Combined chemical and biological approaches are essential for understanding hormone biosynthesis and action. We have used rice and Arabidopsis mutants to study how terpenoid hormones regulate growth and development in plants. Our work in Arabidopsis has shown that gibberellins (GAs) play an important role in growth responses to environmental signals, such as light and temperature. In collaboration with other research groups, we uncovered new GA deactivation mechanisms, including epoxidation by a cytochrome P450 monooxygenase in rice and methylation by methyltransferases in Arabidopsis. Studies on the early stage of GA biosynthesis revealed that ent-kaurene, a hydrocarbon intermediate, can move between plants as a volatile under normal growth conditions. Recently, we set out to study on a new terpenoid hormone; previous studies using shoot branching mutants of pea, Arabidopsis and rice have suggested the involvement of a new hormonal signal in inhibiting shoot branching commonly in these species. We found that strigolactones, a group of terpenoid lactones initially identified as germination stimulants of root parasitic plants, act as the novel shoot branching inhibitor or its biosynthetic precursors. Acknowledgements: I thank laboratory members for their dedicated contributions to these projects. I thank my supervisors and collaborators for their support, advise and discussions.

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Cyanamide (NH_2CN) has been synthesized for over a hundred years for agricultural and industrial uses. In 2001, this compound was isolated from Vicia villosa subsp. varia. A large proportion of the inhibitory activity in the crude extract was explained by the presence of cyanamide. This was the first isolation of cyanamide from natural sources. [^<15>N] Nitrate was administered to V. villosa subsp. varia seedlings, from which cyanamide was purified and subjected to GCMS analysis. The isotopic ratio of ^<15>N in the cyanamide was significantly higher than that of the cyanamide extracted from the seedlings grown in the presence of a natural N source. This ^<15>N-enrichment established the presence of natural cyanamide. Distribution of cyanamide in the plant kingdom was the subject investigated because knowledge of this could enable us to understand the physiological function of cyanamide in plants, obtain plant material suitable for biosynthetic study, and investigate the genetic code of cyanamide biosynthesis. We established a direct quantitative determination method to detect and measure cyanamide by stable isotope dilution gas chromatography-mass spectrometry. We investigated 553 species in total but have so far found only three species with the ability to accumulate cyanamide at detectable levels, V. villosa subsp. varia, V. cracca, and Robinia pseudo-acacia the distribution of the plants that accumulate natural cyanamide appears to be limited.

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Arabidopsis thaliana was thought to contain two spermine synthase genes, ACAULIS5 (ACL5) and SPMS. Current investigations, however, revealed that the ACL5 gene encodes thermospermine synthase. In this study, we have established a simple method to separate two isomers of tetraamine, spermine and thermospermine, in extracts from plant tissues of less than 300mg. Polyamines (PAs) extracted from plant tissues were benzoylated, and the derivatives were completely resolved by high performance liquid chromatography on a C_<18> reverse phase column, by eluting with 42% (v/v) acetonitrile in water in an isocratic manner at 30℃ and monitoring at 254nm. The relevance of the method was confirmed by co-chromatography with respective PAs and by the PA analysis of the single- and double-mutants of acl5 and spms, which could not synthesize thermospermine and/or spermine, respectively. Furthermore, with this method, we monitored the thermospermine contents in various tissues of A. thaliana and found that stems and flowers contain two- to three-fold more thermospermine compared to whole seedlings and mature leaves. The presence of thermospermine was confirmed in Oryza sativa and Lycopersicon esculentum. Finally we addressed whether salinity stress changes the thermospermine content in Arabidopsis.

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Recently, the replanting of roof and wall surface of many buildings is advanced by using Sedum, from the viewpoint of environmental improvement or phytoremediation. Sedum is the plant which does "Crassulacean Acid Metabolism". The Sedum is strong succulent plants which can grow even in slight soil dried place and crack in the rock mass. However, Sedum has also indicated that it does not correspond with having Japanese climate of the season with much rain. By the present, the quantitative relation on cultivation condition and growth situation of Sedum is not completely clear. For the development of the convenient and effective replanting method using Sedum, established of the quantitative diagnostic method of growing condition is necessary. Previous study in our laboratory, a convenient analytical method by high performance liquid chromatography was established in view of the fact that is closely related to malic acid and citric acid production and the growth of Sedum mexicanum. In this study, glucose production was monitored under the open-field culture conditions over time. It became clear that the content of the glucose relevantly increased in the color in the winter than that of the autumn. On UV spectra of extracts which is included for red discolored stock, a new absorbance peak, indicating the anthocyanin, appeared at 530nm. It is of interest that the change of the color of Sedum mexicanum is the mechanism which resembles it of the autumn leaf.

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Phytohormones are key players in the regulation of plant growth and development. Although biosynthetic pathways, receptors and signaling pathways have been uncovered for classical plant hormones, there are still many questions remained unanswered. It is still unclear how these hormones interact each other or function in coordinated manner. Furthermore, new signaling peptide, compounds and hormone have been found, recently. In this study, we focus on the connections and crosstalk of phytohormones to understand how these hormones regulate plant growth in coordinated manner. To explore connection and crosstalk among hormones, we employed analysis of DNA microarray data sets. We collected massive data of Arabidopsis microarray experiments from AtGenExpress and compared gene expression profiles in response to phytohormone treatment or various stimuli such as biotic stress, chemical treatment. To compare microarray experiment data from various laboratories accurately, we developed a method to analyze similarity in gene expression data focusing on marker genes (modules) using network analysis. We also performed microarray experiments for phytohormone-related mutants and characterized them in our system. In this report, we present new information for cross talk of phytohormones and for gene expression profiles of phytohormone mutants. We also introduce AtCAST, which is a web-based database providing the results presented here. It also provides analysis tool for user microarray data.

