Language：Japanese   Publisher：植物化学調節学会  

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Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

Auxin biosynthesis involves two types of enzymes: the Trp aminotransferases (TAA/TARs) and the flavin monooxygenases (YUCCAs). This two-step pathway is highly conserved throughout the plant kingdom and is essential for almost all of the major developmental processes. Despite their importance, it is unclear how these enzymes are regulated and how their activities are coordinated. Here, we show that TAA1/TARs are regulated by their product indole-3-pyruvic acid (IPyA) (or its mimic KOK2099) via negative feedback regulation in Arabidopsis thaliana . This regulatory system also functions in rice and tomato. This negative feedback regulation appears to be achieved by both the reversibility of Trp aminotransferase activity and the competitive inhibition of TAA1 activity by IPyA. The K_m value of IPyA is 0.7 µM, and that of Trp is 43.6 µM; this allows IPyA to be maintained at low levels and prevents unfavorable nonenzymatic indole-3-acetic acid (IAA) formation from IPyA in vivo. Thus, IPyA levels are maintained by the push (by TAA1/TARs) and pull (by YUCCAs) of the two biosynthetic enzymes, in which TAA1 plays a key role in preventing the over- or under-accumulation of IPyA. TAA1 prefer Ala among various amino acid substrates in the reverse reaction of auxin biosynthesis, allowing TAA1 to show specificity for converting Trp and pyruvate to IPyA and Ala, and the reverse reaction.

DOI： 10.1073/pnas.2203633119

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Language：English  

DOI： 10.1093/bbb/zbab052

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Language：English   Publishing type：Research paper (scientific journal)  

p-Phenoxyphenyl boronic acid (PPBo) is a specific inhibitor of auxin biosynthesis in Arabidopsis. We examined the inhibitory activity of PPBo in rice. The activity of OsYUCCA, a key enzyme for auxin biosynthesis, was inhibited by PPBo in vitro. The endogenous indole-3-acetic acid (IAA) level and the expression levels of auxin-response genes were significantly reduced in PPBo-treated rice seedlings, which showed typical auxin-deficiency phenotypes. Seminal root growth was promoted by 1 µM PPBo, which was reversed by co-treatment of IAA and PPBo. By contrast, the inhibition of root growth by 10 µM PPBo was not recovered by IAA. The root meristem morphology and cell division were restored by IAA at 60 µM, but that concentration may be too high to support root growth. In conclusion, PPBo is an inhibitor of auxin biosynthesis that targets YUCCA in rice.

DOI： 10.1093/bbb/zbaa033

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Language：English   Publishing type：Research paper (scientific journal)  

Auxin plays important roles in almost all aspects of plant growth and development. Chemical genetics is an effective approach to understand auxin action, especially in nonmodel plant species, in which auxin-related mutants are not yet available. Among auxin-related chemical tools, we present approaches to utilize auxin biosynthesis inhibitors. The inhibitors are effective not only to understand auxin biosynthesis but also to understand auxin action. The effectiveness of the inhibitors can be assessed based on in vitro or in vivo assays. The in vitro assay employs enzyme inhibition assays. The in vivo assay employs UPLC-MS/MS-based analysis of endogenous IAA and its intermediates or metabolites.

DOI： 10.1007/978-1-0716-0954-5_12

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Language：English   Publishing type：Research paper (scientific journal)  

Although brassinosteroids (BRs) have been proposed to be negative regulators of photomorphogenesis, their physiological role therein has remained elusive. We studied light-induced photomorphogenic development in the presence of the BR biosynthesis inhibitor, brassinazole (Brz). Hook opening was inhibited in the presence of Brz; this inhibition was reversed in the presence of brassinolide (BL). Hook opening was accompanied by cell expansion on the inner (concave) side of the hook. This cell expansion was inhibited in the presence of Brz but was restored upon the addition of BL. We then evaluated light-induced organ-specific expression of three BR biosynthesis genes, DWF4, BR6ox1 and BR6ox2, and a BR-responsive gene, SAUR-AC1, during the photomorphogenesis of Arabidopsis. Expression of these genes was induced, particularly in the hook region, in response to illumination. The induction peaked after 3 h of light exposure and preceded hook opening. Phytochrome-deficient mutants, hy1, hy2 and phyAphyB, and a light-signaling mutant, hy5, were defective in light-induced expression of BR6ox1, BR6ox2 and SAUR-AC1. Light induced both expression of BR6ox genes and petiole development. Petiole development was inhibited in the presence of Brz. Our results largely contradict the early view that BRs are negative regulators of photomorphogenesis. Our data collectively suggest that light activates the expression of BR biosynthesis genes in the hook region via a phytochrome-signaling pathway and HY5 and that BR biosynthesis is essential for hook opening and petiole development during photomorphogenesis.

DOI： 10.1093/pcp/pcaa053

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Language：English   Publishing type：Research paper (scientific journal)  

Long intergenic noncoding RNAs (lincRNAs) play critical roles in transcriptional and post-transcriptional regulation of gene expression in a wide variety of organisms. Thousands of lincRNAs have been identified in plant genomes, although their functions remain mostly uncharacterized. Here, we report a genome-wide survey of lincRNAs involved in the response to low-nutrient conditions in Arabidopsis thaliana. We used RNA sequencing data derived from A. thaliana roots exposed to low levels of 12 different nutrients. Using bioinformatics approaches, 60 differentially expressed lincRNAs were identified that were significantly upregulated or downregulated under deficiency of at least one nutrient. To clarify their roles in nutrient response, correlations of expression patterns between lincRNAs and reference genes were examined across the 13 conditions (12 low-nutrient conditions and control). This analysis allowed us to identify lincRNA-RNA pairs with highly positive or negative correlations. In addition, calculating interaction energies of those pairs showed lincRNAs that may act as regulatory interactors; e.g. small interfering RNAs (siRNAs). Among them, trans-acting siRNA3 (TAS3), which is known to promote lateral root development by producing siRNA against Auxin response factor 2, 3, and 4, was revealed as a nitrogen (N)-responsive lincRNA. Furthermore, nitrate transporter 2 was identified as a potential target of TAS3-derived siRNA, suggesting that TAS3 participates in multiple pathways by regulating N transport and root development under low-N conditions. This study provides the first resource for candidate lincRNAs involved in multiple nutrient responses in plants.

