichia coli as the host organism. A transformant expressing fasciclin-like arabinogalactan protein (FLA) homologue revealed to have enhanced salt tolerance. The role of FLA in the salt tolerance was investigated. This research was supported in part by Industrial Research Grant Program in '03 from NEDO.

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To improve soil environment, utilization of terrestrial cyanobacteria attracts public attention. Nevertheless, the mechanism of desiccation tolerance of this organism has not yet been elucidated. Our previous report revealed that in desiccation tolerant cyanobacterium Abnabaena sp. PCC 7120, expression of genes for trehalose synthesis(all0168 and all0167) and hydrolysis(all0166) was up-regulated by desiccation. Trehalose is a nonreducing disaccharide, which is thought to protect cellular components against various stresses and to be involved in signal transduction as well. A small amount of trehalose was accumulated by desiccation in Abnabaena wild type. Gene disruption of all0168 resulted in a decrease in trehalose level and desiccation tolerance in comparison with wild type, whereas diruption of all0166 resulted in an increase in them. We compared the gene expression profile of wild type and all0168 disruptant by microarray analysis. We will disccuss the role of trehalose in desiccation tolerance of Abnabaena based on these results.

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DREB2A is a transcriptional factor that functions in drought- and high-salinity-responsive gene expression. This protein specifically binds to DRE/CRT in the promoter region of stress-inducible genes. It seems that a post-translational modification is necessary for its activation. We carried out domain analysis of DREB2A and found that a negative regulatory domain exists in the central region of DREB2A and deletion of this region makes DREB2A constitutive active form (DREB2A CA). We generated transgenic Arabidopsis plants overexpressing 35S:DREB2A CA. The transgenic plants showed growth retardation and improved drought stress tolerance. The microarray analysis revealed that many water-stress inducible genes were expressed in the transgenic plants even under non-stress condition. We analyzed the DRE/CRT core sequence in the promoter regions of the DREB2A target genes. We also generated transgenic plants overexpressing DREB2A CA driven by the stress-inducible promoter and analyzed their phenotypes and stress tolerance.

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To analyze the mechanisms of salt-tolerance in a mangrove plant, Bruguiera sexangula, functional screening of the mangrove cDNAs encoding proteins essential for the salt-tolerance mechanisms was done using Eschericha coli. as the host organism. A transformant expression eukaryotic elongation factor 1A homologue (Bs-eEF1A) revealed to have enhanced salt and osmotic stress tolerance. In general, eEF1A is highly abundant protein responsible for the delivery of aminoacyl-tRNA to the accepter site of ribosomes during peptide chain elongation. It could not be explained why enhanced salt-tolerance was acquired in the E. coli transformant by expressing Bs-eEF1A. In this study, we show that Bs-eEF1A give rise to enhance heat shock stress tolerance in E. coli. Furthermore, we demonstrated that Bs-eEF1A has chaperone-like properties in vitro. These data suggest that Bs-eEF1A, in addition to its original function in translation elongation, might play an important role in the salt tolerance mechanisms of B. sexangula.

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Plants are able to perceive moisture gradients, the roots respond by growing towards water. This phenomenon is called hydrotropism. By using our HRS (Hydrotropism Research System) we isolated a mutant (cs2448) that does not exhibit the hydrotropic response. cs2448 plants lack the AKT2 gene, which encodes a voltage-gated K⁺ channel known to localize mainly in the phloem of aerial parts of Arabidopsis.
In this study ,we analyzed AKT2 expression in roots under dehydration stress, by using transgenic Arabidopsis plants expressing an AKT2 promoter::GUS fusion gene. GUS activity was detected at root tips; however, no activity was detected in root caps, which are regarded as the primary sites for hydrosensing. When akt2 plants were subjected to osmotic stress, root elongation was reduced compared to wild type. These results suggest that AKT2 plays a role in root elongation during dehydration stress. Further expression analysis of the AKT2 gene is now in progress.

