White latex in shoots of an oil plant Euphorbia tirucalli contains abundant secondary metabolites such as phytosterols. Using this organism, we have obtained more than 20 cDNA clones related with sterol metabolism by EST analysis and RT-PCR cloning, established plant regeneration by successive cultures on LS medium containing 0.02mg/l thidiazulon and then on hormone-free LS media, and revealed sensitivity of the callus for G418 and hygromycin B. In order to develop transgenic E. tirucalli with higher amount of phytosterols, we have attempted Agrobacterium-mediated introduction of binary vectors for 35S::squalene synthase gene, 35S::squalene epoxydase gene, and beta-amyrin synthase gene RNAi, and are checking corresponding transcripts by RT-PCR. This work was partly performed as one of the technology development projects of the "Green Biotechnology Program" in New Energy and Industrial Technology Development Organization (NEDO).

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When tobacco cells are put under sucrose starvation conditions, the net degradation of cellular components occurs. The degradation of proteins is inhibited by 3-methyladenine (3-MA), an inhibitor of autophagy. Thus autophagy contributes to the degradation of intracellular proteins under sucrose starvation conditions. In contrast, 3-MA does not affect a decrease in the level of phosphatidylcholine (PC), a major phospholipid. Thus autophagy does not seem to contribute to the degradation of PC. In this study, we examined the accumulation of some metabolites relating to PC degradation by thin layer chromatography. We found that several relatively hydrophilic compounds accumulated in cells when the net degradation of PC occurred. 3-MA inhibited the accumulation of these compounds although it did not have any effect on the net degradation of PC. These results suggest that autophagy is involved in a late step of the PC degradation process although it is not in an early step.

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SULTR1;1 and SULTR1;2 are high-affinity sulfate transporters localizing at the epidermis and cortex of Arabidopsis roots. The double knockout mutant, sultr1;1 sultr1;2, was unable to grow on a low-sulfate medium containing 15 μM sulfate; however the growth was partially rescued by the addition of 1.5 mM sulfate to the medium, suggesting that the sultr1;1 sultr1;2 mutant may utilize a low-affinity transport system for sulfate uptake. The cell type-specificities of all sulfate transporters were analyzed by expressing the reporter fusion gene constructs in Arabidopsis. Among them, SULTR3;4 was suggested to localize at the endodermis and phloem of roots. In yeast, SULTR3;4 mediated sulfate import, but showed a linear increase of sulfate uptake activity when the extracellular concentration of sulfate was elevated up to 1 mM. In addition, SULTR3;4 mRNA was accumulated significantly in the roots of sultr1;1 sultr1;2 mutant, suggesting its contribution in compensating the high-affinity uptake of sulfate in Arabidopsis.

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Approximately 40 % of Arabidopsis proteins are predicted to contain membrane-spanning domains, anchoring them to lipid bilayers as integral membrane proteins. In addition to the ion transporters facilitating transport of nutrients and metabolites across the membranes, proteins that may potentially regulate the ion transport systems in response to various environmental stresses can be included in this category. GeneChip analysis of sulfur-starved Arabidopsis plants indicated that several putative membrane-bound proteins as well as the isoforms of functional sulfate transporters accumulate during sulfur limitation. Membrane-bound proteins were selected by a plant membrane protein database, ARAMEMNON (http://aramemnon.botanik.uni-koeln.de./). In particular, we focused on the functions of proteins that stimulate the activity of sulfate transport in yeast-expression system. T-DNA knockouts and over-expressors of these primary candidates are analyzed for identification of their protein functions.

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It is known that plant has two types of nitrate uptake system, a high-affinity nitrate uptake system (HATS) and a low-affinity nitrate uptake system. In barely four nitrate transporter (HvNRT2) genes which is considered to encode low-affinity nitrate uptake system have been identified. We previously reported that HvNRT2 protein was localized in the plasma membrane of barley root cells and the HATS activity was regulated at post-translational levels. It has been considered that the post-translational regulation of HATS activities are mediated by phosphorylation of NRT2 protein at C-terminal region. In this study, we examined the possibility of in vitro phosphorylation of HvNRT2 protein. As a result, C-terminal region of HvNRT2 protein was phosphorylated in vitro by protein kinase in the soluble fraction of barley roots, and its activity was Ca²⁺-dependent and nitrate-inducible.

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Vacuolar (Na⁺, K⁺)/H⁺ antiporter transports Na⁺ and K⁺ from the cytoplasm to vacuoles using a pH gradient generated by two H⁺-pumps, vacuolar H⁺-ATPase (V-ATPase) and H⁺-inorganic pyrophosphatase (V-PPase). In barley, a salt-tolerant crop, salt stress and treatment with ABA and 2,4-D increased the activities of Na⁺/H⁺ antiporter and H⁺-pumps in tonoplast vesicles from roots. We isolated one gene ( HvNHX1) for the antiporter, one (HvVHA-A) for the catalytic subunit of V-ATPase, and two (HVP1 and HVP10) for V-PPase from barley. Salt and osmotic stresses and treatment with ABA and 2,4-D increased the expression of HVP1 in roots. The maximum level of HVP1 transcripts in response to these hormones was much higher than the levels of HVP10 and HvVHA-A transcripts. Furthermore, HvNHX1 expression changed in a pattern similar to that of the HVP1 expression, suggesting that the expression of HVP1 is co-ordinated with that of HvNHX1 in response to environmental changes.