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Seed imbibition is a prerequisite for subsequent dormancy and germination control. Here, we investigated imbibition responses of Arabidopsis seeds by transcriptomic and hormone profile analyses using dormant (Cvi) and non-dormant (Col) accessions. Once imbibed, seeds of both accessions swelled most until 3h, reflecting water uptake. Microarray analysis showed that in both accessions, 15-min-, 30-min-, and 1-h-imbibed seeds were less active in gene expression than at 3h. More than 2000 genes were either up-regulated or down-regulated in 3-h-imbibed seeds. Some 3-h-up-regulated genes were already induced in 1-h-imbibed seeds, suggestive of genome reprogramming early after the onset of imbibition. Imbibition-induced genes in 3-h-imbibed seeds included those up-regulated in both Col and Cvi (common) or unique to either accession (accession-specific). Quantification of plant hormones showed that abscisic acid (ABA) and salicylic acid (SA) contents were higher, but gibberellinA4 (GA4), N6-(Δ2-isopentenyl) adenine (iP), jasmonic acid (JA), JA-Ile, and indole-3-acetic acid (IAA) were lower in imbibed seeds of Cvi compared to Col. In addition, changes in IAA and JA were initiated before 1h, whereas ABA and JA-Ile declined 3h after the onset of imbibition. An increase in GA4 and iP appeared to be correlated temporally with the initiation of secondary water uptake, which marks the completion of germination.

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Plant hormone indole-3-acetic acid (IAA) has various physiological function, but IAA biosynthetic and signal pathway is still unclear. IAA is essential for plant growth, therefore the analysis of IAA biosynthesis knock-out mutants in the main pathway and the selection of double mutant with the knock-out mutant background may have been difficult. There are several pathways in IAA biosynthesis and some of them may function complementarily to each other. Mutants that have defect in such pathway may show no significant phenotype to distinguish mutants from wild type. On the other hand, the use of IAA specific biosynthesis inhibitor may bypass these difficulties and would make it possible to isolate new mutants. Screening of FOX hunting line with the inhibitor is also the powerful combination to find new mutants. We can raise the next point as an overall merit of using IAA biosynthesis inhibitor for isolating IAA mutants: we could isolate double or triple mutant-like mutants by the screening mutants with the inhibitor. Our aim of this study is the screening of mutanized seeds of Arabidopsis by using IAA biosynthesis inhibitor, L-2-aminooxy-3-phenylpropionic acid (L-AOPP) and isolate new IAA mutants (Resistance to Auxin Inhibition: RAI). At present we isolated one mutant that shows no significant phenotype without the treatment of L-AOPP. Therefore it is clear that use of chemicals for mutant screening has an advantage over the conventional mutant screening.

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We established transcriptome data of hormone responses in AtGenExpress project. We have also established correlation analysis to estimate hormone status from microarray data by using hormone-inducible genes as markers. In this study, we analyzed hormone series data from AtGenExpress and found that aminoethoxyvinylglycine (AVG) had the strongest anti-auxin activity in Arabidopsis. It inhibited growth, auxin accumulation, and expression of Aux/IAA genes in Arabidopsis seedlings, which recovered from inhibition of the gene expression after exogenous application of IAA and its intermediates. Since this inhibitor has characteristics to inhibit pyridoxal-phosphate (PLP)-dependent enzymes, we analyzed possible PLP-dependent steps of auxin biosynthesis in enzyme extracts from Arabidopsis and wheat. We also investigated involvement of ethylene using ethylene-insensitive mutants and ethylene-marker genes. As a result, we concluded that this is the first auxin-biosynthesis inhibitor, targeting L-Trp aminotransferase. This work was supported by the Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN).

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We were successful to identify the first auxin-biosynthesis inhibitor, aminoethoxyvinylglycine (AVG), which blocks L-Trp aminotransferase. Additional screening allowed us to identify L-aminooxyphenylpropionic acid (L-AOPP), Aminooxy acetic acid (AOA) and 2-Aminooxy isobutyric acid (AOIBA) as auxin-biosynthesis inhibitors, all of which inhibited L-Trp aminotransferase in enzyme extracts from wheat and Arabidopsis. We used these inhibitors to investigate the conservation and diversity of the auxin biosynthesis pathway in a monocot plant, rice, and dicot plants, tomato and Arabidopsis in vivo. These inhibitors were generally effective both in monocot and dicots, indicating that L-Trp aminotransferase constitutes one of the major auxin biosynthesis pathway conserved among higher plants. However, the inhibitors showed different action spectrum among organs and species. The inhibitors inhibited normal root elongation and the gravitropic response in Arabidopsis seedlings, which recovered from the inhibition by exogenous applications of IAA and its precursor IPyA, almost completely. These results provide novel insights into auxin biosynthesis and action. This work was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN).

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Auxin is plant hormone that regulates diverse developmental processes including apical dominance, gravitropism, and phototropism. Auxin is polar transported to its site of action from its production. This polar auxin transport produces an asymmetric auxin distribution to respond environmental stimulus. Therefore, the auxin transport system plays a crucial role in proper plant development. Auxin is actively transported by auxin influx and efflux carriers/transporters localized in plant cell membrane. Auxin efflux carrier proteins, PIN and auxin transporters ABCB/PGP are thought to play major role in the polar auxin movement. Therefore, inhibitor of PIN and ABCB/PGP proteins would be useful chemical tools for the investigation of physiological role and molecular action of auxin transport. We here demonstrate the new polar auxin transport (PAT) inhibitors with novel mode of action. New PAT inhibitor, auxin analogues directly blocks PIN and ABCB/PGP functions and inhibits IAA movement of Arabidopsis and Maize plants.

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Recent progress on auxin signaling study revealed fundamental molecular mechanism of auxin signaling mediated by SCF(TIR1)-proteasome pathway. In this pathway, auxin was perceived by TIR1/AFB auxin receptor and leads to an SCF ubiquitin-ligase catalyzed degradation of Aux/IAA transcriptional repressors. In addition to SCF(TIR1) auxin signaling, Auxin Binding Protein 1 (ABP1) is believed to be involved in alternative auxin signaling. Auxin Binding Protein 1 (ABP1) was identified as auxin receptor candidate and its physiological role have been extensively studied. However, the detailed physiological function of ABP1 and ABP1 mediated auxin signaling is still unknown. Therefore, ABP1 specific chemical probe would be useful tool to investigate ABP1 function and its involved signaling. We designed ABP1 probe on the basis of ABP1 and TIR1 crystal structures, and synthesized some ABP1 probes. Some designed ABP1 probes showed the promotion on hypocotyl elongation without activating SCF(TIR1)-mediated gene expression. In comparison with the Arabidopsis phenotype treated with various known auxins, these probes exhibited different effects on Arabidopsis from known auxin effects.

