Apical dominance is believed to be induced by IAA. But there are evidences that IAA inhibits growth of axillary buds through an indirect mechanism, and an involvement of a hormone-like compound produced by roots was suggested. In this context, strigolactones were isolated as the causal compound. In the present study we tried to detect a compound that induce dormancy of axillary buds, in other words, inhibit outgrowth of axillay buds, in potato roots cultured in vitro with IAA. For detection of dormancy-inducing activity, single-node segments of potato shoots cultured in vitro were used, since without any additions axillay dormant buds soon begin to grow. Water-soluble fraction obtained by solvent fractionation showed strong activity. Further purification of the active compound was carried out by chromatographies on charcoal and Sephadex G-10, TLC and HPLC. Finally pure compound could be obtained. The spectrum by FD mass spectrometry revealed that the compound had a molecular ion peak at m/z 435 (M+). The compound was different from strigolactones. Structural elucidation and experiments such as effect of IAA on the amount of the compound are now in progress.

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Brassinosteroid (BR) is known as the phytohormone that regulates plant height, although the regulatory system is not fully understood. To identify the novel regulator that control plant height, we screened the Arabidopsis CRES-T lines and found that the transgenic plants that express the chimeric repressor of HR0444 (HR0444SRDX) exhibited dwarf and dark green color phenotype. In HR0444SRDX plants, length of the each cell was much shorter than that of wild type, and the expression of BR inducible genes were decreased. In addition, HR0444SRDX plants were insensitive for BR treatment. Pro35S:HR0444 transgenic plants had similar phenotype to HR0444SRDX plants, and HR0444 does not show repressive activity. These results suggest that HR0444 act as a passive repressor. To approach further mechanism, we isolated transcriptional activators that interact with HR0444. The activators induce cell elongation when it was expressed ectopically. On the other hand, the expression of HR0444 was repressed by BZR1. These results indicate that BR controls the cell size via the repression of the expression of HR0444 that interacts and suppresses the activation activity of cell elongation activators.

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cis-Zeatin (cZ) is a geometric isomer of trans-zeatin (tZ), a highly-active cytokinin, at a double bond of prenyl side-chain. It is thought that cZ might require conversion into tZ to exert cytokinin activity, because cZ showed fairly-low activity in tobacco callus bioassay, low affinity for Arabidopsis cytokinin receptors, and low accumulation level in Arabidopsis. However, cZ and cZ derivates are highly accumulated in some plant species including rice and maize. Furthermore, a maize cytokinin receptor ZmHK1 showed comparable sensitivity to cZ with tZ in a bacteria system, suggesting direct activity of cZ in vivo. In this study, to investigate importance of cZ in rice, we generated transgenic rice plants in which the metabolism of cZ would be perturbed by ectopic over-expression of cZ-O-glucosyltransferase gene. The over-expression rice plants exhibited some visible abnormalities such as dwarf in aerial parts. Based on the phenotypic analysis and profiling of cytokinin metabolism in the over-expression rice, the importance of cZ in rice will be discussed.

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Gibberellins (GAs) are essential endogenous regulators of growth and developmental processes throughout the life cycle of higher plants. We are interested in genetic engineering of the biosynthesis of GAs to control the growth of lombardy poplar (Populus nigra). We isolated twenty-eight genes for the putative GA oxygenase enzymes belonged to the 2-oxoglutarate dependent oxygenase superfamily in lombardy poplar. Phylogenetic analysis revealed that the genes fall into four clades: eight into the GA20ox clade, four into the GA3ox clade, seven into the C19GA2ox clade and six into the C20GA2ox clade. We have isolated transgenic poplar over-expressing the gene for PnGA2ox1 in the C19GA2ox clade. The transgenic poplar showed dwarf phenotypes. To explore the expression of the genes of GA biosynthesis in transgenic poplar, we performed an analysis by real-time RT-PCR using primers specific for each gene.

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CYP78A is a family of cytochrome P450 monooxygenases highly conserved in land plants. In angiosperms, some CYP78As are reported to regulate plastochron length and organ size, but there have been no reports on the role of CYP78As in non-angiosperms. The moss Physcomitrella patens possesses two CYP78As, CYP78A27 and CYP78A28. To investigate the function of CYP78As in P. patens, we have produced single and double mutants and overexpression lines for CYP78A27 and CYP78A28 by gene targeting. Either cyp78a27 or cyp78a28 single mutant did not show any obvious phenotype in the protonema stage and the gametophore formation. On the other hand, the double mutant exhibited severe retardation of protonemal growth and gametophore development. In addition, endogenous levels of some plant hormones were altered in the double mutant. Transgenic lines that overexpress CYP78A27 or CYP78A28 showed delayed and reduced gametophore formation. Our results suggest that CYP78A is an important factor of growth regulation in P. patens.

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Auxinic herbicide, 2, 4-D is a chemical analogue of auxin, IAA. Traditionally it is believed that IAA and 2, 4-D share a common signaling pathway. However, the isolation of 2, 4-D resistant mutant aar1-1, identification of the gene SMAP1, which confers only 2, 4-D sensitivity, and 2,4-D specific degradation of root actin cytoskeleton and inhibition of cell division strongly suggest that 2, 4-D and IAA use both distinct and overlapping pathways to regulate Arabidopsis root growth. To provide a molecular explanation of 2, 4-D specific action, here we characterize the novel protein SMAP1. Our results revealed that SMAP1 acts as a positive regulator for 2, 4-D induced degradation of actin, cell division and cell elongation processes. The ubiquitin proteasome mutants,tir1-1 and axr1-12, which show enhanced resistance to 2, 4-D compared with IAA in root growth, were found to be resistant to 2, 4-D mediated actin degradation. Genetic interaction between SMAP1 and ubiquitin pathway was confirmed by analyzing the double mutant aar1 x tir1, which shows enhanced resistance to 2, 4-D for both root growth and actin degradation compared with their respective parental lines.

