Here, we report that RPK2 is a key regulator of anther development in Arabidopsis. RPK2 knockout mutants, rpk2-1 and rdk2-2 displayed male sterility due to defects in anther dehiscence and pollen maturation. In rpk2 anthers, several defects were observed as follows: the middle layer was not differentiated from secondary parietal cells, the tapetum was abnormally enlarged, developmental homeostasis of anther locules was disrupted, most pollen grains highly aggregated together, and anthers finally crushed without dehiscence caused by inadequate endothecium lignification. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated in the rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate to trigger its degradation and that mutating RPK2 inhibits pollen maturation and anther dehiscence due to disruption of key metabolic pathways.

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Phospholipase D (PLD) plays an important role in plant biotic and abiotic stress responses. To study the function of PLD in rice, we made knockdown plants by introduction of a PLD gene specific RNA interference (RNAi) constructs. OsPLDβ1-RNAi-silenced transgenic rice was found to develop spontaneous lesions on the leaves in the absence of pathogen infection. The transgenic rice exhibited that genes associated with activation of disease resistance, including probenazole- inducible protein (PBZ1), chitinase, ribosome-inactivating protein (RIP2), and thaumatin-like protein (TLP) were significantly up-regulated. The amount of phytoalexine on the leaves of the transgenic rice was strongly increased approximately 100-200-fold relative to the wild-type levels. These results suggest that the transgenic rice is the disease lesion mimic that shows spontaneous hypersensitive reaction in the absence of pathogens. Possible functions of OsPLDβ1 in induction of hypersensitive reaction are discussed.

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In order to elucidate the root elongation mechanism under drought stress, we are studying regulatory mechanism of the expression cell wall-related genes that express in Arabidopsis root tip. Bioluminescence from transgenic Arabidopsis root tips were observed, when promoter of CesA 1, 3, 6, AtEXP11, XTH5, 14, AtFUT6 were fused with luciferase reporter gene. Bioluminescence of the XTH14-LUC reporter gene was reduced under osmotic stress, which inhibits root elongation. Therefore, the XTH14-LUC reporter gene is suitable to elucidate regulatory mechanism of the expression of cell wall-related gene under abiotic stress.
Promoter analysis of XTH14 has revealed that the region between -137 and -205 from the transcription start site is important for the expression in root tip. A deletion of the region between -63 to -137 also reduced the reporter activity. These regions include W-box, G-box. We discuss roles of these cis-elements on the expression of XTH14.

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Gibberellin signaling pathway is composed of a negative regulator, Slender rice 1 (SLR1) and its degradation system via ubiquitin-26S proteasome pathway. We investigated a point of action which rice heterotrimeric G protein α subunit (RGA1) may concern is a regulation of SLR1 activity or its degradation system.
We identified that an approx. 75kDa protein recognized by anti-SLR1 antibody specifically accumulated in normal cultivars, not in RGA1 deficient mutants, d1 , under the dark condition at 30 degrees centigrade. The amount of the protein is decreased after application of gibberellin, in parallel to a response of SLR1, approx.70kDa. This result suggests that an approx. 75kDa protein is SLR1 post-translationally modified or SLR1 relative. An antibody against O-GlcNAc recognized an approx. 75kDa protein in normal cultivars, not in d1 . This observation suggests that RGA1 controls the accumulation of post-translationally modified SLR1 or SLR1 relative.

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PHY3 is composed of a phytochrome chromophore-binding domain in its N-terminal portion and an almost full-length phototropin in its C-terminal half. Because of its structural features, PHY3 must be able to perceive both red- and blue-light signals simultaneously. When we simultaneously irradiated transgenic Arabidopsis expressing PHY3 with both red and blue light, simultaneous irradiation causes greater phototropic curvature than with red or blue light alone. To address the relationship of this synergistic effect with respect to the PHY3 kinase activity, we examined in vitro phosphorylation analysis of microsomal membranes from a PHY3 expressing line. When we applied red and blue light simultaneously, an obvious increase in the phosphorylation level of the 160-kD protein was detected compared with that of single light irradiation. These results clearly demonstrate that two independent light-sensing systems cooperate within PHY3 and that red light and blue light signals are processed synergistically by this single chromoprotein.

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Blue light is an important environmental factor for plant life. Vaucheria, a coenocytic alga, shows several blue light responses such as positive or negative phototropism, light-growth-response, and branch induction.
Two putative photoreceptors for blue light-induced photomorphogenesis were isolated and designated as AUREOCHROME1 (AUREO1) and AUREOCHROME2 (AUREO2). Each of these proteins has one bZip and one LOV domain. LOV domains are known to absorb blue light through flavin, whereas bZip domains are known to bind specific DNA sequences and function as transcription factors. We determined the specific DNA sequence that could bind AUREO1 by random oligonucleotide binding selection assay. Furthermore, we confirmed the DNA-binding specificity of AUREO1 by gel-shift assay. However, the effect of blue light on the DNA-binding activity of AUREO1 has not been analyzed.
We will present our recent results of the effect of blue light on the DNA-binding activity of AUREO1.