DOI： 10.1093/pcp/pcz048

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Language：English   Publishing type：Research paper (scientific journal)  

Melting-flesh peaches produce large amounts of ethylene, resulting in rapid fruit softening at the late-ripening stage. In contrast, stony hard peaches do not soften and produce little ethylene. The indole-3-acetic acid (IAA) level in stony hard peaches is low at the late-ripening stage, resulting in low ethylene production and inhibition of fruit softening. To elucidate the mechanism of low IAA concentration in stony hard peaches, endogenous levels of IAA and IAA intermediates or metabolites were analysed by ultra-performance liquid chromatography-tandem mass spectrometry. Although the IAA level was low, the indole-3-pyruvic acid (IPyA) level was high in stony hard peaches at the ripening stage. These results indicate that YUCCA activity is reduced in ripening stony hard peaches. The expression of one of the YUCCA isogenes in peach, PpYUC11, was suppressed in ripening stony hard peaches. Furthermore, an insertion of a transposon-like sequence was found upstream of the PpYUC11 gene in the 5'-flanking region. Analyses of the segregation ratio of the stony hard phenotype and genotype in F1 progenies indicated that the transposon-inserted allele of PpYUC11, hd-t, correlated with the stony hard phenotype. On the basis of the above findings, we propose that the IPyA pathway (YUCCA pathway) is the main auxin biosynthetic pathway in ripening peaches of 'Akatsuki' and 'Manami' cultivars. Because IAA is not supplied from storage forms, IAAde novo synthesis via the IPyA pathway (YUCCA pathway) in mesocarp tissues is responsible for auxin generation to support fruit softening, and its disruption can lead to the stony hard phenotype.

DOI： 10.1111/tpj.14070

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Increasing grain yield is an endless challenge for cereal crop breeding. In barley (Hordeum vulgare), grain number is controlled mainly by Six-rowed spike 1 (Vrs1), which encodes a homeodomain leucine zipper class I transcription factor. However, little is known about the genetic basis of grain size. Here, we show that extreme suppression of lateral florets contributes to enlarged grains in deficiens barley. Through a combination of fine-mapping and resequencing of deficiens mutants, we have identified that a single amino acid substitution at a putative phosphorylation site in VRS1 is responsible for the deficiens phenotype. deficiens mutant alleles confer an increase in grain size, a reduction in plant height, and a significant increase in thousand grain weight in contemporary cultivated germplasm. Haplotype analysis revealed that barley carrying the deficiens allele (Vrs1.t1) originated from two-rowed types carrying the Vrs1.b2 allele, predominantly found in germplasm from northern Africa. In situ hybridization of histone H4, a marker for cell cycle or proliferation, showed weaker expression in the lateral spikelets compared with central spikelets in deficiens Transcriptome analysis revealed that a number of histone superfamily genes were up-regulated in the deficiens mutant, suggesting that enhanced cell proliferation in the central spikelet may contribute to larger grains. Our data suggest that grain yield can be improved by suppressing the development of specific organs that are not positively involved in sink/source relationships.

DOI： 10.1104/pp.17.01149

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The plant hormone auxin plays a crucial role in plant growth and development. Indole-3-acetic acid (IAA), a natural auxin, is mainly biosynthesized by two sequential enzyme reactions catalyzed by TAA1 and YUCCA (YUC). TAA1 is involved in the conversion of tryptophan to IPA, and YUC catalyzes the conversion of IPA to IAA. We previously demonstrated that yucasin inhibits AtYUC1 enzyme activity and suppress high-auxin phenotype of YUC overexpression plants, although yucasin displayed weak effects on the auxin-related phenotype of wild-type plants. To develop more potent YUC inhibitors, various derivatives of yucasin were synthesized, and their structure-activity relationships were investigated. Yucasin difluorinated analog (YDF) (5-[2,6-difluorophenyl]-2,4-dihydro-[1,2,4]-triazole-3-thione) was identified to be a more potent YUC inhibitor than the original yucasin. YDF caused an auxin-deficient phenotype in Arabidopsis wild-type plants that was restored with auxin application. YDF was found to be highly stable regarding metabolic conversion in vivo, accounting for the potent activity of the inhibition of IAA biosynthesis in planta. Photoaffinity labeling experiments demonstrated that yucasin-type inhibitors bind to the active site of AtYUC1. YDF is a promising auxin biosynthesis inhibitor and is a useful chemical tool for plant biology and agrochemical studies.

