Three phytochrome genes (PHYA, PHYB, PHYC) are known in rice (Oryza sativa), but biochemical analysis of PhyB and PhyC proteins are difficulty. Here, we demonstrated to express holo-phytochromes in phytochomobilin expressed E. coli (Kagawa and Suetsugu 2007) due to photometrical analysis.
All recombinant proteins showed typical red far-red photo-reversibility. Absorbance spectrum of the PhyA purified has 660 nm maximum and the difference spectrum has peaks at 666 and 724 nm. Absorbance spectrum of the PhyC protein shows a peak of around 650 nm, and the peaks of different spectrum indicated 650 nm and 725 nm, respectively. While recombinant phyB protein was purified slightly, a difference spectrum of the PhyB with around 660 and 725 nm peaks were measured. Half-life of Pfr form of the PhyB would be about 5 min, while no dark reversions of the others were observed within a few hours.

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We have indicated that daily oscillations for transcripts of a putative Dof transcription factor gene, Rdd1, were regulated by the circadian clock in rice (Oryza sativa). However, Rdd1 showed arrhythmic expression in etiolated coleoptiles. Experiments to investigate light responsiveness revealed that the Rdd1 transcript accumulated up to 1 h in etiolated coleoptiles after transferring to continuous light conditions and decreased thereafter. We examined Rdd1 expression using phytochrome (phy)-deficient mutants to clarify the regulation of Rdd1 expression by phy. We found that both phyA and phyB played a role in red-responsive Rdd1 expression and that phyA also regulated far-red-responsive Rdd1 expression. However, the expression of endogenous Rdd1 in response to far-red light was found to be modified in transgenic rice plants carried Rdd1 in an antisense orientation. This study demonstrates that Rdd1 is a phytochrome-regulated gene.

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COP/DET/FUS (Constituitive photomorphogensis/De-etiolated/Fusca) proteins negatively regulate photomorphogensis in plants. Recently, they were revealed to degrade some transcription factors, which were positive regulators of photomorphogeneis. In this work, we focus on the proteolysis through a COP/DET/FUS protein, COP10. COP10 composes of CDD complex together with UV-damaged DNA binding protein 1 (DDB1) and De-etiolated 1 (DET1). In addition, CDD complex was found to interact with Cullin4 and Rbx1. Here, we show the results of novel interacting factors with CDD core complex.

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iqm2-1 is a Salk T-DNA insertion line of the IQM2 gene whose T-DNA inserts into the first exon of the gene. IQM2 consists of six exons, and its product contains an IQ motif to which calmodulin (CaM) possibly binds in the absence of calcium ion. Using the yeast two-hybrid system, IQM2 was demonstrated to bind with Arabidopsis CaM2. RT-PCR analyses showed that the IQM2 mRNA was not transcribed downstream of the T-DNA insertion site in the mutant. iqm2-1 showed a long-hypocotyl phenotype in both white- and blue-light conditions, and anthocyanin level of iqm2-1 was lower than that of wild type. These aberrant phenotypes were restored by a complementation experiment using the wild-type IQM2 gene. We also found IQM2 to be localized to the nucleus and its transcription was up-regulated by blue light. Taken together, IQM2 is involved in calmodulin and blue light signaling.

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We have demonstrated that cryptochrome mediate blue light dependent photomorphogenic responses in rice, such as growth inhibitions of coleoptiles, leaf blades and leaf sheaths. But the blue light signaling mechanism mediated by cryptochrome remains unclear.
To investigate the underlying mechanisms, the blue light-induced changes of gene expression were analyzed by microarray experiments (Agilent rice 44k). Among the blue light-induced genes, we focused on GA2ox genes, which inactivate the active GAs. Individual RT-PCR experiments confirmed that GA2ox genes were induced by blue but not by red light.
Next, we made CRY1a gene-repressed transgenic rice in the cry1b background (cry1a-Rcry1b-1). The cry1a-Rcry1b-1 seedlings responded very weakly to blue light and showed little induction of GA2ox genes by blue light. Thus, cryptochrome dependent induction of GA2ox genes seems to be involved in the blue light-mediated growth inhibition of leaf blade and sheath.