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Phototropin is the blue-light receptor that mediates phototropism, chloroplast movement, and stomatal opening in Arabidopsis. Phototropin contains a Ser/Thr protein kinase domain and two LOV domains. The LOV domains function as binding sites for the chromophore FMN. Six genes encoding LOV-containing protein are found in the genome of Arabidopsis. Among them, two encode phototropins and three do LKP-family proteins, which play roles in the circadian clock and photoperiodic-dependent flowering. All LOV domains of phototropins and LKP-family proteins are shown to bind FMN. Other than those above, there exists a unique LOV-containing protein, referred to as PAS/LOV protein. The function of this PAS/LOV protein remains unknown, including FMN binding to its LOV domain. We analyzed FMN binding using a recombinant protein of the PAS/LOV protein homolog of tomato.

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Plants possess several classes of photoreceptors to perceive lights and regulate the development throughout their life cycle. One of these photoreceptors is a phytochrome (phy) family, which is important for a wide range of developmental responses. In this study, we aimed to investigate the differences in metabolic profiles during de-etiolation between wild-type Arabidopsis thaliana and phyA mutant by means of GC-TOF/MS analysis. Etiolated WT and phyA seedlings were irradiated with continuous far-red light (FRc), or white light. Primary metabolites of the seedlings were profiled by GC-TOF/MS to find phyA-regulated metabolic pathways. Comparison of metabolite levels of the seedling under FRc revealed several metabolites that contribute to the difference between WT and phyA seedlings. Several amino acids and major sugars were found in higher amounts in phyA than in WT seedlings under FRc. There were also significant differences in metabolite levels between WT and phyA seedlings during de-etiolating under white light.

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Phototropins (phot1 and phot2) are membrane-bound blue light photoreceptors that function in phototropism, chloroplast relocation, stomatal opening, and leaf flattening in Arabidopsis. The molecular structure of phototropin is well conserved for the distinct biochemical roles; two LOV domains in its N-terminal half as a photosensory domain and a Ser/Thr kinase domain in its C-terminal half as a signal transducer. Here, we studied the structural roles of the N-terminus of phot2 (P2N) in vivo and in vitro. Size exclusion chromatography suggested that phot2 might exist as a multimer form around 500 kDa in vivo. In addition, the recombinant P2N and P2NN (the N-terminus of P2N including LOV1), but not P2NC (the C-terminus of P2N including LOV2) were detected as a tetramer, respectively, suggesting that the region of LOV1 domain was responsible for the multimeric formation of phot2. Physiological roles of the P2N will be also discussed.

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Cyanobacteriochromes are novel phytochrome-like photoreceptor proteins containing GAF domain. SyPixJ1 (sll0041) of Synechocystis sp. PCC6803 was shown to regulate the phototaxis of cells. We studied AnPixJ-GAF2 of Anabaena sp. PCC7120 that shows a photochromism between the green-light absorbing form (Pg, 543 nm) and the red-light absorbing form (Pb, 649 nm). The light-signal conversion process and the regulation mechanisms of absorption spectra are unknown. We investigated the photoreaction by the time-resolved absorption spectroscopy and global analysis. The reaction process from Pg to Pb showed two kinetic components between micro and millisecond time range. The conversions from Pb to Pg showed one kinetic components at millisecond time range. We will discuss the structural-change models based on the absorption peaks of the intermediates obtained by global analysis.

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Plants exhibit various responses to illumination with blue light (BL), such as chloroplast movement and stomatal opening. We found that the nucleus in Arabidopsis thaliana mesophyll cells alters its intracellular position in response to BL.
In dark-adapted cells, the nucleus was positioned at the center of the bottom of the cells. After illumination with strong BL, the nucleus was positioned along the anticlinal wall. This response was induced by BL over 50 μmol m^-2 s^-1 but not under 10 μmol m^-2 s^-1. Red light exhibited little effect. To reveal photoreceptor involved in this response, phototropin mutants, phot1, phot2, and phot1phot2 were analyzed. The response was induced in phot1 but not in phot2 and phot1phot2, strongly suggesting that phot2 is involved in the BL-dependent nuclear positioning. To clarify cytoskeletal components involved in this response, inhibitor experiments are now in progress.

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The phototropic response of Arabidopsis thaliana seedlings is initiated by the blue-light (BL) photoreceptors, phototropin (phot) 1 and phot2, and by their signal transducers, NPH3 and RPT2. Although NPH3 and RPT2 play important roles in phototropic signaling, their biochemical functions remain unclear. Previously, we showed that the NPH3 protein is phosphorylated under dark conditions and is de-phosphorylated under BL. Phot1 is necessary for BL-dependent de-phosphorylation of NPH3. And our immunoprecipitation analysis demonstrated that RPT2 preferentially interacts with the dephpsphorylated NPH3. In this time, we report some phosphorylation sites of NPH3. We generated two polyclonal antibodies against phosphopeptide of NPH3, and analyzed NPH3 phosphorylation state. Under BL condition, two these phosphorylation sites are de-photphorylated mediated by phot1. We also report that PP2A might be involved in de-phosphorylation of NPH3 using phosphatase inhibitors. We will discuss regulation of NPH3 de-phosphorylation and function of NPH3 and RPT2 during induction of phototropism here.