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Cation/H⁺ exchanger (CAX) is an active transporter, which transports divalent cations using a H⁺ gradient. Although the CAX was cloned from yeast, cyanobacteria, Arabidopsis, mung bean, and Zea mays, there is no report about the rice CAX, which is a model plant of monocotyledon.
We identified five CAX from rice, and phylogenetically divided them into two clusters. Gene expression and absolute amounts of mRNA in different organs were analyzed by real-time PCR. OsCAX1a showed high expression in almost all organs. OsCAX1b and OsCAX1c were detected in limited organs and their expression levels were very low. The mRNA levels of OsCAX2 and OsCAX3 varied with the organ. OsCAXs were heterologously expressed in yeast to characterize the ion transport activity. All exchangers, except for OsCAX2, conferred the tolerance to Ca²⁺. OsCAX1a and OsCAX3 conferred the tolerance to Mn²⁺. The diversity of expression sites and substrates suggest the broad range function of CAX.

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Cation/H⁺ exchanger (CAX) is an active transporter, which transports divalent cation using a H⁺ gradient. There are five CAX in rice; OsCAX1a, OsCAX1b, OsCAX1c, OsCAX2, and OsCAX3. OsCAX3 belongs to the Type IB, which might transport heavy metals, such as Mn²⁺ and Cd²⁺. However, the physiological function of OsCAX3 has not been revealed. OsCAX3 was expressed in the whole plant and conferred the Ca²⁺ and Mn²⁺ tolerance to the yeast. The present study aims to elucidate the physiological function of OsCAX3. First, we determined the tissue specificity using promoter-GUS transgenic rice. OsCAX3 was expressed in embryo, aleurone layer, root steles, root tip, shoot apical, vascular bundle and trichome. Second, the subcellular localization was determined using GFP fusion protein (GFP-OsCAX3). When the protein was expressed in protoplast prepared from rice callus, the green fluorescence was observed in dotted organelles moving in the cytosol. Now, we are trying to identify the organelles.

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Plants have two types of H⁺-pyrophosphatase (H⁺-PPase). The type I localizes on vacuolar membranes. Type II H⁺-PPase is different from the type I enzyme in primary sequence and intracellular localization. Previous work using GFP-fusion protein revealed the Golgi apparatus localization of type II H⁺-PPase (FEBS Lett. 2001). In this study, we aim to determine actual localization of endogenous type II of Arabidopsis (AtVHP2;1) and absolute protein amount. The level of AtVHP2;1 was quantified in several organs at different growth stages by immunoblotting. For suspension cultured cells, the content of AtVHP2;1 was less than 0.5% of that of type I enzyme. When the intracellular localization was determined by sucrose density gradient centrifugation, AtVHP2;1 was not co-fractionated with RGP1 (marker of trans-Golgi), AtVSR1/AtELP (PVC/TGN marker), or the other markers of the ER, vacuolar, or plasma membranes. With consideration of its small amount in cells, AtVHP2;1 may be located to small, minor organelle.

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There are several types of K⁺ channel in organisms and they play various roles in cells. In order to investigate physiological meanings of K⁺ channel in rice, we studied expression and localization of putative outwardly rectifying K⁺ channel gene (ROK) cloned from 3 varieties of rice, Hitomebore, Hidekomochi and Nipponbare, Japonica cultivar. Phylogenetic analysis of plant K⁺ channels supported ROK in the outwardly rectifying groups. Specific expression of ROK in anthers revealed higher in the stage at 10 days than 5 days before the heading.
Microsomal membranes of ROK expressing yeast cells were fractionated through sucrose gradient and studied with using antibodies raised against organella marker proteins (PM Pma1, VM Vam3, ER Kar2, Golgi IDPase). Distribution pattern of the ROK protein showed mainly in the ER neither in tonoplast nor plasmamembrane, which was supported by the fusion protein of ROK-GFP expression in suspension-cultured cells of Arabidopsis thaliana.

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Receptor potential of characean plants against mechanical stimuli is accounted for by an activation of Cl^- channels at the plasma membrane. We have demonstrated that the first step of receptor potential is an activation of mechanosensitive Ca²⁺ channels, but the mechanism of Ca²⁺ channels activation is unknown. We hypothesized the following two mechanisms (1) membrane deformation triggers the channel activation and (2) the membrane stretching does.
We first compressed the plasma membrane by plasmolysing the cell, then stretched the membrane by deplasmolysing the cell, and the Ca²⁺ channels activation was monitored by measuring light emission from aequorin microinjected into the cell. Transient increase in cytoplasmic level of Ca²⁺ was induced not only by the plasmolysis but also by the deplasmolysis, but the both patterns of the transient were different from each other. We discussed that activation of the mechanosensitive channels is triggered by the membrane stretching.

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ATP-binding cassette proteins are found in various organisms ranging from bacteria to human. It has been recently reported that their functions involve not only the efflux of xenobiotics but also various complicated physiological functions, such as regulation of other channels or functioning as molecular switch. P-glycoprotein (PGP)-like gene subfamily has drawn a large attention of many plant physiologists because some members are reportedly involved in auxin polar transport in plant body. In this study, we report the expression profile of AtPGP21 analyzed by northern blot and histochemical staining using Arabidopsis plants transformed with the AtPGP21 promoter::GUS fusion construct. These results showed that AtPGP21 was highly expressed both stem and roots, and revealed different response to plant hormones. We are establishing a heterologous expression system with Sf9 cells for the characterization of its biochemical functions.