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Previous studies using rice tillering dwarf mutants suggested that DWARF14 (D14), encoding a member of the Alpha/Beta-hydrolase superfamily, regulates shoot branching in a step downstream of strigolactone biosynthesis. To explore whether D14-related gene(s) acts in the strigolactone-dependent pathway in other plant species, we have been studying the physiological roles of D14 homologs in Arabidopsis through reverse genetics. We found that Arabidopsis mutants defective in a likely D14 ortholog are phenotypically similar to the strigolactone-deficient max1, max3 and max4 mutants, including increased axillary bud outgrowth and reduced petiole lengths in rosette leaves, but are insensitive to strigolactone. In contrast to d14, max2, another strigolactone-insensitive mutant defective in an F-box protein, shows extra phenotypes that are not observed in the strigolactone-deficient max mutants. Our recent results suggest that another D14-related gene might be involved in the MAX1/MAX3/MAX4-independent max2 phenotypes in Arabidopsis.

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Brassinosteroids are growth promoting steroid hormones in plants. But it's signal transduction mechanisms remains unclear. To identify new brassinosteroid signaling mutants, we screened these from FOX (Full-length cDNA over-expressor gene) lines, which were transgenic lines overexpressed normalized Arabidopsis full-length cDNAs at random in Arabidopsis. Brz is an inhibitor of brassinosteroid biosynthesis enzyme DWF4 and it's treatment caused to wild type as shorten hypocotyls in the dark. So we tried to screen about 8000 Fox-lines for mutants that showed longer hypocotyls than wild type with Brz in the dark, bil (Brz-insensitive-long hypocotyls) , and isolated about 20 line mutant candidates. These bil candidate mutants showed not only longer hypocotyls on Brz but also various adult phenotypes in rosette leaves as epinastic and in inflorescence as increased or decreased number. Furthermore three mutants showed longer inflorescence than wild type. It appears that the possible overexpressed gene is brassinosteroid signaling factor and promotes inflorescence elongation. Further analyses are in progress.

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We have shown that chlorophyll biosynthesis was promoted by the application of abscisic acid (ABA) in cucumber plants grown under low nitrogen conditions. Since cytokinin has been well known to promote chlorophyll biosynthesis, we investigated the interaction between ABA and cytokinin in chlorophyll accumulation of cucumber plants under low nitrogen conditions. In addition, we investigated the involvement of reactive oxygen species (ROS) accumulation in chlorophyll degradation.
Chlorophyll contents in the first leaves of cucumber plants grown under low nitrogen conditions were reduced by the application of kinetin (KI). Expression of a rate-limiting enzyme gene in the biosynthesis of chlorophyll which encodes GLUTAMYL-tRNA REDUCTASE was reduced in the first leaves of cucumber treated with KI. On the other hand, the amount of transcripts in relation to ABA biosynthesis was reduced by KI. The amount of H₂O₂ increased in cucumber plants treated with KI, whereas it decreased in the presence of ABA. These results suggest that ROS is one of the limiting factors in degradation of chlorophyll in cucumber plants under low nitrogen conditions, and ABA suppresses the accumulation of ROS.

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Phosphatidate phosphohydrolase 1/2 (PAH1/2) is a soluble phosphatidate phosphatase which produces diacylglycerol (DAG) by dephosphorylating phosphatidic acid (PA). PAH plays an important role in lipid metabolism. In pah1pah2, phospholipids as phosphatidylcholine, phosphatidylethanolamine, PA increase more than wild type (Nakamura et al. 2009 PNAS). PA is a metabolic intermediate playing a central role as a signal transducer of abscisic acid (ABA) stress response in higher plants.
We analyzed various ABA stress responses using pah1pah2 mutant. As the result, germination of pah1pah2 was more severely inhibited than that of wild type particularly with ABA treatment, showing that pah1pah2 seed is hypersensitive to ABA. However, the inhibition of stomatal opening in response to ABA treatment was similar between wild type and pah1pah2. Therefore, we suggest that, in ABA signaling, PAH is differently involved in seed germination and leaf drought stress.

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Gibberellins (GAs) are phytohormones that regulate many aspects of plant development. DELLA proteins are GA signaling repressors whose degradation is promoted by GA. GAF1 was isolated as a transcription factor that interacts with DELLA protein, GAI. DELLA proteins lack a clear DNA binding domain. However, DELLA proteins appear to be involved in the regulation of gene expression because they function as a transcriptional activator in yeast and plants. DELLA proteins accumulate in the nucleus when GA levels decreased. Therefore, we predicted that DELLA proteins participate in the transcriptional regulation of GA biosynthetic genes that are controlled by GA feedback mechanism. Transient assay showed that GAF1-DELLA complex activates GA 20-oxidase gene. BiFC assay showed that the interaction between GAF1 and DELLA protein disappears in plant cell when treated with GA. These results suggested that the transcriptional activation potential of GAF1 is controlled by endogenous GA levels. In this study, we attempted to specify the GAF1 binding site in the promoter of GA 20-oxidase and investigate the mechanism of transcriptional regulation by GAF1 complex.

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NOG1 is a nuclear GTP-binding protein which is conserved among archaea and eukaryotes (including animals, plants and fungi). It has been shown that NOG1 homologues are involved in early steps of 60S ribosomal biogenesis in nucleus of Trypanosoma, yeast and mouse. Here we characterized the plant Nog1 protein in Arabidopsis. Arabidopsis has three NOG1 homologues, named AtNOG1-1, AtNOG1-2 and AtNOG1-3, but only NOG1-1 is constitutively expressed in all the tissues examined. Actually AtNOG1-1 seems to be essential for plant development. AtNOG1-1 is directed to nucleolus and nucleoplasm. FRAP analysis revealed that the distribution of NOG1-1 protein between nucleolus and nucleoplasm was sensitive to transcription and translation inhibitors, also carbon and nitrogen starvation, suggesting its role in early steps of ribosomal biogenesis. NOG1-1 shows a dynamic behavior during mitosis. The NOG1-1 protein disappeared in pre-metaphase, and is rapidly re-accumulated in peripheral chromosomal region in early anaphase. We also found a novel and plant-specific sub nucleolar structure named "nog1 body" which appears in nucleus region during late anaphase and early telophase.