DOI： 10.1038/s41598-017-14332-w

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

The alteration of transcript structure contributes to transcriptome plasticity. In this study, we analyzed the genome-wide response of exon combination patterns to deficiencies in 12 different nutrients in Arabidopsis thaliana roots. RNA sequencing analysis and bioinformatics using a simulation survey revealed more than 600 genes showing varying exon combinations. The overlap between genes showing differential expression (DE) and genes showing differential exon combination (DC) was notably low. Additionally, gene ontology analysis showed that gene functions were not shared between the DE and DC genes, suggesting that the genes showing DC had different roles than those showing DE. Most of the DC genes were nutrient specific. For example, two homologs of the MYB transcription factor genes MYB48 and MYB59 showed differential alternative splicing only in response to low levels of potassium. Alternative splicing of those MYB genes modulated DNA-binding motifs, and MYB59 is reportedly involved in the inhibition of root elongation. Therefore, the increased abundance of MYB isoforms with an intact DNA-binding motif under low potassium may be involved in the active inhibition of root elongation. Overall, we provide global and comprehensive data for DC genes affected by nutritional deficiencies, which contribute to elucidating an unknown mechanism involved in adaptation to nutrient deficiency.

DOI： 10.1111/tpj.13606

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

We previously reported that exogenous application of auxin to Arabidopsis seedlings resulted in downregulation of indole-3-acetic acid (IAA) biosynthesis genes in a feedback manner. In this study, we investigated the involvement of the SCFTIR1/AFB-mediated signaling pathway in feedback regulation of the indole-3-pyruvic acid-mediated auxin biosynthesis pathway in Arabidopsis. Application of PEO-IAA, an inhibitor of the IAA signal transduction pathway, to wild-type seedlings resulted in increased endogenous IAA levels in roots. Endogenous IAA levels in the auxin-signaling mutants axr2-1, axr3-3, and tir1-1afb1-1afb2-1afb3-1 also increased. Furthermore, YUCCA (YUC) gene expression was repressed in response to auxin treatment, and expression of YUC7 and YUC8 increased in response to PEO-IAA treatment. YUC genes were also induced in auxin-signaling mutants but repressed in TIR1-overexpression lines. These observations suggest that the endogenous IAA levels are regulated by auxin biosynthesis in a feedback manner, and the Aux/IAA and SCFTIR1/AFB-mediated auxin-signaling pathway regulates the expression of YUC genes.

DOI： 10.1080/09168451.2017.1313694

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press  

IAA, a major form of auxin, is biosynthesized from l-tryptophan via the indole-3-pyruvic acid (IPyA) pathway in Arabidopsis. Tryptophan aminotransferases (TAA1/TARs) catalyze the first step from l-tryptophan to IPyA. In rice, the importance of TAA/TARs or YUC homologs in auxin biosynthesis has been suggested, but the enzymatic activities and involvement of the intermediate IPyA in auxin biosynthesis remain elusive. In this study, we obtained biochemical evidence that the rice tryptophan aminotransferase OsTAR1 converts l-tryptophan to IPyA, and has a Km of 82.02 µM and a Vmax of 10.92 µM min-1 m-1, comparable with those in Arabidopsis. Next, we screened for an effective inhibitor of OsTAR1 from our previously reported inhibitor library for TAA1/TARs, designated pyruvamine (PVM). Differing from previous observations in Arabidopsis, hydroxy-type PVMs, e.g. PVM2031 (previous name KOK2031), had stronger inhibitory effects in rice than the methoxy-type. PVM2031 inhibited recombinant OsTAR1 in vitro. The Ki of PVM2031 was 276 nM. PVM2031 treatment of rice seedlings resulted in morphological changes in vivo, such as reduced lateral root density. Exogenous IAA rescued this growth inhibition, suggesting that the inhibitory effect is auxin specific. Furthermore, rice roots showed reduced IAA levels concomitant with reduced levels of IPyA in the presence of the inhibitors, suggesting that the IPyA pathway is an auxin biosynthesis pathway in rice. Since PVM2031 showed stronger inhibitory effects on rice auxin biosynthesis than known tryptophan aminotransferase inhibitors, we propose that the hydroxy-type PVM2031 is an effective tool for biochemical analysis of the function of auxin biosynthesis in rice roots.

DOI： 10.1093/pcp/pcx007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

IAA, a major form of auxin, is biosynthesized from L-tryptophan via the indole-3-pyruvic acid (IPyA) pathway in Arabidopsis. Tryptophan aminotransferases (TAA1/TARs) catalyze the first step from L-tryptophan to IPyA. In rice, the importance of TAA/TARs or YUC homologs in auxin biosynthesis has been suggested, but the enzymatic activities and involvement of the intermediate IPyA in auxin biosynthesis remain elusive. In this study, we obtained biochemical evidence that the rice tryptophan aminotransferase OsTAR1 converts L-tryptophan to IPyA, and has a Km of 82.02 mM and a Vmax of 10.92 mu M min(-1) m(-1), comparable with those in Arabidopsis. Next, we screened for an effective inhibitor of OsTAR1 from our previously reported inhibitor library for TAA1/TARs, designated pyruvamine (PVM). Differing from previous observations in Arabidopsis, hydroxy-type PVMs, e. g. PVM2031 ( previous name KOK2031), had stronger inhibitory effects in rice than the methoxy-type. PVM2031 inhibited recombinant OsTAR1 in vitro.The K-i of PVM2031 was 276 nM. PVM2031 treatment of rice seedlings resulted in morphological changes in vivo, such as reduced lateral root density. Exogenous IAA rescued this growth inhibition, suggesting that the inhibitory effect is auxin specific. Furthermore, rice roots showed reduced IAA levels concomitant with reduced levels of IPyA in the presence of the inhibitors, suggesting that the IPyA pathway is an auxin biosynthesis pathway in rice. Since PVM2031 showed stronger inhibitory effects on rice auxin biosynthesis than known tryptophan aminotransferase inhibitors, we propose that the hydroxy-type PVM2031 is an effective tool for biochemical analysis of the function of auxin biosynthesis in rice roots.

