Flowers emit floral scent compounds throughout the initial unfurling to the senescence stages. We have investigated the biosynthesis and the emission of floral scent compounds on Rosa, Osmanthus and Gardenia flowers. During the course of our research, the feeding experiments of labeled precursors with stable isotopes and the molecular biological methodologies were employed. In this paper I will introduce the following research results. 1) 2-Phenylettyl β-D-glucopyranoside (2PEG) and β-glucosidase were involved in the 2PE emission at the late unfurling stage in rose flowers. 2) 2PE emitted from rose flowers was synthesized from L-phenylalanine by the action of two enzymes, aromatic amino acid-decarboxylase and phenylacetaldehyde reuctase. 3) O-Methyl transferase was isolated as the key enzyme involved in the floral scent methoxy-phenol in roses. 4) CCD1 was clarified to be involved in the synthesis of α-, β-ionones from carotenoids in Osmanthus flowers. 5) Rhythmic emission of floral scent was confirmed in rose and Osmanthus flowers under the 12h light-12h dark conditions.

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Abscisic acid (ABA) is a plant hormone that regulates numerous physiological responses including seed maturation, germination, and adaptive responses to drought stress. ABA metabolism (biosynthesis and catabolism) and signaling are regulated tightly in response to environmental and developmental cues. Therefore, mutants defective in its metabolism and signaling fail to properly perform seed maturation, germination, and stress responses. My research group has conducted genetic screening to isolate Arabidopsis mutants, which are able to germinate in the presence of GA biosynthesis inhibitor and of (-)-R-ABA, an ABA analog. These mutants include those defective in ABA biosynthesis enzyme or in transcription factors required for ABA-mediated transcription. We also identified that CYP707A genes encode ABA 8'-hydroxylase, which catalyzes the committed step in a major ABA catabolic pathway. We showed that ABA catabolism is regulated by light, nutrient, drought, and submergence as well as to other plant hormones. Furthermore, identifying the cell types for activation of ABA biosynthesis and catabolism genes indicates that ABA-mediated response is systemic. I acknowledge my colleagues and collaborators involved in these studies.

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In angiosperms, approximately 1%, ca, 4,500 species are parasitic. Parasitic plants can be classified as to their photosynthesis status. Hemiparasites retain photosynthetic ability. On the other hand, holoparasites lack photosynthetic ability and obtain all their carbon from their hosts. In these parasitic species, selective pressure to maintain photosynthetic ability had been released. Photoresponses of plants which are important to optimize photosynthesis for green plants are not necessary in holoparasites. Photoresponses are also involved in the regulation of plant development such as germination process. Studies on photoresponses of parasitic plants, Orobanche minor and Striga hermonthica revealed that the pattern of their photoresponses are unique and had been modified for adaptation to its parasitic life cycle. Molecular analysis of their red/far-red light photoreceptor phytochrome A (phyA) also demonstrated that the alteration of their amino acid sequences and associated molecular function. Elucidation of altered components in phy signaling in the parasitic plants will provide useful information not only about its physiological characteristics but also about general plant photoreception systems.

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Analysis of gibberellin (GA)-deficient dwarf mutants contributed to the identification of GA biosynthesis genes. Meanwhile, additional GA-related genes that have not been discovered by conventional forward genetic approach are likely to be present. We identified such genes by analysis of an activation tagged mutant (1), and by reverse genetic approach (2). (1) A dominant dwarf mutant designated dwarf and delayed-flowering 1 (ddf1) was isolated from a library of activation-tagged Arabidopsis. We found that the contents of bioactive GAs were decreased in ddf1. Genetic and molecular analyses revealed that the ddf1 phenotypes are caused by increased expression of GA 2-oxidase 7 gene (GA2ox7), which encodes a C_<20>-GA inactivation enzyme, through increased expression of a high salinity responsive AP2 transcription factor, DDF1. (2) A rice P450 gene, EUI (CYP714D1) encodes a novel class of GA inactivation enzyme, which catalyzes 16α, 17-epoxidation of GAs. We examined the possible involvement of other CYP714 members of Arabidopsis and rice in GA metabolism. We found the gene products that have GA 13-hydroxylation activity from Arabidopsis (one gene) and rice (two genes). GA measurements of a rice double mutant of these genes indicated that the levels of 13-hydroxy GAs were decreased, whereas those of non 13-hydroxy GAs were increased in the double mutants. These results strongly suggest that these two cytochrome P450 genes play a major role in GA 13-hydroxylation in rice.

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The phototropism has fascinated plant biologists since the work of Darwins using grass coleoptiles. This tropic response is the result of differential growth of the elongating shoot, explained by the IAA redistribution. Here, using detached coleoptile segments from maize seedlings, we confirmed the first pulse positive phototropism were induced by blue light. To reveal the precise sensing region for blue light, effects of partial irradiation on the curvature were tested. The result showed that the photo sensing at the apical 2mm region was required for phototropism. The formation of IAA redistribution was observed after 20min in the top 0-3mm region, which began to be recognized in the lower region after 30min. When NPA was treated at top 2mm, the phototropism was completely inhibited; even after auxin was simultaneously applied at 3-4mm region. These results showed that lateral IAA movement occurred only in top 0-3mm region, and the IAA redistribution was transmitted from the tip into basal region. Further, it suggests the presence of a key component(s) responsible for the photo-sensing or formation of IAA redistribution in the tip region. We therefore investigated expression level of several genes relating in phototropism. NPH3- and PGP-like genes were shown to be exclusively expressed at the apical 0-2mm region. These results indicate that these genes are possibly involved in converting the light-signal to lateral IAA transport at the tip region.