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CYO1 has protein disulfide isomerase activity, and functions as a cotyledon-specific chloroplast biogenesis factor in Arabidopsis thaliana. We screened the CYO1 homolog that function as the chloroplast biogenesis factors at all green tissues by a plastid proteome database and Arabidopsis T-DNA lines. cyo2 mutant has both albino leaves and cotyledons. The maximum quantum yield of photosystem II (PSII) and the electron transport rate in the mutant were much decreased. The amounts of PSI and PSII core subunits in the mutant were significantly decreased compared with those in wild type. The expression of CYO2::GUS was detected at all green tissues. Recombinant CYO2 accelerated disulfide bond reduction in the model substrate insulin, indicating that CYO2 had protein disulfide isomerase activity. These results suggest that CYO2 is a chloroplast biogenesis factor at all green tissues in A. thaliana.

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Vacuole plays an important role in maintaining the homeostasis of plant cells through the regulation of development of turgor pressure, the accumulation of various inorganic ions and metabolites, and the degradation of discarded proteins. However, the molecular mechanisms underlying the diverse vacuolar functions are not fully understood. To get insight into the molecular basis of the vacuolar functions, we conducted the post-genome analyses of the vacuole. We have directly and comprehensively analyzed proteins and metabolites of the intact vacuoles using LC-MS, CE-MS and FT-ICR-MS. The target analyses of metabolites have revealed the existence of organic phosphate compounds in the vacuole. The list of the vacuolar metabolites identified by non-target analyses is also constructed. Proteomic analyses have suggested that the unexpected enzyme reactions exist in the vacuole besides degradative activity. We are also analyzing functions of unknown vacuolar membrane proteins using the transgenic Arabidopsis suspension-cultured cells. In the present report, we would like to discuss the effect of autophagy inhibitor on dynamics of vacuolar metabolites, too.

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To understand the function of new nuclear-encoded chloroplast genes, we isolated the albino mutants (apg11 and apg12) in Ac/Ds transposon tag lines. At this conference, we would like to report about APG11 and APG12. APG11 and APG12 did not have any specific domains and motifs and were only similar to some proteins in higher plants. The amino acid sequence of both proteins were similar each other. The sequence of APG11 showed 51% identity and 65% similarity with the APG12 in the C-terminal 100 amino acid region. They were localized in chloroplast. The chloroplast of apg11 did not have thylakoid, only have vacuoles. On the other hand, one of apg12 contained few internal thylakoid membranes. APG11 and APG12 equally expressed in all tissues that we analyzed in this study. The expression of some photosynthetic genes encoded in chloroplast genome was greatly reduced, but one of nuclear encoded genes was not affected in these mutants. When comparing the accumulation of chloroplast rRNAs in these mutants and wild type, rRNAs were decreased by approximately 25% in these mutants. Now we are finding proteins interacted APG11 and APG12 using the yeast two hybrid system.

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The messenger RNAs of the rice seed storage proteins prolamine and glutelin are targeted to distinct sub-domains of the cortical endoplasmic reticulum via a pathway dependent on both RNA sorting signals and the cytoskeleton. This RNA transport mechanism directly influences the final site of storage protein accumulation, with prolamines remaining within the ER lumen in spherical protein bodies while glutelins are exported to irregularly-shaped protein storage vacuoles. Developing rice seeds therefore represent a leading model for the study of RNA localisation in plants. Previous work using RNA affinity capture from cytoskeleton-enriched developing rice seed extracts has led to the identification of eighteen candidate prolamine RNA binding proteins (RBPs). Of these, RBP-A is a promising candidate for further study since its expression profile, intracellular localisation and in vivo RNA binding specificity all support a direct role for this protein in storage protein gene expression. Recently, mouse antibodies against RBP-A and a second promising candidate, RBP-D, have been generated and co-localisation data will be presented for these two proteins.

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SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor) s play a pivotal role in the membrane fusion between secretory vesicles and target membranes. There are 9 Qa-SNAREs on the plasma membrane of Arabidopsis thalina, and these SNAREs show different expression patterns. Especially, SYP123 is specifically expressed in root hair cells, and is strongly localized to the apical zone of root hair. By contrast, in the root hair cells, SYP132 is ubiquitously localized on the plasma membrane. To investigate the localization mechanism of SYP123 on root hair tip, we searched interacting proteins to SYP123. So far we obtained several candidate of proteins. One of the interacting proteins is SH3P1(SH3 domain-containing protein1) which is known to be involved in the vesicle trafficking,. I will report the expression pattern and localization of SH3P1-GFP in Arabidopsis.

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Expression of nuclear-encoded photosynthesis-related genes is greatly affected by the developmental and functional status of plastids. Retrograde signals (plastid signals) derived from plastids are hypothesized for this regulation. Although the involvement of tetrapyrrole biosynthesis has been strongly suggested, the entity of the signal is still obscure. Mg-chelatase H subunit (CHLH), that is also designated GUN5/ABAR, is a multifunctional protein involved in tetrapyrrole biosynthesis, plastid signaling and ABA signaling as a receptor. We therefore tested possible interaction between plastid signal and ABA. As previously reported (C. Voigt et al. Physiol. Plant, 2009), low dosage of ABA partially restores plastid signal. We are currently testing the effect of ABA in gun mutants including gun5 mutant alleles. To understand the domain structure of CHLH/GUN5/ABAR protein, we expressed modified CHLH protein in Arabidopsis wild type and gun5 mutants, and examined these transgenic plants for tetrapyrrole biosynthesis, plastid signaling and ABA responses. We would also like to present our recent progress on the mutant screening in plastid signaling using FOX-hunting system.

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ATP dependent protease Lon that is first discovered in Escherichia coli, has been identified in all organisms. In Arabidopsis, four genes have been annotated as Lon homologues and a gene At5g47040 encodes peroxisome-localized Lon, AtLon2. Our previous studies showed that the N-terminal domain of AtLon2 has molecular chaperone activities and the C-terminal domain act as a serine protease. The level of transcription of AtLon2 increased 2- to 4-fold by drought and high-salinity stresses and by ABA treatment. To clarify the physiological function of Lon2 in Arabidopsis, we analyzed a T-DNA insertion mutant lon2-1 and a RNA interference mutant lon2i. Transient expression assays using the particle bombardment method showed that the import efficiency of GFP-PTS1 in these mutants was lower than that in the wild type plant. The activity of β-oxidation was inhibited in the seedlings of mutants and the size of the lon2-1 plant was smaller than that of the wild type plant. These results suggested that AtLon2 is involved in protein import into peroxisomes and in establishment of pathway for lipid degradation.