DOI： 10.1093/pcp/pcx007

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Publishing type：Research paper (scientific journal)  

CiNii Research

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Brassinosteroid (BR) is an important plant hormone that is perceived by the BRASSINOSTEROID INSENSITIVE 1 (BRI1) receptor. BRI1 is conserved among dicot and monocot species; however, the molecular mechanism underlying BR perception in monocots is not fully understood. We synthesised two BRs, iso-carbabrassinolide (iso-carbaBL) and 6-deoxoBL, which have different BR activities in Arabidopsis thaliana (Arabidopsis) and rice. Our bioassay indicated that iso-carbaBL has relatively strong BR activity in Arabidopsis, but is inactive in rice and competitively inhibits BR activity. The bioactivity of 6-deoxoBL was similar to that of BL in Arabidopsis, but was much lower in rice. Binding experiments using recombinant Arabidopsis and rice BRI1 protein fragments suggested that iso-carbaBL and 6-deoxoBL bind to both receptors. These results showed that iso-carbaBL and 6-deoxoBL act as an antagonist and agonist, respectively, of BRs in rice. A docking simulation analysis suggested that iso-carbaBL fits deeper in the binding pocket to block the binding of active BR to rice BRI1. The simulated binding energy of 6-deoxoBL with rice BRI1 is much lower than that with Arabidopsis BRI1. The possible structural characteristics of rice BRI1 were determined based on the difference in the BR activities of iso-carbaBL and 6-deoxoBL in Arabidopsis and rice.

DOI： 10.1371/journal.pone.0174015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

We previously reported l-α-aminooxy-phenylpropionic acid (AOPP) to be an inhibitor of auxin biosynthesis, but its precise molecular target was not identified. In this study we found that AOPP targets TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1). We then synthesized 14 novel compounds derived from AOPP to study the structure-activity relationships of TAA1 inhibitors in vitro. The aminooxy and carboxy groups of the compounds were essential for inhibition of TAA1 in vitro. Docking simulation analysis revealed that the inhibitory activity of the compounds was correlated with their binding energy with TAA1. These active compounds reduced the endogenous indole-3-acetic acid (IAA) content upon application to Arabidopsis seedlings. Among the compounds, we selected 2-(aminooxy)-3-(naphthalen-2-yl)propanoic acid (KOK1169/AONP) and analyzed its activities in vitro and in vivo. Arabidopsis seedlings treated with KOK1169 showed typical auxin-deficient phenotypes, which were reversed by exogenous IAA. In vitro and in vivo experiments indicated that KOK1169 is more specific for TAA1 than other enzymes, such as phenylalanine ammonia-lyase. We further tested 41 novel compounds with aminooxy and carboxy groups to which we added protection groups to increase their calculated hydrophobicity. Most of these compounds decreased the endogenous auxin level to a greater degree than the original compounds, and resulted in a maximum reduction of about 90% in the endogenous IAA level in Arabidopsis seedlings. We conclude that the newly developed compounds constitute a class of inhibitors of TAA1. We designated them 'pyruvamine'.

DOI： 10.1111/tpj.13197

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Auxin is essential for plant growth and development, this makes it difficult to study the biological function of auxin using auxin-deficient mutants. Chemical genetics have the potential to overcome this difficulty by temporally reducing the auxin function using inhibitors. Recently, the indole-3-pyruvate (IPyA) pathway was suggested to be a major biosynthesis pathway in Arabidopsis thaliana L. for indole-3-acetic acid (IAA), the most common member of the auxin family. In this pathway, YUCCA, a flavin-containing monooxygenase (YUC), catalyzes the last step of conversion from IPyA to IAA. In this study, we screened effective inhibitors, 4-biphenylboronic acid (BBo) and 4-phenoxyphenylboronic acid (PPBo), which target YUC. These compounds inhibited the activity of recombinant YUC in vitro, reduced endogenous IAA content, and inhibited primary root elongation and lateral root formation in wild-type Arabidopsis seedlings. Co-treatment with IAA reduced the inhibitory effects. Kinetic studies of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA. Inhibition constants (Ki ) of BBo and PPBo were 67 and 56 nm, respectively. In addition, PPBo did not interfere with the auxin response of auxin-marker genes when it was co-treated with IAA, suggesting that PPBo is not an inhibitor of auxin sensing or signaling. We propose that these compounds are a class of auxin biosynthesis inhibitors that target YUC. These small molecules are powerful tools for the chemical genetic analysis of auxin function.

DOI： 10.1111/tpj.13032

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Brachypodium distachyon is a new model plant closely related to wheat and other cereals. In this study, we performed a comprehensive analysis of hormone-regulated genes in Brachypodium distachyon using RNA sequencing technology. Brachypodium distachyon seedlings were treated with eight phytohormones (auxin, cytokinine, brassinosteroid, gibberelline, abscisic acid, ethylene, jasmonate and salicylic acid) and two inhibitors, Brz220 (brassinosteroid biosynthesis inhibitor) and prohexadione (gibberelline biosynthesis inhibitor). The expressions of 1807 genes were regulated in a phytohormone-dependent manner. We compared the data with the phytohormone responses that have reported in rice. Transcriptional responses to hormones are conserved between Bracypodium and rice. Transcriptional regulation by brassinosteroid, gibberellin and ethylene was relatively weaker than those by other hormones. This is consistent with the data obtained from comprehensive analysis of hormone responses reported in Arabidopsis. Brachypodium and Arabidopsis also shared some common transcriptional responses to phytohormones. Alternatively, unique transcriptional responses to phytohormones were observed in Brachypodium. For example, the expressions of ACC synthase genes were up-regulated by auxin treatment in rice and Arabidopsis, but no orthologous ACC synthase gene was up-regulated in Brachypodium. Our results provide information useful to understand the diversity and similarity of hormone-regulated transcriptional responses between eudicots and monocots.