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ABC (ATP-Binding Cassette) proteins constitute a large superfamily found in bacteria, fungi, plants and animals. cDNA array analysis of Lotus japonicus during nodulation was recently reported, in which we found one PDR (Pleiotropic Drug Resistance)-type ABC protein (LjPDR12) gene that shows up-regulation during the early stage of nodulation. LjPDR12 shares amino acid identity of 68%, 70% and 65% to AtPDR12, NpABC1 and SpTUR2, respectively.
We then treated L. japonicus seedlings with various chemicals including plant hormones. The expression of LjPDR12 was strongly increased by methyl jasmonate (MeJA). Detailed time-course analysis showed that the increment of the expression reached to ca. 10-fold from 3 to 48 hours after the treatment. Salicylic acid also increased the mRNA level of LjPDR12 up to 10-fold after 3 hours but the up-regulation was transient. In contrast, absisic acid suppressed the gene expression of LjPDR12. We are preparing RNAi plants to provide phenotype analyses.

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Iron deficiency is a serious nutritional problem especially in the developing countries of Asia. Therefore producing iron-fortified rice will contribute to ameliorate the iron deficiency problem. We have already expressed ferritin gene in rice seeds using an endosperm-specific promoter and obtained transgenic rice seeds whose iron concentrations were increased up to threefold. However, higher levels of ferritin expression in rice seeds hardly increased iron concentration, possibly due to iron exhaustion in leaves. In this study, we aimed to further increase iron concentration in rice seeds by enhancing iron absorption and transport ability. We introduced soybean ferritin gene SoyferH2 connected to two endosperm-specific promoters, along with barley HvNAS1, HvNAATA, B and IDS3 genes encoding biosynthetic enzymes of mugineic acid family phytosiderophores, using a marker-free vector. We obtained 102 T0 transformants. Patterns of ferritin expression and iron concentrations of T1 seeds will be presented.

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NIPs, a member of the major intrinsic protein family, are homologues of NOD26 protein, and specific for plants. NIPs functions as channels of small uncharged molecules such as glycerol as well as water. Boron in solution mostly exists as an uncharged boric acid at neutral pH. Boric acid is the chemical form of B that is taken up by plants. It is possible that NIPs also transport boric acid. We previously reported that a member of NIP gene family, was strongly induced by boron deficiency. We will report updates of the analysis of Arabidopsis thaliana T-DNA insertion lines of NIPs, born transport activities of NIPs using yeast transformants. We obtained evidence suggesting that another member of NIPs which is not induced by born deficiency plays a role in boron transport in planta.

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The major translocating sugar of Rosaceae is sorbitol, a kind of polyol. Polyol transporters of plants are classified into two groups; Group I transporters have been well studied and shown to localize in plasma membrane. Although little information about Group II has been reported, myo-inositol transporters of Mesembryanthemum crystallinum (McITR) was firstly identified as a vacuolar membrane polyol transporter in plants. Thus this homolog of Rosaceae may involve in sugar accumulation of fruit vacuole. We isolated two cDNAs, PcITR1 and PcITR2 from pear belonging to Group II. PcITR1 transcripts are high in all organs except in leaves, whereas PcITR2 transcripts are high in leaves. Sugar transport activities of PcITR1 could not be detected in heterologous expression system of yeast. The GFP-PcITR1 fusion protein localized to the plasma membrane in Arabidopsis cell meaning Group II includes not only plasma membrane polyol transporters but also vacuolar ones.

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It is thought that 2-oxoglutarate (2-OG)/malate transporter (OMT) and general dicarboxylate transporter (DCT) in the inner plastidic envelope membrane cooperatively function in import of cytosolic 2-OG generated from TCA cycle and photorespiration, and export of Glu generated from stromal GS/GOGAT cycle. Therefore, the OMT/DCT transport system is thought to play an important role in mediating between carbon and nitrogen metabolic pathways. Because Arabidopsis OMT knockout plant showed slightly delay of growth, we measured metabolite levels in the leaf tissues. The levels of 2-OG and citrate were lower in the knockout plant compared with wild-type plant. Although some amino acids (Glu, Asp, Ala and Ser) were decreased, Gln was abundantly accumulated in the mutant. The knockout plant showed more prominent photoinhibition than wild-type plant under the high-light stress condition. We will discuss on the importance of OMT in the light of carbon and nitrogen metabolisms, and transport of reducing equivalents.

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Tertiary structures of proteins in aquaporin family have been investigated in detail about ten molecular species including glyceroporin, and the mechanism of substrate transportation based on the structures has been reported that the internal structure of the pore have an important role to transport substrate molecules. However, it is also important to understand the mechanism that there might be a regulation region on the surface of the protein. In this study, we examined in detail how molecular backborn structure would change with/without substrate. Since the tertiary structures of glyceroporin of E. coli. were obteined in different conditions with substrate, we took them from the database, and calculated to compare among them. As a result, existence of glycerol had little effect on the degree in slope of the transmembrane helices, but had a significant effect on the degree in twist and also the N-terminal structure of the sixth transmembrane helix.