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Plant hormone indole-3-acetic acid (IAA) has various physiological functions, but IAA biosynthetic and signal pathway is still unclear. As IAA is essential for plant growth, isolation of knock-out mutants in the main pathway of IAA biosynthesis and the isolation of double mutants in the knock-out mutant background may have been difficult. It has been suggested that there should be several pathways in IAA biosynthesis and some of them may function complementarily to each other. Therefore mutants that have defect in such pathway may show no significant phenotype to distinguish the mutants from wild type. If we have IAA biosynthesis inhibitor targeting the main pathway of IAA biosynthesis, it would make it possible to isolate new mutants because such inhibitors can bypass the difficulties described above by controlling the concentrations at which they are used. That is, the plants can grow with altered phenotypes under chemical treated-conditions, and we can isolate double or triple mutant-like mutants by the mutant screening with the inhibitors. Our aim of this study is the isolation of new IAA mutants (rai: resistance to auxin inhibition) from mutanized seeds of Arabidopsis by using IAA biosynthesis inhibitor, L-2-aminooxy-3-phenylpropionic acid (L-AOPP). At present we isolated one.

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The natural auxin IAA (indole-3-acetic acid) is one of very precisely studied subjects, and its biosynthesis mechanism has been also well investigated. However, since the biosynthesis system consists of several pathways and genes playing a role in those pathways are not fully identified, the biosynthesis mechanism of IAA remains to be elucidated more precisely. Chemical genetics approach with biosynthesis inhibitors is a reliable means for studies of hormone biosynthesis. However, biosynthesis inhibitors for IAA had not been available. Recently, we reported the inhibitory activity of L-2-aminooxy-3-phenylpropionic acid (L-AOPP) to IAA biosynthesis in Arabidopsis, although its specificity to the activity remained to be shown. In this study, we demonstrated biochemical study for the inhibitory activity of L-AOPP to TAA1 (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1), which has been reported to be an IAA biosynthesis enzyme. Furthermore, physiological effects of L-AOPP were examined, and its mode of action was analyzed.

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Auxins have critical roles in plant growth and development. Arabidopsis has multiple pathways for biosynthesis of a naturally occurring auxin, indole-3-acetic acid (IAA): the YUCCA (YUC) pathway, the CYP79B pathway, the indole-3-pyruvic acid (IPA) pathway and the indole-3-acetamide pathway. The YUC and IPA pathways widely exist in plant kingdom. Recently, we found that the CYP79B pathway operates in the crucifer plants producing indole glucosinolates like Arabidopsis, but may not in non-crucifers. It has been demonstrated that cyp79b2 cyp79b3 mutants show IAA-deficient phenotype, but IAA levels are not greatly reduced in these mutants under the normal growth condition. yuc1D mutants show IAA-overproduction phenotype, but IAA levels are slightly increased in yuc1D. To address this question, we analyzed IAA metabolites in Arabidopsis IAA biosynthesis mutants. We found that IAA-amino acid conjugates were significantly decreased in the CYP79B-deficient mutants. In contrast, the levels of IAA-amino acid conjugates were dramatically increased in the YUC-overexpression lines. These results indicate that the IAA-amino acid conjugates may have important roles to maintain IAA levels in IAA biosynthesis-deficient mutants.

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TIR1/AFB auxin receptors play a central role in the perception of auxin and regulate the many aspects of auxin-regulated developmental processes in plant. Therefore, specific auxin antagonists on TIR1/AFB receptors are promising chemical tools for auxin biology. We have found that α-alkyl-IAA function as specific auxin antagonists to block TIR1/AFB function. Our efforts for the development of new potent auxin antagonists using virtual screening and structure-based drug design lead to the identification of potent lead compounds, PEO-IAA that showed a high affinity to TIR1 and anti-auxin activities in planta. We then optimized the structure of PEO-IAA to show higher auxin antagonistic activity. As the results of derivatization, mXO-IAA, new antagonistic probe exhibited potent and specific inhibitory activity on SCF (TIR1) signaling.

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Auxin is polar transported by the transporter proteins, AUX1, PINs and ABCBs, and the transport system establishes an auxin gradient to regulate proper plant development. Thus, the spatial and temporal effect of the auxin gradient on plant development is highly important for the understanding of plant auxin responses. The caging group, 2-nitrobenzyl group was found to be unstable in plant cells and released original auxin without photolysis due to the enzymatic hydrolysis by esterases. Thus, three auxins, indole 3-acetic acid, naphthalene 1-acetic acid and 2,4-dichlorophenoxy acetic acid were caged with the MNI (4-methoxy-7-nitroindolinyl) caging group to form an amide bond between the sec-amino group of MNI group and the carboxylic acid in auxins. MNI-caged auxins, therefore, were very stable within a plant cell, but release auxins with photo-irradiation to activate auxin-responsive reporter gene expression. We evaluated a spatial and temporal control of intracellular auxin levels by photo-irradiation using MNI-caged auxin system, demonstrating that the photo-control of the physiological auxin response in Arabidopsis plants.

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Cytokinins primarily promote cell division to regulate cell growth and differentiation, and also affect apical dominance, axillary bud growth, and leaf senescence. To investigate the detailed responses to cytokinins in plant tissues and cells, the accurate application of cytokinin are essential for the manipulation of cytokinin effects. Caged cytokinins are chemical tools to achieve the accurate application of cytokinins, because the cytokinin level can be temporally and spatially controlled by the photo-irradiation. We therefore synthesized the caged cytokinin, 2-nitrophenylethyl carbonate linked caged dihydro zeatin that release the zeatin molecule in response to photo-irradiation. However, the caged zeatin with carbonate linker still showed cytokinin activity without photo-irradiation. We then designed and synthesized N9-caged cytokinins (dihedro zeatin and benzyl adenine caged at N9 position in adenine). These N9-caged cytokinins were inactive until photo-irradiation and release active cytokinin when light is irradiated.