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Plants perceive light signals to adjust their growth and development to environmental conditions. To better understand the mechanism of blue-light response in plants, we used one of the most basal land plants, Marchantia polymorpha, as a model. Blue light induced phototropism in protonemata and young thalli. Blue as well as red light pulse promoted regeneration from edges of cut thalli. Far-red light pulse following blue light pulse did not affect thallus regeneration, indicating that this response is independent of phytochrome-mediated signaling. M. polymorpha cryptochrome gene, MpCRY was isolated. Southern-blot analysis suggested that MpCRY is a single-copy gene. The expression of MpCRY was downregulated by red and blue lights and upregulated by Far-red light and dark. The expression of photosynthesis genes, such as RbcS and Cab, were upregulated by blue light. M. polymorpha shows obvious blue-light responses, and, therefore, can be a model suitable for analysis of blue-light signaling.

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Phototropins (phots) are autophosphorylating protein kinases of plant-specific blue light receptors. They regulate many blue light responses, including phototropism, chloroplast relocations, leaf flattening, and stomatal opening in Arabidopsis. A blue light-dependent autophosphorylation of the kinase activation loop in phot1 is commonly required for these responses. However, the physiological role of the phosphorylation in phot2 has not been understood. Here, we substituted Ser-761 and/or Ser-763 with Ala in the loop, and analyzed their roles by investigating the phot2-mediated responses after the transformation of the phot1phot2 double mutant. We found that the phosphorylation was required for all responses tested. The results suggest that the blue light-dependent autophosphorylation of the kinase activation loop is a common biochemical step for downstream signaling in both phot1 and phot2. We further discuss the some different contribution of the phosphorylation between in phot1- and phot2-mediated responses.

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Phototropins (phot) are one of the important blue light receptors in plants and are involved in phototropism, stomata opening, chloroplast relocation, leaf expansion and etc. The N-terminal half has two light-receptive domains (LOV1, LOV2) each of which binds an FMN and the C-terminal half forms a Ser/Thr kinase (KD). The kinase activity is inhibited by LOV2 in the dark. Blue light cancels the inhibition resulting in both auto- and substrate-phosphorylation. In Arabidopsis, there are two phots. phot1 and phot2 act as low- and high-light sensor, respectively. We have reported that bacterially expressed Arabidopsis phot1 LOV2-KD showed kinase activity on the peptide of phot1-N-terminal region as an artificial substrate. In this study, we performed biochemical analyses for phot2 LOV2-KD peptide. It phosphorylated the phot1-N-terminal region. We will discuss the differences of light-regulation mechanisms of kinase activity between phot1 and phot2.

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Actin filament dynamics during chloroplast photorelocation were analyzed in thallus cells of Marchantia polymorpha expressing tdTomato-talin. When thullus cells were irradiated with unilateral light, blue light effectively induced chloroplast movement towards the light. Red light, on the other hand, had almost no effect on the chloroplast movement. Prolonged red light irradiation however, induced non-directional movement and chloroplasts located along all the periclinal and anticlinal cell walls. In the experiments using cytoskeletal inhibitors, both movements were found to depend on actin filaments. Accumulation and avoidance movements were induced under microbeam irradiation of the cell with low and high fluence-rate blue light, respectively. When actin reorganization during chloroplast movement was examined in the system, biased localization of actin filaments on the leading edge of chloroplast was revealed in both accumulation and avoidance movements. The evidences indicate the significance of the biased actins in the mechanism of chloroplast movement.

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Chloroplasts change their positions in the cell depending on light condition. In the fern, Adiantum capillus-veneris prothallia, chloroplasts locate along the periclinal wall when the light comes vertically to the surface of the prothallus (Light position). In contrast, chloroplasts locate on the anticlinal wall in the dark (Dark position). In the photorelocation movement of chloroplasts, phy3/neo1 is assigned as the red light receptor. Nevertheless, we found chloroplasts in phy3/neo1 mutants (rap mutants) still relocate from dark position to light position under red light. We tested several rap mutants (rap2, rap7 and rap31) and all of those showed the red light-induced chloroplast relocation. Further, it was found that the light-positioning of chloroplasts occurred independent from the direction of red light. Photosynthesis inhibitor, DCMU, blocked this movement. These results indicate that chloroplast relocation under red light in rap mutants occurs through photosynthesis-dependent, non-directional movement of chloroplasts.

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In C₄ plants, mesophyll (M) chloroplasts are randomly distributed in the cells, but bundle sheath (BS) chloroplasts are located in a centripetal or centrifugal position. In the last conference, we reported that only M chloroplasts showed accumulation movements to the BS side in response to extremely high light (3,000-4,000 μmol m^-2 s^-1) or environmental stresses such as drought and salinity (light intensity: 250-500 μmol m^-2 s^-1). In this study, we report that M chloroplast movement of finger millet (Eleusine coracana) in response to environmental stresses was induced by light irradiation but did not need direct irradiation. Additionally, M chloroplast movement was also observed in plants growing in the field in the daytime full-summer but not before dawn. We conclude that M chloroplast movement in response to environmental stresses is occurred in a light-dependent manner and will discuss its physiological mechanism.