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Auxin is an important phytohormone and acts in many physiological events in plants. Especially, it has long been studied about its role in tropic curvature in glass coleoptiles. However, the production/biosynthesis of IAA at the coleoptile tip region was not approved in general. Our previous studies showed that the maize coleoptile 2-mm tip sections have an ability of de novo IAA biosynthesis at least until 2hrs after the cutting. However, when the tissues were chopped or homogenized, the increase of IAA amount was almost suppressed. To maintain the activity in the chopped samples, we had checked about the effects of buffer conditions, enzyme stabilizers, co-factors, metals, metal chelating agents, and other chemicals. However, we could not find any conditions to keep the IAA synthetic activity. Therefore, it was postulated that some specific cell/tissue structures are required for the IAA biosynthesis in the tip region. In the present work, we investigated whether the tip structure is important for the IAA biosynthetic activity. When the 2-mm tips were chopped into 1-mm or 0.5mm blocks, the activity was decreased about 40% or 70%, respectively. One or two cutting in longitudinal direction caused about 30% reduction, but in horizontal cutting would not show such large decrease. We are now investigating the relationship between IAA biosynthetic activity and cell/tissue structure in maize coleoptile tip regions.

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The phototropism has fascinated plant biologists since the pioneering work of Charles and Francis Darwin using grass coleoptiles. This tropic response is the result of differential growth on the two sides of the elongating shoot, which can be explained by the asymmetrical IAA distribution. We have previously shown that the coleoptile 2-mm tips can synthesize IAA and the synthesized IAA is immediately transported basipetaly. However, it is still unclear what events happen from the lateral blue light irradiation to formation of asymmetric IAA distribution. Here, we established system to analyze phototropic curvature by using excised coleoptiles. The 2-cm segment of coleoptiles detached from maize seedlings was irradiated with unilateral blue light for 8s at 0.33μmol・m^<-2>・s^<-1> (first positive curvature), for continuity at 10μmol・m^<-2>・s^<-1> (second positive curvature) or for 200s at 0.25μmol・m^<-2>・s^<-1> (no curvature). As the results of observation, it was shown that each response were similar to those of previous reports with maize seedlings. In the first positive curvature, coleoptiles began to be straight in vertical direction after 60min irradiation. To investigate the possible participation of gravity stimulus on this negative curvature, we observed first positive curvature under nominally zero-gravity condition by using clinostat. Continuous curvature was observed under this condition, it was suggested that the negative curvature occurred due to gravity.

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Since the early days of Darwin, monocot coleoptiles have been used to investigate indole-3-acetic acid (IAA) production, polar transport, and tropisms. This tropic response, as explained by the Cholodny-Went Hypothesis, is the result of differential growth on the two sides of the elongating shoot, which can be explained by the asymmetrical IAA distribution. Here, using maize coleoptiles, we first showed that the asymmetric IAA flow after gravi-stimulus directly affects the TIR/AFBs-mediated auxin signaling pathway, which results in tropic curvature. ZmPIN1(s) immunohistochemical analyses revealed that ZmPIN1(s) was present in almost all cells, but a basal cellular localization at lower regions. Application of the IAA transport inhibitors NPA and BFA at the very tip region almost completely inhibited IAA movement from the tip. These inhibitors also severely suppressed gravitropic bending. PEO-IAA (α-(phenylethyl-2-one)-IAA), an auxin antagonist that binds to TIR1/AFBs, suppressed not only the expression of an auxin responsible ZmSAUR2 gene, but also gravitropic curvature. Expression of ZmSAUR2 was up-regulated on the lower side and down-regulated on the upper side of the elongation zone, corresponding to the asymmetric IAA distribution. These results indicate that the asymmetric downward streams of IAA control the differential growth rate of the cells by attenuating TIR1/AFBs-mediated auxin response genes, including ZmSAUR2, and therefore result in tropic curvature.

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The plant hormone auxin (IAA) plays important roles in many plant physiological events. In the study of IAA biosynthesis, genetic approaches have identified several IAA biosynthetic genes, but endogenous IAA levels are not significantly reduced in the proposed IAA biosynthetic mutants. Thus, its biosynthetic pathways remain a mess to sort out. We have previously shown that maize coleoptile tips involve active sites for biosynthesis of IAA from tryptophan. To identify compounds which affect the amount of IAA synthesized in the tip, we carried out an IAA quantification-based screening of chemicals by using HitFinder library (10,000 chemicals). Apical 2-mm region of coleoptiles (4-day-old) were sectioned and incubated for 2hrs in a K^+-phosphate buffer (KPB) with or without chemical. The amounts of IAA diffused into a KPB were measured by HPLC-fluorescent photometer. The levels of IAA were about 800pg/tip/2h in the control (KPB without chemical). After the first screening, we selected out 105 candidates that reduced the amounts of IAA to less than 300pg/tip/2h, and 41 candidates that increased the levels to more than 1400pg/tip/2h. Among those, 43 chemicals significantly reduced the levels of IAA in a dose-dependent manner and one chemical increased the levels of IAA. We are currently investigating the effect of these chemicals on IAA production in maize coleoptile tips.

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Plant hormone auxin has an important role in many aspects of plant physiological events. From the study of C. Darwin, glass coleoptiles have been used for the research in tropism and IAA biosynthesis. Our previous works indicated that IAA was synthesized from tryptophan at the coleoptile 2-mm tip region and the synthesized IAA is immediately transported basipetaly. However, the mechanisms of IAA biosynthesis remain largely uncharacterized. Recently, using maize coleoptiles, we showed that IAA transported from the tip is necessary for the gravitropic curvature. Here, by using the chemical library (HitFinder, Maybridge), we have screened novel chemicals affecting IAA biosynthesis rate by observing the effect of the chemicals on gravitropic curvature in maize coleoptiles. The effects of chemicals on the degrees of curvature of coleoptiles were compared to those of K^+-phosphate buffer (KPB) and NPA in KPB treatments as curved and non-curved controls, respectively. As a result, 42/10000 chemicals showed inhibitory effect on gravitropic curvature. To verify the possibility whether these chemicals have an inhibitory effect on IAA transport, IAA amounts of both free IAA in the tip tissue and transported from the tip were determined by using GC-MS. The results suggest that most of these chemicals may inhibit IAA biosynthesis or IAA transport or both in maize coleoptiles.