DOI： 10.1038/srep14476

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

KEY MESSAGE: The IPyA pathway, the major auxin biosynthesis pathway, is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels. The phytohormone auxin plays an important role in plant growth and development, and levels of active free auxin are determined by biosynthesis, conjugation, and polar transport. Unlike conjugation and polar transport, little is known regarding the regulatory mechanism of auxin biosynthesis. We discovered that expression of genes encoding indole-3-pyruvic acid (IPyA) pathway enzymes is regulated by elevated or reduced active auxin levels. Expression levels of TAR2, YUC1, YUC2, YUC4, and YUC6 were downregulated in response to synthetic auxins [1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)] exogenously applied to Arabidopsis thaliana L. seedlings. Concomitantly, reduced levels of endogenous indole-3-acetic acid (IAA) were observed. Alternatively, expression of these YUCCA genes was upregulated by the auxin biosynthetic inhibitor kynurenine in Arabidopsis seedlings, accompanied by reduced IAA levels. These results indicate that expression of YUCCA genes is regulated by active auxin levels. Similar results were also observed in auxin-overproduction and auxin-deficient mutants. Exogenous application of IPyA to Arabidopsis seedlings preincubated with kynurenine increased endogenous IAA levels, while preincubation with 2,4-D reduced endogenous IAA levels compared to seedlings exposed only to IPyA. These results suggest that in vivo conversion of IPyA to IAA was enhanced under reduced auxin levels, while IPyA to IAA conversion was depressed in the presence of excess auxin. Based on these results, we propose that the IPyA pathway is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels.

DOI： 10.1007/s00299-015-1791-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

In transcriptome experiments, the experimental conditions (e.g. mutants and/or treatments) cause transcriptional changes. Identifying experimental conditions that induce similar or opposite transcriptional changes can be useful to identify experimental conditions that affect the same biological process. AtCAST (http://atpbsmd.yokohama-cu.ac.jp) is a web-based tool to analyze the relationship between experimental conditions among transcriptome data. Users can analyze 'user's transcriptome data' of a new mutant or a new chemical compound whose function remains unknown to generate novel biological hypotheses. This tool also allows for mining of related 'experimental conditions' from the public microarray data, which are pre-included in AtCAST. This tool extracts a set of genes (i.e. module) that show significant transcriptional changes and generates a network graph to present related transcriptome data. The updated AtCAST now contains data on >7,000 microarrays, including experiments on various stresses, mutants and chemical treatments. Gene ontology term enrichment (GOE) analysis is introduced to assist the characterization of transcriptome data. The new AtCAST supports input from multiple platforms, including the 'Arabisopsis gene 1.1 ST array', a new microarray chip from Affymetrix and RNA sequencing (RNA-seq) data obtained using next-generation sequencing (NGS). As a pilot study, we conducted microarray analysis of Arabidopsis under auxin treatment using the new Affymetrix chip, and then analyzed the data in AtCAST. We also analyzed RNA-seq data of the pifq mutant using AtCAST. These new features will facilitate analysis of associations between transcriptome data obtained using different platforms.

DOI： 10.1093/pcp/pcu174

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

To develop a growth inhibitor, the effects of auxin inhibitors were investigated. Application of 30 μM L-α-aminooxy-β-phenylpropionic acid (AOPP) or (S)-methyl 2-((1,3-dioxoisoindolin-2-yl)oxy)-3-phenylpropanoate (KOK1101), decreased the endogenous IAA levels in tomato seedlings at 8 days after sowing. Then, 10-1200 μM AOPP or KOK1101 were sprayed on the leaves and stem of 2-3 leaf stage tomato plants grown under a range of environmental conditions. We predicted plant growth and environmental response using a model based on the observed suppression of leaf enlargement. Spraying AOPP or KOK1101 decreased stem length and leaf area. Concentration-dependent inhibitions and dose response curves were observed. Although the effects of the inhibitors on dry weight varied according to the environmental conditions, the net assimilation rate was not influenced by the inhibitors. Accordingly, the observed decrease in dry weight caused by the inhibitors may result from decreased leaf area. Validation of the model based on observed data independent of the dataset showed good correlations between the observed and predicted values of dry weight and leaf area index.

DOI： 10.1038/srep04556

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Previously we identified indole-3-acetic acid (IAA) biosynthesis inhibitors that act on the conversion of l-tryptophan to indole-3-pyruvic acid in the IAA biosynthesis of Arabidopsis. In the present study, we synthesized a new compound, indole-3-oxoethylphosphonic acid (IOEP), and found that IOEP had an inhibitory effect on IAA biosynthesis in Arabidopsis. The results suggest that IOEP is a novel inhibitor of auxin biosynthesis in Arabidopsis.

DOI： 10.1080/09168451.2014.877183

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Bioscience Biotechnology and Agrochemistry  

Brassinosteroids are plant steroid hormones that regulate plant organs and chloroplast development. The detailed molecular mechanism for plant development by BR signaling is yet to be revealed, and many points regarding the relationship between BR signaling and chloroplast development remain unknown. We identify here the dominant mutant Brz-insensitive-pale green3-1D (bpg3-1D) from the Arabidopsis FOX lines that show reduced sensitivity to the chlorophyll accumulation promoted by the BR biosynthesis inhibitor, Brassinazole (Brz), in the light. BPG3 encodes a novel chloroplast protein that is evolutionally conserved in bacteria, algae, and higher plants. The expression of BPG3 was induced by light and Brz. The inhibition of electron transport in photosystem II of the chloroplasts was detected in bpg3-1D. These results suggest that BPG3 played an important role in regulating photosynthesis in the chloroplast under BR signaling.