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Glycine betaine accumulates at high level as an osmoprotectant inside cells of several plant families which have tolerance to salt or drought stresses. Betaine aldehyde dehydrogenase (BADH) catalyses the last reaction of two-step oxidation of step in biosynthesis of glycine betaine from choline.To understand the role of BADH during stress response, we have isolated a BADH gene from oat (Avena sativa), and investigated changes in levels of activity and transcripts of BADH. A partial cDNA was amplified using primers designed from a region conserved in several known BADH sequences and a full-length cDNA was obtained by RACE methods. RT-PCR analysis showed that the transcription level of BADH was increased by salt, drought and ABA treatments. The salt induction of BADH transcript varied, depending upon the developmential status of plants. The predicted amino acid sequence had unique SKL tripeptide at C-terminus, indicating that oat BADH is localized in peroxisome.

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Three new genes induced by flash flood were cloned from flash flood-tolerant rice cultivars and their full length cDNA sequences were determined. By homology research, the first is homologous to monogalactosyldiacylglycerol synthase (MGDGS), the second to glycogenin glucosyltransferase (glycogenin) and the third to cation transport protein ChaC (CTP). The expressions of their genes were increased in response to flash flood, drought, high salt, ethylene, abscisic acid and gibberellin, but methyljasmonic acid and cold stress had no effect on their expressions. Among these three genes, the expressions of MGDGS and CTP gens were increased by flash flood not only in flash flood-tolerant rice cultivar but also in flash flood- sensitive one. However, the expression of glycogenin gene was increased in response to flash flood only in flash flood-tolerant cultivar. The flash flood-tolerance of rice may relateto the capacity for polysaccharide synthesis.

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Under high salinity condition, plant growth is inhibited due to osmotic and ionic stresses. Especially, the effect of osmotic stress is mainly observed during the initial phase of salt stress treatment. To investigate the effects of osmotic stress caused by salt stress, we compared expression profilings at 1 h and 24 h of 20% PEG and 200 mM NaCl stresses using barley cDNA microarray carrying 460 stress-responsive genes. During the initial phase of osmotic and salt stresses, 22 and 62 genes were up-regulated, and 30 genes were down-regulated, respectively. Furthermore, we found the commonly regulated candidates, 18 up-regulated and 16 down-regulated genes, suggesting these expressions are modulated by osmotic stress signaling under salt stress. On the other hand, we also found the differentially regulated genes under osmotic or salt stress, and this indicated the complexity in regulation of gene expression under osmotic and salt stresses.

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Proline is a common osmolytes in osmotic-stressed plants. We previously reported that an ACTCAT sequence acts as a cis-acting element involved in hypoosmolarity- and proline-responsive expression of an Arabidopsis proline dehydrogenase (ProDH) gene.
Search of the database for cis-acting elements revealed that the ACTCAT sequence is similar to the GCN4 motif recognized by bZIPs. We examined whether Arabidopsis bZIPs function as transcription factors for the ACTCAT sequence. Transient assay revealed that the four bZIPs, named an ATB2 subgroup, activate the expression of a reporter gene driven by the ACTCAT sequence. The transactivation activity of the ATB2 subgroup was enhanced by hypoosmolarity. The recombinant ATB2 subgroup proteins specifically bound to the ACTCAT sequence. The sGFP::AtbZIP11/ATB2 fusion protein is localized in the nucleus. These results suggest that the ATB2 subgroup functions as a transcriptional activator for hypoosmolarity-inducible expression of ProDH. We are currently analyzing transgenic >Arabidopsis plants overexpressing the ATB2 subgroup cDNAs.

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We have reported that submergence decreases the osmotic concentration of cell sap by decreasing the levels of sugars and amino acids, thereby inhibiting elongation growth in azuki bean epicotyls. The present study was undertaken to elucidate the mechanisms by which submergence decreases the levels of sugars and amino acids in epicotyls by examining the effect of submergence on the activities of proton symport, which is involved in the unloading of these osmotica. The levels of protons in apoplastic solutions of epicotyls grown underwater were significantly lower than those grown in air. Also, under water, the levels of ATP in epicotyl cells decreased by half. These results suggest that submergence decreases activities of proton pumps by decreasing the levels of ATP in epicotyl cells. Such a decrease in activities of proton pumps may be involved in inhibition of the unloading of both sugars and amino acids into epicotyl cells under submergence.