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The appropriate leaf position is essential to optimize photosynthesis and plant growth. In this study, we show that the Arabidopsis leaf position is appropriately regulated by blue light-mediated growth response. When 1-week-old seedlings were transferred to under red light and grown for 5 days, new petioles appeared almost horizontally and their leaves dangled. However, if a very weak blue light was superimposed on red light, the new petioles emerged obliquely to upward and the leaf surface faced toward light. This leaf positioning required both phototropin1 and NON-PHOTOTROPIC HYPOCOTYL 3 and resulted in a dramatic enhancement of plant growth. In nph3 mutant, neither proper leaf positioning nor leaf flattening by blue light was found, and blue light-induced growth enhancement was drastically reduced. We conclude that NPH3-mediated phototropin1 signaling optimizes the light perception through inducing both appropriate leaf positioning and leaf flattening, and enhances plant growth.

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Root hair elongation induced by acidic stress requires high fluence blue light (3.6 Wm^-2, more than 9hr). This fact suggests the possibility that photosynthesis or its products are essential for induction of root hair elongation.
One day old roots grown under blue light for 9hr slightly contained chlorophylls (1.8 μg/ Fw g), while roots grown under red light did not. One hundred μM DCMU and 10 μM DBMIB inhibited the elongation completely. However, this inhibition was not recovered by addition of sugars. Pulse amplitude modulation fluorometer (Walz, PAM101/102/103) could not detect photosynthesis activity in roots grown under red or blue light. Furthermore, sugars could not induce root hair elongation in the darkness although main root elongation was enhanced (Suc; 3 times, Glc; 1.5 times).
Considering these results and that red light cannot induce root hair elongation, photosynthesis does not promote root hair elongation.

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One of the most unique promoters in the plastid genome is the psbD light responsive promoter (psbD LRP). The psbD LRP is transcribed by a eubacteria-type PEP and contains conserved AAG-box and -10 elements. We analyzed the upstream sequences of the psbD among 9 ferns and 17 gymnosperms. The psbD LRP sequences are found in Cycadopsida, Ginkgoopsida and Conferopsida of gymnosperms, but not in ferns and in Gnetopsida (gymnosperm), suggesting that the psbD LRP sequences probably emerged early during the evolution of the gymnosperms. We subsequently examined expression of the light-inducible psbD transcripts in several plants. The psbD gene is constitutively expressed at high levels irrespective of light condition in Adiantum (fern), Cycas (gymnosperm) and Black Pine (gymnosperm), whereas psbD gene expression is induced by light in Arabidopsis (angiosperm). These results may suggest that the evolution of the psbD LRP sequences preceded the establishment of the light-responsive psbD transcription system.

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The flower of Portulaca is ephemeral and generally opens early in the morning. It is well known that flowers of Portulaca open in response to a temperature rise and a light exposure, but the little is known of the cellular and molecular mechanisms behind the enviromental control. We established the flower-opening system using with isolated flower buds. In this system, blue light (470 nm) is more effective on the induction of flower opening than red light (660 nm) at 50 μE. The longitudinal elongation of epidermal cells of the petals was enhanced by a temperature rise and a light irradiation. CytochalasinB, DPI, BrefeldinA, IAA and K-252a inhibited the flower opening and the longitudinal elongation of epidermal cells without the inhibition of the fresh weight increase. During the flower-opening process, the vanadate-sensitive ATPase activity in crude microsome fraction of the petal increased about 1.5 times, on the basis of protein.

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Chloroplast avoidance movement is mediated by phototropin 2 (phot2). The velocity of the avoidance movement in PHOT2/phot2-1 heterozygote is slower than that in WT. Because the PHOT2 amount in the heterozygote is less than that in WT, it is supposed that the velocity of the avoidance is dependent upon the PHOT2 amount. Thus, we investigated whether the phot2-dependent responses were controlled by the PHOT2 amount.
PHOT2-GFP (P2G) was introduced into phot1phot2 double mutants of Arabidopsis. We isolated transgenic plants that showed 0 to 20 fold accumulation of PHOT2 than that in WT and examined phot2-related responses. While the velocity of the chloroplast avoidance movement was dependent on the P2G amount, almost leaves of the transgenic plants grew to flat despite the P2G amount under natural light condition. These results suggested that threshold levels of the PHOT2 amount were dependent upon light induced physiological responses.

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State transition is a mechanism that redistributes excitation energy between PSI and PSII. When an excess energy is accumulated in PSII, LHCII is detached from PSII and reassociate with PSI, resulting in the increased excitation energy populates on PSI. Recently, we reported three monomeric LHCIIs (CP29, CP26 and LHCII-typeII) act as linkers between trimeric LHCII and PSI in State 2. In this study, we generated a RNAi mutant of CP29 (b41) to investigate on a specific role of CP29 in state transitiosn in Chlamydomonas reinhardtii. RT-PCR and western blot analysis indicated that LHCB4 mRNA level and the amount of CP29 in the b41 strain decreased to about one-fifth of that of wild type. The b41 strain did not grow under high light, indicating that the down-regulation of CP29 caused reduction of the ability of photoprotection. Additional results from physiological and biochemical studies on the b41 mutant will be presented.