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Two phototropins, PHOT1 and PHOT2, function as blue light photoreceptors. Here, we investigate the effect of phototropins on the leaf photomorphogenesis. The downward curling in the photo1phot2 leaves has been already reported. Hence, we measured the size of epidermal cells in the phot1phot2 mutant and found that the size decreased in the abaxial side, while it was normal in the adaxial side. Hence, the downward curling in the photo1phot2 leaves can be explained by asymmetrical growth of the epidermal cells. We also found that the phot1phot2 mutant showed elongation defect in palisade cells, suggesting that phototropin regulates the polar cell elongation in the palisade tissues. We then asked in which tissue of the leaves PHOT2 regulates the leaf flatness and the palisade cell elongation. One the basis of present results, roles of PHOT2 in different tissues to control the leaf morphogenesis will be discussed.

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Stomata surrounded by a pair of guard cells in the epidermis regulate gas exchange between plants and atmosphere. Stomata open through activation of plasma membrane H⁺-ATPase in guard cells in response to blue light. Under drought condition, stomata close through activation of plasma membrane anion channel in response to abscisic acid. In this study, to elucidate signaling pathways in response to blue light and abscisic acid, we screened stomatal aperture mutants by water loss in detached leaves from EMS-treated Arabidopsis. We isolated 2 mutants named std1 and std2 and 2 mutants named ftd1 and ftd2. std1 and std2 showed closed stomata under light illumination. In contrast, ftd1 and ftd2 showed open stomata even in the dark condition. we are trying to identify the FTD2 locus by map-based cloning. We'll report the results of mapping and analysis of detailed phenotype in ftd2.

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In most amphistomatous leaves, stomata on the adaxial surfaces (upper surfaces) and those on the abaxial surfaces (lower surfaces) inhabit different light environments in terms of both intensity and wavelength composition. According to our recent study, adaxial stomata were always less sensitive to both white light and monochromatic light than abaxial stomata (Wang et al., 2008, PCE, 31:1307-16). In this study, to investigate the influence of growth light environment on stomata light response, we artificially inverted the leaves of sunflower (Helianthus annuus L.) at their expanding stage, and measured the gas exchange properties after their expansion, using a laboratory-constructed system. Compared with the normal leaves, abaxial stomata of inverted leaves became more sensitive to white light and blue light, but red light sensitivity did no change. On the other hand, adaxial stomata of inverted leaves went into less sensitive to light.

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The flower of Portulaca generally opens early in the morning in response to a temperature rise and a light exposure. We had reported that the light irradiation could induce the flower-opening of Portulaca hybrid alone at 30C and that the Plasma membrane (PM) H⁺-ATPase activity in the petal increased during flower-opening process. In this study, we examined the effect of the timing of light irradiation on the flower-opening and the enzyme activity using the plants cultivated under 12L (8:00-20:00, 30C) /12D(20:00-8:00, 25C) condition. At 30C, the flower-opening never began before 3:00 regardless of the timing of light irradiation and when the dark period was less than 4 hours, the flower did not open completely. On the other hand, the activity and amount of the enzyme increased from 2:00 to 5:00. We now investigated whether the timing of light irradiation and inhibitors of flower-opening influence the behavior of the enzyme.

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We previously reported that, (1) increasing UVB radiation inhibited growth, yield, and grain development, (2) such UVB-induced damage caused by UVB-induced cyclobutane pyrimidine dimers (CPDs), and that (3) increasing CPD photolyase activity can significantly alleviate UVB-caused growth inhibition. In this study, to evaluate experimentally the influence of the solar-UVB on rice, we have conducted field experiment in Miyagi (2006-2008) and Kagoshima (2008) Pref., using UVB-resistant rice Koshihikari (japonica) with higher photolyase activity and the UVB-sensitive chromosomal segment substitution line (SL-229), substituted chr.10 by UVB-sensitive rice Kasalath (indica), as experimental plant: CPD photolyase gene locates on chr.10. As a result, grain size and pollen fertility of SL-229 grown under current outdoor conditions were reduced, and the degrees of SL-229 grown in Kagoshima Pref. were higher than in Miyagi Pref. These results mean that growth and yield of rice grown under current environmental conditions would be inhibited by UVB in natural sunlight.

