Control of plant growth depends on the comprehensive understanding of phytohormone function and establishment of technologies to regulate the function. Here I report two basic studies and one application-oriented study. Pharbitis 'uzukobito' is arecessive mutant showing extremely dwarfphenotype. Exogenous application ofbrassinosteroidbiosynthetic intermediates and analysis of endogenous brassinosteroids suggested a defect of PnDET2, a homologous enzyme of arabidopsis DET2. The cloning and function assay demonstrated the mutation and the functional loss of the enzyme of uzukobito. AGPs constitue a family of glycoproteins. The specific binder of AGPs (Yariv) showed the involvement of AGPs in GA function in barley aleurone cells. Microarray analysis showed that Yariv is an overall represser of GA-responsive gene expression, and the reagent also activated defense-related signaling. Well-established defense system inducers such as jasmonic acid and chitin elicitor also inhibited GA-inducible events in aleurone cells, indicating that GA signaling is under the regulation of defense-related signaling. Immunomodulation is a mean to modulate organism function by antibody production to capture either endogenous or exogenous antigens. I applied this method to plants. Production of single-chain antibodies both against bioactive GAs and against their biosynthetic precursors gave clear dwarf phenotype, by repressing GA action and GA biosynthesis, respectively.

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Arabidopsis F-box protein TIR1 function as an auxin receptor. TIR1, a component of SCF-class E3 ubiquitin-ligase, is responsible for the regulation of the 26S proteasome-mediated destruction of the Aux/IAA family of transcriptional repressers, thereby altering the expression of hundreds of genes. We have designed a series of a novel probes for auxin signaling. BH-IAA is the most potent anti-auxin probe and antagonized all known genomic auxin responses at the molecular and whole-plant level, such as auxin-responsive gene expression, auxin-induced Aux-IAA degradation, auxin-promoted interaction of Aux/IAA-TIRl and physiological auxin responses of Arabidopsis plant. To test the utility of these probes for the analysis of TIR1-mediated auxin signaling in species other than Arabidopsis, we examined the effect of BH-IAA on development in the monocot Oryza saliva (rice) and the lower plant Physcomitrella patens (moss). In both cases, BH-IAA antagonized axuin responses of plants, suggesting that BH-IAA is a useful tool to probe auxin signaling in a variety of plant species. Further, BH-IAA anti-auxin activity in moss demonstrates that P. patens SCF^<TIR1> pathway plays a crucial role in moss development, suggesting SCF^<TIR1> pathway is an ancient mechanism. Our auxin probe provides a new useful tool for plant chemical biology.

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We developed alpha-alkyl-auxin as a novel auxin probe to specifically modulate TIR1 function, an auxin receptor. TIR1 is F-Box protein, a component of an SCF-class E3 ubiquitin-ligase. To reveal the molecular mechanism of auxin probes, we solved crystal structure of three complexes of TIR1-ASK1-probe (an agonist or two antagonists). Crystal structure of three complexes revealed the probes bind to an auxin-binding site of TIR1 and modulate the TIR1 recognition process for Aux/IAA represser. Furthermore, no conformational change of TIR1 was observed among the three complexes. Additionally, molecular docking calculation between TIR1 and probe also support the molecular mechanism of probe action. These evidences clearly indicate that auxin actually works as 'a molecular glue'in TIR1 complex proposed by a recent study. This is the first report on a molecular mechanistic study for anti-auxin probe and a modulator of the substrate recognition in SCF-class ubiquitin-ligase complex.

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Epicotyls of etiolated pea (Pisum sativum L. cv. Alaska) seedlings grow toward the vertical direction on earth. On the other hand, those of agravitropic mutant, ageotropum, and Alaska treated with auxin polar transport inhibitors of TIB A (2,3,5-triiodobenzoic acid), HFCA (9-hydroxyfluorene-9-carboxylic acid) and NPA (N-(1-naphtyl)phthalamic acid) showed automorphosis-like growth and development. These results strongly suggest close relationships between growth and development, and auxin polar transport in etiolated pea seedlings. Activities of auxin polar transport were asymmetrical in epicotyls, being extremely higher in the proximal side of epicotyls than in the distal one in etiolated Alaska and ageotropum pea seedlings. These results in etiolated Alaska pea epicotyls were true in expression of PsPIN1 encoding a facilitator protein of auxin polar transport and of an auxin-inducible gene, PsIAA4/5. Activities of auxin polar transport in ageotropum and Alaska pea epicotyls treated with auxin polar transport inhibitors were significantly reduced. These results strongly support the idea that normal activities of auxin polar transport is essentially required for normal growth and development in early growth stage of etiolated pea seedlings.

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Hydrotropism is a response of root to a moisture gradient. Because of the necessity of water acquisition, root hydrotropism has been considered as an important tropism. Recent analyses using Arabidopsis have emphasized the contribution of polar auxin transport to differential auxin distribution as well as to gravitropism. Our previous results also suggested that differential auxin distribution plays a pivotal role in root hydrotropism. Nonetheless, how auxin distribution is controlled in this phenomenon remains unclear. In search of the molecules that control auxin redistribution in hydrotropic response, we found that inhibitors of auxin efflux significantly reduced hydrotropism in cucumber seedlings. Moreover, our immunohistochemical analyses revealed that the expression of putative auxin efflux facilitator CsPIN5 was diminished at the dry side of the distal elongation zone of hydrotropically responding roots, whereas it was maintained at the corresponding region of the humid side. Considering the previous results that auxin-responsive gene expression is mainly occurred at the humid side of distal elongation zone of cucumber seedlings, our present results suggests that CsPIN5-mediated auxin efflux plays definite role in regulating auxin redistribution during hydrotropic response in cucumber seedlings.