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Ultraviolet radiation (UV) is toxic to organisms. Higher plants grown under solar radiation are irradiated with UV at the same time. DNA damages induced by UV, pyrimidine dimers, cause an inhibition and error of DNA replication, leading to mutation. We investigated the specificity of UV-induced mutation in Arabidopsis thaliana with the use of the system for mutation detection in plants we have developed. E. coli rpsL gene introduced into Arabidopsis is a target gene for mutation. Infrequent base substitution mutations of AT to TA and GC to CG were detected in UV-B-irradiated Arabidopsis in addition to GC to AT transitions which are frequently observed in E. coli and mouse skin. Insertion of long sequence was also observed. Mutational spectrum obtained was different from other organisms, suggesting to be specific to higher plants. We are analyzing mutation in Arabidopsis in which CPD photolyase gene was silenced by RNAi.

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In the wild-type and colorless mutant cells of Chlorella kessleri grown with glucose as a sole carbon source in the dark, the uptakes of hexose and of acetate were reduced by up to 50% by irradiation with white light. Irradiation of the colorless mutant cells with white light resulted in photo-bleaching of Chl(lide) a. On the contrary, the photo-inhibitions of hexose and acetate uptake were not observed in a chlorophyll-less yellow mutant of Chlorella kessleri. Thus the photo-inhibitions of hexose and of acetate uptake observed in the Chlorella cells are assumed to be caused by active oxygen produced in the process of photo-bleaching of Chl(lide) a.

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In deciduous trees, immature leaves in early spring and senescing leaves in autumn have low photosynthetic activity. In addition, they sometimes suffer from low temperature in cool temperate forests. To examine whether defensive responses to photo-oxidative stress are observed in such leaves, we harvested leaves of 5 deciduous tree species from the experimental forests of Hokkaido Forestry Research Institute once in every month from May to October for 3 years. Xanthophyll cycle pool size was larger in early spring and autumn. Changes in de-epoxidation rate of xanthophyll cycle were different among species and years, however, the de-epoxidation rate was higher in autumn, especially in broad leaved species. Membrane-bound APX activity was relatively higher in early spring and increased in autumn. These results suggest that xanthophyll cycle and APX activity involve in defensive responses to photo-oxidative stress in immature leaves in early spring and senescing leaves in autumn.

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It is known that the pool size of xanthophyll cycle pigments increases on light-harvesting complex-II (LHCII) of plants grown under high light conditions. However, little is known about the change of the carotenoid composition of LHCI during the process of adaptation to high intensity light. In the present study, plants and photoautotrophic (PA) cells of tobacco were grown under high light (HL) or low light (LL) conditions, and the pigment composition of LHCI and LHCII were analyzed.
Results indicate that, in leaves, the ratio of xanthophyll cycle pigments in total carotenoids of PSI increased in HL in comparison with that in LL. The number of xanthophyll cycle pigments per LHCI in HL was also higher than in LL. Besides, since the Chla/Chlb ratio was higher in HL compared with LL, we suggested that the proportion of LHCI to reaction center is different between HL and LL.

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Ultraviolet (UV) damages the plants by targeting the DNA, photosynthetic apparatus and a wide variety of physiological processes. Our recent study on ozone-induced cell death in tobacco cells propounded the involvement of reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), and hydroxyl radical (HO^•), and calcium signaling in the ozone-induced cell death induction. By analogy to the ozone-induced cell death, we attempted to evaluate the involvement of oxidative and calcium signaling mechanisms in UV-C (254 nm)-dependent cell death induction using both the ozone-sensitive and ozone-tolerant tobacco cells. Data suggested the involvement of ROS such as H₂O₂, HO^•, ¹O₂ and superoxide (O₂^•-), enhancement of cell death by Fenton catalysts, and relay of signals mediated by calcium influx during the UV-induced cell death induction.

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Plants were exposed to the various DNA-damaging stress in daily life. To survive in such hostile environments, plants developed many mechanisms to avoid or to repair the DNA damage. Yeast and mammals have a mechanism named translesion syntheses (TLS), in which DNA damage is bypassed by specific DNA polymerases. However, TLS generates mutations as a result of incorrect replication (error-prone TLS).
We have previously isolated AtREV3, AtREV1 and AtREV7 genes, which are homolog of yeast TLS polymerases. We also showed that disruption of these genes made the plants sensitive to UV-B, gamma-ray and MMC.
To determine whether AtREV3 acts in the error-prone TLS, we utilized the β-glucuronidase (uidA) gene containing single nonsense mutations. Transgenic plants with inactivated uidA were treated with UV-C and reversion events were counted. As a result, mutation frequency was reduced in the rev3 background, indicating that AtREV3 acts in the error-prone TLS in Arabidopsis.

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In order to study the mechanism of light adaptation, we determined the chloroplast pigment composition and photosynthetic properties of Begonia semperflorens plants cultivated under16 h light/8h dark photoperiod, and either low light (LL) or high light (HL) conditions. Regarding the composition of photosynthetic pigments, even though an increase of xanthophylls cycle pigments was observed, both LL and HL plants had very low Chla/Chlb ratio value, 0.6 and 1.2, respectively. Based on these results, we analyzed the pigment-protein complex composition of the thylakoids by non-denaturating electrophoresis, and found that the Photosystem I band was very weak. We were almost unable to detect the PsaA even when thylakoidal proteins were analyzed by SDS-PAGE. On the other hand, the Photosystem II protein level of Begonia was comparable with those from other plants such as tobacco similar to that other plants, which suggest that the Photosystem I/Photosystem II ratio is lower in B. semperflorens.