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Aquaporins are membrane proteins which facilitate water movement in plants. We've already identified 33 rice aquaporin genes from the database and showed their organ specific expression patterns. To clarify the possible role of each aquaporin member in water movement in rice plants, we conducted (1) expression analysis at protein level using specific peptide antibodies and (2) analysis of water permeability of each aquaporin member. OsPIP2;1 and OsTIP1;1 were expressed both in leaf blades and roots, while OsTIP2;1 was only in roots. Several aquaporin members were localized in endodermis, rhizodermis, exodermis, vascular bundles or cortex, but distribution patterns were different among aquaporin members. Analysis of water channel activities revealed that OsPIP2;1, OsPIP2;2, OsPIP2;3, OsPIP2;4, OsPIP2;5, OsTIP1;1 and OsTIP2;1 had high water permeabilities, while OsPIP1;1 and OsPIP1;2 did not. From these results, we will discuss the function of each aquaporin members.

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Regulation of water permeability of plasma-membrane (Pf1) and vacuolar-membrane (Pf2) is important for plant growth, water supply from root to shoot, and survival under drought condition. To understand how Pf1 and Pf2 should be regulated for satisfying these different demands, a three-compartment model which we developed previously, was used for simulating swelling/shrinking of a protoplast in response to osmotic changes. The results indicate that: (1) In a mature cell with large central vacuole, high values of both Pf1 and Pf2 are required for quick cell-enlargement, and for quick water transport across the cell. By contrast, whole-cell dehydration can be delayed most effectively when both Pf1 and Pf2 are low, but severe dehydration from the cytoplasm can be avoided when Pf1 is low and Pf2 is high. (2) In a young cell which has smaller vacuole, only Pf1 but not Pf2 is important for the optimal adjustment of cell water relations.

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To understand the functions and properties of phytochromes in lower plants, we analyzed phytochrome functions and photomorphogenesis in liverwort Marchantia polymorpha. Most of the life cycle of M. polymorpha is haploid and with a simple body structure, and therefore it can be a unique model plant. We identified a phytochrome gene in M. polymorpha and designated it MpPHY. MpPHY consisted of four exons and three introns, similar to typical phytochrome in higher plants. Southern-blot analysis suggested that M. polymorpha has a single PHY gene, unlike from higher plants that have several PHY genes. We analyzed the properties of MpPHY using an expression system in E. coli, and in M. polymorpha using gene targeting by homologous recombination. In M. polymorpha, it is known that the transition into the reproductive stage is repressed under usual light conditions. We found that M. polymorpha rapidly develops into the reproductive stage under FR rich conditions.

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Phototropin is a plant blue-light sensor protein that possesses a flavin mononucleotide (FMN) as the chromophore. Upon light absorption, structural changes of the FMN-binding LOV domain activate serine/threonine kinase domain, leading eventually to various functions such as tropic responses, relocation of chloroplast, and stomata opening in plants.
Phototropin is a water-soluble protein. Therefore hydration conditions are presumed to affect structural change of LOV domains. Recently, we found that hydration condition significantly affected light-induced structural change of Phy3-LOV2 by means of FTIR spectroscopy. Structural changes of secondary structures, such as α-helices and β-sheets, depend on hydration volume. Furthermore, the decay of the S390, intermediate, also depends on hydration volume. From these results, it is concluded that hydration is necessary for the structural change of Phy3-LOV2, which probably causes protein fluctuation for conformation transition of protein.

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In general, phototropic response in plants was induced by blue light, and wasmediated by a blue light receptor phototropin. In the fern Adiantum capillus-veneris, however, red as well as blue light are effective to induce positive phototropic response in protonemal cells. The red light effect on the tropistic response is mediated by phytochrome3 (phy3), which is a chimeric photoreceptor between phytochrome chromofore binding domein and full length phototropin. Recently, we have found negative phototropism in Adiantum rhizoid under red light. When half side of apical part of rhizoid cell was irradiated with red microbeam, the rhizoid bent towards the opposite side of light irradiation. Similar experiment was performed using rap mutants which are defective of PHY3 gene, and the lack of negative phototropism in rhizoid cell, was revealed meaning that positive and negative phototropisms in protonema and rhizoid, respectively, are mediated by the same photoreceptor phytochrome3.

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There are five species of phytochrome, phyA to phyE, in Arabidopsis thaliana. Among them, phyB is the major photoreceptor that regulates photomorphogenesis under light conditions. Despite large effort, the molecular mechanisms by which phyB signals regulate the seedling development are still largely unknown. As previously reported, the phyB signals are fully induced by the N-terminal domain of phyB. To identify components that are involved in the signaling pathway of N-terminal domain of the phyB protein, we have started a new genetic screen by using mutagenized transgenic plants that expressed the N-terminal domain of phyB (Matsushita et. a, 2005, JSPP Annual Meeting). The M3 seedlings showing enhancement of hypocotyl elongation under weak red light condition and normal elongation of hypocotyl under far-red light condition were newly selected. Consequently, recessive phyB N-terminal domain signaling4 (pns4) and pns5 mutants were isolated. Genetic and physiological analysis of these mutants are in progress.

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Phytochromes (phy) are red(R)/far-red(FR) light photoreceptors. Phys are encoded by a small gene family that is composed of phyA-C in rice. To elucidate specific function of each phy, we analyzed gene expression in phyA, phyB, phyBC, phyAC and phyABC mutants with oligo-microarray. Dark-grown seedlings of wild type (cv. Nipponbare) and phy mutants were irradiated with an R or FR pulse. RNAs were isolated 30min and 4hrs after the irradiation and analyzed. Genes whose expression levels showed more or less than twice of control were selected. They were classified in 4 groups, early induced, late induced, early repressed and late repressed genes, depending on the expression levels at 30min and 4hrs after the irradiation. Most of the early-induced genes were not induced in phyAC mutants. We compared expression patterns between the mutants and found that phyA is a main photoreceptor for R as well as for FR in rice.