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Auxin is an important plant hormone for morphogenesis. Cucumber seedlings form a specialized protuberance, called peg, on the transition zone (TR-zone) between the hypocotyl and root. When cucumber seeds germinate in a horizontal position, seedlings develop a peg on the lower side of the TR-zone. We previously demonstrated that auxin was more abundant in the lower side than in the upper side of the gravistimulated TR-zone and suggested that this asymmetric auxin distribution induced differential auxin response to contribute to peg formation. Moreover, application of auxin or auxin-polar-transport inhibitor (TIBA) induced both expression of auxin-inducible gene expression and peg formation at both upper and lower side of the TR-zone, whereas application of anti-auxin (PCIB) induced neither. Recently, we found CsGRPl whose mRNA accumulated more abundantly in the upper side than in the lower side of the TR-zone. We found that this accumulation of CsGRPl mRNA was decreased by the application of either auxin or TIBA. On the other hand, application of PCIB induced CsGRPl gene expression in both upper and lower side of the TR-zone. These results suggest that gravistimulation increases not only the expression of auxin-inducible genes on the lower side of the TR-zone but also the expression of auxin-repressed genes, such as CsGRPl, on the upper side in cucumber seedlings to contribute to the one-sided formation of the peg.

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The leading shoot dominates the growth of the axillary buds. After decapitation of the shoot apex, outgrowth of axillary buds begins. This phenomenon is known as apical dominance. Auxin basipetally derived from apical bud has an inhibitory effect on the growth of axillary buds, whereas cytokinin promotes axillary bud outgrowth. It is also proposed that graviresponse is involved in apical dominance. When the upper part of main shoot of morning glory (Pharbitis nil) is bent down, an axillary bud situated on the uppermost node starts to elongate. Using an agravitropic mutant of morning glory, weeping, we previously demonstrated that this release from apical dominance requires graviresponse. However, the molecular mechanism of this phenomenon is unclear. In this report, we examined the involvement of cytokinin and auxin in gravity-regulated axillary bud growth in comparison with that induced by decapitation. The newly isolated cytokinin biosynthetic gene, PnIPT2, was up-regulated by decapitation, whereas it was suppressed by auxin exogenously treated to the stump. PnIAAl, an auxin inducible gene, was down-regulated by decapitation. In contrast, neither PnIPT2 nor PnIAAl expression changed with shoot bending treatment. These results suggest that mechanism for gravity-influenced release from apical dominance is independent of that of decapitation.

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The plant hormone auxin (indole-3-acetic acid (IAA)) plays important roles in response to external factors such as light and gravity. We have reported using maize coleoptiles that IAA was biosynthesized mainly at 0-2 mm tip region and that the synthesized IAA was continuously transported to basal part. We also showed that IAA distribution rapidly changed at upper and lower side of the coleoptile at 0-10mm region within 20 minutes after gravi-stimulation, and IAA level at top region was temporary increased and then decreased. Here, we examined details about the changes of IAA level and movement after gravi-stimulation. The amount of IAA at 0-3 mm tip was increased from about 200 to 250pg/tip during 20 min after gravi-stimulation, and then gradually decreased to the initial level until 120 min. Before and after the stimulation, 0-3 mm tips were excised and placed on agar blocks in a horizontal direction, and diffusible IAA levels were determined. The amount of IAA diffusing from tip to agar was decreased by gravi-stimulation. Thus, increasing level of endogenous IAA in the tip after gravi-stimulation could be explained by the decrease in the rate of IAA transport. To know details for IAA movement after gravi-stimulation we are also investigating the effect of NPA and BFA treatment comparing between before and after gravi-stimulation.

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Indole-3-acetic acid (IAA) is an important hormone in almost all aspects of plant growth and development. From our previous work using maize coleoptiles, it has been indicated that the tip region of coleoptiles is the site of IAA biosynthesis from tryptophan (Trp) and the IAA synthesized in the tips is transported to the lower regions. However these studies were performed with sectioned maize coleoptiles. In the present study, we tried to investigate the movement of synthesized IAA at the tip in intact coleoptiles. The results showed the high efficient incorporation of stable isotope label from [^^<13>C_<11>^^<15>N_2]-Trp into IAA molecules during 15 to 30 min feeding. The rate of labeled IAA was about up to 15% of endogenous free IAA. The labeled IAA did not stay at the tip region but immediately transported to the lower parts at the rate of approximately 7 mm/hr. These results suggest that IAA continuously synthesized from Trp at the tip region is transported to the lower region in a polar transport manner. Even the different concentration of [^^<13>C_<11>^^<15>N_2]-Trp was used for the feeding to the tip, the total amount of free IAA in the coleoptile was not changed. These results suggest that this system is reflected in vivo IAA biosynthetic situations.

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Indole-3-acetic acid (IAA) is a plant hormone that plays an important role in many physiological events. Recent studies suggest that such events are accurately controlled through the regulation of IAA transport. To know the IAA function, uncovering the regulation of IAA biosynthesis is the most important issue. We have reported that maize coleoptiles tip is a main site of de novo IAA biosynthesis from tryptophan and produced IAA moves to the lower parts. However, IAA biosynthetic cell(s) in coleoptiles tips and route of IAA transport from the cell(s) are still unknown. Our recent works based on immunolocalization of PIN have revealed IAA transport route(s) in maize coleoptiles. In tip region (0-1mm) of coleoptiles, the putative IAA efflux carrier, PIN, was detected at almost all cells, where it localized in whole plasma membrane. In subapical region, polar staining patterns were observed, and the signals were intensely detected at epidermal and subepidermal cells. When BFA was treated on coleoptile tips, IAA transport was inhibited to about 65% after 40 min and IAA accumulated in the tip region. In addition, internalization of PIN was observed in intra cellar compartment. These data indicated that IAA could be transported in every direction in top 0-1 mm of coleoptiles, and polarly toward lower parts in subapical region mainly through epidermal and subepidermal cells. It was concluded that BFA induces internalization of PIN, which results in inhibition of IAA transport.