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Eucalyptus globulus that is one of the major afforestation species is known to be difficult for vegetative-propagation. Therefore, it was necessary to develop novel method for promotion of adventitious root (AR) formation in E. globulus. First, we measured endogenous hormones at basal part of stem of easy-rooting line and poor-rooting line by UPLC-ESI-qMS/MS. As a result, the Indole-3-acetic acid (IAA) level of easy-rooting line was two times higher than that of the poor-rooting line, suggesting that endogenous IAA level may regulate ability of AR formation. Next, we forcused on the cytochrome P450s that are involved in a vast array of reactions of many different metabolic pathways. A chemical library of triazole derivatives to find chemicals which have the effect of promoting AR formation was screened. Consequently, five compounds effectively promoted AR formation. Finally, we investigated how these chemicals affected the growth of Arabidopsis thaliana. Arabidopsis seedlings were grown on agar medium containing chemicals. One of the selected chemicals, MA65 increased the number of roots in wild-type Arabidopsis seedlings and this phenotype was similar to a mutant superroot2 (sur2). The amounts of endogenous IAA in 14-d-old Arabidopsis grown in the presence of MA65 were analyzed. The IAA content was increased two-fold in the presence of MA65 as compared with untreated. Taken together, MA65 may increase endogenous IAA level in a plant cell and promotes an AR formation.

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Insect galls are the highly distinctive plant structure formed by some herbivorous insects as their own microhabitats. Galls are often formed when insect larva suck the plant tissues or female insect deposit its egg to the plant tissue. As its unique phenomenon, galls have been studied by many researchers, but its formative mechanism is still unknown. Currently, it is thought that galls are usually induced by chemicals that are secreted by salivary gland of larva or ovipositor. It has been suggested that amino acids and auxin present in this secretion are important, notably auxin, for gall formation. But the evidence so far obtained that auxin exists in the secretion is not fully reliable, and the ability of gall-maker insects to synthesize auxin has not been demonstrated. In this research, we used a gall that is made on Artemisia by a gall midge larva, and tried to reveal the gall formation mechanism. Here, we show that larvae contain auxin. Its auxin concentration was 10 times higher than plant tissue, and young larvae showed higher auxin concentration than old larvae. This result suggests that auxin is important for gall formation. We are currently under investigation to know if the auxin in larvae is synthesized by larva or is derived from plant ingestion, and also to reveal the relationship between auxin and gall formation.

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Galls are abnormal plant tissues formed by parasitic insects on various plant parts. It is generally assumed that the insect secretes chemicals at oviposition or ingestion, which induce abnormal cell growth and proliferation leading to gall formation. The active substances, however, have yet to be identified. In this study, we selected a willow (Salix japonica) gall formed by a sawfly of the genus Pontania. The detailed observation by Hovanitz (1959) indicated that the gall formation is initiated by the liquid (accessory gland fluid) at oviposition and is further developed by saliva at larval ingestion. Here we examined the possibility that auxin takes part in the gall formation. Although the involvement of auxin in gall formation has long been argued, there was no evidence that the gall-forming insects have ability to synthesize auxin. LC-MS/MS analysis indicated that accessory gland fluid taken from female adult abdomen contained indole-3-acetic acid (IAA) at several times higher concentration than that in normal willow leaves. On the other hand, the level of IAA in larvae was more than 100 times higher than that of the willow leaves. The feeding experiment clearly indicated that the larvae can synthesize IAA from tryptophan. These results suggest that the IAA plays a key role in gall formation.

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Tetraneura nigriabdominalis is distributed in Japan and known as an insect pest against the Gramineous crops such as the upland rice plant, wheat, and barley. The female nymphs hatching from eggs in the spring on the primary hosts, Ulmus tree, produce galls on the leaf. After development of winged aphids in a gall, the winged aphids are dispersing in the field and infest the root of the Gramineous plant. Here, we investigated a gall formation by aphids on leaves of Ulmus parvifolia and change of metabolites in gall. Ionic chemicals contained in galls and uninfected leaves were extracted with MeOH and extracts were analyzed by the CE-TOF (Agilent G3250AA LC/MSD TOF system). Separations were carried out using a fused silica capillary (50um i.d.×100cm) filled with 1M formic acid for cation analyses or with 20mM ammonium formate for anion analyses as the electrolyte. The concentrations of amino acids in galls were increased than those in intact leaves, and among them the concentration of Asn in galls was 15 times higher than that in intact leaves. The time course experiments revealed that the concentration of Asn in galls was increased with gall development and it was decreased after dispersing of winged aphids. On the other hand the concentration in intact leaves was decreased during leaf development. These results suggest that the nutrition conditions of plant for aphids have been improved by gall formation.

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The hydroxylation of gibberellin (GA) by GA 2-oxidase (GA2ox), a 2-oxoglutarate dependent dioxygenase, is known to be one of its major inactivations. The regulation of this catabolic step is very important for a fluctuation of endogenous GA in plants, and therefore is a good chemical target to control the gibberellin action. At present, prohexadione is known as a commercially available inhibitor to GA2oxs, however prohexadione has a characteristic that it inhibits other 2-oxoglutarate dependent dioxygenases like GA 3-oxidases. In this report, we performed in vitro random screenings to obtain novel inhibitors more specific to GA2oxs, by using radio-labeled GA_4-derivative and recombinant Arabidopsis GA2ox2. As a result, we discovered an inhibitory effect of methyl 6-chloro-3H-1,2,3-benzodithiazole-4-carboxylate 2-oxide (CBTC) to the enzymatic activity of GA2ox2, and we subjected it to the successive in planta evaluations. The CBTC promoted both the germination of Arabidopsis seeds and their growth. This strongly suggests that CBTC specifically inhibits GA2oxs, not GA3oxs, in Arabidopsis.