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Bryophytes are widely distributed on the earth and some bryophytes are known to have high desiccation tolerance. In order to find out the mechanism of the tolerance, we have investigated changes in various photosynthetic activities during drying using bryophytes grown under different water environments. We found that PSII fluorescence is quenched and the PSII activity is lost by dehydration, which seems to be important to protect PSII under dry conditions. Here, we will report on the changes in fluorescence Work Integral (WI) due to dehydration, which reflect changes in antenna size of PSII or the rate of Q_A reduction, in a desiccation-tolerant bryophyte Bryum argenteum. The data show that the moss also has PSIIα and PSIIβ, and the ratio was changed by drying. Relationship among fluorescence quenching, changes in Fv/Fm, and changes in the Q_A reduction rate will be discussed.

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Contrary to the thus far accepted knowledge we have found that light enhances hook curvature in many dicots including tomato through low fluence and very low fluence responses of phytochrome. Since hook curvature is a growth response at the apical part, the role of phytohormones was examined. Evolution of endogenous ethylene was not altered by R or FR, which enhances curvature. Application of ACC, an ethylene biosynthesis precursor, or of ethylene to dark-grown tomato seedlings caused hook to open. Treatment with PAC, gibberellin biosynthesis inhibitor, enhanced hook curvature as markedly as R or FR. When auxin transport was blocked by NPA, oppositely no hook was formed in not only non-irradiated seedlings but also R- or FR-irradiated ones. These results suggest that gibberellin and auxin, rather than ethylene, may negatively or positively be involved in hook curvature.

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It is well known that apical hook openings occur upon exposure to light in dicotyledonous seedlings. We confirmed these phenomena in Arabidopsis, cabbage etc. However, under the same experimental conditions, we found that tomato, cucumber, carrot, etc. exhibited enhanced hook curvature upon exposure to red light (R) and far-red light (FR) through phytochrome-mediated low-fluence-response (LFR) and very-low-fluence-response (VLFR), respectively. In tomato seedlings, the phyA-deficient mutant lacked FR-induced hook curvature of VLFR. Although phyA- or phyB1-deficient mutant was sensitive to R pulse irradiation of LFR, they partially lost sensitivity to continuous irradiation of R. PhyA and phyB may regulate hook curvature via R-high-irradiance-response.
The hook curvature is a growth response of the apical part of the hypocotyl. Time-dependent analyses of hook structure during curvature will be reported in LFR, VLFR, or HIR, and the process of growth will be discussed in relation to phytohormones.

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By comparison of light-sensing systems in non-photosynthetic with those in photosynthetic plants, we intend to get new insight into light-sensing systems in higher plants. Previously, PHYA in Orobanche minor, a holoparasitic plant, had been cloned and characterized. In this study, we confirmed phyA protein expression in O. minor. Plants were cultivated in darkness for 1 week, then protein was extracted and phyA was detected by zinc-blot. Additionally, phytochrome responses were checked during germination and conditioning periods by monochromatic light (red (R), far-red (FR)). It was found that germination rates were affected by light quality during both periods and the effect was remarkable during conditioning. Under continuous R and FR, germination rates were 34% and 85%, respectively. With brief irradiation by R/FR, FR/R, the germination rates were 77% and 44%, respectively. As a conclusion, germination of O. minor is inhibited by R while FR recovered the inihibitory effect of R.

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We are interested in how different are the light signaling pathways in non-photosynthetic plants from those in photosynthetic plants. We had cloned phytochrome A (PHYA) homologous cDNA (OmPHYA) from holoparasitic Orobanche minor. It was found that 26 amino acids, which are well conserved in other phyAs from photosynthetic plants, had been substituted in OmphyA. In this study, OmphyA was expressed in protoplasts prepared from Arabidopsis phyA null mutant, phyA-211, and the effects of the transient expression on the expression of phyA-regulated genes were relatively quantified using real-time RT-PCR. Under continuous far-red light, OmphyA induced the expression of two transcriptional factors, HY5 and CO. But expression of other three transcriptional factors, CCA1, LHY and RPT2, and four photosynthesis-related genes, FtsH, Lhcb2, MgCh and PSY, was not induced.