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Chloroplasts have their own DNA and gene expression systems. In order to study transcriptional regulation, genetic approaches have been historically used. However, this approach may include some indirect effects, which make it difficult to understand specific regulation by the transcription factors. Chromatin immunoprecipitation (ChIP) is a powerful and useful tool that can obtain information for binding sites for transcription factors, and directly detect dynamic changes of their interaction patterns in vivo.
To further understand the roles of plastid sigma factors in Arabidopsis thaliana, we here developed ChIP-based method, and analyzed binding pattern of SIG5, a stress-induced chloroplast sigma factor. We found SIG5 specifically binds to novel target promoters as well as psbA or psbD BLRP that are already known, and this binding depends on several kinds of stress conditions. We further analyzed and identified target promoters for SIG1, an essential sigma factor by ChIP analysis. These results suggest that ChIP analysis is useful to understand transcriptional regulation of chloroplast genes, which can overcome several problems from traditional methods.

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Nuclear positioning is important for cell division and cell differentiation in plants. In tobacco BY-2 cells, it has been reported that the pre-mitotic nuclear migration from the periphery to the central region of the cell requires both microfilaments (MFs) and microtubules and that the migration of daughter cell nuclei away from the newly formed cell wall is depend mainly on MFs. However, further molecular mechanisms of these nuclear migration events are not known. Here we report the identification of a strong candidate for the nuclear migrating motor, designated NMK-1, by molecular cloning of cDNAs encoding kinesin related proteins and localization studies using GFP-fused proteins. The full-length NMK-1:GFP fusion protein localized to nuclear surface through out the cell cycle in BY-2 cells. The expression of various truncated NMK-1 proteins revealed that NMK-1 associates to nuclear surface via its coiled coil domain. The induced expression of a rigor mutant of NMK-1 resulted in an accumulation of cell pairs in which daughter nuclei were associated with newly formed cell wall, indicating that the rigor mutant NMK-1 inhibited the migration of daughter nuclei after cytokinesis.

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The cell cytoskletal component actins, which regulate diverse biological functions during plant growth and development, are classified into vegetative and reproductive classes. Among the vegetative actins in Arabidopsis, ACT7 and ACT8 have been shown to regulate the root and root hair morphology respectively. However, the distinct role of ACT7 and ACT8 on root meristem development remains elusive. By analyzing the single mutant of ACT7 and ACT8, here we show that ACT7 but not ACT8 predominantly regulates the root meristem formation as judged by the root growth, cell length and cell production rate. On the other hand, ACT8 plays a major role in lateral root meirstem formation. A two-fold induction in lateral root number was observed in act8 mutant, suggesting that ACT8 is a negative regulator of lateral root development. ACT7, which is active in primary root meristem, affected the lateral root meristem development mildly. Further, act8 mutant but not act7 showed a reduced response to IAA or IBA mediated lateral root induction. Taken together, these results suggest that these two isovariants of actin play distinct role in root meristem formation.

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Intermediate filaments (IFs) are known as a kind of major cytoskeletons in animal cells. However, IFs in plant cells have not been identified yet. The deduced amino acid sequence of Arabidopsis IFMoP1 gene has IF motifs and long α-helical structure which are conserved among animal IFs. Previously, we found that IFMoP1-GFP fusion protein, which was over-expressed in transgenic tobacco BY-2 cells, formed cytoskeleton like filamentous structures in cytoplasm around nuclei. We regard IFMoP1 as a candidate for a gene encoding the IF protein in higher plants.
By fluorescence microscopy, it is revealed that structures which were formed from IFMoP1-GFP protein were fragmentated in the cell cortex and were arranged transversely to the cell axis. Additionally, time-lapse analysis showed that the filamentous structures moved randomly in cytoplasm around nuclei, and then were fragmentated. After the fragmentation of the structures, these were distributed and localized in cell cortex. The arrangement of fragments was similar to cortical microtubules arrays. It might co-localize with cortical microtubules.

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After fruit development has been triggered by pollination, abscission zone in the pedicel strengthens its adhesion to keep the fruit in tact with the plant body. The unpollinated flower sheds from the abscission zone, while the enlargement of the same tissue can be observed in pollinated flower. After fruit has developed and fully ripened, shedding can easily occur from the abscission zone, indicating acceleration of abscission. Cell wall degradation and synthesis may play an important role in these processes, however not much is understood about the shedding process of the floral organs or fruits. In our research, we have attempted to visualize the changes of polysaccharide distribution in abscission zones of pollinated/ unpollinated flowers and ripened fruits by using cell wall staining reagents and monoclonal antibodies specific to some cell wall epitopes. In abscising flower, pectic galactan, arabinan, and xyloglucan content increased particularly in the abscission zone.

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Salicornia herbacea is one of the halophytes whose growth is promoted by the presence of NaCl. In this study, we investigated parameters regulating the growth of hypocotyl in Salicornia seedlings treated with NaCl under continuous light or dark conditions.
Hypocotyl elongation of Salicornia seedlings was promoted by the addition of 100 mM NaCl in both continuous light and dark conditions. Viscous and elastic extensibility of cell wall in the hypocotyls of both light-grown and dark-grown seedlings were decreased by the exposure to 100 mM NaCl. The content of pectin in cell wall of hypocotyls was significantly decreased in seedlings treated with 100 mM NaCl under continuous light conditions. On the other hand, the content of pectin and hemicellulose in cell wall of hypocotyls were decreased in dark-grown seedlings treated with 100 mM NaCl.
These results suggest that NaCl reduces the content of polysaccharides such as pectin and hemicellulose of the hypocotyl cell walls, and increases the extensibility of hypocotyl cell wall as a result of decrease of the viscosity in Salicornia.