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Recently, chemical genetics approaches are often used in plant biology. Identification of target protein is an integral, but rate-limiting step in chemical genetics. Here we show the rapid target analysis system using the Arabidopsis full-length cDNA over-expressing (FOX) lines. When anti-malarial fosmidomycin (FSM) that causes albino phenotype on Arabidopsis wild-type plants was tested as a model, FSM-resistant mutants that over-express a gene coding target enzyme in the methylerythritol phosphate pathway was promptly isolated from the FOX lines. We also isolated FSM-resistant mutants that over-express an Arabidopsis UDP-glycosyltransferase, which likely inactivates FSM by glycosylation. This method is useful for chemical genetics studies in plant biology when inhibitor causes visible phenotype on Arabidopsis.

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Plant growth largely depends on the chemical energy produced by photosynthesis in the chloroplast. Chloroplasts are differentiated from proplastids in response to light and/or hormonal signals, although the overall mechanisms are not clarified. When OsGLK1 encoding a GARP-type transcriptional regulator was ectopically overexpressed, rice calli exhibited green color on a 2,4-D-containing selection (N6D) medium, on which wild-type calli are ivory yellow. The green calli accumulated transcripts for a set of genes involved in chloroplast functions. These results showed that OsGLK1 is a key factor regulating chloroplast development. We also observed that the endogenous OsGLK1 mRNA level increased synchronously with the greening of wild-type calli after transfer to regeneration medium containing a low level of auxin and high concentration of cytokinin. Therefore, we hypothesize that auxin and cytokinin control chloroplast development by regulating the expression of OsGLK1. So we carefully examined the relationship between auxin/cytokinin concentrations and the expression of OsGLK1, and found that auxin repressed and cytokinin promoted the expression of OsGLK1. As a consequence, possible changes in OsGLK1 activity may affect chloroplast development. To elucidate the mechanisms of the hormonal regulation of OsGLK1 expression, we are currently investigating whether and which factor(s), involved in the cytokinin signal transduction, regulate the expression of OsGLK1.

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Plant high-temperature (HT) injury has become a serious problem associated with global warming. The early phase of anther development is especially susceptible to HT injury in wheat, barley and other crops. Here, we have found that HT conditions showed a high correlation coefficiency with transcriptional repression of replication-related and auxin-induced genes. The application of auxin to barley completely reversed HT-induced male sterility. Similar reductions in auxin levels and reversibility by exogenous auxin application occurred in the developing anther cells of HT exposed Arabidopsis. In addition, YUC2 and -6 gene transcripts decreased by HT treatment in developing anther. These data suggest that tissue-specific auxin reduction causes comprehensive transcriptional inhibition including anther specific auxin biosynthesis genes. It is a primary cause of HT injury, which leads to the abortion of pollen development, and moreover suggests a novel use for auxin treatment to sustain steady yields of crops under conditions of climate change.

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When cucumber seeds germinate in a horizontal position, resulting seedlings develop a specialized protuberance, termed the peg, on the lower side of the root-hypocotyl transition zone, due to gravistimulation. We have shown that exogenously applied auxin induces peg formation and that content of a free form of auxin, indole-3-acetic acid (IAA), in the upper side of the gravistimulated transition zone is less than that in the lower side. In addition, we have shown that auxin efflux carrier CsPIN1 proteins are expressed in endodermal cells and after 120min gravistimulation, the localization pattern of CsPIN1 changed to transport more auxin to lower side than upper side. To reveal the contribution of CsPIN1 to auxin dynamics during gravistimulation in the transition zone, we analyzed the content and distribution of endogenous IAA in the gravistimulated transition zone. After 30min gravistimulation, a significant accumulation in IAA content was detected in lower side of transition zone compared with its upper side. In addition, our immunohistochemical analysis using anti-IAA antibody revealed that the distribution of IAA-signals changed within 2min after gravistimulation. These results suggest that during peg formation, the change in localization pattern of CsPIN1 due to gravistimulation has the secondary effect and the other mechanisms exist to induce an immediate asymmetric IAA distribution in the transition zone of cucumber seedlings.

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The coordination of cell polarity within the plane of a single tissue layer (planar polarity) is a crucial task during development of multicellular organisms. Mechanisms underlying establishment of planar polarity, however, differ substantially between plants and animals. In Arabidopsis thaliana, planar polarity of root-hair positioning along epidermal cells is coordinated towards maximum concentration of an auxin gradient in the root tip. This gradient has been hypothesized to be sink-driven and computational modeling suggests that auxin efflux carrier activity may be sufficient to generate the gradient in the absence of auxin biosynthesis in the root. Here, we demonstrate that the Raf-like kinase constitutive triple responsel (CTR1) acts as a concentration-dependent repressor of a biosynthesis-dependent auxin gradient that modulates planar polarity in the root tip. We analysed auxin biosynthesis and concentration gradients in a variety of root-hair-position mutants affected in CTR1 activity, auxin biosynthesis and transport. Our results reveal that planar polarity relies on influx- and efflux-carrier-mediated auxin redistribution from a local biosynthesis maximum. Thus, a local source of auxin biosynthesis contributes to gradient homeostasis during long-range coordination of cellular morphogenesis.