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Phytochrome apoprotein is existing in roots of many plants, but its molecular specific function has not been well characterized except for phototropism. We examined photoregulation of root growth using phytochrome mutants in rice. When standard rice plants (Nipponbare) were grown for 7days in darkness, the seminal root grew straight towards geotropic orientation and reached a length of 8cm or more. In contrast, when the seedlings were grown under the continuous white light irradiated condition, the root growth was inhibited when the length reached approximately 2cm. In addition, the distal part of the seminal root showed a peculiar coiled shape. This response depended on the nitrogen concentration in the range of 0.1-10mM. These and the other results of physiological experiments and the Western blot analysis strongly suggested that phytochrome molecular species existing in seminal root perceive light and control their morphogenesis. This work was partly supported by a grant of IP-1006.

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We performed the night-break (NB) experiments and carried out molecular analysis of flowering time genes in rice. We found that a single NB causes the down-regulation of Hd3a without affecting OsGI or Hd1 expression pattern. We also found that phyB transduces the NB signal to down-regulate Hd3a and inhibit the flowering in rice. (Ishikawa et al 2005). In order to understand the role of phytochrome in rice flowering, we focused on the light quality causing NB effect. Down-regulation of Hd3a and delay of flowering was observed by red light mediated NB, but not by far-red light. This result indicates that Pfr-form of phyB negatively regulates expression of Hd3a. Expression mechanism of Hd3a by light-regulated phytochrome will be discussed.

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Phytochrome B (phyB) consists of two domains; the chromophore binding N-terminal domain and the C-terminal domain. The C-terminal domain plays a role in dimerization and nuclear localization of phyB. Yeast two-hybrid analyses have suggested that C-terminus of phyB interacts physically with cryptochromes. We have reported that over-expression of phyB C-terminus enhanced plant's responses to blue light (Usami et al. 2005 JSPP Annual Meeting).
We observed the phyB C-terminus over-expressing plants on various photoreceptor mutants background. The results suggested that phyB C-terminus enhanced the signaling of cryptochrome 1 (cry1). However, we could not detect physical interaction between phyB and cry1 in the co-immunoprecipitation assay. The C-terminal domain of phyB is constitutively localized to the nucleus and forms nuclear speckles. To elucidate how this domain enhances cry1 signal, we are examining various phyB C-terminus variants, which show different intracellular localizations from the wild type C-terminus, by over-expressing them in Arabidopsis.

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Rice has two Cryptochrome 1 (CRY1) genes (OsCRY1a and OsCRY1b) and one CRY2 gene (OsCRY2).
In order to elucidate the function of cryptochromes in rice, we isolated all CRY genes from rice and constructed transgenic rice in which OsCRY1a, OsCRY1b or OsCRY2 gene was overexpressed. When these overexpressers were grown under white light, transgenic plants harboring any of the constructs showed shorter leaf sheaths and blades than the wild type. We further examined the effects of different light qualities (blue-, red- or far-red light) on the growth of leaf sheath and blade.

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The leaf of kidney bean moves in response to blue light. The movement is induced by a decrease in the turgor pressure of motor cells on the irradiated side of pulvinus. Previous study showed that the plasma membrane H⁺-ATPase was inactivated by blue light in pulvinar motor cells. In this study, we investigated the initial event of the movement with respect to function of phototropin and the H⁺-ATPase in pulvini. In darkness, phototropin existed in a dephosphorylated state and the H⁺-ATPase existed in a phosphorylated state. A blue light-pulse induced the phosphorylation of phototropin and subsequently the dephosphorylation of the H⁺-ATPase. These reactions exhibited similar fluence-rate dependencies on blue light. The specific flavoprotein inhibitor diphenyleneiodonium and the protein kinase inhibitor K-252a inhibited both phototropin phosphorylation and H⁺-ATPase dephosphorylation in the same degree in pulvini. These results suggest that phototropin mediates the inactivation of H⁺-ATPase.

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Phototropins, autophosphorylating protein kinases, mediate stomatal opening through activation of the plasma membrane H⁺-ATPase via phosphorylation in response to blue light. However, the mechanism by which the signal from phototropins is transduced into H⁺-ATPase activation remains unknown. Here we show that protein phosphatase 1 (PP1) mediates stomatal opening by blue light. Transient expression of guard cells with mutant type of catalytic subunit of PP1 that lost the catalytic activity, or inhibitor-2, a specific inhibitor protein of PP1 suppressed blue light-dependent stomatal opening. Furthermore, tautomycin, a cell-permeable inhibitor of PP1, impaired the phosphorylation of the H⁺-ATPase in response to blue light without affecting that of phototropins. These separate lines of evidence indicate the function of PP1 as a positive regulator between phototropins and the H⁺-ATPase in the blue light signaling of guard cells.

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Arabidopsis ht 1 was isolated as a hot phenotype under low CO₂ concentration. Its stomatal response to CO₂ was significantly inhibited. The mutated HT1 gene encodes a protein kinase expressed in stomatal guard cells. Phosphorylation assays demonstrate that HT1 has both autophosphorylation and phoshprylation activity. HT1 protein carrying the ht 1-1 or ht 1-2 mutation is greatly impaired or abolished, respectively. The kinase activities of the WT HT1 and its two mutants corresponded nicely to the phenotypes observed in whole plants. Dominant negative transgenic plants in which HT1 kinase activity was disrupted showed suppressed sensitivity to CO₂. These results indicate that the HT1 kinase plays an important role in the CO₂ signaling. Furthermore, in the ht 1 mutants, ABA responses were comparable to those of WT plants. This suggests that the HT1 kinase would function in CO₂ signaling pathway independent of ABA signaling.