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HCF164 is a membrane anchored thioredoxin-like protein faced to thylakoid lumen side. In this study, we found that chloroplast stroma m-type thioredoxin is the source of reducing equivalents for reduction of HCF164 in the thylakoid lumen. In order to probe the function of HCF164 in the lumen, we screened the reducing equivalent acceptor proteins of HCF164 using a resin-immobilized HCF164-single cysteine mutant, and successfully identified the putative target thylakoid proteins. Among the newly identified target protein candidates, the reduction of the PSI-N subunit of photosystem I by HCF164 was confirmed both in vitro and in isolated thylakoids. Two components of the cytochrome b6f complex, the cytochrome f and Rieske FeS proteins, were also captured as novel potential target proteins by our chromatography method. We will discuss the physiological role of HCF164 in the thylakoid lumen.

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Thioredoxin (Trx) is small ubiquitous protein that catalyzes thiol-disulfide reaction by two catalytic cysteine. Green sulfur bacterium Chlorobium tepidum TLS require strict anaerobic condition for growth and use various inorganic sulfur compounds as well as H₂S as electron donors. The complete genome of C. tepidum was determined in 2002 (The Institute for Genomic Research) and two Trx homologues were found. To elucidate Trx system in C. tepidum, we constructed recombinant Trx expression vector and cysteine mutant which one cysteine at the active site was substituted with serine. By immobilized cysteine mutant to resin, Trx target protein of C. tepidum were efficiently acquired.

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Brassica napus leaf protoplasts become swollen and finally degrade during culture. The process through which the death occurred were subjected to apoptotic-like manner. Glutamate dehydrogenase (GDH) catalyzes the amination of 2-oxoglutarate and the deamination of L-glutamate. GDH of B. napus consists with a and b subunits that are encoded by two genes (GDH1, 2), respectively and forms seven isoforms (GDH1~7).
de novo synthesis of a subunits was post-translationally regulated in B. napus leaf protoplasts. While, activation of a subunits in wounded tissue were regulated by reduction of disulphide bond. Leaf protoplasts were subjected to oxidative stresses and in different redox state, which led to prevent translational inhibition. Leaves treated with SA and JA in an acidic conditions showed the same GDH pattern as protoplasts.
These results suggest that a subunits of GDH are stress responsive and could be involved in redox regulation and pH stability.

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Acylamino Acid Releasing Enzyme (AARE) catalyzes the N-terminal hydrolysis of N^α-acylpeptide, which releases N^α-acylated amino acids. In mammal it is also active as Oxidized Protein Hydrolase (OPH) which might play an important role in destroying glycated and oxidatively damaged proteins. Using plants expressing high AARE, we analyzed its function in order to clarify whether plant AARE has the OPH activity like animal AARE.
Arabidopsis AARE incorporated transgenic tobacco was generated by Agrobacterium-mediated method. The incorporation of the gene into tobacco genome was confirmed by immunoblot analysis. Transgenic lines of plants showed higher AARE activities than wild-type ones. Subcellular fractionation analysis indicated that AARE of transgenic tobacco localized in cytosol. The presence of AARE in cytosol of nontransgenic Arabidopsis was also confirmed. The levels of oxidized proteins in transgenic lines were compared with those in wild-type. We are examining correlation between acylamino acid releasing activity and accumulation of oxidized proteins.

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It was reported that sfr2 (sensitive to freezing 2) gene plays an important role in freezing sensitivity in Arabidopsis (Thorlby et al., 2004). Arabidopsis sfr2 protein can be classified into family 1 glycosyl hydrolase based on deduced amino acid sequence and shows a specific β-glucosidase activity. However, expression of Arabidopsis sfr2 gene was observed to be constitutive rather than cold stress inducible. We detected a rice cDNA (OsSFR2) in the public databases, which encodes homolog of Arabidopsis sfr2 gene. Rice OsSFR2 protein has 60% homology at amino acid level to Arabidopsis SFR2 protein. Southern analysis with OsSFR2 full-length probe revealed that more than two closely related genes might exist in rice genome. Northern analysis with OsSFR2 gene specific probe showed that the gene is expressed strongly in rice seedling leaves in response to cold stress. These results indicate that OsSFR2 may be involved in cold stress response in rice seedlings.

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Some plants in the family of Gramineae accumulate fructan in their tissues as a temporary storage carbohydrate. The accumulation of fructan is associated with freezing tolerance of the plant. Rice is a cold-sensitive plant that is not able to synthesize fructan. We have reported that an increase in cold tolerance at the booting stage was found in transgenic rice expressing wheat fructosyltransferase (Sato et al., Breeding Science Supple, 2005). A rice transformant with 1-SST (sucrose:sucrose 1-fructosyltransferase) accumulates β(2,1) linkage oligomer (DP3-5) and that with 6-SFT (sucrose:fructan 6-fructosyltransferase) accumulates mainly β(2,6) linkage fructan. Cold tolerance of rice seedlings in which fructan accumulates was higher than that of non-transgenic plants. While sucrose content in rice seedlings was remarkably increased by cold treatment, an increase in fructan content and a decrease in the degree of rise in sucrose content were detected in rice seedlings with wheat fructosyltransferase under a low temperature condition.