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We found that novel 5-arylpyrimidine derivatives with fluoroalkyl groups at 2- and 4-position on the pyrimidine ring showed high level of herbicidal activity, and obvious necrotic symptoms were observed. The substituted phenyl group of the 5-position of the pyrimidines, exhibited herbicidal activity, was similar to a highly functionalized phenyl group of typical protopophynogen IX oxidase (protox) inhibitors. So, we checked the protox inhibitory activitiy of the pyrimidine derivatives presented, and their intensity of each inhibitory activity was evaluated and calculated as the pI_<50> values. The pyrimidinone I (I: R^2=CF_3, Xn=4-Cl) showed moderate level of inhibitoy activity and the pI50 value of was 5.5. Protox inhibitory activity of 4-methoxypyrimidine derivative II (II: R^1=Me, R^2=CF_3, Xn=4-Cl) was not observed at all. On the other hand, the pI_<50> value of 4-methoxypyrimidine derivative III (III: R^1=Me, R^2=CF_3, R^3=propargyl) bearing a 1,4-benzoxazin-3-one ring was 7.1. These findings indicated that introduction of a 1,4-benzoxadin-3-one ring at 5-position on the 4-methoxypyrimidine ring significantly enhanced the protox inhibitory activity. Among the compounds evaluated, the pyrimidinone derivative IV (IV: R^2=CHF_2, R^3=propargyl) exhibited the most potent protox inhibition (pI_<50>=8.4), and completely controlled all weeds tested at 1.9g a.i./ha application.

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The plant enzyme, 4-hydroxyphenyloyruvate dioxygenase (4-HPPD), plays an important role in the plastoquinone biosynthesis. We synthesized 4-benzoyl-5-hydroxypyrazole derivatives including practical 4-HPPD inhibitors such as sulcotrione and active form of pyrazolate, and examined their 4-HPPD inhibitory activity. Some compounds exhibited moderate to high level of activity. By the way, it is known that 4-HPPD inhibitors cause the inhibition of enzyme activity by forming a chelate combination with Fe^<2+> in the active site. Iron (II) complexation ability of benzoylpyrazoles was demonstrated by means of UV-spectrophotometric analysis under different concentration of iron (II) sulfate aqueous solution. Sulcotrion and the active form of pyrazolate showed large spectral changes in the presence of Fe^<2+>, by contrast, no apparent change were observed under Fe^<2+>-free condition. Benzoylpyrazoles, exhibiting moderate 4-HPPD inhibitory activity and bearing a substituent such as a chlorine atom or a nitro group on the benzene ring at the 2-position made change in absorbance in the presence of Fe^<2+>, respectively. On the other hand, benzoylpyrazoles, which exhibiting no activity such as Ib and Ic, didn't give any changes. These findings indicated that UV-spectrophotometric analysis using iron (II) sulfate aqueous solution presented in this session was a useful method in finding the novel candidates to be 4-HPPD inhibitors.

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Cabbage plug seedlings at 4 to 5-leaf stage were treated by foliar spray of PKS (liquid fertilizer containing ALA) diluted at 3,000-fold (160L/10a). Four days after the treatment, the seedlings were soaked in 0.3% and 0.6% NaCl solutions once (1 minute) a day for 4 days. Then the seedlings were grown for 4 days. The treatments with 0.3% and 0.6% NaCl solution decreased the length of 3rd to 4th and 4th to 5th-internodes and increased dry matter ratio (dry weight/fresh weight in the seedling). But the treatments decreased the total dry weight. In contrast, the combination treatment of PKS and NaCl decreased the internode length and increased the dry matter greater than NaCl treatment.

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Rice seedlings were grown in nursery boxes treated with PKS diluted at 3000-fold (15L/a), inabenfid (120g.a.i/a), or the combination of these compounds for three weeks. The combination treatment increased weight of underground part of the plants, number of the tillers, and thickness of the shoots much more than the treatment of PKS, or inabenfide. The combination retarded perfectly the spindly growth. It was shown that combined application of PKS and a GA synthesis inhibitor can be very helpful for growing the healthy rice seedlings.

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Peel puffing of Satsuma mandarin (Citrus unshiu Marc.) is a serious problem in the citrus industry. High temperature and high humidity during ripening are known to enhance peel puffing. The puffy fruits are easily damaged to fall in value during storage and transportation. It is required that stably effective methods are developed. It has been found that combined application of gibberellin (GA) and prohydrojasmon (PDJ) in September or early October reduces peel puffing in a late maturing cultivar, 'Aoshima-unshu'. In this study, to develop the method for early or medium maturing cultivars, time for combined application of GA (5ppm) and PDJ (50ppm) was examined in extremely early maturing 'Iwasaki-wase', early maturing 'Okitsu-wase', and medium maturing 'Silver hill-unshu'. As a result, by the application in September for those cultivars, the alleviatory effects on peel puffing were very influential, but the delay of peel coloring was serious. Overall, the application in August or before that was considered to be practical for those cultivars. On the other hand, quantitative changes of endogenous phytohormone were investigated in order to elucidate the mechanism of peel puffing alleviation by the application. The combined application of GA and PDJ in September 20 for 'Aoshima-unshu' significantly suppressed the increase of abscisic acid in the peel of fruits. It is possible that the suppression may be related to the effects of the application.

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Seasonal timing of seed germination is crucial for winter- and summer-annual seeds to establish vegetative and reproductive growth. The upper-limit temperature for germination of winter annual seeds gradually rise from spring to autumn, but their germination is suppressed by ambient high temperature during summer. This germination suppression by high temperature (thermoinhibition) enables the seeds to germinate in autumn. In this study, we selected chemicals which alleviate or enhance thermoinhibition of Arabidopsis seeds. We used three chemical libraries which containing un-annotated 12,000 diverse chemicals (Landscape2, Tripos), 280 natural compounds (TimTec) and annotated 2,000 chemicals (Spectrum, MicroSource) for the screening. We used tt3-1 seeds along with Columbia accession since the mutation enhances permeability of testa for the applied chemicals. Thermoinhibition resistant and thermoinhibition susceptible mutants seeds were also used. This screening allowed us to identify forty thermoinhibition alleviating chemicals including gibberellin. One of the chemicals induces germination ten times more effective than GA_3 at restrictive temperature condition. We also identified thermoinhibition-enhancing chemicals which do not inhibit germination of the seeds at optimal temperature conditions, in addition to known germination inhibitors such as abscisic acid and cycloheximide.