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Phototropism is caused by the accumulation of growth inhibitory substance(s) in the illuminated sides, interfering with the action of evenly distributed auxin. Based on this concept, benzoxazinoids (DIMBOA and MBOA) have been isolated as candidates for phototropism-regulating substances in maize coleoptiles. In our previous studies, it is suggested that blue light can induce the activity of β-glucosidase in the illuminated halves, resulting the up-regulation of DIMBOA level from its precursor DIMBOA glucoside. As a result, growth inhibition occurs in the illuminated sides that causes phototropic curvature. In this study, the transcriptional level of β-glucosidase and H₂O₂ level between the illuminated and shaded halves was investigated. Moreover, the level of benzoxazinoids with β-glucosidase inhibitor in both halves was also studied. The mechanism of growth inhibition in the illuminated sides in response to phototropic stimulation in maize coleoptiles will be discussed.

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The chloroplast movement in spinach mesophyll cells, which was induced by high-intensity blue light (460 nm, 50 μmol/m² sec), was inhibited by an actin depolymerizing reagent cytochalasin B (100 μM) reversibly. BDM (2,3-butanedione monoxime), a myosin inhibitor, also inhibited the chloroplast movement reversibly in a concentration-dependent manner (0-100 mM). BDM did not seem to inhibit the chloroplast movement by disruption of the actin organization. Before actinic light irradiation, actin bundles looked like thin, mesh-like configuration surrounding each chloroplast. In contrast, actin filaments in cells irradiated with blue light became thick, straight bundles traversing the cell. On the other hand, red light (660 nm, 30 μmol/m² sec) neither induced chloroplast movement nor affected the actin organization. Our results suggest that the actomyosin system plays a crucial role in the chloroplast movement in spinach mesophyll cells.

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Dynamic behavior and light strength dependency of the blue light induced SIG5 transcription were examined in Arabidopsis thaliana, being compared with other five SIG genes. Transcription of SIG1 and SIG5 in wild type plants was induced by blue-light with the induction time of 30 min and the threshold intensity of 2 μmol/m²s. The SIG1 transcripts accumulated after 90 min illumination kept constant between 2 and 50 μmol/m²s. While that of SIG5 kept constant between 2 and 10 μmol/m²s, but increased with increasing light-intensity beyond 10 μmol/m²s. In the cryptochrome mutant, the SIG5 transcription was not induced by blue-light at all. While the SIG1 transcripts were induced at 30% of the wild-type. These results indicate that cryptochrome mediates the transcription of SIG5 via two pathways differently responding to weak and strong blue-light, and cryptochrome-dependent and independent pathways are involved in the blue light induced SIG1 transcription.

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PixD from Thermosynechococcus elongates is a flavin-binding BLUF protein relevant to phototaxis. In the X-ray structure of PixD, Gln50 and Tyr8 form a H-bond network from the flavin C4=O group, which was proposed to exhibit a large structural change upon photoreaction. The role of this H-bond network in the PixD function, however, has yet to be clarified. In this study, we have examined the relevance of Tyr8 to the reaction mechanism of PixD by means of FTIR spectroscopy. In the light-induced FTIR spectrum of PixD, the CO and COH bands of Tyr were identified using the PixD in which Tyr residues are replaced with 4-13C-Tyr. Theoretical analyses revealed that the OH of Tyr8 is H-bonded to the C=O of Gln50 and this H-bond is strengthened upon photoreaction. This result suggests that the H-bond network from flavin C4=O to Gln50-Tyr8 plays an important role in the signal transduction mechanism of PixD.

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Cyanobacteria have been known to drastically change gene expression pattern in response to change in photic environment suggesting the presence of machinery for transcriptional regulation in response to light stimulus. In order to identify such components, we carried out DNA microarray analysis in mutants of transcription factor whose expression was light responsive. In consequence, disruption of light-repressive slr0741 (phoU-homolog) and light-inducible sll1594 (ndhR) were shown to attenuate responsiveness to light in subset of gene expression. Disruption of slr0741 reduced induction rate of 57 genes including rbc operon, ribosomal protein genes and repression rate of ten genes including dnaJ. Disruption of sll1594 reduced induction rate of four genes including ribosomal protein genes and repression rate of 22 genes including trxM and nblA. This result suggested that slr0741 and sll1594 function as an activator and a repressor of gene expression upon dark to light transition.

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Seedlings of sunflower, tomato and lettuce were cultivated under blue-, red- monochromatic LED lights and white light by fluorescent lamp. Stem elongation of sunflower was promoted by blue light, and those of tomato and lettuce were promoted by red light. Length and number of epidermal cells in each plant were increased under the respective promotive light in stem elongation. Main factor of 1st internode growth was cell division in sunflower under blue light and tomato under red light, but that of 3rd internode growth was cell elongation. In lettuce under red light, each of the factors for 1st and 3rd internode elongation was cell growth and cell division, respectively. Thus, different stem elongation under different light quality was controlled by both of cell growth and cell division. However, the change in cell growth and cell division was different, according to plant species and stem position.