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While Arabidopsis YUCCA, AAO, NIT, CYP72B2 and CYP79B3 genes have been reported as IAA synthesis genes, there is no direct evidence that they actually work in plants. In monocot coleoptiles, it has been shown for a long time that IAA is produced in very tip region and transported in a polar manner, and our previous works revealed that the IAA is synthesized de novo from tryptophan in the tip region of maize coleoptiles. We here tried to identify the IAA biosynthesis genes through Microarray analysis using rice coleoptiles. Apical (0-1.5 mm) and subapical (3-4.5 mm) parts of coleoptiles were sectioned from 4-day-old rice seedlings and mRNA was extracted from them. Microarray was performed according to Rice Genome Resource Center's protocol for Rice Oligo Microarray Kit, 22K from Agilent. We found 133 genes strongly expressing in the apical region. RT-PCR on 20 genes of them revealed that at least 15 genes expressed specifically in the apical region. Also, among six YUCCA-like genes, five AO-like genes, two NIT-like genes and forty-two P450-like genes found in the 22K rice Microarray targets, only three P450-like genes belonging to CYP71, not CYP79, family had the apical specific expression. Since 22K rice Array Slide does not cover all genes of rice (37554 genes), we are planning to perform 44K rice Microarray analysis using coleoptiles treated with IAA transport inhibitor NPA, adding to apical and subapical region of coleoptile.

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Indole-3-acetic acid (IAA) is an active natural auxin that is involved in the most aspects of plant growth and development. Recently, the important roles of the YUCCA pathway in IAA biosynthesis have been reported. In this pathway IAA is generated from tryptophan (TRP) through tryptamine (TAM) and N-hydroxy-tryptamine (HTAM). As for the intermediates between HTAM and IAA, indole-3-acetaldoxime (IAOX), indole-3-acetonitlile (IAN) and indole-3-acetaldehyde (IAAld) have been proposed as candidate compounds. To elucidate the YUCCA pathway in plants, we have developed a new method for the analysis of IAA intermediates by LC-TOF-MS/MS. Using ^^<15>N or ^^2H-labeled IAA intermediates as internal standards, we have detected TAM and IAOX from Arabidopsis seedlings. In addition, the identification of each compound was confirmed by a feeding of [^^<13>C_8, ^^<15>N]indole to the TRP-auxotroph mutants of Arabidopsis. Our data clearly demonstrated that both TAM and IAOX were efficiently labeled from [^^<13>C_8, ^^<15>N]indole. On the other hand, we have not yet detected HTAM and lAAld from this plant due to the unstable property of these indole compounds. We are currently analyzing the IAA intermediates in various IAA biosynthesis mutants.

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Plasmodiophora brassicae parasitizes cruciferous plant roots and manipulates indole-3-acetic acid (IAA) biosynthesis system of host plants to induce hypertrophy, called clubroot. We isolated three cDNAs encoding nitrilase from Brassica rapa (turnip), i.e. BrNIT-T1, BrNIT-T2 and BrNIT-T4. Enzyme analysis of recombinant proteins showed that BrNIT-T1 and BrNIT-T2 possess IAA synthetic activity, but BrNIT-T4 is involved in the plant cyanide detoxification pathway downstream of ethylene production. Realtime PCR analysis revealed specific involvement of BrNIT-T1 in clubroot initiation phase: in early-growing clubroot, BrNIT-T1 was strongly up-regulated but BrNIT-T2 and BrNIT-T4 were down-regulated. In maturing clubroot, not only BrNIT-T1 but also BrNIT-T2 and BrNIT-T4 were up-regulated. Free IAA level was transiently elevated at the early and later phases, despite a high IAA conjugation activity throughout disease development. Ethylene-forming activity was reduced during clubroot formation but subsequently increased, just like BrNIT-T4 expression. These results indicate a close relationship of the nitrilases to auxin and ethylene biosynthesis. Treatment of roots with cytokinin or jasmonic acid activated BrNIT-T1 or BrNIT-T2 expression, respectively. Thus, the two phytohormones may trigger IAA production in clubroot. Based on these observations, we propose a scheme regarding individual roles of the nitrilases in hormone crosstalk during clubroot initiation and maturation in turnip.

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The formation and degradation of IAA-amino acid conjugates (IAA-aa) play an important role in auxin homeostasis. GH3s encode IAA-aa synthetases, but their functional redundancy has hampered the genetic approach to study auxin homeostasis. GH3s belong to the acyl-activating enzyme superfamily that activates IAA by adenylation. According to this catalytic mechanism, we designed and synthesized N-sulfamoyladenosine (SA) derivatives of auxins (IAA-SA, PAA-SA, NAA-SA and 2,4-D-SA) as intermediate analog inhibitors of GH3s. These compounds not only served as potent in vitro inhibitors of GH3s with IC_<50> values of 3 to 45 μM, but also exhibited in vivo activities. In the excised Arabidopsis leaves treated with exogenous IAA, the endogenous levels of IAA-Asp, IAA-Glu and IAA-Gln greatly increased. When the SA derivatives were applied together with IAA, the increase was significantly repressed. Thus, the SA-derivatives of auxins were adsorbed in plant tissues and inhibited GH3s involved in lAA-aa synthesis, thereby serving as highly effective chemical tools to probe otherwise inaccessible auxin homeostasis. The cell permeability was suggested to be an important factor that affected the in vivo activities of SA derivatives. We therefore designed and synthesized a series of N-acylsulfamide derivatives that are chemically stable and more hydrophobic than SA derivatives. The results on N-acylsulfamide derivatives will also be presented.

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In Arabidopsis thaliana, CURLY LEAF (CLF) and SWINGER (SWN) are factors of the Polycomb complex (Pc) that plays key roles in chromatin-remodeling by modulating histone methylation. Although it is suggested that PC regulate repression of embryonic trait from the fact that clfswn forms somatic embryos after germination, the molecular mechanisms are still unclear. We found that the formation of somatic embryos in shoot apical meristem (SAM) of the clfswn was suppressed by culitvation at low temperature (10℃). The LEAFY COTYLEDON1 (LEC1) that is an embryo specific gene was mis-expressed in a whole part of the clfswn at 21℃. By contrast, the LEC1 expression in SAM of the clfswn was reduced at 10℃. The formation of somatic embryos at 10℃ was dose-dependently restored by 2,4-D. To identify developmental stage for repressing embryonic trait, the clfswn was transferred from 21℃ to 10℃ at various days after sowing. When the clfswn was transferred to 10℃ at 12 days after sowing, ratio of somatic embryo formation was reached about 50%. The cotyledon of elf swn was not expanded at 12 days after sowing, yet. These results suggest that auxin is involved in somatic embryo formation in clfswn and the mis-expression of the embryo specific genes may occur before cotyledon expansion.