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Cyclobutane pyrimidine dimer (CPD) photolyase is a crucial factor for determining UVB sensitivity in plants. CPD photolyases are categorized into two groups, class I in microorganisms and class II in higher organisms, on the basis of their deduced amino acid sequences. Plant CPD photolyase is classified as class II group. However, biochemical characteristics of class II CPD photolyase of plant are unclear. To analyze the characteristics of CPD photolyase, we purified the CPD photolyase from rice leaves, and found the native rice CPD photolyase is phosphorylated. There is no report that CPD photolyase is phosphorylated in organism other than rice. Then, we established the new method for purification of CPD photolyase from rice leaves using a UV irradiated DNA-conjugates magnetic beads, and analyzed the phosphorylation on CPD photlyase in several plants. In this study, we discuss about the significance of phosphorylation on CPD photolyase in plants.

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To development gene resources related to UVB tolerance of rice, UVB-resistant rice cultivar Sasanishiki was irradiated with carbon ion beam (320 MeV:¹²C⁶⁺, 80Gy). We isolated three UVB hyper-resistant and two hyper-sensitive mutants, compared with UVB-resistant Sasanishiki. In this study, we focused one of UVB hyper-resistant mutant, UVTSa-319 (UV-tolerant Sasanishiki-319) and analyzed the characteristics of the mutant. Consequently, there were no differences in the amount of UV-absorbing compounds, the activities of cyclobutane pyrimidine dimers and (6-4) photoproducts photorepair and dark repair activity between wild type Sasanishiki and UVTSa-319. Next, we performed Array Comparative Genomic Hybridization (CGH) to estimate mutated region. As a result, we found that about 40 kbp on chromosome 7 containing 2 genes which has unknown function was deleted in UVTSa-319. We discuss about possibility of new UV tolerance gene resource.

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Ultraviolet-B radiation (UV-B: 280-320 nm) has pleiotropic effects on plant development. We have previously demonstrated that alignment of chloroplast in palisade cells become irregular and palisade cells become like spongy cells by UV-B irradiation in cucumber cotyledon. Recently, the Cs1-MMP gene was isolated from a cucumber cotyledon. Cs1-MMP encodes a putative matrix metalloproteinase (MMP) and is expressed at the boundary of senescence and programmed cell death (PCD) of the cotyledon. In animals, MMPs make up a major group of enzymes that degrade the extracellular cell matrix (ECM). To investigate the possibility that UV-B irradiation induces PCD in cucumber cotyledon, Cs1-MMP expression and DNA fragmentation were analyzed. Cs1-MMP expression was induced by UV-B irradiation. However, DNA fragmentation was not detected in cucumber cotyledon irradiated by UV-B for 15 days. Further study is necessary to characterize cell death in cucumber cotyledon irradiated by UV-B.

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Thioredoxin (Trx) localized in stroma, is reduced by ferredoxin and ferredoxin-thioredoxin reductase. The required reducing equivalents are provided by photosynthetic electron transfer system. Reduced form Trx activates various thiol-enzymes in stroma by way of reduction and directly supplies reducing equivalents required for the reaction cycle of peroxiredoxin as well, which functions as detoxification system of reactive oxygen species. In addition, we showed that stroma Trx functions as a part of a trans-membrane reducing equivalent transfer system in higher plant chloroplasts. HCF164 that is a membrane anchored thioredoxin-like protein faced to thylakoid lumen side, receives reducing equivalents from stroma Trx. In this study, we analyzed the components of this reducing equivalents transfer system itself. We discuss the electron transfer mechanism across the thylakoid membranes.

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Thioredoxin occurs in all living cells, and regulates the function of various target proteins by way of the dithiol-disulfide exchange reaction. In many organisms, thioredoxin is involved in protection from the oxidative stress. In phototrophs, thioredoxin has essential regulatory roles that link photosynthesis reaction and other reactions in the chloroplasts. In chloroplasts of Arabidopsis thaliana, 10 thioredoxin isoforms were revealed from the genome analysis. These isoforms were supposed to have their own preference for the target proteins. We considered that the selectivity of the target proteins by each of isoforms will be explained in term of affinity between "thioredoxin" and "target". We then measured affinity between thioredoxin and the target using surface-Plasmon-resonance method. Thus interaction between chloroplast thioredoxin isoforms and target proteins, FBPase and PrxQ, were analyzed. We discuss the affinity of target protein to thioredoxin isoforms based on these interaction studies.