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We are interested in how different are the light signaling pathways in non-photosynthetic plants from those in photosynthetic plants. We have been investigating phytochrome A from a holoparasitic plant, Orobanche minor. In this study, we observed its physiological responses to red/far-red light. Germination of O. minor was inhibited by continuous and pulse red light whereas far-red light reversibly counteracts the effect of red light. From the result, it is indicated that phytochrome-mediated signaling systems work in the non-photosynthetic plant. The hypocotyl elongation was inhibited by white and far-red light but not by red light. Anthocyanin level was increased by white and far-red light and not by red light. So, it seems that high irradiation response to red light is lacked in O. minor during the stem elongation process. These results suggest that O. minor respond to red/far-red light in different way from that in photosynthetic plant such as Arabidopsis.

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To elucidate the mechanism of plant responses to the shade, we have previously identified a promoter trap line in which the GUS reporter gene expression in hypocotyls is regulated by the end-of-day far-red light and auxin treatments (Tanaka et al., PCP 43:1171, 2002). Examination of the photoreceptive sites in this response revealed the signaling from phytochromes in cotyledons to the target genes in hypocotyls. To identify molecular components of this inter-organ signaling, we screened for mutants showing different patterns of GUS expression. We found 2 mutant lines which showed morphological phenotype in darkness. Both the mutants had shorter hypocotyls in complete darkness compared to the parental line. However concerning the GUS expression, two lines showed an opposite phenotype. One line, named as reg1, showed lower GUS expression in darkness whereas the other, named as rig1, showed higher expression. We also observed abnormal response to end-of-day far-red light treatments in rig1.

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Light signals initiate hook opening and cotyledon unfolding in dicotyledonous seedlings. Surprisingly, we have found that light enhances apical hook closing (curvature) rather than opening in the early stage of tomato seedlings. Our results: 1) Red light (R) or far-red light (FR) enhances hook curvature. 2) phyA mediates the FR action. 3) The FR-absorbing form of phytochrome (Pfr) causes hook curvature in the presence of IAA. 4) Pfr reduced the ethylene production in the hook portion. 5) Ethylene or gibberellin causes hook opening. Additionally, we recently found that the angle of hook curvature greatly increased under continuous irradiation of R or FR when cotyledons were tightly covered by seed coats. Here, we also report the influence of cotyledons and seed coats on phytochrome-mediated apical hook curvature.

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Active phototropic bending of radially growing woody stems which have finished elongation has not been documented, while negative gravitropic bending is known. Firstly, we examined existence of active phototropic bending in woody stems and its bending mechanism. We inclined 1-year-old Quercus crispula seedlings and unilaterally irradiated them perpendicular to a vertical plane running parallel to the inclination azimuth to analyze the active phototropic bending separately from negative gravitropic bending and deflection caused by shoot's own weight. One-year-old stems significantly bent with asymmetrical formation of tension wood toward the light source. It is demonstrated that woody stems bend through phototropism with contribution of tension wood formation. Secondly, we examined photoreceptive site in the active phototropic bending. Seedlings were artificially inclined toward south, and east side of 1-year-old stems were masked with aluminum foil. The 1-year-old stems significantly bent toward west. Woody stem itself is shown to contribute photoreception in phototropic bending.

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Under low-light conditions, C₄ photosynthesis would be partly limited due to dysfunction of the CO₂ concentrating mechanism. In this study, we investigated the light dependency of CO₂ leakiness and in vivo activities of C₄ photosynthetic enzymes in Amaranthus cruentus L. (NAD-ME subtype, dicot).
CO₂ leakiness was estimated at 0.3 and was constant at irradiances over 200 μmol photon m^-2 s^-1. On the other hand, at irradiances less than 200 μmol photon m^-2 s^-1, CO₂ leakiness increased with the decrease of irradiances, and it was 0.45 at 80 μmol photon m^-2 s^-1. In contrast, the ratio of Rubisco initial activity to its total activity (Rubisco activation state) decreased with the decrease of irradiances at irradiances below 200 μmol photon m^-2 s^-1. These results indicate that the increase in the CO₂ leakiness under low light conditions would be explained by the decrease of Rubisco activation state.

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Cyclobutane pyrimidine dimers (CPDs) constitute a major portion of UVB-induced DNA lesions. Cucumber plants adapt to the solar radiation by expressing CPD photolyase, which catalyzes rapid restoration of CPDs, synchronously with the UVB content of sunlight. Such an expressional pattern is attributable to light-dependent transcriptional activation of the CPD photolyase gene (CsPHR). The purpose of this research was to elucidate the molecular mechanisms underlying the light-driven transcriptional activation. Irradiation of monochromatic light revealed that UVB with wavelengths around 310 nm maximally induces the transcription of CsPHR. Furthermore, we irradiated transgenic Arabidopsis plants expressing β-glucuronidase (GUS) under the control of the 2.5-kbp CsPHR promoter and confirmed that the GUS expression was induced maximally by UVB with wavelengths around 310 nm. UVB-driven transcription of CsPHR, therefore, seemed to be mediated by a UVB-specific photoreceptor. The results of promoter deletion experiments will also be presented.