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Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins present at plasma membrane and extracellular space. A synthetic chemical reagent called β-glucosyl Yariv reagent (β-GlcY) binds specifically with AGPs. We have reported that gibberellin (GA) signaling was specifically inhibited by β-GlcY treatment in barley aleurone protoplasts. In this study, we examined the specificity of inhibitory effect of β-GlcY on GA signaling using microarray analysis and found that β-GlcY was largely effective in repressing both the GA-induced- and repressed-gene expressions. In addition, quite a number of genes were up-regulated by β-GlcY in a GA-independent manner, and many of them were categorized to defense-related genes including two WRKYs and Esi47, which were recently shown to repress GA signaling. Plant defense signaling triggered by several plant defense system inducers such as jasmonic acid or chitin elicitor could inhibit GA-inducible events such as α-amylase secretion, programmed cell death, and gene induction of some GA-inducible genes in aleurone cells. These results indicate that GA signaling in aleurone cells is under regulation of defense-related signaling, which is presumably mediated by WRKYs and Esi47 at least in part. It is also probable that AGPs are involved in the perception of stimuli causing defense responses.

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Gibberellin (GA) participates in many aspects of plant growth and its development. Following the identification of rice GA receptor gene (OsGIDl), we also found three receptor genes (AtGIDla, AtGIDJb, AtGIDl c) in Arabidopsis. Each recombinant protein of the AtGIDl gene showed a high affinity to biologically active GAs, and the expression of each AtGIDl gene could complement the dwarfed phenotype of rice gidl-1 mutant. In this study, we elucidated the functional redundancy and specificity of each AtGIDl by using some T-DNA or Ds insertion mutants. The phenotype of the homozygous single knock-out mutants (that is, atgidla-1KO, atgidlb-1KO, or atgidlc-1KO) are all similar to that of wild type plants while we found distinctive phenotypes in 2KO mutauts: (i) the atgidla atgidlb-2KO showed shorter stamens, and then low fertileity and (ii) the atgidla atgidlc-2KO showed a dwarfed phenotype. Moreover, it was observed that the atgidla atgidlb atgidlc-3KO mutant did not germinate, however it began to grow after the removal of seed coat. Its seedlings showed a severely dwarfed phenotype, and a complete loss of their ability to respond to GA. Taken together, we concluded that three AtGIDls function as GA receptors in Arabidopsis with high redunduncy, but partially have specific role(s) for growth and development of Arabidopsis.

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We have previously expressed single-chain variable fragments (scFvs) against bioactive gibberellin (GA), designated as 8/E9 and 21/D13, as fusions with GFP in plants with subcellular localization. In the study, GFP-scFv was most stably accumulated when targeted to endoplasmic reticulum (ER), and the transgenic plants producing GFP-scFv in ER showed dwarf phenotype. Although both 8/E9- and 21/D13-scFvs gave the phenotype, we found as-yet-unrecognized filamentous shape of fluorescence in the leaf epidermal cells only of 21/D13 lines. Because the GFP-scFv has an KDEL ER-retention signal, the structure was considered to be an ER-derived compartment, and therefore we here tentatively call it filamentous ER (f-ER). The appearance of f-ER is like an long thread entwining complicatedly. The f-ER is present in the inner part of the cells compared to ER networks locating beneath plasma membranes, and has much stronger fluorescence than ER networks. Interestingly, the filamentous structure was fragmented immediately after treatment with GA4, the ligand of the scFv, suggesting that the structure is induced and maintained by binding of the scFv with an unknown target in the ER. Analysis of this phenomenon might give an insight to the mechanism of ER development.

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An antibody (Ab) which recognizes another Ab binding with its ligand is called an anti-metatype Ab. Only limited numbers of anti-metatype Abs have been reported. In this study, we screened a phage display Ab library to obtain anti-metatype Ab of antibioactive gibberellin (GA) Ab. We also screened phage display peptide libraries to obtain peptides which bind anti-GA Ab in the GA dependent manner (tentatively designated as anti-metatype peptide). We screened anti-metatype Abs or peptides from a phage display single-chain variable-fragment (scFv) library and three kinds of phage display peptide libraries against anti-bioactive GA antibody (8/E9) in the presence of GA1 or GA4. Although anti-metatype scFv could not be obtained because of extensive non-specific and GA-non-dependent binding, two different anti-metatype peptides which bind to 8/E9 Ab in the presence of GA4 were obtained (designated as A peptide and I peptide). The I peptide, which showed clearer GA4 dependency for the binding, did not bind to 8/E9 in the presence of GA1, another ligand of 8/E9, suggesting that the structure of GA4 molecule directly participated in the binding. I peptide was not reactive with 21/D13, another anti-bioactive GA antibody, regardless of GA existence, indicating that both GA4 and the partial structure of the 8/E9 are recognized by the peptide. Using 8/E9 and I peptide, an ELISA system equivalent to sandwich ELISA was established to detect as low as 30 pg of GA4 specifically.