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In leguminous plants, beyond the circadian rhythmic movements of their leaves, blue-light irradiation induces changes in turgor pressure of the pulvinar motor cells and leaf movement. The molecular mechanism of blue-light induced leaf movement has remained unknown, although blue-light receptors and the signaling pathway have become clear in Arabidopsis thaliana. To know the above-mentioned mechanism, we have isolated a Lotus japonicus mutant showing a slow leaf-movement to blue-light irradiation, laziness. laziness was also a stomatal-opening defective mutant. We are in the process of positional cloning the LAZINESS locus. The LAZINESS locus was positioned to the upper arm of chromosome 5 and not linked to Lotus CRYPTOCHROMEs (LjCRYs) and PHOTOTROPINs (LjPHOTs), which loci encode blue-light receptors in Arabidopsis. Functional analysis of LjCRYs and LjPHOTs in Arabidopsis transgenic plants showed LjCRYs and LjPHOT1 could complement the phenotypes of the blue-light receptor mutant of Arabidopsis partially.

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Plants are classified into short-day plants (SDP) and long-day plants (LDP) based on their flowering response to day-length. Recently, signaling cascades of flowering have been intensively studied. Analysis of flowering-time genes in Arabidopsis (LDP) and rice (SDP) suggests genes controling photoperiodic flowering are conserved between them. To investigate the molecular mechanisms of rice flowering, we employed the night break (NB) experiment.
We found that Hd3a mRNA was dramatically suppressed by a single NB without affecting OsGI and Hd1 expression. Suppression of Hd3a by NB is highest in the middle of night and recovers next day with SD treatment. NB treated on se5 mutants, defected on the phytochrome pathway, exhibited no suppression of Hd3a, indicating Se5 is essential for NB effect. However, which phytochrome plays a role in NB is not known. We are investigating the effects of phytochromes on the photoperiodic flowering in rice by using phytochrome mutants.

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We have been developing a plant growth imaging system for recording visible phenotypes over a long term in high-throughput performance. Using this system, we cultivated phytochrome-deficient rice mutants grown in a long-day condition(14h light 30°C/10h dark 25°C). Standard material plants (Nipponbare,WT) showed that the 5th to 11th new leaves of main culm emerged in an average interval of 84±4.5 h. The appearance cycle of new leaves in a phytochrome A-deficient(phyA)mutant, was 12 h shorter than that in WT. On the other hand, the cycle in a phytochorome B-deficent(phyB)mutant was evidently longer (8th-11th leves:127.2±19.2h) than that in WT. The rate of a new leaf elongation in the phyB mutant was also affected. These results suggest that phyA delayed the appearance cycle of a leaf and phyB promoted leaf appearance and leaf elongation.

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Rice phytochrome family is comprised of three members, phyA, phyB and phyC. We analyzed relative levels and interactions of three phytochromes in rice (Oryza sativa cv. Nipponbare). Percent ratios of three phytochrome proteins, PHYA : PHYB : PHYC, were 84:9.5:6.5 and 57:21.5:21.5 in coleoptiles and leaves of dark-grown seedlings, respectively. However, in white light-grown seedlings the ratio was 5:65:29. The physical interactions among rice phytochromes were analyzed by co-immunoprecipitation experiments using plant extracts. Not only PHYB but also PHYA was found to be co-immunoprecipated with PHYC, which is not consistent with the results obtained in Arabidopsis. No direct physical interaction between PHYA and PHYB was detected. Formation of heterodimers between PHYB and PHYC was confirmed by gel filtration analysis. We will discuss the implications of the physical interactions between different phytochrome species in regulating plant photomorphogenesis.

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Phytochrom B (phyB) is a dimeric chromoprotein that detects the quantity, quality, and duration of red or far-red light throughout the entire life cycle of plants. Upon absorption of red light, phyB translocates from the cytoplasm to nucleus, and regulates gene expression through interaction with transcription factors such as basic-helix-loop-helix proteins. The PAS domain within the phyB C-terminal domain contains determinants necessary for nuclear translocation and signal transduction. Here we showed the solution structure of PAS1 domain, one of two PAS domains, determined by multidimensional NMR spectroscopy. The structure of a loss of function missense mutant was partially denatured. Our structural and biochemical data revealed that the N-terminal region of the PAS1 domain is necessary to form homodimer, and the PAS1 domain does not interact with the other PAS domain, PAS2.