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Hybrid lethality, a major reproductive isolation, hinders gene exchanges among taxa. Identification of genes involved in this mechanism is important not only for studies on evolution but also for plant improvement. To find out the genes, we have started to screen cDNA library which enriched expressed genes of GTH4, lethal cultured cells of F₁ hybrid of N. gossei x N. tabacum, compared with that of GTH4S, non-lethal cultured cells. Further screening of 2,016 cDNA clones came out new 15 genes which expressed in GTH4 more strongly than GTH4S. However, only three were verified as preferentially expressed genes in GTH4 by semi-quantitative RT-PCR. These included Pre-pro-proteinase inhibitor I (Acc.#, Z12619), Glycine-rich protein precursor (Acc.#, GU177460) and Inhibitor of microbial serine proteinase (Acc.#, X67075). Analysis for functioning of these genes in hybrid lethality is in progress.

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Fagus crenata produce intermittently large numbers of seeds or flowers, not annual reproduction. Several hypotheses have been developed to explain the mechanism of intermittent reproduction; i.e. internal resources, climate cues. These proximate factors assume that the plant sets flowers as a result of that these factors induce differentiation of floral meristem as a starting signal. However, there are no direct evidences about whether these factors function as a signal of floral initiation, because it cannot be detected when the first switch of flowering on. In order to explore these factors on floral initiation, we quantified expression level of flower identity gene; LEAFY homolog (FcLFY) as well as process of flower bud differentiation using mature F. crenata. We measured seasonal pattern of FcLFY expression in bud using real-time quantitative PCR. In addition, we observed the relationship between FcLFY expression in bud and process of flower bud differentiation. We also analyzed relationship between FcLFY expression and internal resources. This approach could allow us to evaluate which proximate factor works as a signal of floral initiation in F. crenata.

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Gentian is a perennial plant and known as ornamental flowers. In gentian plant, there was no study about flowering. To identify the machinery of the flowering initiation, we isolated and characterized the gentian FT/TFL1 family genes. First, we obtained three genes possessing high homology to FT/TFL1 and denoted GtFT1, GtFT2 and GtTFL1, respectively. The seasonal expressions of these genes were analyzed. The expressions of GtFT1 and GtFT2 were remarkably increased prior to the flowering initiation, whereas GtTFL1 was highly expressed at vegetative stage and was remarkably decreased just before the flowering initiation. We compared the expression pattern of GtFT/GtTFL1 family with early-flowering (EF) and late-flowering (LF) lines. Both lines showed remarkable increase of GtFT1 and GtFT2 at the flowering initiation. The result suggested that GtFT might play roles in the flowering initiation in gentian. However, the GtTFL1 expression was higher in LF line than that in EF line. Therefore, we compared genomic region of GtTFL1 between EF and LF lines and found that EF line had a 320 bp insertion in promoter region, implying that the insertion may be related to the flowering time.

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Plants are sensitive to changes in their ambient environment and respond to these to determine the optimal timing of the floral transition. Under shade or high density of vegetation, plants positively change their morphology to compete for sunlight, by for example elongating their stems and leaves. Acceleration of flowering is also a typical shade avoidance response. Detection of changes in light quality is a major signal in responding to the surrounding shade condition. Plant leaves absorb red light, whereas far-red light is transmitted or reflected. Therefore the R:FR ratio of shady conditions is lower than that of unfiltered sunlight. This change of light quality is perceived by phytochrome, and particularly phyB represses the shade response. We showed previously that inactivation of phyB by mutation stabilizes CONSTANS (CO) protein, a transcription factor that promotes early flowering. This suggests that stabilization of CO in shade could be responsible for the observed early flowering. We are interested in the mechanism by which phyB and CO functions promote early flowering in shade and are using a combination of biochemical and genetic approaches to address these issues.

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Plant circadian clock is synchronized to daily changes in light and temperature. We monitored the responses of the circadian gene expression to light pulses by introducing the bioluminescent reporter system to Lemna plants (duck weeds).
In 1960, Hillman analyzed the photoperiodic flowering of the shortday plant Lemna paucicostata in response to "skeleton photoperiods". Skeleton photoperiods are composed of two light pulses with a 24-h period in prolonged darkness. He found the bistability phenomenon that plants recognized the intervals between the pulses as day or night depending on the timing of the first pulse. Recently, Ito-Miwa and Oyama measured the circadian gene expression under skeleton photoperiods by the bioluminescent reporter system, and directly observed the bistability of the rhythm. In order to analyze the mechanisms of the circadian clock system to light pulses, we monitored responses of the circadian gene expression to various light pulses. We also measured the circadian bioluminescent reporter activities at the single cell level. In addition, we observed several clock deficient cells.

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Cyanobacteria are photosynthetic prokaryotes with a circadian system. In cyanobacteria, kaiA, kaiB and kaiC are known as the central genes of circadian clock genes. KaiC has an autokinase and an autophosphatase activity. KaiA and KaiB enhance KaiC autophosphorylation and autodephosphorylation, respectively. These regulatory systems form a KaiC phosphorylation cycle with a ~24-h period. The circadian changes in molecular states are converted to the transcriptional regulation in the cell. The transcriptional activities of kaiB,kaiC are under the feedback regulation of KaiC and they show a circadian rhythm. These results have been obtained from researches using a freshwater cyanobacterium, Synechococcus elongatus PCC 7942.
It was reported that a group of marine cyanobacteria, Prochlorococcus species lack kaiA in their genomes, and their circadian rhythms disappear in constant conditions. In order to verify the function of their kaiB and kaiC in diurnal system, we have tried to analyze their physiological roles in a heterologous circadian systems by introducing various constructs of Prochlorococcus kaiB and kaiC into Synechococcus cells.

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Japanese cedar (Cryptomeria japonica), a major species forestry in Japan, attracts a great deal of attention as a carbon sink to cope with global warming. Since photosynthesis plays important role in carbon capture and storage, its mechanism is desire to be investigate. To elucidate light-response mechanisms of photosynthesis in Japanese cedar, Suppression Subtractive Hybridization (SSH) and qRT-PCR were used to detect differences of gene expression between the day and night. Most of photosynthesis-related genes showed no difference in expression level between the two periods (e.g. Lhca4, PRK, PetC). A statistical analysis indicated significant differences in some genes (e.g. rbcL, ChlL, PsaB), however, the ratios of expression level were no more than 2-fold. Results of SSH indicated that genes function in cell organization and biogenesis, and kinase activity were expressed at higher level in the night, but showed no difference in photosynthesis-related except aldolase. Since most of photosynthesis-related genes are light-responsive in other higher plants, regulation of the gene transcription estimated to be specific to Japanese cedar.