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In Arabidopsis, seed germination is regulated by light via phytochrome. Once phytochrome is activated by light in dark-imbibed seeds, expression of the gibberellin (GA) biosynthesis genes GA3oxl and GA3ox2 is upregulated, whereas the GA-deactivation gene GA3oxl is downregulated, resulting in an increase in concentrations of bioactive GAs. PHYTOCHROME-INTERACTING FACTOR3-LIKE5 (PIL5) is a bHLH transcription factor that is involved in the regulation of GA biosynthesis and deactivation genes via phytochrome. Besides GA content, GA responsiveness also is modulated photo-reversibly via phytochrome in Arabidopsis seeds; in the absence of active phytochrome, more exogenous GA is necessary for the induction of germination of GA-deficient gal-3 mutant seeds. To understand the molecular mechanisms underlying phytochrome-regulation of GA responsiveness, we set out to identify phytochrome/PIL5-regulated genes in gal-3 mutant seeds using microarray analysis. We found that genes encoding DELLA proteins, negative regulators of the GA response pathway, are direct targets of PIL5, and that they contribute to altering GA responsiveness under varying light conditions. In addition, experiments using ga2ox2 mutants seeds and 2,2-dimethylGA_4, which is resistant to deactivation by GA2ox, indicated that elevated GA2ox2 expression in the dark also contributes to decreasing apparent sensitivity to exogenous GA_4.

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RSG is a bZIP transcriptional activator that regulates endogenous amounts of gibberellins. The endogenous level of GA decreased in the transgenic tobacco expressed of dominant negative form of RSG (dnRSG). The transgenic tobacco exhibited dwarf phenotypes.We investigated the expression levels of six GA biosynthetic genes. The expression of ent-kaurene oxidase is decreased in the transgenic tobaccos. RSG regulates GA biosynthesis through the transcriptional control of ent -kaurene oxidase gene. When endogenous level of GA decrease, the expression level of GA20-oxidase gene and GA3-oxidase gene increased by feedback regulation. Interestingly, in dnRSG plants, though decrease of endogenous GA, did not occur the feedback regulation of GA20-oxidase. dnRSG inhibits the feedback regulation of the GA20-oxidase (Ntc12) gene. GAs regulates intracellular localization of RSG. To search where is necessary to feedback regulation in Ntc12 promoter, we produced transgenic tobacco that GUS gene fused to Ntc12 promoter. We identified a cis element that is important of GA feedback regulation. RSG binds this element directly. The point mutation of this element in Ntc12 promoter is disrupting the feedback regulation. Moreover ChIP analysis suggests that RSG bind to Ntc12 promoter dependent on endogenous GA level.These results indicate that RSG regulates feedback regulation of GA in Ntc12 promoter.

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Gibberellins, a phytohormone, are biosynthesized from geranylgeranyl diphosphate (GGDP) via ent-kaurene. In higher plants, entkaurene was formed from GGDP via ent-copalyl diphsphate (ent-CDP) by two distinct diterpene cyclases, ent-CDP synthase and ent-kaurene synthase. Rice (Oryza sativa L.) produces diterpene phytoalexins, such as oryzalexins, momilactones and phytocassanes, of which biosynthetic hydrocarbon intermediates are converted from GGDP by the similar manner as ent-kaurene synthesis. We have shown that rice has two ent-CDP synthase genes; OsCPSl is for gibberellin biosynthesis and OsCPS2/OsCyc2 is for oryzalexins A-F and phytocassanes A-E. Although ent-CDP synthases for gibberellin biosynthesis from other plant species were well characterized, we have known little about properties of rice ent-CDP synthase for phytoalexin biosynthesis.Here, we enzymatically characterized recombinant putative mature OsCPS2/OsCyc2 as well as OsCPS4/OsCycl, syn-CDP synthase for mimilactones A and B and oryzalexin S, and compared to OsCPS1. The optima of pH (around neutral), temperature (around 30 oC), and divalent ion (0.1 mM Mg^<2+>) on OsCPS2/OsCyc2 and OsCPS4/OsCyc1 were almost the same as OsCPS1. Interestingly, the inhibition rate of activities of OsCPS2/OsCyc2 and OsCPS4/OsCyc1 by substrate, GGDP (50-60μM), and Amo-1618 (10^<-5>-10^<-4>M), a gibberellin biosynthetic inhibitor which suppresses ent-CDP synthase activity, was less than that of OsCPS1.

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Germination of lettuce (Lactuca sativa L. cv. Grand Rapids) seed is regulated by phytochrome. We have shown that endogenous levels of bioactive gibberellin (GA_1) increased after red light irradiation during germination of lettuce seeds. In order to investigate the molecular mechanism on control of endogenous GA_1 levels, we have isolated 11 cDNAs encoding GA metabolic enzymes (LsCPS, LsKS, LsKO1, 2, LsKAO, LsGA20ox1, 2, LsGA3ox1, 2, LsGA2ox1 and 2) and carried out expression analysis by semiquantitative RT-PCR. We reported previously that expression levels of LsGA3ox1 increased and these of LsGA2ox2 decreased after red light irradiation, and the effects of red light were partially cancelled by application of ABA. In this study, the functions of translated products of lettuce diterpene cyclases (LsCPS and LsKS) and P450 monooxygenases (LsKO1, 2 and LsKAO) were confirmed by in vitro assay using recombinant enzymes. And expression analysis of 11 GA metabolic enzyme genes will be performed by real time QRT-PCR. Recently, it was reported that the expressions of GAI and RGA encoding DELLA proteins, negative regulators for GA signaling, were down-regulated by phytochrome in Arabidopsis (Oh et al., 2007). Then, we successfully isolated two GAI/RGA homologue cDNA fragments from lettuce (LsGAI/RGA like1, 2), and will discuss regulation of these genes by phytochrome.

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In the growth chamber, foliar applications of 5-ALA enhanced NR activity in leaves of Komatsuna seedling, resulting in the increases of plant growth. On the other hands, nitrate concentrations in shoots were hardly changed by these treatment.

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In tea fields, the effect of spray-applied fertilizer containing ALA (Pentakeep-V; PKV) on the growth of tea plant was investigated. The application of PKV not affected on the yield and the contents of total-nitrogen and fiber of first crop, but significantly increased the color index of tea leaves, comparing to that of Urea. Moreover, the color index of tea leaves was increased with the increment of the application times of PKV. Theses suggested that PKV contained ALA was useful fertilizer to enhance the color of tea leaves.