DOI： 10.1080/09168451.2014.885831

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The unfolded protein response (UPR) is a cellular response highly conserved in eukaryotes to obviate accumulation of misfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring enzyme 1 (IRE1) catalyzes the cytoplasmic splicing of mRNA encoding bZIP transcription factors to activate the UPR signaling pathway. Arabidopsis IRE1 was recently shown to be involved in the cytoplasmic splicing of bZIP60 mRNA. In the present study, we demonstrated that an Arabidopsis mutant with defects in two IRE1 paralogs showed enhanced cell death upon ER stress compared with a mutant with defects in bZIP60 and wild type, suggesting an alternative function of IRE1 in the UPR. Analysis of our previous microarray data and subsequent quantitative PCR indicated degradation of mRNAs encoding secretory pathway proteins by tunicamycin, DTT, and heat in an IRE1-dependent manner. The degradation of mRNAs localized to the ER during the UPR was considered analogous to a molecular mechanism referred to as the regulated IRE1-dependent decay of mRNAs reported in metazoans. Another microarray analysis conducted in the condition repressing transcription with actinomycin D and a subsequent Gene Set Enrichment Analysis revealed the regulated IRE1-dependent decay of mRNAs-mediated degradation of a significant portion of mRNAs encoding the secretory pathway proteins. In the mutant with defects in IRE1, genes involved in the cytosolic protein response such as heat shock factor A2 were up-regulated by tunicamycin, indicating the connection between the UPR and the cytosolic protein response.

DOI： 10.1073/pnas.1219047110

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Cytokinin (CK) is a plant hormone that plays important regulatory roles in many aspects of plant growth and development. Although functions of CK and its biosynthesis pathway have been studied extensively, there is still no efficient biosynthesis inhibitor, which would be useful for studying CK from a chemical genetic approach. Here, CK biosynthesis inhibitor candidates were searched for using a systematic approach. In silico screening of candidates were carried out using genome-wide gene expression profiles and prediction of target sites using global CK accumulation profile analysis. As a result of these screenings, it was found that uniconazole, a well known inhibitor of cytochrome P450 monooxygenase, prevents the biosynthesis of trans-zeatin, and that its target is CYP735As in Arabidopsis.

DOI： 10.1016/j.phytochem.2012.11.023

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Biosynthesis of indole-3-acetic acid (IAA) is crucial for the regulation of plant growth and morphological changes. However, the pathways of IAA biosynthesis have not been completely understood. Although indole derivatives have been proposed to be intermediates of the pathway, it has not been thoroughly elucidated whether the proposed intermediates are indeed converted into an active auxin, IAA, or intermediate metabolites other than IAA. In the present work, we examined 11 indole derivatives whether they are indeed IAA intermediates. Te indole derivatives were examined by measuring their efects on recovery from auxin-defciency conditions caused by l-aminooxyphenylpropionic acid (AOPP), which we previously identifed as an IAA biosynthesis inhibitor. We estimated auxin activity of each indole derivative by analyzing auxin-inducible marker gene expression and root morphology in Arabidopsis. We found that indole-3-pyruvic acid most effectively recovered seedlings from auxin deficiency among the indole derivatives examined, followed by N-hydroxyl tryptamine and indole-3-acetonitrile, with effective recovery from the auxin deficiency caused by AOPP. We also found that tryptophol recovered the root growth defects. Among the indole derivatives examined, only indole-3-acrylic acid did not show effective recovery from auxin defciency. These results provide information on which compounds are reliable intermediates of IAA biosynthesis pathways in Arabidopsis. © 2013 The Japanese Society for Plant Cell and Molecular Biology.

DOI： 10.5511/plantbiotechnology.13.0128c

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The plant hormone auxin is a master regulator of plant growth and development. By regulating rates of cell division and elongation and triggering specific patterning events, indole 3-acetic acid (IAA) regulates almost every aspect of plant development. The perception of auxin involves the formation of a ternary complex consisting of an F-box protein of the TIR1/AFB family of auxin receptors, the auxin molecule, and a member the Aux/IAA family of co-repressor proteins. In this study, we identified a potent auxin antagonist, alpha-(phenylethyl-2-oxo)-IAA, as a lead compound for TIR1/AFB receptors by in silico virtual screening. This molecule was used as the basis for the development of a more potent TIR1 antagonist, auxinole (alpha-[2,4-dimethylphenylethyl-2-oxo]-IAA), using a structure-based drug design approach. Auxinole binds TIR1 to block the formation of the TIR1 IAA Aux/IAA complex and so inhibits auxin-responsive gene expression. Molecular docking analysis indicates that the phenyl ring in auxinole would strongly interact with Phe82 of TIR1, a residue that is crucial for Aux/IAA recognition. Consistent with this predicted mode of action, auxinole competitively inhibits various auxin responses in planta. Additionally, auxinole blocks auxin responses of the moss Physcomitrella patens, suggesting activity over a broad range of species. Our works not only substantiates the utility of chemical tools for plant biology but also demonstrates a new class of small molecule inhibitor of protein-protein interactions common to mechanisms of perception of other plant hormones, such as jasmonate, gibberellin, and abscisic acid.