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The chromophore of phytochrome is a linear tetrapyrrole, phytochromobilin. Phycoerythrobilin is one of the chromophores of phycobilin which is a cyanobacterial light-harvesting protein. These are biosynthesized through a common pathway. Although apo-phytochrome can bind with phycoerythrobilin as a chromophore, it no longer shows photoreversibility in vitro, because phycoerythrobilin lacks a double bond between C15 and C16 which is important for photoconversion and maintenance of phytochrome structure. To understand the role of phytochrome chromophore structure on plant development, we introduced the genes for phycoerythrobilin biosynthesis into the hy2 mutant which lack phytochromobilin. The plants expressing phycoerythrobilin (PEB plants) show long hypocotyl under light. Some mutants for the tetrapyrrole biosynthetic genes show the gun (genome uncoupled) phenotype which lose cooperativeness between nuclear gene expression and plastid development. PEB plants also show the gun phenotype. We discuss the relationship between chromophore structure and phytochrome function, and between tetrapyrrole biosynthesis and gun phenotype.

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Adiantum phytochrome3 (phy3) is a chimeric photoreceptor that contains both phytochrome and phototropin sequences and controls red light-induced phototropism and chloroplast photorelocation movement. phy3 has a serine/threonine kinase domain in its phototropin-related region. To examine the role of the kinase domain in phy3 signaling, we carried out biochemical and physiological analyses. We tested the kinase activity of a GST-kinase domain fusion protein expressed in E. coli and showed that the recombinant protein has autophosphorylation activity. Replacement of histidine at position 1183 (located in the kinase subdomain IV) by leucine (H1183L) impaired the autophosphorylation activity. Introduction of a PHY3(H1183L)-GFP fusion gene into prothallial cells of a PHY3-deficient mutant of Adiantum could not restore red light-induced chloroplast accumulation, whereas that of the wild-type PHY3-GFP fusion gene could complement the response. These results suggest that the phosphorylating activity of the phy3 kinase domain is involved in this phy3-mediated response.

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In order to elucidate the function of cryptochromes in rice, we had isolated all rice CRY genes (OsCRY1a, OsCRY1b and OsCRY2) and have analyzed their expression patterns at transcription level.
According to Shalitin (2002), blue-light dependent phosphorylation of AtCRY2 triggers photomorphogenic responses and eventually causes degradation of AtCRY2. We examined whether OsCRY2 underwent light-induced phosphorylation in vivo. OsCRY2 proteins were labeled by Phosphorus-32 in vivo and immunoprecipitated from the crude extract by using anti-OsCRY2 antibody. OsCRY2 was found to be phosphorylated under white-light. OsCRY2 protein and radioactively labbelled immunoprecipitation product were found at higher levels in transgenic rice overexpressing OsCRY2 than in WT. We also investigated the phosphorylation of OsCRY2 under blue- or red-light because rice CRY2 protein was degraded under red-light as well as blue-light.
We transformed rice with OsCRY2-overexpression vector or -antisense vector to analyze the roles of cry2 on the flowering time determination in rice.

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Phototropins (phot) as a blue-light receptor regulate chloroplast movement, phototropism, stomatal opening, and leaf expansion. In previous studies, the velocity of the chloroplast avoidance response was limited by amounts of PHOT2 protein. To demonstrate a relationship between the blue light responses mediating by phot2 and expression level of PHOT2 protein, we prepared the CaMV35S::PHOT2-GFP transgenic lines under the Arabidopsis phot1phot2 double mutant. Therefore, several transgenic lines that showed different expression level of PHOT2-GFP protein were obtained. We will report the analysis of the blue light response in the transformants.