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Four cultivars of potatoes were grown in soil in pots from May 31 to July 20.The plants were treated with or without NaCl solustions of 0.3% three times, 0.6% twice and 1.0% 6 times by the pot-dipping treatment. The plants were also treated with or without a fertilizer containing ALA at the dilution of 1/3,000 by spray treatment at every week. By the fertilizer treatment the salt injury was recovered completely in cv. Kitamurasaki and nearly completely recovered in cv. Chelse, but the injury was not recovered in cv. Kitaakari and Star Ruby.

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Radish plants were grown in soil in pots, and treated with mineral nutrients (Ca, Mg, Fe, Mn, Co, Cu, Mo) by soil treatment and ALA by folier treatment. The fresh weight of the radish decreased with the increase of ALA concentrations in the pots of insufficient Mg application, but it increased with the increase of ALA in the pots of sufficient Mg treatment. The fresh weight increased with ALA at low concentrations in the pots of sufficient Fe application, and it did not increase with AlA at high concentrations in the pots of sufficient Fe application.

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Senescence in monocarpic plants, such as legume and wheat, is dramatic because the entire resources of the plant are mobilized and redirected to support seed development. We found that the level of senescence promoting activity in extract of soybean pods changed greatly during the plant growth. Abscisic acid (ABA) was first identified as senescence promoting factor. However, senescence promoting activity in pod extract could not be explained by endogenous ABA content because soybean pod did not contain enough amounts of ABA. As a result of further purification, it was found that jasmonic acid (JA) played a role as synergist against ABA and exhibited the synergic effect at 10^<-6> M. We investigate whether JA regulates the monocarpic senescence of soybean.

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RSOsPR10 was originally found as the root specific protein induced by drought and salt treatment in rice. The mRNA was induced specifically in roots by drought, salt, probenazole, rice blast fungus and jasmonic acid (JA), but not by low temperature, abscisic acid and salicylic acid (SA) (Hashimoto et al., PCP, 2004). In this study, it was confirmed that the expression of RSOsPR10 protein was induced by salt, JA, and ethylene precursor (ACC), and the induction was suppressed almost completely by SA. Moreover, using RT-PCR method we revealed that the suppression occurred at a transcriptional level. From these results, we present a signaling pathway for RSOsPR10 expression as shown in Fig.1.

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The enzyme 12-oxophytodienoate (OPDA) reductase, which is involved in the biosynthesis of jasmonic acid (JA), catalyses the reduction of OPDA to yield 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid. The rice OsOPRl gene encoding OPDA reductase (OPR) converts (-)-cis-OPDA preferentially, rather than (+)-c/s-OPDA, a natural precursor of JA; therefore, another OPR to convert the natural substrate is assumed to function in the JA biosynthetic pathway in rice. Here, we provide evidence that an OPR family gene in rice chromosome 8, designated OsOPR7, encodes the enzyme involved in the JA biosynthesis in rice. Recombinant OsOPR7-His protein efficiently catalysed the reduction of both enantiomers of cw-OPDA, similar to the Arabidopsis OPR3 protein. The expression of OsOPR7 mRNA was induced and reached maximum levels within 0.5 h of mechanical wounding and drought stress, and the endogenous JA level started to increase in accordance with the increase in OsOPR7 expression. Furthermore, complementation analysis using an Arabidopsis opr3 mutant indicated that the OsOPR7 gene, but not OsOPR1, was able to complement the phenotypes of male sterility in the mutant caused by JA deficiency, and that JA production in the opr3 mutant was also restored by the expression of the OsOPR7 gene. We conclude that the OsOPR7 gene encodes the enzyme catalysing reduction of the natural (+)-cis-OPDA for the JA biosynthesis in rice.

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Jasmonic acid (JA) is a plant hormone that acts as a secondary signaling molecule in plant defense responses. RERJ1 that encodes a bHLH transcription factor was isolated as a JA-responsive gene from rice suspension cells. Previously, we reported that RERJ1 localized nuclear and functions as a transcriptional activator, and that a rice chitinase gene was screened as a RERJ1-taraget gene by the microarray analysis using RERJ1 overexpressor. The chitinase mRNA levels peaked at 4-6 h after JA treatment in rice cells. Besides, the expression of chitinase gene was induced by in a RERJ1-dependent manner in the transgenic rice in which RERJ1 expression could be controlled by an estrogen treatment. Here, we identified JA-responsive cis element in the promoter region of the chitinase by reporter-gene assay. We conducted the deletion analysis of the chitinase promoter regions located from -2415 to -265 bp upstream of the translation start site. Deletions from -2415 to -515 bp did not have a significant effect on responsiveness to JA, but a further deletion to -265 bp rendered the promoter incapable of responding to JA. This result suggests that the region from -515 to -265 bp contains JA-responsive cis element. Indeed, the region contains three E-box elements, which are putative binding sites for bHLH transcription factors. The mutation analysis showed that an E-box at -448 to -443 bp is essential for responding to JA. Direct binding of RERJ1 to the E-box is now being examined.

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The preparation of both enantiomers of 8-[1-(2, 4-dichlorophenyl)-2-imidazol-1-yl-ethoxy] octanoic acid heptyl ester (JM-8686), a potent inhibitor of allene oxide synthase, has been achieved from 2, 4-dichlorophenacyl bromide as a starting material. The key step was the asymmetric reduction of 1-(2, 4-dichlorophenyl)-2-imidazol-1-yl-ethanone. The synthesized optical active (R)-(-)-JM-8686 and (S)-(+)-JM-8686 were purified by chiral HPLC. The inhibitory activities and binding affinities of these enantiomers against allene oxide synthase were determined. We found that the inhibition potency of the (R)-(-)-JM-8686 was approximate 200 times to that of (S)-(+)-JM-8686, with an IC_<50> value approximate 5 ± 0.2 nM and 950 ±18 nM, respectively. The spectral dissociation contents of (R)-(-)-JM-8686 and (S)-(+)-JM-8686 to the recombinant allene oxide synthase were approximate 1.4 ± 0.3 μM and 4.8 ± 0.6 μM, respectively.