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Filamentous cyanobacterium Anabaena (Nostoc) sp. PCC 7120 forms heterocyst for nitrogen fixation under nitrogen starved conditions. In order to understand the roles of thioredoxin (Trx) especially in the heterocyst cells, we examined the Trx-affinity chromatography method using the immobilized Anabaena Trx mutant, in which an internal cysteine at the active site was substituted with serine. The target proteins for thioredoxin in Anabaena were efficiently acquired from the vegetative cells and the heterocyst cells. The obtained proteins were separated by two-dimensional gel electrophoresis and analyzed by MALDI-TOF mass spectrometry. We have identified 38 proteins for Trx potential target proteins in the vegetative cells. In addition, we have identified several specific proteins including the nitrogenase in the soluble proteins of isolated heterocysts. Based on the obtained proteomics data, the role of thioredoxin is discussed.

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Among ascorbate peroxidase (APX) isoenzymes, cytosolic APX plays an important role to control cellular redox conditions in response to photooxidative stress. In this study, we have evaluated the effect of cytosolic APX overexpression (cAPX-OE) or suppression (cAPX-KO) on cellular H₂O₂ accumulation and gene expression levels under photo-oxidative stress conditions. We obtained 7 independent Arabidopsis plants transformed with pBI/cAPX1 and one cAPX-KO line from TAIR. The APX activities in cAPX-OE and cAPX-KO plants were approximately 300-600 % and 30%, respectively, compared with that in wild-type plants. When exposed to high-light stress at 1,000 μmol m^-2 s^-1, the H₂O₂ levels in both wild-type and cAPX-KO transiently increased within 1 h, but the level was almost constant in cAPX-OE. The results of comprehensive gene analysis in these plants will be reported.

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Recently, we have been engaged to development of novel system for preparation of water rich in reactive oxygen species (ROS), by passing the water through the photochemical apparatus equipped with thin alumina-coated aluminum fibers further coated with titania, UV-A lamps & ultrasonic devices. Data suggested that the processed water contained superoxide, singlet oxygen & hydroxy radicals. In the present study, we treated the growing plants & leaf disks of tomato, tobacco and other species. We observed that expression of PR-genes & other defense & redox-related genes by treatment with photochemically processed water.

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Aluminum (Al) ions have toxic effects on plants in acid soil area. Contamination of soil with heavy metals is also a big problem for both agriculture and our health. To characterize high-tolerant mechanisms in wild plants, isolation of high tolerant genes from Andropogon was carried out by the two approaches shown below.
A) Isolation of Al-induced genes; Total-RNA was extracted from Al treated plants and then used for fingerprint method. Nine of Al-inducible candidates were obtained. After DNA sequencing, highly homologous full-length cDNA for each clone was obtained from maize or rice. To identify which clones can confer Al tolerant phenotype, these clones were introduced into yeast. B) Direct screening; The total-RNA was also used for a construction of cDNA library, and tolerant genes were directly screening by yeast system. Six candidates conferring tolerance to various stresses were independently isolated. Further characterizations are proceeding for the isolated clones.

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Betalains are plant pigments, consisting of red betacyanins and yellow betaxanthins. Distribution of betalains is limited in Caryophyllales except for Caryophyllaceae and Molluginaceae, and betalains and anthocyanins are mutually exclusive. It has been reported that flavonoids including anthocyanins have antioxidant functions in plant. However, the physiological role of betalains in plant is not clear. Here we examined betalains as antioxidant agents to elucidate their functionality.
Two major betalains, beet betaxanthin(BBx) and beet betacyanin(BBc), were isolated from red beet(Beta vulgaris L.). Both BBx and BBc were rapidly bleached by the addition of peroxynitrite(ONOO^-). This bleaching reaction was dependent on concentration of ONOO^-, while suppressed by glutathione, a well-known ONOO^- scavenger. These results indicated that betalains have an ability to quench ONOO^-. In this present study, we also discuss antioxidant activity of betalains against reactive oxygen species.