DOI： 10.1021/cb200404c

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOL  

Phytohormones have vigorous crosstalk relationships. For example, abscisic acid (ABA), a hormone involved in abiotic stress responses, has antagonistic interactions with plant hormones that play pivotal roles in defense responses, including salicylic acid (SA) and methyl-jasmonic acid (MeJA). Evidence indicates that the relationships among these plant hormones extend beyond simple antagonism. To explore the interplay between hormones in detail, we analyzed the effects of double hormone treatment on gene expression. By contrast to the antagonistic effects reported previously, our data indicates that ABA interacts with SA and MeJA cooperatively as well. Particularly many genes responded only to double hormone treatment, and, interestingly, the loci that responded to ABA+SA also responded to ABA+MeJA. The expression of early-response genes following double hormone treatment did not fit the linear superposition of individual hormone treatments, in contrast to mammalian and prokaryotic cell responses to multiple chemical stimuli. Thus, synergies in these plant hormone signalings are not simply the sum of individual responses. ABA and SA collaboratively down-regulated the expression of genes involved in cell cycle progression at G2/M phase. Presumably, plants interpret combined hormone signals differently from individual signals in order to respond appropriately to their environmental conditions.

DOI： 10.5511/plantbiotechnology.11.1130a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：11  

The jasmonate receptor COI1 is known to facilitate plant defense responses against necrotrophic pathogens, including the ascomycete Sclerotinia sclerotiorum. However, it is not known to what extent jasmonates contribute to defense nor have COI1-indepenent defense pathways been sufficiently characterized. Here we show that the susceptibility to S. sclerotiorum of the aos mutant, deficient in biosynthesis of jasmonic acid (JA) and its precursor 12-oxophytadienoic acid, was elevated to a level reminiscent of that of hypersusceptible coi1 mutants. In contrast, susceptibility of the JA-deficient opr3 mutant was comparable with that of the wild type. A set of 99 genes responded similarly to infection with S. sclerotiorum in wild-type and coi1 mutant leaves. Expression of this COI1-independent gene set correlated with known differences in gene expression between wild-type plants and a mutant in the transcriptional repressor auxin response factor 2 (arf2). Susceptibility to S. sclerotiorum was reduced in two arf2 mutants early during infection, implicating ARF2 as a negative regulator of defense responses against this pathogen. Hypersusceptibility of an axr1 mutant to S. sclerotiorum confirmed the contribution of auxin action to defense responses against this fungal pathogen. © 2011 The Author.

DOI： 10.1093/pcp/pcr127

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Plant secondary metabolites are known to facilitate interactions with a variety of beneficial and detrimental organisms, yet the contribution of specific metabolites to interactions with fungal pathogens is poorly understood. Here we show that, with respect to aliphatic glucosinolate-derived isothiocyanates, toxicity against the pathogenic ascomycete Sclerotinia sclerotiorum depends on side chain structure. Genes associated with the formation of the secondary metabolites camalexin and glucosinolate were induced in Arabidopsis thaliana leaves challenged with the necrotrophic pathogen S. sclerotiorum. Unlike S. sclerotiorum, the closely related ascomycete Botrytis cinerea was not identified to induce genes associated with aliphatic glucosinolate biosynthesis in pathogen-challenged leaves. Mutant plant lines deficient in camalexin, indole, or aliphatic glucosinolate biosynthesis were hypersusceptible to S. sclerotiorum, among them the myb28 mutant, which has a regulatory defect resulting in decreased production of long-chained aliphatic glucosinolates. The antimicrobial activity of aliphatic glucosinolate-derived isothiocyanates was dependent on side chain elongation and modification, with 8-methylsulfinyloctyl isothiocyanate being most toxic to S. sclerotiorum. This information is important for microbial associations with cruciferous host plants and for metabolic engineering of pathogen defenses in cruciferous plants that produce short-chained aliphatic glucosinolates. © 2011 Blackwell Publishing Ltd.

DOI： 10.1111/j.1365-313X.2011.04578.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Catabolism of brassinosteroids regulates the endogenous level of bioactive brassinosteroids. In Arabidopsis thaliana, bioactive brassinosteroids such as castasterone (CS) and brassinolide (BL) are inactivated mainly by two cytochrome P450 monooxygenases, CYP734A1/BAS1 and CYP72C1/SOB7/CHI2/SHK1; CYP734A1/BAS1 inactivates CS and BL by means of C-26 hydroxylation. Here, we characterized CYP734A orthologs from Oryza sativa (rice). Overexpression of rice CYP734As in transgenic rice gave typical brassinosteroid-deficient phenotypes. These transformants were deficient in both the bioactive CS and its precursors downstream of the C-22 hydroxylation step. Consistent with this result, recombinant rice CYP734As utilized a range of C-22 hydroxylated brassinosteroid intermediates as substrates. In addition, rice CYP734As can catalyze hydroxylation and the second and third oxidations to produce aldehyde and carboxylate groups at C-26 in vitro. These results indicate that rice CYP734As are multifunctional, multisubstrate enzymes that control the endogenous bioactive brassinosteroid content both by direct inactivation of CS and by the suppression of CS biosynthesis by decreasing the levels of brassinosteroid precursors.

DOI： 10.1111/j.1365-313X.2011.04567.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

IRE1 plays an essential role in the endoplasmic reticulum (ER) stress response in yeast and mammals. We found that a double mutant of Arabidopsis IRE1A and IRE1B (ire1a/ire1b) is more sensitive to the ER stress inducer tunicamycin than the wild-type. Transcriptome analysis revealed that genes whose induction was reduced in ire1a/ire1b largely overlapped those in the bzip60 mutant. We observed that the active form of bZIP60 protein detected in the wild-type was missing in ire1a/ire1b. We further demonstrated that bZIP60 mRNA is spliced by ER stress, removing 23 ribonucleotides and therefore causing a frameshift that replaces the C-terminal region of bZIP60 including the transmembrane domain (TMD) with a shorter region without a TMD. This splicing was detected in ire1a and ire1b single mutants, but not in the ire1a/ire1b double mutant. We conclude that IRE1A and IRE1B catalyse unconventional splicing of bZIP60 mRNA to produce the active transcription factor.