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Phototropins (phot1 and phot2) are blue light receptors, mediating phototropism, chloroplast relocation, and stomatal opening in Arabidopsis. In this study, we show that 14-3-3 proteins bind to phot1, but not to phot2 when phototropin is phosphorylated by blue light in vivo. The 14-3-3 binding to phot1 was found in dark-grown seedlings, guard cells, and green leaves within 1 min after the illumination. We determined the sites of 14-3-3 binding and found two sites: phosphorylated Ser-376 and Ser-350 in the hinge1 region of phot1. The binding of 14-3-3 proteins to phototropin was very fast and reversible, and the phototropin-14-3-3 protein complex may have some unknown functions in all of these tissues. To clarify the physiological role of 14-3-3 binding to phot1, we transformed genomic phot1 with amino acid substitution of 14-3-3 binding sites into phot1 phot2 double mutant. We are now analyzing phot1-mediated responses in these transformants.

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In Arabidopsis, blue light (BL) receptors phototropin1 (phot1) and phot2 mediate phototropic response. NPH3 is an essential signal transducer in the induction of phototropic response by phot1 and phot2. Whereas expression of NPH3 protein does not change in response to in vivo irradiation, molecular size changes in response to irradiation with blue or red light. But biochemical function of NPH3 has been not clear. Here we analyzed protein expression in seedlings irradiated with various wavelength of light involved in phototropic response. We demonstrate that NPH3 is modificated by red light irradiation or dark-grown, and de-modificated by BL irradiation. The de-modification of NPH3 is sensitive to BL, and depends on BL intensity. We also show that the phot1 is essential to de-modification of NPH3, but not phot2. These results suggested that function of de-modification of NPH3 is essential to induce phototropic response by phot1.

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Arabidopsis COP1 has been reported to localize in nucleus and acts as ubiquitin E3 ligase to target photomorphogenesis-promoting transcription factor degradation via 26S proteasome pathway. To examine the relationship between nuclear localization and photomorphogenesis repressive activity, GFP-COP1 and GFP-COP1 (NLS mut) fusion genes were constructed and introduced into Arabidopsis. NLS was mutated in the latter to prevent nuclear import of GFP-COP1. Their overexpression didn't cause significant biological effects on skotomorphogenesis, though cotyledon expansion observed in both transgenic plants irrespective to wild type NLS or mutated NLS. Overexpressors were crossed to cop1-4 mutant, and the phenotypes of their F2 populations were examined. From segregation data, it appears that GFP-COP1 partially complements cop1-4 but GFP-COP1 (NLS mut) doesn't. This partial complementation is mostly on cotyledon phenotypes, but not on hypocotyl phenotypes. This might be interpreted as cotyledon folding and hypocotyl elongation in darkness require distinct threshold or different aspect of COP1 activity.

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1-aminocyclopropane-1-carboxylate oxidase (ACO) is one of key enzymes on the biosynthetic pathway of ethylene, gaseous phytohormone, in plant and several isozymes exist in rice. Phytochromes (phy) are red/far-red light photoreceptors. There are three members of phytochromes (phyA, phyB and phyC) in rice. In microarray experiments, we found that one of ACO genes (AK058296) showed altered expression (3-10 times higher than wild type) in phyAB and phyABC mutants. Expression levels of other ACO isozymes and ACC synthase genes were not changed. The AK058296 expression was repressed by far-red light pulse in wild type but not in phyA mutant. Contrary to our exception, ethylene production was not accelerated in phyAB nor phyABC mutants. The roles of phytochromes on the regulation of ACO gene expression are under investigation.

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The photosynthesis-related plastid genes are transcribed mainly by PEP, in which plastid-encoded core enzyme (α₂ββ'β") assembles with a nuclear-encoded σ factor to recognize the specific promoters for transcription initiation. In Arabidopsis, there occur six σ factor genes, Sig1-6, which might development- and environment-dependently regulate the plastid-gene transcription through their differential expression. We examined the effect of lights on Sig1, Sig2 and Sig5 transcript induction in Arabidopsis leaves. In contrast to the cryptochrome-mediated (470nm,10 μmolm^-2s^-1) rapid induction of Sig5 and Sig1 transcripts, red-light illumination (660nm) more slowly induced Sig1 and Sig2 transcripts to their blue light-levels. The red-light induction of Sig1 and Sig2 was stronger at 5 μmolm^-2s^-1 than 50 μmolm^-2s^-1. Under either light illumination Sig2 transcripts were slowly induced, but more stable compared with Sig5 and Sig1 transcripts which were rapidly degraded upon the subsequent dark-treatment. Thus, these Sig genes differ in the light-induced transcription profile and the transcript stability.