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To monitor the expressions of clock genes, we developed two types of reporter strains. One is clock-promoter reporter strains that were transformed with reporter genes consisting of the promoter regions of clock genes and the firefly luciferase gene. Another is clock-protein reporter strains that were done with the protein-coding regions of clock genes and the luciferase gene. Bioluminescence rhythms of all clock-promoter reporter strains oscillated with the same phase, although the endogenous clock genes have different phases in their mRNA expression rhythms. Comparison of several reporter strains developed in other wild-type strains suggested that these results may be due to a feature of the host strain. On the other hand, bioluminescence rhythms of clock-protein reporter strains showed phases corresponding to the expression rhythms of clock proteins. For example, expression levels of the clock protein of ROC40 and ROC75 peaked at subjective dawn and subjective mid-day, respectively. Bioluminescence rhythms of these protein reporters well reflected the corresponding clock protein rhythms. These protein-reporters will be useful to monitor the expressions of ROC40 and ROC75.

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Cyanobacteria are the simplest organisms known to possess a circadian oscillator. The central oscillator of the cyanobacterium Synechococcus elongatus PCC. 7942 comprises three clock proteins termed KaiA, KaiB, and KaiC. In vitro reconstruction of the KaiC phosphorylation cycle achieved simply by incubating KaiA, KaiB, and KaiC in the presence of ATP, has provided a means of studying the detailed mechanisms of the Kai-protein oscillator.
We used time-resolved fluorescence spectro scopy, small-angle X-ray scattering, and mutational analyses to track and visualize the ticking of KaiC in solution. During the phosphorylation cycle, the C-terminal domains of KaiC were repositioned in a stepwise manner to effect global expansion and contraction motions of the C-terminal ring. We found that Arg393 of KaiC played a critical role in expanding the C-terminal ring and its replacement with Cys affected the temperature compensation of the period, a fundamental property of circadian clocks. We will discuss mechanisms of KaiC oscillator based on structural information.

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How cell fate is specified according to its position during plant embryogenesis is not well understood. Laser ablation studies and sector analyses suggested that the position of the cell rather than the lineage plays an important role in determining cell fate during plant development. However, it still remains unknown what factors provide the positional information. To elucidate the mechanism of position-dependent gene activation, we analyzed the regulation of ATML1 which was expressed in the outermost cell layer. Our expression studies suggested neither auxin signaling nor cell lineage was required for epidermis-specific expression of ATML1. Promoter deletion experiments revealed several cis-regulatory elements including ATML1-binding site contribute to the expression of ATML1, suggesting that several unknown factors as well as ATML1 itself regulate ATML1 expression. To identify factors that determine epidermis-specific expression of ATML1, we examined the effects of overexpression of several candidate genes that appeared to be involved in the regulation of ATML1 in the embryo. Possible roles of these genes in determining epidermis-specific gene expression will be discussed.

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Dormancy of the bulbils of Dioscorea is induced by GA treatment (GA-induced dormancy).ABA treatment also inhibited the sprouting of the bulbils. So far we have tried to isolate the key genes of ABA metabolism, and succeeded in cloning the full lengths of the ORFs of DjNCED1 and 2, and DjABA8'ox1, 2 and 3 from the young sprouts of D. japonica. The recombinant proteins from the yeasts into which DjABA8'ox1 and 2 were transformed possessed the enzymatic activities of ABA8'-hydroxylase. Here,we analyzed the endogenous levels of ABA and its catabolites by LC-MS/MS as well as the key gene expression levels of ABA metabolism by semi-quantitative RT-PCR in the chilled nondormant bulbils of D. japonica incubated with aqueous GA₃.The GA₃ treatment increased the expression levels of DjNCED1 and 2, and decreased those of DjABA8'ox2 and 3 in the bulbils.The endogenous level of ABA in the bulbils was clearly increased by the GA₃ treatment. The GA treatment might increase the endogenous level of ABA through the regulation of gene expression of ABA metabolism, leading the Discorea bulbils to GA-induced dormancy.We are grateful to Dr. Y. Jikumaru (RIKEN) for analyzing ABA and its catabolites.

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How plant cells determine the direction of polar auxin transport is a central question in plant development. PIN auxin efflux transporters often show polar localization, and are considered to play a central role in determining auxin distribution patterns. However, signaling factors governing polar localization of PINs remain to be elucidated. We have previously shown that auxin promotes cell interdigitation in leaf epidermal pavement cells by activating Rho-like GTPase family from plants (ROP). The activation of the interdigitation by ROP requires PIN1-dependent polarized auxin export at the tip of lobes. We found that the polar localization of the auxin efflux carrier PIN1 protein is altered in rop mutants. Our results suggest that ROPs regulate endocytosis as well as recycling of PIN1, which participates in the establishment of PIN1 polarity. We propose that a positive feedback regulation of auxin activation of ROP, which involves polar PIN1 distribution, is critical for the interdigitation of pavement cells in Arabidopsis leaves.

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Cyclamen (Cyclamen persicum Mill.) is a main horticultural plant, and we have been developing for breeding cyclamens based on molecular genetics. It is important to elucidate the exact mechanisms regulating flower formation for the modification of flower traits. AGAMOUS (AG) proteins, which are an important transcription factor for pattern formation of the floral organs, play a role in specification of stamen and carpel.
We isolated the two kinds of AG homolog genes (CpAG1 and CpAG2) in cyclamen and performed functional analysis of these genes. Our results suggested, despite their high sequence similarity, CpAG1 functions in stamens while CpAG2 have acts in carpels. To clarify the function of two types of CpAG transcription factors, we transformed a chimeric repressor gene construct for CpAG1 and CpAG2 into Arabidopsis and cyclamen. We show here that the function of two cyclamen AGAMOUS genes are different.