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Introduction Gibberellin (GA) is one of the phytohormones that promotes seed germination, stem elongation and flower development. GID1 was identified as GA receptor in 2005. We selected GID1 binding compounds by in silico screening and evaluated their binding activity to GID1 to develop a molecular probe for the analysis of GA-GID1 interaction. Methods and Results We used Surflex docking program for the analysis of the interaction between known GAs and GID1 receptor. Then we selected potent compounds from chemical library data set by in silico virtual screening and evaluated their in vitro binding activity to GID1 with tritium labeled GA. Finally we found a variety of compounds that bind to GID1. The structure-activity relationship study was performed to improve their in vivo and in vitro activity.

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In Arabidopsis, three receptors exist for the gibberellins (GA). Of the three, only a double loss-of-function mutant (atgid1a atgid1c) shows a dwarf phenotype. We show that no enzymatic activity exists in inflorescence stems of AtGID1b-GUS expression lines driven by AtGID1b promoter. The fact strongly suggests that atgid1a atgid1c lacks sufficient GA receptor for normal stem growth. On the other hand, we detected enzymatic activities of AtGID1c-GUS gene's product in stamens of the transformants, even though atgid1a atgid1b has short stamens and shows quite low fertility. Then we evaluated the affinity of the AtGID1-DELLA interaction by a competitive yeast three-hybrid system. It was elucidated that AtGID1c showed quite low affinities to major DELLA proteins in floral buds, which may be responsible for short stamens of atgid1a atgid1b. We propose that in a double loss-of-function mutant of GA receptors, the emergence of any phenotype(s) depends on the abundance of the remaining receptor and its preference to DELLA proteins existing at a target site.

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We have been working on regulating plant growth by using antibodies, and have succeeded in creating transgenic plants producing single-chain variable fragments (scFvs) against bioactive gibberellins as fusions with GFP, in endoplasmic reticulum (ER). In this study, we used two anti-GA_4 scFv designated as 8/E9 and 21/D13, and in only 21/D13 lines, we found filamentous shape of fluorescence in the leaf epidermal cells. The structure was considered to be an ER-derived compartment, and therefore we here tentatively call it filamentous ER (f-ER). In this study, we examined the applicational possibility of f-ER to produce substances by using <Arabidopsic thaliana>___- transgenic lines. By Western and ELISA analyses, we found that GFP-21/D13 scFv lines keep enormous amount of bioactive GFP-scFv compared to GFP-8/E9 scFv. This result suggested that it could be possible to produce large amount of any antibody in plants by using this structure. We crossed the transgenic lines producing anti-chlorpropham (CIPC) scFv with a GFP-21/D13 scFv line, and analyzed the amount of anti-CPIC scFv when two scFvs were coexpressed. Although we expected that anti-CIPC scFv production level would increase by the filamentous structure, both scFvs production level decreased compared with their original lines, presumably due to decreased gene expression by cosuppression. We are currently analyzing the gene expression level and also trying to produce anti-CPIC scFv as fusion protein with GFP-21/D13 scFv.

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We isolated two cDNAs (LsDELLA1 and LsDELLA2) encoding DELLA proteins (570 and 591amino acids, respectively), negative regulators of gibberellin (GA) signals, from Lettuce (Lactuca sativa L. cv. Grand Rapids). We have also identified a cDNA derived from a splicing-variant mRNA of LsDELLA1 (LsDELLA1sv). LsDELLA1sv mRNA encoded a truncated protein (357amino acids) missing C-terminus GRAS domain as a result of frame-shift caused by 82bp splicing. In this study, we characterized the LsDELLA1, 2 and 1sv transcripts. We first isolated two lettuce cDNAs (LsGID1a and LsGID1b) encoding GID1s, soluble gibberellin receptors, for binding assay and pull-down assay. Binding assay and pull-down assay showed that both of recombinant LsGID1a and b bound physiologically active GA in the presence of recombinant LsDELLA1 or 2, and interacted to the recombinant LsDELLAs. The recombinant LsDELLA1sv, truncated LsDELLA1, also interacted to the recombinant LsGID1s, accompanied with GA binding. QRT-PCR analysis indicated that transcript levels of LsDELLA1 and LsDELLA2 were higher in early-stage flowering organs than those in other organs examined. On the other hands, transcript levels of LsDELLA1sv were higher not only in early-stage and but also in late-stage flowering organs. We will show the phenotype of transgenic Arabidopsis plants expressing LsDELLA1, 2 or LsDELLA1sv.

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Gibberellins (GA), plant hormone are large family of tetracyclic diterpenoids that are biosynthsized from ent-kaurene, the key intermediate of GA biosynthetic pathway. Recent studies proposed that GA act as plant hormone from the land plant lineage after the bryophyte divergence. The fern biosynthesize GA and has functional components of GID-DELLA gibberellin signaling. On the other hands, any GAs and functional GID and DELLA orthologues were not found in the moss P. patens. However, if physiologically active GA differs among the moss and higher plants, it would be possible that alternative GA-related diterpenes might work as active growth regulators of the moss. To assess the biological role of GA-related hormones in the moss Physcomitrella patens, the disruption of ent-kaurene biosynthesis in P. patens would be a straightforward approach. We performed the insertion knock-out of ent-kaurene synthase gene, PpCPS/KS in P. patens. The phenotype analysis demonstrate that Ppcps/ks Knock-Out plant has defect in the protonema development. This defects was recovered by an exogenous ent-kaurene and ent-kaurenoic acid application. Our results demonstrate that P. patens utilize diterpene metabolites synthesized from ent-kaurene as endogenous growth regulator.

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Two kinds of bioactive gibberellins (GAs), 13-hydroxy and non 13-hydroxy GAs are frequently found in a given plant tissue. However, physiological meaning of the GA 13-hydroxylation is still unclear. We have previously identified a cytochrome P450 gene that encodes steviol synthase (ent-kaurenoic acid 13-hydroxylase) in Arabidopsis. Transgenic Arabidopsis plants overexpressing this steviol synthase gene showed semi-dwarfism like mild GA deficient mutants. GA analysis indicated that the levels of non 13-hydroxy GAs including GA_4 were decreased, whereas those of 13-hydroxy GAs including GA_1 (which is less active in Arabidopsis) were increased in the transgenic plants. To identify rice ortholog(s) of this gene, we made transgenic Arabidopsis plants that overexpress each rice gene whose function is unknown. We found that two of these overexpressor plants show semi-dwarfism. GA analysis indicated that the endogenous GA profiles of these plants were similar to those of the overexpressor of the Arabidopsis steviol synthase gene. Using a yeast expression system, we so far found one of these recombinant rice proteins catalyzed the conversion of GA_<12> into 13-hydroxy GA_<12> (GA_<53>). These results suggest that rice has at least two cytochrome P450 genes that are involved in GA 13-hydroxylation.