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The genes associated with various stress responses seem to be particularly prone to alternative splicing in higher plants. Arabidopsis contained 21 types of SR protein homologues as a splicing factor. We have shown that expression of a SR protein homologue 'SR41.2' is rapidly induced by high light and the SR41.2 protein has an ability to specifically bind to the RNA aptamer containing a GU_2-5-repeated sequence. Here we analyzed the physiological function of SR41.2 as a regulatory factor of stress-responsible alternative splicing events in Arabidopsis. We generated the transgenic Arabidopsis plants overexpressing SR41.2 in order to identify a gene whose splicing efficiency was regulated by the protein. No difference in the splicing efficiencies of various genes that contained GU_2-5-repeated sequence and were involved in stress tolerance was observed between the transgenic and wild-type plants. Therefore, we are trying to identify the target genes using differential display.

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We isolated a paraquat-resistant mutant (pqr-216) from Arabidopsis activation-tagged lines and found that the tolerance to oxidative stress was responsible for overexpression of a MutT-like protein (AtMutT1). Among eleven types of cytosolic MutT-like protein homologues (AtMutT1-11) in Arabidopsis, we found that AtMutT1 hydrolyses ADP-ribose and AtMutT8 hydrolyses 8-oxo-dGTP. Here we explored the molecular properties of cytosolic MutT-like proteins. Using His-tagged recombinant proteins of AtMutTs, we analyzed substrate specificities to various types of nucleoside diphosphate derivatives and deoxyribonucleoside triphosphates. The recombinant proteins of AtMutT5 and AtMutT6 also showed the ability to hydrolyse ADP-ribose, like AtMutT1. The transcript levels of AtMutT1, 5, and 6 were increased by various stress conditions, such as high light, paraquat treatment, drought, and salinity. These results suggest that MutT-like proteins in Arabidopsis are associated with the maintenance of level of ADP-ribose in the cells under stress conditions.

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The damage to plants by ozone is an environmental problem. The ozs1-1 mutant is an ozone-sensitive mutant we isolated aiming at the elucidation of the underlying molecular mechanisms, and has a T-DNA-tagged responsible gene encoding a transporter-like protein of the TDT family. Microarray data showed that expression of the OZS1 gene was not induced by ozone. The OZS1 transcripts were detected in the wild-type plants, but not in the ozs1-1 mutant plants. The ozs1-1 mutant showed normal jasmonate-sensitivity. Results of comparisons of chlorophyll content, ion leakage, and growth inhibition under ozone exposure between ozs1-1 and wild-type plants, and a comparison of the growth inhibition between ozs1-1 and other ozone-sensitive lines will be presented. Tolerance to high light or low temperature was normal in the ozs1-1 mutant. However, it showed sulfur dioxide sensitivity. The results indicate that OZS1 plays a role in the tolerance mechanism common to ozone and sulfur dioxide.

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A paraquat-resistant Arabidopsis mutant, radical-induced cell death1 (rcd1; Overmyer et al., 2000) is caused by mutation in the gene At1g32230. Expression of the rcd1-1 cDNA under the CaMV 35S promoter also increases tolerance to paraquat (Ahlfors et al., 2004), suggesting that rcd1 is a gain-of-function mutation. rcd1-2 was a missense mutation that created a premature stop codon. We carried out genetic complementation test between rcd1-2 and RCD1 over-expression lines. rcd1-2 harboring 35S::RCD1 exhibited weaker defects with respect to paraquat resistance and abnormal morphology than rcd1, indicating that rcd1 mutation was complemented by RCD1 gene. On the other hand, RCD1 over-expression lines also exhibited weak paraquat resistance like the rcd1-1 over-expression lines. These results may suggest that proper regulation of expression level of RCD1 gene is necessary for the wild-type phenotype.