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We have previously reported that Klebsormidium flaccidum, a green alga (Charophyceae), which lives in cold regions and belongs to sister group of land plants, increases freezing tolerance during cold acclimation. The increase is associated with increases in the content of sucrose and γ-aminobutyric acid (GABA), both of which are closely associated with in freezing tolerance of land plants. We here isolated genes encoding key enzymes of biosynthetic pathways of sucrose (sucrose phosphate phosphatase, SPP) and GABA (glutamate decarboxylase, GAD) in K. flaccidum by PCR amplification using degenerate primers constructed with information on conserved amino acid sequence segments of SPPs and GADs of land plants. The deduced amino acid sequences of isolated cDNA fragments of KfSPP and KfGAD showed high similarities to those of SPPs and GADs of land plants. KfSPP and KfGAD may be associated with the accumulation of sucrose and GABA during cold acclimation in K. flaccidum.

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Exposure of rice plants at booting stage to moderate low temperature results in dramatic decrease in fertility. The phenomenon is associated with male sterility, due to the disfunction of tapatum cells. To address molecular events during this process, we carried out transcriptome analysis using a 22K chip (Agilent).
Since we observed notable difference in fertility in plants chilling (12°C) treated for 2d (70%) and 4d (25%). RNAs were prepared from anthers of 2d and 4d plants and plants with 1 day recovery at normal temperature (2d+1 and 4d+1), and were subjected to microarray analysis. Analysis of gene expression profiles revealed no significant change in major metabolic pathways, whereas genes involved in photo-system I, II are dramatically diminished in 4d+1 plants, indicating that there is a correlation between disorder of photo-system and diminished fertility by 4d chilling-treatment.

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Arabis serrata is a perennial plant that grows on Mt. Fuji. Although most of Arabis abscise leaves before winter, this plant winters with green leaves. It has been observed that the winter leaves accumulate high level of sugars, and the accumulated sugars are thought to enhance cold tolerance and supply energy for growth in early spring.
To investigate the mechanism of accumulation of sugars in leaves in detail, we did quantitative analysis of sugar components in leaves sampled at Mt. Fuji at May, September and November. The results showed that sucrose accumulated in leaves from May through November, while significant accumulation of fructose and glucose wasn't observed. Analysis of gene expression of sucrose-phosphate synthase(SPS), which is a key enzyme of sucrose synthesis, revealed that expression of SPS gene also increased from May through November. These results suggested that sucrose synthesis was promoted by an increase of expression of SPS gene.

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FOX Hunting System is useful tool for large-scale functional gene analysis. By using this system, we attempt to isolate rice genes involved in high-temperature stress tolerance. So far, we screened about 17,000 lines of transgenic Arabidopsis overexpressing rice full-length cDNA (rice FOX lines), and isolated a thermotolerant line R04333 that can survive after heat shock (42°C for 90 min). The introduced rice cDNA in R04333 encodes putative heat shock transcription factor (HSF). We generate transgenic Arabidopsis plants expressing the HSF gene (retransformant lines). Retransformant lines showed thermotolerance as R04333, concluding that thermotolerance phenotype of R04333 is caused by over-expression of HSF gene. In rice seedling, HSF gene responded to heat shock. In R04333 and retransformant plants, constitutive expression of some heat shock protein (HSP) genes was observed. Therefore, the HSF gene characterized by FOX Hunting system should be involved in thermotolerance with up-regulating HSP genes.

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Arabidopsis transcriptional factors DREB1s/CBFs specifically interact with a cis-acting element DRE/CRT/LTRE involved in cold-, drought- and high-salinity-inducible gene expression. Expression of the DREB1C gene induces rapidly under low temperature conditions. Using a GUS reporter gene fused to the DREB1C promoter, we identified a 67-bp region that contains cis-acting elements involved in low temperature-responsive gene expression of the DREB1C gene. Using yeast one-hybrid system, we isolated a cDNA clone encoding a protein that binds to the 67-bp promoter region of DREB1C. The cDNA encodes a protein named bHLH072, which belongs to the bHLH-type transcription factor family. We analyzed function of bHLH072 in the expression of the DREB1 genes in response to low temperature stress using transgenic plants.

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Low-temperature storage of potato tubers leads to an accumulation of reducing sugars. We have investigated that there are three types of sugar change among cultivars: an increased level of reducing sugars; low levels of reducing sugars; and increased sucrose, not reducing sugars. These patterns are related to the activities of vacuolar acid invertase. In this study, we performed cloning of cDNA for this enzyme, and genomic southern analysis among cultivars having different type of sugar changes (May Queen, White Fryer and Inca-no-mezame). cDNA from May Queen contained one open reading frame including β-fructosidase motif, peptide domain consisting of a Cys, and four potential glycosylation sites, which are highly conserved in acid invertases of plant origin. Southern blot analysis showed that the gene exists as a single-copy gene in all cultivars, and promoter region and/or non-coding region in Inca-no-mezame seem(s) to be different from those of other two cultivars.