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Direct interaction of a gibberellin receptor GID1 with a signal repressor DELLA triggers the transduction of gibberellin signal in plants. The binding of the two molecules occurs only in the presence of gibberellin. In this study, we developed a system for screening of the chemicals which inhibit the GID1-DELLA binding, based on a yeast two-hybrid system. When biologically active gibberellin exists, the yeast can grow even in the undernourished medium. By using its growth response in the co-presence of gibberellin and each examined chemical, we performed a random screening of inhibitors to the interaction between GID1 and DELLA. We found that some chemicals inhibited this binding. Then, we examined their inhibitory effects in vitro and in planta. Finally one chemical which showed inhibitory effects to both Arabidopsis and Barley, was selected.

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On the signaling pathway for gibberellin (GA) in plants, the GA-dependent GID1-DELLA interaction is a main event for its transduction. After their interaction, the GID1-bound DELLA shows an affinity to a GA-specific F-box protein, which is a member of E3 ubiquitin ligase complex. In Arabidopsis, SLY1 is known as the protein for GA signaling. We had already established the yeast three hybrid system (Y3H) consisted from GID1, DELLA, and SLY1. By using this system, we can monitor the SLY1's activity to receive a DELLA molecule from the GID1-DELLA complex. In this report, we apply this Y3H system to screen inhibitors to the SLY1's activity by using commercially available chemical library. We selected 21 chemicals as candidates, and now in planta evaluations are proceeding.

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Terpenoids are widely distributed in nature and contain many bioactive secondary metabolites. The enzymatic total synthesis and isotope labeling of natural terpenoids will be powerful probes for studying the biosynthesis of terpenoids. Here, we successfully synthesized a bioactive giberrellin from acetate with biosynthetic enzymes. When [U-^<13>C_2]acetate was used for the starting material, [U-^<13>C_<20>]ent-kaurene was enzymatically synthesized via mevalonate. The ^<13>C-NMR of [U-^<13>C_<20>]ent-kaurene observed signals to assign all peaks from chemical shifts values and ^<13>C-^<13>C coupling constants. GA_4-biosynthetic genes encoding two P450 monooxygenases and two dioxygenases were expressed in E. coli or Pichia yeast, respectively. [U-^<13>C_<20>]GA_<12> was synthesized from [U-^<13>C_<20>]ent-kaurene containing two P450 monooxygenases. Finally, addition of two soluble dioxygenases into the [U-^<13>C_<20>]GA_<12> synthetic cocktail led to synthesis of [U-^<13>C_<19>]GA_4. In addition, we will report also ^<31>P-NMR monitoring of intermediates during enzyme reactions from mevalonate to isopentenyl diphosphate. We optimized the enzyme reaction condition by ^<31>P-NMR technique.

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The P450 enzyme CYP701A is known as ent-kaurene oxidase (KO) in higher plants and it catalyzes the oxidation reaction of ent-kaurene to form ent-kaurenoic acid. We found a CYP701B1 gene instead of CYP701A in PHYSCObase database of Physcomitrella patens. We obtained the full-length cDNA of CYP701B1 from Riken BRC and tried heterologous expression for analysis KO activity. The CYP701B1 ORF in pPICZ vector was used to transformation of Pichia pastoris harbouring Arabidopsis NADPH cytochrome P450 reductase 1 (ATR1). A transformant was incubated and induced the recombinant protein by addition of methanol. The microsome fraction was incubated with ent-kaurene and NADPH, and metabolite was analyzed by GC-MS. From the GC-MS analysis, the recombinant CYP701B1 catalyzes the oxidation of ent-kaurene to produce ent-kaurenoic acid. These results indicated that the GYP701B1 gene encodes ent-kaurene oxidase in P. patens (PpKO). We tested tolerance activity against uniconazole-P of PpKO. PpKO showed higher resistant activity rather than AtKO at 50μM of uniconazole-P.

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The developing fruit undergoes phase of cell division and expansion followed by numerous metabolic changes leading to ripening. In Japanese pear, exogenous GAs have been widely used to increase fruit enlargement. Exogenous application of GA 3+4 paste until 40 days after anthesis to the young fruit results in increased fruit size. However, the molecular mechanism of fruit development has not understood yet. Here, we have searched genes related to GA-induced fruit enlargement in pear by microarray analysis. Pear fruit was treated with GA1, GA3, GA4, GA4+Prohexadione calcium (GA4P) in lanolin paste at five weeks after full bloom (WAF) and the fruit were sampled at two week after the treatment (at 7 WAF). Total RNA was extracted from the above samples. Among these treatments, GA4 and GA4+Prohexadione calcium resulted in getting larger fruit. A custom oligoarray of Japanese pear based on 9,812 independent ESTs was used for comprehensive investigation of gene expression after GA4 and (GA4P) treatments. Up-regulated 4 genes and down-regulated 7 genes were identified. Expression of up- and down-regulated genes was examined by quantitative RT-PCR during fruit development. Expression of up-regulated 3 genes showed remarkable increase after full bloom and that of down-regulated 4 genes showed decrease trend during fruit development. Total 7 (3 up-regulated and 4 down regulated) genes were selected for further transgenic experiment to clarify the role of fruit cell elongation.