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Gibberellins regulate germination, elongation and flowering. DELLA proteins are members of the plant-specific GRAS protein family and act as repressors of the GA signaling pathway. DELLA proteins are rapidly degraded in the presence of GA. The degradation mechanism is beginning to be uncovered by the discovery of GA receptor and F-box protein. To understand the downstream signaling of DELLA proteins, we have identified GAF1 that interacts with GAI. GAF1 is a novel transcriptional factor that binds to DNA with sequence specificity. Arabidopsis plants that overexpress GAF1 are early flowering with larger leaves. In contrast the gaf mutant exhibits a semi-dwarf and late flowering phenotype. Exogenous GA cannot restore these phenotypes of the gaf mutant. BiFC analysis indicated that the interaction of GAF1 and GAI disappeared after GA treatment. Our trans-activation assays in yeast and plant cells suggest that GAI protein regulates the function of GAF1. These data suggest that GA controls transcriptional activity via alteration of GAF1 complex components. Based on this model, we are searching for the target genes of GAF1.

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Fungi produce diterpenes exhibiting phytohormone-related activities, such as gibberellin and fusicoccin. We identified a diterpene synthase gene PaFS, which is responsible for fusicoccin biosynthesis, in the mycelium of fusicoccin-producing fungus Phomopsis amygdali (Toyomasu et al., PNAS, 104: 3084-3088, 2007). PaFS (719 amino acids) possesses the diterpene cyclase domain at N-terminus and prenyltransferase domain at C-terminus. Our database search suggested that PaFS homologues exist in genome DNA of several fungal species. Among them, we found two PaFS homologous genes in Aspergillus oryzae genome database. It has been believed that A. oryzae produces few secondary metabolites including terpenoids. In this study, we characterize the two homologues, A. oryzae diterpene synthase like 1(AoDSL1) and AoDSL2. AoDSL1 contained a stop codon in region encoding prenyltransferase domain probably because of naturally occurred nonsense mutation, and is perhaps expressed as a truncated enzyme composed of N-terminal 412 amino acid residues. AoDSL2 encoded 728 amino acid residues, in which a successive Asp of DDxxD motif in the prenyltransferase domain was substituted by AN (position 482-483). These possible naturally occurred mutations perhaps resulted in loss of prenyltransferase activities. Recombinant AoDSL1 converted GGDP into two unknown diterpene cyclic hydrocarbons. The effects of site directed mutagenesis of possible naturally occurred mutation on enzyme activity will be presented.

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ent-Kaurene is a diterpene hydrocarbon compound and an intermediate of plant hormone gibberellins. This diterpene is biosynthesized from GGDP via ent-CDP. In flowering plants, two distinct cyclases, CPS and KS, are involved in the biosynthesis of ent-kaurene. Interestingly, lower vascular plants such as moss and liverwort biosynthesize ent-kaurene by bifunctional ent-kaurene synthases having both CPS and KS activities in single polypeptides. On the other hands, little is known about ent-kaurene synthetic genes and enzymes in ferns. Here, we cloned cDNAs of diterpene cyclases from Selaginella moellendorffii, and analyzed their function. Expression analysis showed that four genes were expressed during the growth of S. moellendorffii, which has eight diterpene cyclase genes in the genome. These recombinant proteins were produced by in vitro protein expression kit and used for functional analysis. SmDTC1 has DxDD motif and converted GGDP to ent-kaurene in the presence of Arabidopsis KS protein. SmDTC7 has DDxxD motif and converted GGDP to ent-kaurene in the presence of SmDTC1. These results indicated that SmDTC1 and SmDTC7 are CPS and KS, respectively. Interestingly, SmDTC3 used GGDP as substrate and slightly produce ent-kaurene. Hence, we concluded that SmDTC3 is a bifunctional ent-kaurene synthases, SmCPS/KS. From the mass spectrum analysis, SmDTC4 synthesized a novel diterpene hydrocarbon. The structure determination of the product by SmDTC4 is in progress.

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Artemisia spp. have been used for medicine and food. Artemisia annua contains anti-malarial sesquiterpenoid artemisinin. Therefore intensive studies on both chemical and molecular genetics have been done in A. annua. However, there were few such studies in other Artemisia spp. We previously demonstrated that sesquiterpenoid profile was correlated with gene expression of sesquiterpene synthase among A. annua, A. afra, and A. absinthium, suggesting that comparative approach is useful to find new gene function. Toward systematical understanding of molecular basis for chemical differences among Artemisia spp., sesquiterpenoid profiling was conducted. In this work, leaves of 33 Artemisia spp. were collected and their sesquiterpenoid profile was analysed by GC-MS. Germacrane, copaene, and caryophyllane were commonly detected. In contrast, some spp. had characteristic sesquiterpenoid profiles. Based on sesquiterpenoid profiles, biosynthetic mechanism of each sesquiterpenoid was proposed. For sesquiterpenes commonly detected, genes for corresponding sesquiterpene cyclases should be commonly existed in Artemisia spp. On the other hand, novel sesquiterpene synthase genes should be expressed in the restricted species which showed characteristic sesquiterpenoid profile. A. abrotanum contained silphiperfolane compounds in major proportions. This suggests that yet unidentified gene encoding silphiperfolane synthase is highly expressed. Molecular cloning of this gene is currently in progress.