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Plants are exposed with many environmental stressors that are accompanied with generation of ROS. It is important to know early responses of plants against the oxidative stressor. We found that expression of Glutathione S-transferase 3 (AtGST3) has occurred within 1hr when plants were expose to ozone. Although it has reported that AtGST3 expression is regulated with SA and ethylene, it can't explain early response of AtGST3 because more than several hours were needed to induce AtGST3 expression with treatment of phytohormones. Alternatively, we employed regulation system of GST gene expression in animal as a model for regulation of AtGST3 expression. In the model, Keap1-Nrf2 system regulates GST expression through a cis-element, designated as antioxidant responsible element (ARE). We found an ARE in AtGST3 promoter region and analyzed its function using GST3::GUS transformants. Moreover, we analyzed AtGST3 expression in knockout mutants of Keap1, MAPKs, and transgenic plants introducing animal Nrf2 gene.

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In this study we investigated the role of the CAO gene coding for the chloroplast recognition particle cpSRP43 in the acclimation to photooxidative stress. The Arabidopsis mutant chaos that lacks cpSRP43 is known to have reduced PSII antennae proteins. We revealed that the mutant has lower growth rates but significantly higher tolerance to photooxidative stress under both tightly controlled laboratory conditions and highly variable conditions in the field. This greater tolerance of the mutant was associated with a lower production of H₂O₂, lower ascorbate levels and less induction of ascorbate peroxidases. Expression of the CAO gene may be regulated by a light-dependent chloroplastic redox-signalling pathway, and was suppressed during acclimation to high light and chilling temperatures. It is concluded that presence/absence of the CAO gene has an impact on photooxidative stress tolerance, and the regulation of CAO gene may be part of the plants system for acclimation to high light.

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Ascorbic acid (AsA) has the important role of the antioxidant action and the control of the transcriptional induction of the defense gene in the plant. We reported the increase of the AsA on a tomato by JA treatment, and it was suggested that this depends on the increase of the transcription on L-galactono-γ-lactone dehydrogenase (GLDH) in the AsA biosynthesis. It is thought that GLDH was important about the control in the biosynthesis. Thus GLDH antisense tomato was created and the mutants show about 30-90% of activity of the wild strain. AsA quantity of these mutants was low generally compared with the content of the wild strain but isn't always correlated with GLDH activity. Moreover, the ratio of the reduced and the oxidized type didn't show a constant tendency and it suggested the complexity of the controlling mechanism to the AsA accumulation and the anti-oxidant action.

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Superoxide dismutase (SOD) consists of three isozymes of CuZn-SOD, Mn-SOD and Fe-SOD. Although most algae lack CuZn-SOD, the green alga Spirogyra possesses this type of isozyme. Previously, we have reported the molecular evolution of CuZn-SOD by comparison of exon-intron structure of the genes from Spirogyra, moss and fern. Here, to characterize the remaining isozymes in Spirogyra we have cloned cDNAs of SOD isozymes.
PCR using isozyme-specific primers with cDNA libraries from Spirogyra, the moss Pogonatum, the fern Equisetum and maize confirmed isozyme-specific amplification of cDNA fragments. Two types of Mn-SOD cDNAs were cloned by RACE method. The clone of 1047 bp encoded 231 amino acid residues of mitochondrial SOD, most resembled to the moss gene. The other (917 bp) encoded Mn-SOD similar to Chlamydomonas enzyme lacking mitochondrial transit peptide. However, alignment of both SODs revealed their resemblance in signal region, indicating their mitochondrial localization.

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We have characterized chloroplastic fructose 1,6-bisphosphate aldolase (FBA), one of the glutathionylated proteins previously found in the suspension-cultured cells of Arabidopsis [Plant Cell Physiol. 44:655-660]. The activity of recombinant FBA was upregulated by the conditional transition from pH 7 to 8, and reached the peak level at 10 min after transition. CD spectrometry showed the pH-dependent activation of FBA was accompanied with some structural changes. GSH inhibited the FBA activity at pH 7, but promptly activated it to the peak level at pH 8.
At pH 7, GSSG little affected the FBA activity, while it had a strong activating effect at pH 8. Thioredoxin reduced with DTT inhibited the FBA activity more strongly than DTT alone at between pH 7 and 8, this inhibition being abolished by GSSG. These suggest that the activity of FBA is specifically regulated by glutathione and that the mechanism is not a simple redox regulation.

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