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Agriculturally important grasses contain numerous diazotrophic bacteria inside their bodies, which interaction is thought to have some other benefits to host plants. In this study, we analyzed the effect of a bacterial endophyte, Azospirillum sp. (NITE BP-194) on disease resistance of host plants. Rice treated with Azospirillum sp. exhibited resistance against diseases caused by the virulent rice blast fungus, Magnaporthe grisea. This result indicated that the bacterial endophyte, Azospirillum sp., was able to induce disease resistance in rice. Azospirillum sp. was also colonized in Arabidopsis thaliana and induced disease resistance against a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. The Arabidopsis plants treated with Azospirillum sp. did not exhibit the accumulation of antimicrobial substances. These results indicated that the bacterial endophyte, Azospirillum sp., was able to induce disease resistance in Arabidopsis. The investigation of the detailed mechanisms of the induced resistance was in progress.

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Pharbitis nil, a short-day plant (SDP) , can be induced to flower by poor nutrition stress under long days (LD). Phenylalanine ammonia-lyase (PAL) was involved in this stress-responding flowering. Perilla frutescens var. crispa, a SDP, can be induced to flower by low-light intensity under LD. This may be stress-responding flowering. Accordingly, we analyzed the regulating mechanism of flowering under LD in both species. For poor nutrient-stress treatment, P. nil, Violet was cultured in tap water. Red-leafed P. frutescens was grown under different light intensities. In P. nil, the stress-responding flowering was inhibited by a PAL inhibitor, aminooxyacetic acid (AOA) , and this inhibition was overcome by salicylic acid (SA). Exogenous SA alone did not induce to flower indicating that some other factor (s) must be necessary. Because DNA demethylation can induce flowering, the stress could stimulate DNA demethylation. In fact, the treatment of P. nil with 5-azacytidin, a DNA demethylating reagent, together with SA induced flowering. P. frutescens was induced to flower by low-intensity light. This flowering was inhibited by aminooxypropionic acid (AOPP) , another PAL inhibitor. AOA or AOPP did not induce to flower under non-inductive condition. These suggest that PAL activity increase under low-intensity light. Thus, the metabolic pathway regulated by PAL may be involved in the regulation of stress-responding flowering in the both species.

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We found that the treatment with a DNA demethylating reagent, 5-azacytidine (azaC) induced flowering in Perillafrutescens, suggesting that the flowering of P. frutescens was epigenetically regulated. The progeny of the plants flowered by azaC treatment did not flower under non-inductive photoperiod. The flowering-related genes activated by azaC treatment may be deactivated by remethylation during the generation change. Thus, we hypothesized that the expression of flowering-related genes is regulated by DNA methylation. Accordingly, we compared the genomic DNA extracted from photoperiodically induced P. frutescens and that from non-induced control plants by MS-AFLP technique, and detected some fragments specific to the DNA sample of photoinduced plants. This suggests that the inductive photoperiod changed DNA methylation status. Some flowering-specific fragments were cloned, and some of them contained CpG island-like regions which are known to play an important role in epigenetical regulation in mammals. We found a fragment homologous to the gene encoding Zn-finger domain-containing protein of rice. Zn-finger domain is contained in the protein important to vernalization, suggesting a relationship to epigenetics. Interestingly, this fragment had a CpG island-like region. These results suggest that the photoperiodic flowering of P.frutescens may be regulated by DNA methylation in CpG islands and the genes for Zn-finger containing proteins.

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Most leguminous plants close their leaves in the evening, as if to sleep, and open them in the morning, called nyctinasty. The circadian rhythmic movements of their leaves are controlled by a pair of leaf-closing and -opening factors to vary the ratio between the two compounds. Five pairs of leaf-movement factors were identified from five legumes, respectively, and recent study revealed that each leaf-movement factor interacts its specific receptor. But, it seemed to be difficult to elucidate the detail of the signal transduction of nyctinasty, since these legumes have less genetic information. Here we reported to identify a novel leaf-opening factor from Lotus japonicus, possessing major advantages as a model legume for the study of genetic information. We screened the leaf-opening factor from extract of L. japonicus by a bioassay system using a leaf of L. japonicus. After solvent partition, the bioactive aqueous layer was purified by four steps of column chromatography and HPLC to give a pure active compound (136μg), which kept opening the leaf at 1 mg/L. The structure of the compound was identified as eucomic acid based on 800 MHz NMR with cold prove and MS/MS analyses. We synthesized eucomic acid, and its NMR spectrum and retention time of LC/MS analyses were identical to those of natural one. Furthermore, synthetic eucomic acid showed a significant biological activity at 10μM. We have thus confirmed that the leaf-opening factor of L. japonicus is eucomic acid.

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Albizzia plants close their leaves in the evening, as if to sleep, and open them in the moerning according to the circadian rhythum. Compound 1 was isolated as leaf-closing factor 1 (LCF) of Albizzia plants. We developed molecular probes consisting of modified LCF in order to identify its mode of action. We synthesized enantio pair-type photoaffmity-labeling probes 2 and 3, and used them for photoaffinity labeling of receptor for LCF. By using protoplasts of motor cell, we found 35 kDa membrane protein strictly recognizes the stereochemistry of LCF, and it is highly likely that the protein is the specific receptor for LCF.

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Pyrroloquinoline quinone (PQQ), an element first discovered as a novel cofactor in bacterial dehydrogenase, has been identified as an important player in improving reproductive performance in mammals and now recognized as a new vitamin. We demonstrated the existence of PQQ in model plant Arabidopsis thaliana grown in germ-free environments on MS agar plates and yeast by LC/MS/MS analysis. In addition, PQQ conferred distinct stress tolerance to Arabidopsis which grown on medium including salt. This effect of PQQ treatment on Arabidopsis stress tolerance was also observed in yeast. So, we have started the screening of PQQ-related mutants to find new PQQ biosyntheses genes in the plant.