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Artemisia is a large, diverse genus of plants with between 250-400 species belonging to the family asteraceae. A. momiyamae is endemic to Miura peninsula and Inamuragasaki in Japan. Both chemical and genetic information from these plants is quite limited. These plants are presumed to be the variety of A. princeps from morphological characteristics. We have been clustering Artemisia species based on initial closure mechanism from their common substratel. In this study, this method was applied A. momiyamae. Leaves of 33 Artemisia species were collected and analyzed their terpnoid profiles by GC-MS (Refer M. Shimomura's poster). Sesquiterpenoids are separated into four group base on initial closure mechanism from the substrate farnesyl diphosphate. We chose a group of compounds (total 22) biosynthesized through initial 1,6 closure, and clustered 17 Artemisia species using these compounds. Cluster I: Amorpha-4,11-diene and these analogs were included (A. annua). Cluster II: β-sesquiphellandrene and α-zingiberene were common compounds (4 Artemisia species). Especially, sub-cluster was found between A. momiyamae and A. stolonifera because of the presence of bisabolone. Cluster III: Various monocyclic sesquiterpenoids were included (12 Artemisia species). From chemometrics cluster analysis, A. momiyamae is closer to A. stolonifera than to A. princeps.

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Radish (Raphanus sativus L. var. hortensis f. gigantissimus Makino) seedlings were grown under various gravity conditions in the dark. A compound content in the etiolated radish hypocotyls was decreased under simulated microgravity conditions on a 3-dimensional clinostat, but increased under 500g gravity produced by centrifugation, comparing to 1g conditions. It was isolated from etiolated radish hypocotyls grown on 1g conditions and identified to be 1-β,4-di-0-(4-hydroxy-3,5-dimethoxycinnamoyl) gentiobiose, raphanusol A which showed a potent growth-inhibiting activity for radish hypocotyl growth, based on its spectral data. The distribution of endogenous raphanusol A between the epidermis of the upper and lower flank of radish hypocotyls during gravitropic curvature was analyzed using a physicochemical assay. Gravitropic stimulation increased raphanusol A content in the upper flank within 30min after the onset of gravitropic stimulation, whereas that in the lower flank was similar to that in the controls. Previously we reported that a major factor in gravitropism in radish hypocotyls is the suppression of growth on the upper side of hypocotyls, but not the acceleration on the lower side. These results suggest that raphanusol A plays a role in controlling radish hypocotyl growth in response to various gravistimuli, especially in differential growth in gravitropism of radish hypocotyls.

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Mesquite (Prosopis juliflora) is the leguminous shrubs growing in the tropical or subtropical regions. In this study, we conduct screening of biologically active substances produced by mesquite. Dried mesquite leaves (250g) were extracted with methanol. The extract (48.6g) was partitioned into the acidic and neutral fraction (22.2g) and the basic fraction (3.9g). The acidic and neutral fraction exhibited the inhibitory activity for root and shoot growth of lettuce seedlings. Furthermore, the basic fraction and the acidic and neutral fraction inhibited seed germination of Striga hermonthica, S. gesnerioides and Orobanche minor. The purification of these bioactive substances is in progress.

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Most leguminous plants close their leaves in the evening, as if to sleep, and open them in the morning according to the circadian rhythm. Potassium isolespedezate 1 is a leaf-opening factor of leguminous plant, Cassia obtusifolia. We planed to purify the target protein of 1, by labeling with FLAG peptide-tag. First, we labeled the target protein with azide group by incubating the iodoacetamide-type probe (2) with the motor cell protoplasts, which is the target cell of 1, and then, succeeded in introduction of FLAG using click chemistry. Then, the 83kDa cytoplasmic protein (CTPL; cytosolic target protein of lespedezate 1) was detected and purified as a target protein of 1. The analysis of CTPL by using LC-MS/MS revealed that CTPL shares homology with MetE (5-methyl-tetrahydropteroyltrigultamate-homocysteine methyltransferase).

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Most leguminous plants close their leaves in the evening, and open them in the morning. Glycosylated jasmonate 1 was isolated as leaf-closing factor (LCF) of Albizzia plants. We detected the target membrane protein of 1 by using molecular probe 2. Here, we designed and synthesized more powerful molecular probe 3. Using click chemistry, we could introduce a variety of tags such as biotin and FLAG etc. into a molecular probe. In this way, we can examine the best combination of reactive functional group and tag.

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Coumarins are ubiquitously found as the plant secondary metabolites in plants. They are thought to play important roles in plant defense due to their antimicrobial and antioxidative activities. Coumarins are biosynthesized via ortho-hydroxylations of cinnamates (C2'H). This is a key step in the lactone ring formation in coumarin biosynthesis, and C2'H is classified into the 2-oxoglutarate-dependent dioxygenase (2OGD) family. Arabidopsis roots accumulate scopoletin β-glucoside, and Arabidopsis C2'H shows the high substrate specificity to feruloyl-CoA but does not accept ferulic acid as a substrate. Sweet potato (Ipomoea batatas L.) tubers accumulate the β-glucosides of scopoletin and umbelliferone, and C2'H of sweet potato can accept feruloyl-CoA and p-coumaroyl-CoA as its substrates to form scopoletin and umberiferone, respectively. These results suggest that the substrate specificity of C2'H determines the accumulation patterns of coumarins in each plant species. In this study, we have found that there are C2'H homologs in the EST database of citrus and legume (Medicago truncatula and Lotus japonicus). Citrus fruits are known to accumulate furanocoumarins, but little is known about coumarin synthesis in M. truncatula and L. japonicus. In order to explore coumarin biosynthesis in citrus and the legume plants, we have performed the functional analysis of the C2'H homologs of these plants.

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In the biosynthesis of isoquinoline alkaloids, several unusual monooxygenation reactions such as the intermolecular C-O phenol-coupling and the intramolecular C-C phenol coupling reactions are involved, and cytochromes P450 such as CYP80A1 and CYP80G2 have been shown to catalyze these reactions. Root cultures of Stephania cephatantha produce several isoquinoline alkaloids, and, in this study, we have isolated five P450 cDNAs from the root cultures by RT-PCR with the degenerated primers specific to CYP80A1 and CYP80G2. The isolated P450s showed high amino-acid sequence identities with the CYP80 family members, suggesting their involvement in isoquinoline biosynthesis in S. cepharantha. Further analyses to determine the function of these P450 cDNAs with heterologous expression are now in progress.

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