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Modulation of cellular energy homeostasis is an attractive alternative to improve crop yield and plant performance under stressful conditions. Previously, we reported that overexpression of Arabidopsis NAD kinase2, which produces chloroplastic NADP, promoted nitrogen assimilation in Arabidopsis. Consequently, metabolites associated with the Calvin cycle were also accumulated. In this study, we generated transgenic plants overexpressing Arabidopsis NAD synthetase gene (NADS). NADS overexpressor plants did not show any remarkable changes in vegetative growth and the level of NAD(P)(H) was unexpectedly unchanged albeit enhanced NADS activity. However, NADS overexpression caused early ageing coincidentally with bolting. Then, the levels of NAD⁺ was drastically decreased and the levels of intermediates of NAD biosynthesis (NaMN and NaAD) and NAD derivatives (nicotinate and nicotinamide) were dramatically increased in NADS overexpressors after bolting. These results suggest that the levels of NAD(P)(H) could be kept properly during vegetative phase even under NADS overexpression and that bolting may act as a signal for the alteration of NAD homeostasis.

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An Arabidopsis membrane-bound transcription factor bZIP60 translocates to the nucleus from the ER and activates the genes related to the ER quality control such as BiP under the ER stress. In animal cells, prolonged ER stress eventually induces programmed cell death (PCD). Although induction of PCD after ER stress has been also reported in plants, involvement of bZIP60 in PCD has not been clarified. We found transcriptional activation and proteolysis of bZIP60 by FumonisinB1 (FB1), an agent inducing PCD in both animal and plant cells. A T-DNA insertion mutant of bZIP60 was found to be more sensitive to FB1 than wild type. However, FB1 did not induce genes encoding proteins for the ER quality control. Microarray analysis using wild type and the T-DNA insertion mutant of bZIP60 identified several genes that were induced by FB1 dependent on bZIP60. Most of these genes were different from genes activated in the tunicamycin-induced ER stress response. These observations suggested that bZIP60 regulates novel signaling pathway different from the ER stress response.

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The application of plant biomass is one of the promising ways to realize the Zero-Waste Society. Although plant seed oil is a valuable resource of biodiesel fuels and bioplastics, only few favorable results of molecular breeding have been reported. Arabidopsis thaliana is the relative species of the major oil crop, Brassicae napus. Then we searched factors that increase seed oil content using the chimeric repressor gene silencing technology (CRES-T) and measured the oil content of the seeds of the independent Arabidopsis CRES-T plants employing nondestructive analysis using 1H-pulse NMR. One of the CRES-T line in which the chimeric repressor against unknown function of AP2/ERF transcription factor encoded by At5g07580 showed increased seed oil content average 6% with a maximum of 11%. Although oil content was not markedly increased in first-generation, oil yield per plant showed average increase of 32% with a maximum of 70% in second-generation due to increase in aerial biomass and seed yield. We are going to discuss the role of At5g07580-SRDX on the result of transcription analysis, own promoter-GUS assay and plant oil assay in rice.

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Perennial plants continue the growth for several years repeating annual growth cycle, active growth or dormant phase. In autumn plants cease their growth and buds enter dormancy state called endodormancy, then in spring, they resume their growth. Endodormancy allows plants to survive under severe environmental conditions such as low temperate and drought. However, the molecular mechanism of endodormancy in perennial plants is yet to be well understood. Recently, we revealed that SVP-like MADS-box genes of Prunus mume, named DORMANCY ASSOCIATED MADS-box (PmDAM), are involved in endodormancy. For further understanding the molecular basis of endodormancy, we performed comprehensive expression analysis using custom microarray of P.mume (180Kx3) which is designed from sequences of EST and peach genome database. We will report the results of microarray analysis and discuss several genes which showed the dormancy-related expression patterns similar to PmDAM.

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We have established polyploid series of four strains of Arabidopsis thaliana (Columbia, Landsberg erecta, Wassilewskija, and C24), and with these, quantitatively analyzed the ploidy effects on primary root growth by the kinematic method. In all strains, there were increases in cell size and cell volume growth rate of tetraploid plants compared to diploids, while no significant differences were found in the rate and location of cell proliferation.
We additionally analyzed the root growth of the hexaploid and octoploid of Columbia strain. The cell size and cell volume growth rate of hexaploid were larger than those of tetraploid, and thus the ploidy changes from tetraploid to hexaploid and from diploid to tetraploid had a similar effect on root growth. The root growth of octoploid, however, decreased compared to that of hexaploid and two groups were recognized in octoploid plants; one group showed a slightly lower volume growth rate than hexaploid and the other showed a much lower growth rate. The ploidy level was reduced to tetraploid in the next generation of the latter group, suggesting that some chromosomal disorder may affect the volume growth rate in octoploid.

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Aluminum (Al) causes inhibitions of plant-growth in acid soil area and various genes change their expressions under the stress. As Arabidopsis AtGST11 gene is induced by Al stress, heavy metal stress and oxidative stress, we believe that this is a good model system to characterize the common response mechanism for these stresses. Isolation and characterization of transcription factors (TF) related to the expression of AtGST11 are performed in this study. We have already isolated 4 candidates for TF #11-1-1, #13, #11-1-3 and #43. First, candidates were confirmed their DNA binding capability to the promoter of AtGST11 by gel-shift assay. Furthermore, dual Luciferase assay was performed to estimate the effect of the 4 clones in the gene-expression of the AtGST11. The result suggested that #11-1-1 and #11-1-3 are activators and #13 and #43 are repressor. Real-time PCR showed that #11-1-1 activated the gene-expression of AtGST11 gene under Al and Cd stresses, and that #13 and #43 repressed the expression under cold and heat stresses. These TFs may contribute to the expression of AtGST11 under each condition.