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Cytokinin (CK) is a plant hormone that plays important roles in many aspects of plant growth and development. There are several type of CKs in nature but at least two CK species, t-zeatin and isopentenyl adenin, are considered to be active forms in Arabidopsis. However much unclear matters still remain about biosynthesis pathway and functions of CK. One of the main reasons making difficult for CK research is lack of tools for molecular biology such as CK deficient mutants or biosynthesis inhibitors. Therefore, we challenged to search for candidates of CK inhibitor. First, we searched for candidates of CK inhibitors using correlation analysis of public microarray database AtCAST. From the result, we focused on Uniconazole (Uni) as a candidate of CK inhibitor. It has been reported that Uni affects amounts of other hormones, Brassinosteroid, Gibberellin and Abscisic acid. After Uni treatment, CK response of each hormone insensitive mutant was suppressed except for a CK insensitive mutant. This result suggested Uni inhibit CK responses independently from other hormones. Next, we investigated the changes of CK and intermediates contents after Uni treatment. CKs contents in downstream of CYP735As were significantly reduced by Uni treatment. In order to examine whether Uni prevents CYP735As activity directly, we carried out an in vitro enzyme assay. As a result, CYP735As activity was suppressed by Uni treatment dose-dependently. Thus, we concluded that Uni inhibits CK biosynthesis.

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Bentgrasses (Agrostis stolonifera) sods grown in pots were treated with PKS diluted at 3000-fold (15L/a), Prohex-adione-Ca (1.25g.a.i/a), or the combination of these compounds by foliar-treatment and grown for 6 weeks. The combination treatments increased the number and greenness of the leaves much more than the treatment of PKS, or Prohex-adione-Ca alone. Manila-grassses (Zoysia matrella Merr.) sods grown in pots were treated with PKS, GA-biosynthesis inhibitors or the combinations of PKS and the GA-biosynthesis inhibitors and grown 6 weeks. Their combination treatments retarded the spindly growth and increased the growth of basal part of the stems (including stolons). These results show that combined application of PKS and GA synthesis inhibitors can be very helpful for growing cool-season and warm-season turf grasses healthily in summer.

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Two experiments were conducted to examine the effectiveness of the fertilizer. Preparation of the seeds: The seeds were selected by specific gravity with salt and then sterilized in sodium hypochlorite solution before the seeds were treated with water or the diluted solutions of PKS for 1 or 2 days. Experiment 1: Test on the emergence and establishment of rice seedlings grown in submerged soil The seeds were seeded at 1cm or 2cm depth in soil covered with water at 3.5cm depth and grown at 15℃ or 20℃ for 10 days or 19 days. The rate of emergence and establishment of the seedlings increased with PKS treatment in the both tests conducted at 15℃ and 20℃. Especially the effectiveness was striking in the test conducted at 15℃. Experiment 2: Test on the growth of the rice seedlings grown in deep water The seeds were seeded in glass tubes filled with water at 3cm or 5cm depth and grown at 20℃ for 14 days. The length (2nd-leaf and root) and dry weight (aerial part and underground part) of the seedlings increased with PKS treatment in both tests conducted at 3cm and 5cm water-depth. Especially the promotive effectiveness was striking in the test conducted at 5cm waterdepth. These experiments show that the treatment with PKS enhanced the emergence and growth under low temperature, low oxygen and deep water conditions.

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Citrus fruit abundantly contain various kinds of carotenoids that play important roles in pigmentation of the fruit and dietary functions for human health. Carotenoids are metabolized into the precursor of abscisic acid (ABA) or other compounds by enzymes such as 9-cis-epoxycarotenoid dioxygenase (NCED). Therefore, in this study, we investigated the effects of a new NCED inhibitor, SAR29, on carotenoids contents in the fruit of Satsuma mandarin (Citrus unshiu Marc.). In addition, the effects of combination of SAR29 and ethephon that up-regulated carotenoid biosynthesis were also investigated. The levels of ABA in the flavedo decreased by the treatment of SAR29 (100μM) regardless of ethephon treatment. The levels of xanthophylls such as lutein, all-trans-violaxanthin and β-cryptoxanthin in the flavedo increased with the combined treatment of SAR29 and ethephon (100ppm). Accumulation of total carotenoids in the flavedo was also enhanced (about 140% of the level in control) by the combined treatment. These results suggested that SAR29 treatment inhibited biosynthesis of ABA in the flavedo. Moreover, it was thought that the combined treatment of SAR29 and ethephon was effective in increasing carotenoid contents in the flavedo.

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Root parasitic plants and arbuscular mycorrhizal fungi receive strigolactones as host recognition signals in the rhizosphere. In host plants, strigolactones play a key role in shoot branching. Several lines of genetic evidence have illustrated that strigolantones are derived from the carotenoid pathway in plants. However, the biosynthesis pathway of strigolactones has not yet been fully elucidated. Establishment of their biosynthesis pathway and subsequent studies on regulation of their biosynthesis will provide important clues to control their physiological functions. To better understand the biosynthesis of strigolactones in plants, we investigated the occurrence of strigolactones and regulation of their biosynthesis in plant cultured cell lines provided from RIKEN BioResource Center of Japan. Feeding experiments using deuterium-labeled strigolactones in the plant cell cultures are now being conducted to unveil unestablished pathways in strigolactone biosynthesis.

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Strigolactones function as germination stimulants of root parasitic plants and hyphal branching factors of symbiotic arbuscular mycorrhizal fungi in the rhizosphere, and in plants they act as a novel class of plant hormones inhibiting shoot branching. In this study, characterization of strigolactones in rice (Oryza sativa L.) root exudate was conducted. Rice root exudate was found to contain strigolactones including orobanchol, 2'-epiorobanchol, 7-oxoorobanchyl acetate, two 5-deoxystrigol isomers and some novel strigolactones. One of the 5-deoxystrigol isomer was identified as ent-2'-epi-5-deoxystrigol by spectroscopic analyses, and purification and structure elucidation of the other novel strigolactones will be presented.