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Glutaredoxin catalyzes the reduction of protein disulfide using the reducing power of glutathione and is supposed to participate in the defense against oxidative stress. However, its physiological role in higher plants is largely unknown. We previously found that rice glutaredoxin protein is abundantly expressed in developing and mature seeds, but not in seedlings or leaves. In this study, we investigated the antioxidant function of glutaredoxin from rice using yeast expression system and transgenic rice plants. Yeast cell lines expressing rice glutaredoxin showed increased tolerance against menadione but not against hydrogen peroxide compared with control. In contrast, expression of glutaredoxin decreased the paraquat tolerance and glutathione reductase activity in green leaves in transgenic rice plants. This result suggests that ectopically expressed glutaredoxin in green leaves might cause the perturbation of cellular redox balance which resulted in the decreased tolerance to oxidative stress.

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Ascorbate (AsA) functions as an antioxidant to scavenge reactive oxygen species (ROS). Monodehydroascorbate reductase (MDAR) catalyzes the regeneration of AsA from its primary oxidant monodehydroascorbate. One of five MDAR genes in the Arabidopsis genome encodes the region of the transit peptide for dual targeting to mitochondria and chloroplasts. Because mitochondria and chloroplasts are the main sites of the generation of ROS in plant cell, chloroplastic/mitochondrial (cp/mt) MDAR must contribute to the maintenance of redox state of AsA pool significantly. Under optimal growth condition, the appearance of cp/mt MDAR knockout line of Arabidopsis selected from the Tag-line pool of the Kazusa DNA Research Institute did not differ from that of wild type. Moreover, MDAR specific activity and AsA content were not different between the wild type and the knockout line. However, the knockout line had about 2-fold higher specific activity of superoxide dismutase than the wild type did.

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Programmed cell death (PCD) plays an essential role to the development and maintenance of bodies. Dysfunction of DAD1 (Defender against Apoptotic cell Death) has been linked to PCD in animals and plants. One type of PCD in animals is triggered by cytochrome c release from mitochondria via pores formed by BAX proteins. This type of PCD can be prevented by expression of BAX inhibitor 1 (BI-1). In last report, the promotor-GUS assay had shown that the genes of AtDAD1 and AtBI-1 express the root cap, a vascular bundle of the root, an abscission zone of the silique and a septum of the maturity silique. These results suggest the involvement of the AtDAD1 and AtBI-1 in the developmental programmed cell death. In this report, we analyzed effect of stress on the activation of ATDAD1 and AtBI-1 in Arabidopsis T87 cultured cells.

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In order to establish a culture method for a long-term preservation of microalgae, we investigated physiological responses of a model alga, Chlamydomonas reinhardtii, to low temperatures. When the cells pre-cultured at 25^oC were exposed to temperatures below 25^oC, growth rate of the cells was decreased with decreasing temperatures but they still grew at over 8^oC. After cultured at 4^oC for 7 days, the cells did not grow, but viability was more than 90%. When a cultural period at 4^oC was extended to 80 days, viability was decreased to about 3%. Furthermore, viability was decreased to about 50% after 2 days and to below 1% after 7 days at -2^oC. These results suggest that growth of C. reinhardtii cells are prevented, keeping physiological function to grow, at low temperatures over 4^oC, and that the cells at non-freezing temperatures below 0^oC are rapidly and severely injured and become no longer viable.

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Fructan is a fructose-based polymer associated with freezing tolerance in overwintering plants. We have reported an increase in cold tolerance of transgenic rice expressing wheat sucrose:sucrose fructosyltransferase, 1-SST (Kawakami et al. JXB, 2008). To clarify the mechanism of this enhancement of cold tolerance by fructan, we examined the changes in sugar content and the expression levels of OsSUT genes in the source and sink tissues of booting-stage rice exposed to a temperature of 12^oC. Remarkable increases in sucrose contents in source leaves and its sheaths and decreases in those in panicles were observed in plants under the low temperature. Those changes recovered after exposure to a temperature of 26^oC. In the transgenic rice, fructan increased under the cold treatment condition. Among the OsSUTs, transcript levels of OsSUT1 in the leaf sheath in both fructan-acumulated rice and non-transgenic rice decreased at 12 ^oC and increased at 26 ^oC after cold treatment.

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Cowpea (Vigna unguiculata [L.] Walp.) is used as important sources of proteins and carbohydrates in the tropics and subtropics because it is well adapted to hot and dry environment. But the grain yield of cowpea is severely inhibited by high night temperature.
We reported last year that retentivity of pollen proline concentration at stressed condition could be an important factor of heat-sensitivity of cowpea genotype. In the present study, we have undertaken gene expression analysis of proline transporters, VuProT1 and Vu ProT2 by RT-PCR.
Both ProT1 and ProT2 gene expression were observed in all organs we analyzed. Expression of ProT2 was almost equal level in all organs. However, ProT1 was abundantly-expressed in anthers and flower stalks. It seems ProT1 play important roles for transport proline into flower organs. ProT1 was expressed more in anthers of heat-tolerant line TVu4552 than heat-susceptible lines CB5 and Bambey23 in heat-stressed condition.