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We have isolated and characterized an ultraviolet-B resistant mutant, uvi4, of Arabidopsis. The fresh weight of uvi4 plants grown under supplemental UV-B light was more than twice of that of the wild type. No significant difference was found in the ability to repair the UV-B-induced cyclobutane pyrimidine dimmers or in the amount of UV-B absorptive compounds, both of which are well known factors that contribute to UV sensitivity. Positional cloning revealed that the UVI4 gene encodes a novel basic protein of unknown function. The UVI4 gene is expressed mainly in actively dividing cells. We found that the hypocotyls cells in uvi4 undergo one extra round of endoreduplication. The uvi4 mutation also promoted the progression of endoreduplication during leaf development. Tetraploid Arabidopsis was hyperresistant to UV-B than diploid Arabidopsis. These results suggest that the enhanced polyploidization is responsible for the increased UV-B tolerance of the uvi4.

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Little is known about the translesion synthesis (TLS) in higher plants. We have isolated and characterized an Arabidopsis homolog (AtREV1) of the yeast REV1 gene that is involved in the error-prone TLS. An AtREV1-disrupted mutant was sensitive to UV-B and DNA crosslinking-agents, suggesting that the error-prone TLS is important for tolerance to DNA damages. To clarify the functions of AtREV1 in error-prone TLS, we overexpressed the AtREV1 in E. coli and purified the recombinant protein by affinity-column and anion-exchange column chromatographies. The activity of the purified AtREV1 was analyzed by primer extension assay using four kinds of oligonucleotides annealed with a ³²P-labelled primer. As a result, the AtREV1 efficiently transferred a dCMP opposite guanine, adenine, thymine and cytosine. In addition, AtREV1 inserted a dTMP opposite guanine with a similar efficiency to transfer dCMP. These results demonstrate that AtREV1 is a member of low-fidelity DNA polymerases with a deoxycytidyl transferase activity.

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Endosymbiotic algae in reef-building corals are often exposed to extreme light fluctuation. The ripples on the surface water can concentrate light up to 9000 μmol photons m^-2s^-1. Recent reports suggest that photoinhibition of endosymbionts can induce the dissociation of coral-algae symbiosis, also known as Coral Bleaching. We investigated the effects of light fluctuation on symbiotic algae in hospite of a shallow inhabiting coral, Acropora digitifera. Pulse amplified modulated (PAM) fluorescence measurements showed less dynamic photoinhibition under fluctuating light treatments (1 hour repetition of 3 sec of 1300 μmol photons m^-2s^-1 and 7 sec of 155 μmol photons m^-2s^-1) compared to that measured under the constant light (500 μmol photons m^-2s^-1). The results suggest potential stress-amelioration by ripple-induced light fluctuation in shallow inhabiting reef-building corals. Future experiments will focus on the effects of higher-frequency light fluctuation (sub-second fluctuation) on dynamic photoinhibition in symbiotic algae.

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A photoautotrophic cell culture presents the advantages of simplified system for photosynthetic research. We have established a photoautotrophic cell suspension culture of Arabidopsis thaliana and studied the cellular acclimation mechanisms to high light conditions. Arabidopsis cells grown under high light conditions (HL) showed a higher non-photochemical quenching of chlorophyll fluorescence, a higher carotenoids/chlorophylls ratio, and a higher content of xanthophyll-cycle pigments compared to low light grown cells (LL). We also analyzed the pigment-protein complexes composition of thylakoids from HL cells and LL cells. After the photoinhibitory treatment (1000 μmol photons m ^-2 s ^-1 for 30 min), both HL and LL cells had more zeaxanthin in LHCII compared to non-treated cells. HL cells had more xanthophylls in LHCII and a higher Lhcb3/Lhcb1 ratio than LL cells. Our results indicate that photoautotropnic Arabidopsis cultured cells could be a good model system to study high light acclimation mechanisms at the cellular level.

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It has been recognized that plants accumulate the specific carotenoids in chloroplast to cope with high light stress. We have studied the high light adaptation of photoautotrophic culture of Petunia hybrida and found that the composition of carotenoids associating light harvesting complex II (LHCII) changed during adaptation. Further analyses were made on the pigments in major LHCII and minor LHCII. We also examined the pigment composition in other LHCs in intact leaves and cultured cells, and discussed the differences between them.
The results showed that LHCII in cultured cells had more violaxanthin and less lutein compared with LHCII in leaves, indicating that violaxanthin binds to the site of lutein in LHCII. In case of LHCI, both leaves and cultured cells had almost the same lutein contents, but LHCI in high light grown cultured cells contained extra zeaxanthin, suggesting extra zeaxanthin binds to LHCI during high light adaptation in cultured cells.

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We have reported that Arabidopsis transcription factors DREB1s control expression of many stress-responsive genes. In rice, we isolated cDNAs for DREB1-type proteins, OsDREB1s.
In this study we analyzed function of OsDREB1F, which has high homology to Arabidopsis DREB1E/F, using transgenic Arabidopsis and rice plants. Expression of OsDREB1F was induced strongly but transiently by touch, though OsDREB1A was induced strongly by cold stress. In transgenic Arabidopsis overexpressing OsDREB1F, the same downstream genes were up-regulated as those in transgenic Arabidopsis overexpressing DREB1E/F.
Using transgenic rice plants overexpressing OsDREB1F, we performed microarray analysis and found that many genes were up-regulated as compared with those in wild-type plants. On the other hand, using RNA interference (RNAi) we generated loss-of-function mutants of OsDREB1F, which showed promoted growth as compared with wild-type plants. These results indicate that OsDREB1F plays an important role in growth regulation. Currently we are analyzing downstream genes in the OsDREB1F loss-of-function mutants.

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