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Striga spp., devastating root parasitic weeds, attack monocot crops including sorghum, millet and maize in semi-arid tropics. Their seeds require germination stimulants (mainly strigolactone, SL) released from host roots to germinate. In the present study, characterization of SLs in the root exudates from maize plants including the Striga susceptible cultivar Pioneer 3253 and tolerant cultivars KST94 and WH502, grown hydroponically was conducted by comparing retention times of germination stimulants on reversed phase HPLC with those of synthetic standards and by using LC/MS/MS. The susceptible cultivar mainly exuded larger amounts of 5-deoxystrigol and the tolerant cultivars exuded mainly strigol, sorgomol and orobanchol. 5-Deoxystrigol is more stable than hydroxy-SLs like strigol and thus susceptible cultivars might induce more germination of Striga seeds in the fields.

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At the previous meeting, we reported that chloronema of Physcomitrella patens subsp. patens produces strigolactones (SLs). In this study, WT and the CCD8 loss-of-function mutant (Ppccd8) of P. patens were grown in liquid BCD culture for about 30 days to obtain gametophores during which the media were replaced with fresh one weekly. Then, they were grown for additional 3 days and media collected were extracted with ethyl acetate and SLs in the extracts were analyzed by LC-MS/MS. Both WT and Ppccd8 were found to produce 7-oxoorobanchyl acetate and strigol at similar levels, while fabacyl acetate, orobanchyl acetate, 7α-hydroxyorobanchyl acetate and orobanchol were produced only by WT. Although there is no MAX1 homolog in the moss genome, P. patens produces SLs, suggesting that MAX1 functions in later steps of the active branching inhibiting hormone biosynthesis, i.e, conversion of SLs to the branching inhibitor. Alternatively the moss may have other P450(s) which replace the function of MAX1 for SL biosynthesis.

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Strigolactones (SLs) are released from plant roots to soil and work as communication signals for symbiosis and parasitism in the rhizosphere. Also, SLs inhibit shoot branching of the host plants. SLs consist of tricyclic-lactone (ABC-ring) and methyl butenolide (D-ring), connected with an enol ether bridge. Studies using chemical inhibitors and mutants have suggested that SLs are derived from carotenoids. However, the isoprenoid origin of the SL carbon skeleton has not been proved directly in a metabolism study. Especially, little is known about the biosynthetic origin of D-ring. To address this question, a ^<13>C-labeled precursor specific to the mevalonate or methylerythritol phosphate (MEP) pathway was fed to Lotus japonicus cultured roots in the presence of a chemical inhibitor that blocks each endogenous pathway. We found that the MEP pathway contributes to SL biosynthesis. Here, we discuss the results of ^<13>C-NMR analysis of ^<13>C-labeled SL.

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Parasitic weeds are responsible for large-scale crop devastation all over the world. Parasitic weeds initiate germination by recognizing the secondary metabolites, strigolactones, which are derived from carotenoid and secreted from the roots of the host crops. Strigolactones are also rhizosphere signaling for the symbiotic arbuscular mycorrhizal fungi, and recently, it has been reported that strigolactones and their metabolites act as a novel plant hormone in shoot branching. For the control of parasitic weeds, utilizing the strigolactone biosynthesis inhibitor is one of the promising ways. To screen for the chemicals that inhibit strigolactone biosynthesis, we used chemical library constructed in our laboratory. By LC/MS-MS analysis, we selected some chemicals reducing the level of 2'-epi-5-deoxystrigol in rice and found some chemicals. In addition, these chemicals inhibited the Striga infection. These results suggest that stigolactone biosynthesis inhibitors can be new tool for controlling the parasitic weeds.

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Strigolactones (SLs) have been known as germination stimulants of root parasitic plants and hyphal branching factors of symbiotic arbuscular mycorrhizal fungi. Recent studies suggest that they act as a new plant hormone and regulate shoot blanching, however functions of SLs in other stages of development and the regulation of SL-related gene expressions are mostly unknown in Arabidopsis. MAX2 is a F-box protein and proposed as a candidate for the SL receptor. We performed yeast two-hybrid screening to identify the target of MAX2 and isolated some proteins that probably interact with Leu rich repeat of MAX2. As they belong to the same family, we examined the interaction of other proteins in this family with MAX2 by yeast two-hybrid system. We also identified some SL-responsive genes by using microarray analyses and real-time RT-PCR. On the basis of this result, we overexpressed SL-responsive genes in rice and analyzed the phenotype of these mutants.

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Strigolactones (SLs) or their metabolites act as endogenous inhibitors of axillary bud outgrowth. SLs induce seed germination not only parasitic weed seeds but also some dicot seeds, and inhibit hypocotyl elongation in Arabidopsis thaliana. We have been evaluated structural requirements for inhibition on hypocotyl elongation using structural analogs of SL. Synthetic SL, GR24, inhibited hypocotyl elongation with dose-dependence in Arabidopsis seedlings germinated in the dark, whereas the tricyclic lactone and the hydroxy methylfuranone little exhibited the inhibition. This results show the significance of the core structure of SL (a tricyclic lactone connected with a methylfuranone via an enol ether bridge) on inhibition of hypocotyl elongation in Arabidopsis seedlings. In tested compounds, GR24 was the most effective inhibitor. The C ring-modified compounds were weaker inhibitors than GR24, which act as an effective bud outgrowth inhibitor of rice d10 mutant. In our assay, GR24 also inhibit hypocotyl elongation in Arabidopsis max2 and max3 mutants. These results imply that the inhibition of hypocotyl elongation will mediate a new signal transduction pathway of SL.