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An Arabidopsis transcription factor gene, DREB2A, is known to play an important role in environmental stress responses. DREB2B was isolated as a DREB2A homologue with highest similarity. But function of DREB2B under environmental stress conditions was still unclear.
Expression analysis using qRT-PCR revealed increased expression levels of DREB2B under drought, heat and high salinity stress conditions. Transient assay using GFP-fused protein indicated that DREB2B protein localized in nuclei of plant cells. Phenotypic analysis of DREB2B overexpressors and T-DNA tagged mutants did not show any morphological difference compared to wild-type plants. Microarray analysis using DREB2B overexpressors showed that 41 genes were upregulated in plants. Most of these were related to sugar metabolisms. Thermotolerance test revealed improved tolerance of overexpressors. DREB2B possesses the sequence with high homology to negative regulatory domain of DREB2A. We are currently analyzing the activity of this regulatory domain.

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Overwintering plants are capable of exhibiting high levels of cold tolerance, which is acquired through cold acclimation. It was reported that plants have common cold tolerance mechanisms with prokaryotes. They are post-transcriptional or translational regulation mechanisms, which control mRNA structure and stability. Recently degradation mechanisms of RNA by miRNA or siRNA are well studied, however stabilization mechanism of mRNA is not fully understood during cold stress adaptation or deacclimation step. In order to understand this system during cold stress, we focus on the RNA masking system, which is a translational regulation mechanisms involved in RNA silencing and stability. Translational masking of RNA is a system to control protein expression by repressing translation. As the first step to understand the RNA masking system, we performed comparative analysis between transcriptome and proteome to identify the targets of RNA masking. According to these analyses we identified several candidates of RNA masking targets.

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We found, by the T-DNA activation-tagging method, that the Arabidopsis AtFIB5 gene may be involved in enhancing freezing tolerance. Two transcripts (FIB273 and FIB259), which are generated by pre-mRNA alternative splicing of the AtFIB5, are presumed to encode the proteins composed of 273 and 259 amino acids, respectively. To elucidate the function of these transcripts, we have studied involvement of the AtFIB5 gene in freezing tolerance with FIB5-overexpressing and -knockout plants. FIB5-knockout (fib5) plants exhibited a dwarf phenotype and a remarkable decline in freezing tolerance. These abnormalities were complemented by the introduction of cDNA of FIB273 into fib5 plants, while they were still observed in the FIB259-introduced plants. Meanwhile, FIB259-overexpressing plants were much more tolerant to freezing than FIB273-overexpressing ones. These results suggest that two transcripts derived from AtFIB5 have different functions, which are maintaining the photosynthesis apparatus for FIB273 and improving freezing tolerance for FIB259.

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Higher plant mitochondria are known to respond to cold temperature in terms of cytologically, physiologically, and biochemically. To investigate the relationship between mitochondrial gene expression and cold temperature, alterations in the accumulation of transcripts and the extent of RNA editing were examined in all the mitochondria-encoded genes bearing group II introns when transferred under cold temperature. We used rice and wheat as models for a cold-sensitive plant and a cold-acclimatable plant, respectively. In rice treated at 12˚C for 14 days, the accumulation of transcripts with introns increased or remained unchanged, and RNA editing was not occurred at some sites where usually edited under 25˚C. The accumulation of transcripts without introns decreased or remained unchanged. In wheat treated at 0.5-2.0˚C for 14 days, alteration of the accumulation of transcripts with introns and the occurrence of RNA editing was similar to that of rice, but the transcripts without introns generally increased.

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In northern part of Japan, rice crop production is occasionally damaged severely by cool temperature in summer due to the disturbance in pollen formation. Rice is most chilling sensitive at the onset of microspore release. Through the application of chilling stress in rice anther, the expression of OPDAR1 and Radc1 was repressed and the expression of SAMDC1 was induced. Expression patterns of these genes were histochemically examined in anthers from transgenic rice plants bearing individual gene promoter::GUS fusions. A new cis element that includes a DNA transposon sequence found in the 5' upstream region of Radc1, and functional importance of polyamine or jasmonate under chilling-stress during rice pollen formation will be discussed.