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This study examined how the root temperature affects water uptake and transpiration of rice plants (Oryza sativa L.). Over a range of root temperature, T_R from 35 to 13 ^oC, transpiration E_T and the leaf water potential Ψ_L gradually decreased with decreasing T_R. Below a critical root temperature 13 ^oC, E_T and Ψ_L abruptly decreased with decreasing T_R, which is due to an abrupt decrease in hydraulic conductance of plants. The bulk stomatal conductance of whole plants under experimental conditions was expressed as a unique function of only Ψ_L. Using the experimental result, we constructed a model for water uptake and transpiration of rice plants, which consists of the energy balance equation for plant leaves and the water transport equation by the root. We also found the possibility that the reduction in the activity of aquaporins causes a decrease in hydraulic conductance of plants at low root temperatures.

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When environmental conditions such as temperature or salt concentration etc. are changed, photosynthetic organisms attenuate their metabolic systems and acclimate to the new environment. The cyanobacterium, Synechocystis sp. PCC6803 maintains constant growth rates between 25ºC and 40ºC. This is supported by constant photosynthetic rates at each cultivation temperatures. The cyanobacterium changes photosynthetic activity depending on temperature change. It is reported that photosynthetic activity gradually changes when cultivation temperature is changed in light conditions and reaches to its optimal rates at the temperature. But it is unclear if acclimation takes place in darkness. In this study, we investigated time-dependent activity changes on the shift of cultivation temperature between 25ºC and 35ºC. It was found that acclimation was not observed by temperature shift in darkness but occurred by the following light period. We will also report on the acclimation site in the electron transport system.

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Cold acclimation is the phenomenon in which plants increase in freezing tolerance upon exposure to low nonfreezing temperatures for an appropriate duration. Expression of cold-induced genes serves as a key during cold acclimation. In this study, we adopt an activation-tagging method to search novel Arabidopsis genes that are involved in cold acclimation.
We found that one of the mutants obtained, the line 18-16, possessed a significant capability of freezing tolerance: the survival rate of calli of the mutant was 13 times higher than that of the wild type at -15 ^oC. The analyses of Southern blot and TAIL-PCR revealed that only one copy of T-DNA was inserted near the genes of ACT7/2 and fibrillin-like protein (FIB) in the mutant. Furthermore, we made the mutants overexpressing the FIB gene, of which the calli showed a remarkable freezing tolerance.

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Protonema cells of the moss Physcomitrella patens develop freezing and dehydration tolerance upon treatment with abscisic acid (ABA). To understand mechanisms underlying physiological processes leading to development of stress tolerance, we isolated P. patens mutant lines with reduced sensitivity to ABA. Among these lines, AR7 showed growth similar to wild type but did not develop freezing or dehydration tolerance by ABA treatment. Analysis of low molecular-weight soluble sugars indicated that AR7 protonema cells accumulated less but significant amounts of sucrose upon ABA treatment. In contrast, AR7 accumulated very little amounts of trisaccharide theanderose. Results of analysis of LEA-like heat soluble proteins in the AR7 mutant will also be presented.

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Spores of Osmunda japonica are suitable for the material of biochemical studies, because it is possible to prepare plentiful spores. However, we have some experience getting the spore preparation with low viability. If the viability of spores depends on the humidity at the time of spore preparation, this phenomenon has been verified in this study. Spores were treated by various relative humidity (RH) for 24 hours, and then used to determine the viability. The viability of the spores treated by 0.3% RH was very low. The viability became larger as RH increases from 0.3% RH to 60% RH and fell off to the bottom at 81% RH. It was elevated again to the largest viability by increasing toward 100% RH. Clearly the difference of humidity in the time that prepared spores led to the dispersion of viability. The transfer speed of water may relate to the viability of spores.

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ABA plays important roles in plant development and responses to various environmental stresses. ABA is synthesized in response to drought, and ABA promotes the closing of opened stomata and inhibits the opening of closed stomata. The resulting stomatal closure reduces transpirational water loss and maintain water homeostasis in the plants. Proteomics will be extremely useful approach to investigate signal transduction pathway, because protein-protein interaction and post-translation modification including phosphorylation often plays a major role in determining the final effectiveness of signal transduction pathways. The crude protein mixtures were prepared from epidermal tissues of Arabidopsis thaliana Col-0.
They were subjected to 2D-PAGE. The gels were stained with Pro-Q Diamond for detecting phosphorylated proteins and with CBB for all proteins. The spots were digested with lysyl endopeptidase and characterized by MALDI-TOF MS. Several proteins were up regulated in response to ABA treatment (50uM for 5 min).

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In the Arabidopsis thaliana genome, there are at least 11 distinct Arabidopsis histidine kinases classified into two groups: the ethylene receptors consist of 5 members and the other six AHKs. Recently, it has been shown that ATHK1 and a cytokinin receptor CRE1/AHK4 can function as osmosensing receptors in yeast, here we further showed that AHK2 and AHK3 as well. However, the ethylene receptors do not. Expression of ATHK1, AHK2, AHK3 and AHK4 is induced by dehydration. Transgenic plants overexpressing ATHK1 have delay in bolting and display shorter inflorescence, however the morphological phenotype of the athk1 T-DNA insertion mutant is unchanged. Callus induction assay suggested that ATHK1 may also function as cytokinin receptor. Germination assays demonstrated that these 4 kinases may have important function in ABA and glucose signalings. Northern blot analysis of stress-inducible marker genes suggested that these kinases also play an important role in abiotic stress signaling.