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Pyrroloquinoline quinone (PQQ) had been discovered as a coenzyme for dehydrogenase in bacteria, and recognized as a new vitamin in mammals in 2003. Recently, we detected the PQQ from germ-free plants and yeasts with LC-MS/MS. To know the function of PQQ in plants and yeasts, we treated plants and yeasts with PQQ and found that PQQ confers salt stress resistance to both plants and yeasts. These result suggests that plants and yeasts should have PQQ biosynthesis pathway and use it for their survival in various circumstances. So, first we tried to identify the genes that are committed to PQQ biosynthetic pathway in yeasts by screening mutants from yeast deletion lines. Here we reported some of mutants which are less tolerant to salt stress than wild type and their intolerance to salt stress was recovered by the application of PQQ.

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There are many kinds of horticultural flowers showing various coloration patterns. These patterns are reflected by operation of sitespecific regulation of pigments biosynthesis. We are studying generation mechanism of marginal picotee pattern in petunia petal, where anthocyanins are major pigments. We found that treatment of some pesticides changed the pattern in a marginal picotee cultivar with white margin and colored center, resulting in blooming pattern-less colored flowers. The pattern-less flowers bloomed from the second to the third week after treatment, suggesting that these pesticides act in buds at the early growth stage. We have indicated that marginal site-specific repression of mRNA of chalcone synthase is involved in the formation of white marginal tissue and the repression probably operates at post-transcriptional steps. These pesticides seem to have activities to release the repression.

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Changes in free amino acid levels in the root tip were examined when R/S-1-α-methylbenzyl-3-p-tolylurea (R/S-MBTU) enantioselectively inhibited root elongation of rice and wheat. Total free amino acids and free glutamine, asparagine, alanine and leucine levels were lower in the 20μM R-MBTU treatment than in the control in rice, and total free amino acids and free glutamine, alanine, valine, leucine and isoleucine levels were reduced in the 20μM S-MBTU treatment in wheat. Changes in free glutamine, asparagine, aspartate, alanine, valine, leucine and isoleucine levels were different between the 20μM R-MBTU treatment of rice and the 20μM S-MBTU treatment of wheat. In particular, changes in asparagine and isoleucine levels differed markedly. These results suggest that enzymes involved in the turnover of free asparagine and isoleucine in the root tip participate in the enantioselective inhibition of root elongation by R/S-MBTU.

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The urea compound R-1-α-methylbenzyl-3-p-tolylurea (R-MBTU) shows very interesting property, R-MBTU inhibits root growth of Cyperus paddy weeds. It also inhibits root growth of Beckmannia syzigachne, a companion weed of wheat (Triticum aestivum L.), but not that of wheat. Thus, R-MBTU enantioselectively shows herbicidal activity, but there has been little information on the biochemical and molecular biological aspects for R-MBTU mode of action. The elucidation of the R-MBTU mode of action would provide key information in the development of highly selective herbicides. The aim of this study was to elucidate the mode of inhibited action of R-MBTU on rice (Oryza sativa L.) roots using the PCR-based SSH technique. Variations of mRNA levels of five genes was identified by quantitative real-time RT-PCR. These genes were related to disruption of amino acid biosynthesis, cellulose synthesis, mitosis, and repression of transcriptions of various genes.

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In contrast to the case of dormancy of many plants, dormancy of bulbils of D. japonica is induced by gibberellins (GAs) as well as abscisic acid (ABA), which is referred to as GA-induced dormancy. It has been proved that ABA is, in bulbils of D. japonica, metabolized not only to dihydrophaseic acid through 8' -hydroxyABA but also to 7'-hydroxyABA. Along the line of investigation on ABA metabolism in GA-induced dormancy in Dioscorea, we isolated from the bulbils of D. japonica the fragments of two homologs ofNCEDs (DjNCED1 & 2) as well as the full sequences of two homologs of ABA8'oxs (DjABA8'ox 1 & 2) which we had already cloned. We preliminarily tried to analyze expression of these ABA metabolic genes in the bulbils of D. japonica at different dormant state. The treatment of uniconazol, which induces the sprouting of half-dormant bulbils, decreased the expression level of DjNCEDl in the bulbils, while increased the level of DjABA8'ox2. This is not contradictory to the hypothesis that GAs keep the endogenous level of ABA higher to induce and maintain bulbil dormancy. However, further detailed analysis of the gene expression levels in the course of dormancy development of the bulbils remains to be needed.

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The phytohormone abscisic acid (ABA) plays an important role for seed dormancy. Indeed, ABA-deficient mutant, aba2, shows reduced seed dormancy, whereas ABA-over-accumulation mutant, cyp707a1 cyp707a2 cyp707a3 triple mutant, exhibits strong seed dormancy. Thus, ABA regulates a large number of genes that is involved in seed dormancy. To explore genome-wide expression patterns and regulatory mechanisms in Arabidopsis seeds, comprehensive expression analysis was performed using whole-genome tiling array. Although there was large difference in the endogenous ABA levels between aba2, and cyp707a triple mutant dry seeds, a large number of genes was not drastically changed in these mutants, indicating that the difference of endogenous ABA levels in dry seeds does not influence global gene expression. In contrast to dry seeds, the transcript levels of many genes in both mutants were drastically changed compared to those of wild type. Transcriptome analysis identified 380 ABA-upregulated and 540 ABA-downregulated AGI code genes at 24 h after imbibition. Moreover, whole-genome transcriptome analysis identified 4884 non-redundant new transcription units (TUs) in Arabidopsis seeds. Among new TUs, 4559 TUs probably encoded hypothetical non-coding RNAs (ncRNAs). We identified 127 ABA-upregulated and 13 ABA-downregulated new TUs at 24 h after imbibition. Furthermore, 26 ncRNAs were conserved in either rice or poplar. At present, we're proceeding analysis of functional ncRNA.

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