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We already found the desiccation-responsible genes from a terrestrial cyanobacterium Anabaena sp. PCC7120 using DNA segment array. To clear these genes function, the genes selected in typical gene-expression patterns were disrupted and desiccation tolerance of the gene-disruptants was tested. Although selected genes were expressed in nitrogen-rich condition, the gene-disruptants decreased desiccation tolerance in nitrogen-free condition. In cell growth, some of the gene-disruptant decreased growth rate in nitrogen-free condition. In accumulation of trehalose and sucrose known as desiccation-protectant, the gene-disruptants were almost the same tendency as wild-type irrespective of nitrogen content, suggesting that desiccation-responsible genes were larger influence than desiccation-protectant. These results may suggest that 1) desiccation-tolerant genes are containing nitrogen fixation relating genes, 2) many of the genes are expressed irrespective of nitrogen content to protect desiccation sensitive nitrogen fixing heterocyst and 3) complementary genes hide the mutant phenotype under desiccation in nitrogen rich condition.

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We introduced about 13,000 full-length cDNAs of rice into Arabidopsis by Agrobacterium-mediated transformation method and constructed a large-scale library of transgenic Arabidopsis (rice-Arabidopsis FOX lines). By screening T2 seeds from about 20,000 FOX lines under salinity stress, we have isolated more than 200 candidate lines resistant to salinity stress. Here, we report characterization of a line named R07047. R07047 showed high salinity tolerance in various aspects including germination, root growth, and survivability of seedlings. In addition, R07047 showed tolerance to dehydration and oxidative stresses. Genomic PCR revealed that rice cDNA encoding unknown protein was inserted in R07047. Newly generated transgenic Arabidopsis expressing the cDNA showed high-salinity tolerance as well as R07047. The encoded protein has a C2 domain known as a calcium binding domain. It is interesting whether the cDNA can confer salt tolerance to plants other than Arabidopsis.

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We have found that overexpression of BZF1, which is a member of the BZF subfamily of a B-box zinc finger family, confers enlargement of vegetative growth, increase in viability under severe drought condition, and improvement of irrigation efficiency in Arabidopsis. In the BZF1-overexpressed plants, transpiration was reduced and decrease of soil water content after stop of irrigation was delayed. Expression of BZF1 was not responsive to drought and ABA. In the BZF1-overexpressed plants, expression of genes related to drought tolerance was not enhanced and expression of genes in the central metabolism was generally reduced. BZF1 is possibly involved in the function of sink leaves. These results suggested that the overexpressed BZF1 confers improvement of drought adaptation of plants by its unique mode of action that is different from that of known regulatory factors, which improve the drought tolerance by enhancement of drought response and/or dehydration resistance.

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Although xanthine dehydrogenase (XDH) is known as the rate-limiting enzyme in purine catabolism, the plant physiological role of the function of this enzyme remains still unclear. We previously reported that RNAi-mediated silencing of the two Arabidopsis XDH genes (AtXDH1 and AtXDH2) resulted in xanthine accumulation and pleiotropic phenotypes including growth retardation, impaired fertility and precocious senescence, possibly due to the metabolite deficiency in the catabolic pathway. In this study, we investigated a possible role of XDH in plant acclimatization to drought because the expression of AtXDH1, the major XDH gene in Arabidopsis, is known to be activated by abscisic acid and during the progression of drought stress. Compared with wild-type plants, RNAi plants exhibited significantly more cell death and less chlorophyll contents, and markedly reduced biomass production when subjected to drought stress. These results suggest the functional importance of XDH in the acclimatization to drought.

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Using CRES-T system, we isolated transgenic Arabidopsis which exhibited tolerance to salt and osmotic stresses. We selected transcription factors in which expression was responsed to these stimuli, we converted them to chimeric repressor by fusion with repression domain (SRDX) and expressed them in Arabidopsis. The seedlings of 35S:AtMYB102-SRDX, 35S:ANAC047-SRDX and 35S:GARP-SRDX survived on MS plate containing 225 mM NaCl, under which condition wild-type Arabidopsis were unable to be alive.
Chimeric repressors against ZAT6 and AtERF5 exhibited tolerance to osmotic stress since both transgenic plants sustained viability even at high concentration of mannitol, 650 mM where most of all plants could not survive and germinate. Microarray analysis showed that chimeric AtMYB102 repressor enhanced expression of stress responsible genes such as DREB1A and TINY genes and suppressed expression of ZAT11 and AtMYB15. We will discuss a mechanism of improving plants on abiotic stresses and possibility for agricultural application.

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