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By using yeast two-hybrid screening, we identified a C3HC4-type ring domain containing protein which interacts with DREB2A and named D2AIP1. In Arabidopsis genome another two homologs were found and named D2AIP2 and D2AIP3. The three proteins were characterized to interact with DREB2A through the protein C-terminals. In vitro ubiquitination assays demonstrated that D2AIP1 ubiquitinates itself in the presence of ubiquitin, E1 and E2, and the C3HC4 ring domain was essential for its activity. Promoter activity analysis of D2AIP1 showed that it expresses in roots, cotyledons and flowers. DREB2A transcripts were also found in roots and cotyledons under normal condition, but the DREB2A protein could be hardly detected. When the transgenic plants harboring DREB2Apromoter:GFP-DREB2A were treated by a proteasome inhibitor MG132, a clear GFP inflorescence was observed. These results suggest that D2AIP1 interacts and ubiquitinates DREB2A under unstressed condition.

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A SNF1-related kinase 2 homolog (LeSRK2C) cDNA was isolated from tomato Micro-Tom (Lycopersicon esculentum Mill.). LeSRK2C has a kinase domain in the amino terminal domain and an acidic and hydrophobic amino acid-rich region in the carboxyl terminus. LeSRK2C mRNA is expressed in leaves, flowers and fruits but not in roots. A recombinant LeSRK2C expressed in E. coli cells phosphorylated myelin basic protein but not histone H1. LeSRK2C is detected mainly in leaves, but not in flowers, fruits nor roots by immunoblot using an anti-LeSRK2C specific antiserum. Transient expression assay of LeSRK2C by agroinfiltration method demonstrated that LeSRK2C is stimulated strongly by chilling stress and by NaCl stress. These observation shows that LeSRK2C has an important role in stress signaling of tomato leaves in response to salt and chilling stress.

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The common ice plant is a halophyte and a model plant for salt tolerance. We have studied on the ice plant for understanding molecular mechanisms of salt tolerance, especially focusing on genes encoding protein phosphatases type 2C (PP2C) that are thought to be negative regulators of the MAPK signal transduction pathway. In this study, we have cloned 11 PP2C genes from the ice plant and named MPC1~11. Gene expression of MPC2 and MPC8 are up-regulated and that of MPC6 is down-regulated by salt stress. Since we have not yet established a transformation method of the ice plant, we introduced MPC2, MPC6 or MPC8 genes into Arabidopsis plants under 35S promoter, allowing over-expression of these heterogous genes to investigate functions of these PP2C genes concerning the signal transduction pathways against salt tolerance.

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A transcription factor DREB2A interacts with a cis-acting DRE sequence and activates expression of downstream genes involved in drought and salt stress response in Arabidopsis thaliana. OsDREB2B is one of DREB2 homologues in rice, and it has high homology to HvDRF and ZmDREB2A. OsDREB2B transcripts were accumulated by low temperature, dehydration and salt stresses. OsDREB2B produced two forms of transcripts and quantitative real-time PCR analyses demonstrated that the functional transcription form of OsDREB2B was significantly induced by stresses compared with the other form. Using transient transactivation experiments we found that functional transcription form of OsDREB2B exhibited high transactivation activity in Arabidopsis protoplasts but the other form did not. These data suggest that splicing mechanisms play an important role in regulating OsDREB2B gene activity under stress conditions. We are also analyzing the function of OsDREB2B using transgenic Arabidopsis and rice.

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A cDNA encoding a transcription factor DREB1A was introduced into lettuce (Lactuca sativa L.) by Agrobacterium tumefaciens-mediated transformation to improve salt and drought stress tolerance. First we analyzed activity of CaMV35S and rd29A promoters using a GUS reporter gene. GUS activity driven by the rd29A promoter was induced by dehydration and that driven by the 35S promoter showed constitutive induction. We also generated transgenic plants overexpressing DREB1A using the 35S and rd29A promoters. The 35S:DREB1A-18 transgenic plants showed enhanced survivability under salt stress condition by 200mM NaCl compared to control plants. The rd29A:DREB1A-31 plants are likely to increase the drought stress tolerance. Expression of DREB1A target genes was analyzed by hetero-probed micro array and some of the genes were induced by DREB1A overexpression. These results indicate that the Arabidopsis rd29A promoter and DREB1A gene might be useful for improvement of salt and drought stress tolerance in transgenic lettuce.

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Protein phosphorylation/dephosphorylation are major signaling events induced by osmotic stress in higher plants. Recently, we found that the SNF1-related protein kinase 2 (SnRK2) family was activated by ABA or hyper osmolarity. There are ten members (SRK2A-J) of SnRK2 in Arabidopsis and one of these, SRK2E/OST1, is an ABA-activated protein kinase which regulates stomatal closure. In addition, SRK2C is an osmotic stress-activated protein kinase that can significantly impact drought tolerance in Arabidopsis. Although both SRK2E and SRK2C are classified into SnRK2a subfamily, there are four SnRK2b subfamily in Arabidopsis and they have not been fully investigated yet. Here, we investigated one of Arabidopsis SnRK2b, SRK2G, for its activation or expression pattern in Arabidopsis. In-gel kinase assay showed that SRK2G was rapidly activated by osmotic stress within 30 seconds, and expression analysis revealed constitutive expression of SRK2G in Arabidopsis. We are now analyzing SRK2G-overexpressing plants and its T-DNA insertional mutants.

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