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Strigolactone was found as a stimulant for parasitic weeds. Recent papers demonstrated that strigolactones induce hyphal branching in AM fungi and inhibit the outgrowth of axillary buds. Based on the chemical structure of strigolactones isolated from plants, such as strigol and orobanchol, several kinds of artifacts have been synthesized. Among them, GR24, GR7 and GR5 were known as analogues that show similar activity as natural strigolactones. These compounds have both D-ring and enol ether bond as common structural features. Recently we found that new compound 5-(4-Chlorophenoxy)-3-methylfuran-2(5H)-one (CPMF) interrupted brunching of rice d10 mutant as GR24, although CPMF does not have enol ether moiety in its molecule. We were very much interested in the structure of CPMF and started the structure-activity relationship study on CPMF. As a result, we found a new compound (BPMF) without enol ether moiety was 10 times more active than GR24. BPMF reduces the branching of both d10 mutant and max3 mutant of Arabidopsis to the level of wild type in the long-term test. Interestingly, these phenol ether type compounds did not promote the germination of O. minor.

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Strigolactones are the allelochemicals which act as host recognition signals for both root parasitic plants and arbuscular mycorrhizal fungi in the rhizosphere. Additionally, they regulate shoot branching as a novel class of plant hormones. In this study, germination stimulants produced by dokudami (Houttuynia cordata) were searched. Root exudates of dokudami showed germination stimulating activity against Orobanche minor, and so far five known strigolactones (strigol, sorgomol, orobanchyl acetate, fabacyl acetate, and 5-deoxystrigol) were identified by LC-MS/MS analysis. Further purification and structural elucidation of germination stimulants are now in progress.

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Orobanche minor seedlings were attached onto red clover (Trifolium pretense) roots grown in rhizotrons. Approximately 30% of the O. minor seedlings formed tubercles on red clover roots 2 weeks after attachment. Root primordia were appeared on the surface of the tubercles, and crown roots were developed 19 days after attachement. A shoot bud was differentiated from the crown roots 5 weeks after attachment. O. minor parasitism induced little morphological changes in red clover roots during the development process from tubercle formation (de-differentiation) to shoot bud formation (re-differentiation). These observations suggest that O. minor produces growth regulators in its own metabolism to control its differentiation. In addition, the crown roots and the shoot bud were differentiated without the seed embryo of O. minor, suggesting that the developmental process seems to be the process of adventitious root and shoot formation. To investigate the involvement of phytohormone homeostasis in the differentiation process, the endogenous levels of various plant hormones during the developmental process from seed germination to flowering were measured. Furthermore, gene expression involved in the developmental process of O. minor has been extensively studied with the next-generation sequencing platform.

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Orobanche minor is an obligate root parasitic weed belonging to Orobanchaceae. Some root parasitic weeds of Orobanchaceae cause serious damage to many agronomically important crops worldwide. A novel and effective strategy for the parasitic weed control is desired for economical and humanitarian reasons. Since the life cycle of parasitic weeds is significantly different from that of host plants, understanding of the parasite-specific biological events is important for design of selective control strategies. We focused on the germination process of parasitic weeds to find biological events specific to these species and conducted metabolic profiling of O. minor seeds. Consequently, we revealed that gentianose was decreased immediately after GR24 treatment. Gentianose is a trisaccharide consisted of two glucoses and a fructose. An inhibitor of glycosyl hydrolases, nojirimycin bisulfite (NJ), decreased the germination rate. From these results, we hypothesize that gentianose metabolism is essential for germination of O. minor seed and the key enzyme in the gentianose metabolic pathway could be a novel target for selective control of parasitic weeds. In this study, we extracted crude enzymes from O. minor seeds and measured glycosyl hydrolyzing activity. We revealed that the enzyme involved in germination did not exist in the soluble fraction, but associated with the insoluble components, most likely the cell wall.

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Mesquite (Prosopis juliflora) is the leguminous shrubs growing in the tropical or subtropical regions. In this study, we are searching for biologically active substances produced by mesquite leaves toward root parasitic plants. Dried mesquite leaves (250g) were extracted with methanol. After partitioning, the extract (48.6g) yielded crude alkaloids (3.9g), which exhibited the potent inhibitory activity for seed germination and radicle elongation of root parasitic plants Striga hermonthica and Orobanche minor. Bioassay-guided separation of the crude alkaloids by silica gel column chromatography, TLC and HPLC gave two active principles. Structural analysis of the compounds is in progress.

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Arabidopsis cut flowering stems were analyzed to elucidate its molecular mechanism. Microarray analysis demonstrated differential gene expression between cut and non-cut flowering stems and their changes in expression during reunion process. Genes determined to be up-regulated in the reunion process include those involved in cell division, cell wall modification, phytohormone-related gene and transcription factors (TF). To understand how phytohormone involves in the tissue-reunion process, we focused on the phytohormone related genes and putative downstream TFs. Tissue-reunion was inhibited by decapitation or application of TIBA, and pin1 mutant, in which auxin transport was suppressed, showed lower efficiency in the reunion process. Gene expression analysis showed that AP2- and NAC-type TFs were upregulated at 1 and 3 days after cutting and the expression was depend on jasmonic acid (JA) and auxin, respectively. The gene-suppressing transformants for AP2-TF or NAC-TF using CRES-T method did not show normal reunion process. Ethylene-signaling deficient mutant also showed lower efficiency in tissue-reunion and gene expression analysis showed that AP2-TF and NAC-TF were also ethylene-regulated. These results suggest that JA/auxin/ethylene signaling pathways and putative downstream TFs have an important role in the regulation of gene expression during tissue-reunion of Arabidopsis cut flowering stems.