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The Smirnoff-Wheeler pathway is the pivotal pathway for the biosynthesis of L-ascorbic acid (AsA) in Arabidopsis. Reproduction of GDP generated by phosphorylation of GDP-L-galactose in this pathway to GTP is catalyzed by nucleoside diphosphate kinase (NDPK; EC 2.7.4.6). We found the activity of NDPK to be light responsive with correlation to changing AsA levels in Arabidopsis. The expression levels of genes in the AsA pathway were analyzed using an Arabidopsis ndpk1 knock-out mutant. The mutant showed 25-30% lower AsA content compared to the wild type. The reduction in AsA level was found to correlate with NDPK activity as well as the expressions of some vital AsA biosynthetic genes in various tissues of the plants. The redox status, but not oxidized AsA, of the wild type was significantly different from that of the mutants. The mutant was not impaired in the activities of its major antioxidant enzymes. The activity of L-galactono-1,4-lactone dehydrogenase activity was reduced in the mutant but feeding with AsA precursors caused AsA level to increase. These results suggest that NDPK1 may be implicated in the overall biosynthesis of AsA in Arabidopsis.

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C. merolae is a photoautotrophic unicellular red algae living in acid hot springs, and has the simplest cell architecture among eukaryotes. In addition to the minimally redundant gene content, many nuclear and chloroplast genes are regulated by light conditions. These features provide a good model for studying light signal transduction in plant cells.
In higher plants, DET1 is known as a negative regulator of light-dependent transcription, and widely influences cellular functions as photomorphogenesis. DET1 binds nuclear chromatin and associates with CUL4-DDB1. While these lines of evidence suggest that DET1 might affect transcriptional regulation via chromatin modification, the underlying mechanism remains unclear.
In this study, we constructed a null mutant strain lacking the DET1 homolog gene in C. merolae. This mutant showed growth inhibition under light-dark cycles. As observed in higher plants, several light-regulated nuclear and chloroplast genes were highly expressed even under dark. Currently, we are analyzing the relationship between DET1 and histone H3 lysine 9 di-methylation as a representative chromatin mark for transcriptional silencing.

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In Synechococcus elongatus PCC 7942, the LysR-type transcriptional regulator CmpR activates transcription of the genes involved in the CO₂-concentrating mechanism (CCM) , including the cmpABCD operon and the sbtA gene, which encode the ABC-type high-affinity HCO₃^- transporter BCTI and the Na⁺-dependent high-affinity HCO₃^- transporter, respectively. Low concentration (~10μM) of 2-phosphoglycolic acid (2-PG), the product of the oxygenase reaction of RubisCO, enhances the binding of CmpR to the upstream region of the target genes, thus, suggesting that 2-PG is a co-inducer of CmpR (Nishimura et al,. 2008). In previous studies, we identified several 2-PG dependent CmpR-binding sites in the regulatory region of the cmpABCD operon. In this study, we found that 600μM of D-riburose-1,5-bisphosphate (RuBP) also enhances the binding of CmpR to each of these sites. This indicates that CmpR binds to the same sets of DNA sites in response to 2-PG and RuBP, suggesting that accumulation of RuBP would enhance the low-CO₂ responsive activation of the cmpABCD operon.

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Cyanobacteria possess a CO₂-consentrating mechanism (CCM), which enables efficient uptake and assimilation of CO₂. Some CCM-related genes (e.g. cmp opren, ndhF3 operon and sbtA) are activated under CO₂-limited growth conditions. CmpR is a LysR family protein, which senses 2-phosphoglycolate (2PG) as a low CO₂ signal and activates transcription of the cmp operon. Lately we found that CmpR binds to the ndhF3 promoter in a 2PG-independent manner, repressing ndhF3 operon transcription under high-CO₂ conditions. The gene encoding CmpR (cmpR) is also repressed by CmpR under high-CO₂ conditions and the short intergenic region upstream of cmpR was found to have a similar sequence to that of the 2PG-independent CmpR-binding site in ndhF3. EMSA analyses confirmed 2PG-independent binding of CmpR to this region. Moreover, EMSA revealed the presence of a second CmpR-binding site in the ORF located upstream of cmpR. The binding of CmpR to this site is observed in the absence of 2PG but was enhanced by 2PG. We are trying to identify the second binding site to gain insight into the self-regulation mechanism of cmpR.

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The moss Physcomitrella patens has eight genes coding for the NRT2 nitrate transporters. Among the PpNRT2s, PpNRT2;1-PpNRT2;4 are the major NRT2 species in the protonemata, while the others are minor components. Although expression of all of the NRT2 genes is induced by nitrate and repressed by glutamine, preferential expression of PpNRT2;3 is observed in the protonemata grown under nitrate-sufficient conditions. On the other hand, protonemata incubated in nitrogen-free medium expressed PpNRT2;1, PpNRT2;2 and PpNRT2;4 mRNAs as major NRT2 mRNA species. Promoter-LUC fusions constructed using PpNRT2;1 and PpNRT2;3 promoters also showed nitrogen-responsive differential expression of the reporter gene, indicating that the differential accumulation of PpNRT2;1 and PpNRT2;3 transcripts is ascribed primarily to regulation of transcription. PpNRT2;1 and PpNRT2;3 promoters of 1169 bp and 1124 bp in length, respectively, showed their specific response to nitrogen. However, the PpNRT2;3 promoter truncated to 633 bp showed only poor response to nitrate. This indicated that the -1243 to -634 region of PpNRT2;3 contains necessary elements for nitrate-responsive expression.

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A double-AP2 domain transcription factor WRINKLED1 (WRI1) of Arabidopsis thaliana is expressed during the mid-stage of seed development, and it plays an essential role in seed oil synthesis. WRI1 binds to AW-box conserved among the upstream region of genes involved in fatty acid synthesis in plastids, and activates their expression to facilitate the conversion of incoming sugar to oil¹⁾. Mutations in two Aw-boxes in the upstream region of PlPKβ1 abolished the expression of PlPKβ1p::GUS in not only in developing seeds but also in vegetative tissues and floral organs, suggesting that AW box-binding factors other than WRI1 are involved in the expression in tissues other than seeds. WAT1 and WAT2, closely related to WRI1, were expressed in vegetative tissues and floral organs, and activated genes involved in fatty acid synthesis in protoplasts. To clarify differential role of WRI1, WAT1, and WAT2 in the regulation of fatty acid synthesis, we characterized single, double, and triple mutants of wri1,wat1 and wat2, and introduced PlPKβ1p::GUS to these mutants. In addition, we compared effects of overexpression of WAT1, WAT2 or WRI1. ¹⁾Maeo et al., Plant J. 60: 476 (2009)

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