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1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is the rate-limiting enzyme of the ethylene biosynthesis pathway. ACS is regulated both transcriptionally and post-translationally. We previously reported that LeACS2 is phosphorylated in vivo, and suggested the possibility that phosphorylation regulates protein stability rather than enzymatic activity. Here, we demonstrate that phosphorylation/dephosphorylation of LeACS2 regulates its stability. Pulse-chase experiments coupled with treatment with protein kinase/phosphatase inhibitors clearly demonstrated that LeACS2 is stabilized by phosphorylation and immediately degraded after dephosphorylation. The amount of LeACS2 affected by the protein kinase/phosphatase inhibitors significantly influenced cellular ACS activity, ACC content, and ethylene production levels in tomato fruit tissue, suggesting that post-translational regulation by phosphorylation plays an important role in the control of ethylene production as well as in transcriptional regulation. LeACS2 was immediately phosphorylated after translation by CDPK and MAP kinase at different sites in response to wound signaling. CDPK inhibitor, calmidazolium, and MAPKK inhibitor, U0126, were decreased the amounts of phosphorylated form and total ACS protein. Furthermore, the remaining ACS proteins were phosphorylated at both sites; suggesting that type 1 ACS required both phosphorylation sites to be stabilized.

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Stem cells are maintained in the niche by intercellular interactions and signaling networks. Here we study extracellular signals required for maintenance of the root stem cell niche in higher plants. We identify a family of functionally redundant homologous peptides that are secreted, tyrosine-sulfated, and expressed mainly in the stem cell area and the innermost layer of central columella cells. We name these peptides root meristem growth factors (RGF). RGFs are required for maintenance of the root stem cell niche and transit amplifying cell proliferation in Arabidopsis. RGF1 defines expression levels and patterns of the stem cell transcription factor PLETHORA mainly at the post-transcriptional level. The RGFs function independently of the auxin pathway. These peptide signals play a crucial role in post-embryonic root development.

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Background) Nostoc sp., a cyanobacterium establishes symbiosis with cycad (Cycus revolute) root. Nostoc sp. firstly recognizes the host plant root, next moves to the root tip, and eventually induces the coralloid root. Generally Nostoc sp. does not possess motility, but it differentiates to hormogonia, a motile form. Hypothetically, cycad root releases hormogonia-inducing factor (HIF) to establish the symbiosis between them. In this research, we screened HIF-like principle from the coralloid roots. Materials and Method) The coralloid roots collected at Yaku Island in late March, 2009 (1.08kg), and at Tanegashima Island, in early May, 2010 (6.25kg), were soaked in MeOH. It was concentrated and re-suspended in water (ca. 1000ml) to be extracted with equal volume of EtOAc to give 2.58g of EtOAc solubles. In a paper disc assay, aggregated colonies of Nostoc sp. was impregnated in 0.6% Winogradsky's agar medium, and the fraction-charged paper disc (9mm diameter, 0.25mm thick) were put onto the agar plates and incubated at 25oC. When the aggregated colonies of Nostoc sp. around the paper disc started to be dispersed within 2 days, the fraction was judged to contain HIF. The active fraction was further purified to elucidate structures of HIF. Results and Discussions) After 12-h of incubation, Nostoc sp. around 40% EtOAc fraction started to be dispersed. Under a light microscopic observation, some chain-like cells turned into hormogonia, to acquire motilitiy. Using this bioassay system, pure active principle was obtained 1mg of the colorless syrup from the 1kg fr. wt. of coralloid. In spectroscopic analyses and chemical derivatization using FD-MS, ^1H-NMR and treatment with lipase followed by released fatty acid analyses by FD-MS, it was concluded that the HIF is a 1,2-diacylglycerol possessing linolic acid and palmitic acid as constitutive fatty acids.

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The short-day plant Pharbitis nil (synonym Ipomoea nil) can flower under long-day conditions when stressed. P. nil, var. Violet was induced to flower by poor-nutrient stress whereas var. Tendan was not. The plants induced to flower by stress produced fertile seeds and the progeny developed normally. The defoliated Violet scions grafted onto rootstocks of Violet or Tendan were induced to flower under the stress conditions, but the Tendan scions grafted onto Violet rootstocks were not. The results indicate that a transmissible flowering stimulus is involved in the stress-induced flowering. Tendan produces the flowering stimulus, but does not respond to it. Anthocyanin was accumulated in the roots of P. nil grown in tap water suggesting that the stress enhanced the activity of phenylalanine ammonia-lyase (PAL). In fact, higher PAL activity was detected in the plants which flowered by stress. The PAL Inhibitor inhibited the stress-induced flowering, and this inhibition was reversed by salicylic acid (SA). The endogenous SA level increased when flowering was induced by stress. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was strongly expressed under poor-nutrient stress conditions, but PnFT1, another ortholog of FT, was not. The expression of PnFT2 and a weak expression of PnFT1 were induced by cold stress. PnCO, a P. nil ortholog of CONSTANS (CO) of A. thaliana, was not involved in stress-induced flowering of P. nil.

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Spermidine (Spd) is a well-known plant polyamine involved in disease resistance responses, especially hypersensitive response (HR). HR is a powerful resistance system plants have developed against pathogen attack, occurred with production of reactive oxygen species including hydrogen peroxide (H2O2). It is already reported that, H2O2 is generated during Spd oxidation by polyamine oxidases in plant and the induction of HR by Spd infiltration into tobacco is suppressed with guazatine, a polyamine oxidase inhibitor, but the reactive mechanism of HR still remains unclear. Here we synthesized some Spd derivatives to examine the effect of this chemical modification on disease resistance responses, and compared their inductive activities with that of Spd to investigate the possibility if Spd derivatives induce disease resistance more efficiently. The target amino group is definable with using appropriate protective group, and Spd is easily acylated with acylchloride. Thus, we synthesized some Spd mono acyl derivatives. We prepared 1mM solution of them including 0.1% tween20, then treated them as 5μl drop on leaf blades of 4 week soil-cultured rice (nihonbare). As a result, we found some Spd derivatives which caused HR-like cell death more efficiently than Spd. Now we examine the effects of them using real-time PCR on the expression of OsPR1b and PBZ1, representative resistance marker gene of rice, and recently discovered WRKY45, a resistance gene elevated by benzothiadiazole.

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