DOI： 10.1038/srep00029

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The comparison of gene expression profiles among DNA microarray experiments enables the identification of unknown relationships among experiments to uncover the underlying biological relationships. Despite the ongoing accumulation of data in public databases, detecting biological correlations among gene expression profiles from multiple laboratories on a large scale remains difficult. Here, we applied a module (sets of genes working in the same biological action)-based correlation analysis in combination with a network analysis to Arabidopsis data and developed a 'module-based correlation network' (MCN) which represents relationships among DNA microarray experiments on a large scale. We developed a Web-based data analysis tool, 'AtCAST' (Arabidopsis thaliana: DNA Microarray Correlation Analysis Tool), which enables browsing of an MCN or mining of users' microarray data by mapping the data into an MCN. AtCAST can help researchers to find novel connections among DNA microarray experiments, which in turn will help to build new hypotheses to uncover physiological mechanisms or gene functions in Arabidopsis.

DOI： 10.1093/pcp/pcq185

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Previously we identified aminooxy compounds as auxin biosynthesis inhibitors. One of the compounds, aminooxyacetic acid (AOA) inhibited indole-3-acetic acid (IAA) biosynthesis in rice and tomato. Here, we found that AOA induced auxin over-accumulation in Arabidopsis. The results suggest that auxin-related metabolic pathways are divergent among these plant species.

DOI： 10.1271/bbb.100463

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Language：Japanese   Publisher：公益社団法人 日本農芸化学会  

DOI： 10.1271/kagakutoseibutsu.48.485

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Despite its importance in plant growth and development, the auxin biosynthetic pathway has remained elusive. In this study, we analyzed hormone series transcriptome data from AtGenExpress in Arabidopsis and found that aminoethoxyvinylglycine (AVG) had the strongest anti-auxin activity. We also identified other effective compounds such as l-amino-oxyphenylpropionic acid (AOPP) through additional screening. These inhibitors shared characteristics in that they inhibited pyridoxal enzymes and/or aminotransferases. They reduced endogenous IAA levels in both monocots and dicots. l-AOPP inhibited root development of Arabidopsis in main root elongation, gravitropism, root skewing and root hair formation. This inhibition was generally recovered after exogenous IAA treatment, and the recovery was almost completely to the level of non-inhibited seedlings. The compounds inhibited conversion from tryptophan to indole-3-pyruvic acid in enzyme extracts from Arabidopsis and wheat. Our data collectively suggest that the inhibitors directly blocked auxin biosynthesis, and that the major target site was tryptophan aminotransferase. This enzyme probably makes up one of the major biosynthesis pathways conserved among higher plants. Each inhibitor, however, demonstrated a different action spectrum in shoot and root of rice and tomato, indicating diversity in biosynthesis pathways between organs and species. Our results provide novel insights into auxin biosynthesis and action, and uncover structural characteristics of auxin biosynthesis inhibitors.

DOI： 10.1093/pcp/pcq032

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Plants possess many metabolic genes for the production of a wide variety of phytochemicals in a tissue-specific manner. However, the metabolic systems behind the diversity and tissue-dependent regulation still remain unknown due to incomplete characterization of phytochemicals produced in a single plant species. Thus, having a metabolome dataset in addition to the genome and transcriptome information resources would enrich our knowledge of plant secondary metabolism. Here we analyzed phytochemical accumulation during development of the model plant Arabidopsis (Arabidopsis thaliana) using liquid chromatography-mass spectrometry in samples covering many growth stages and organs. We also obtained tandem mass spectrometry spectral tags of many metabolites as a resource for elucidation of metabolite structure. These are part of the AtMetExpress metabolite accumulation atlas. Based on the dataset, we detected 1,589 metabolite signals from which the structures of 167 metabolites were elucidated. The integrated analyses with transcriptome data demonstrated that Arabidopsis produces various phytochemicals in a highly tissue-specific manner, which often accompanies the expression of key biosynthesis-related genes. We also found that a set of biosynthesis-related genes is coordinately expressed among the tissues. These data suggested that the simple mode of regulation, transcript to metabolite, is an origin of the dynamics and diversity of plant secondary metabolism.

DOI： 10.1104/pp.109.148031

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Language：English   Publishing type：Part of collection (book)   Publisher：Nova Science Publishers, Inc.  

An international research effort, the AtGenExpress consortium was organized to make an important contribution to science community by collecting large-scale transcriptome data sets. The consortium detailed the developmental process (Schmid et al., 2005), stress responses (Kilian et al., 2007), hormone responses (Goda et al., 2008) and etc. in Arabidopsis. In this manuscript, we will summarize activities in AtGenExpress project. The data have been distributed from primary databases, such as, TAIR, GEO or NASCArrays. The data have been utilized actively to develop secondary web-based databases, data browsers (digital northern analysis tools), coexpression databases, or sample-wise correlation analysis tools. These web-tools are further facilitating plant science. We will also introduce data utilization in current plant biology especially in the area of plant hormone research.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Strigolactones (SLs) have recently been found to regulate shoot branching, but the functions of SLs at other stages of development and the regulation of SL-related gene expression are mostly unknown in Arabidopsis. In this study, we performed real-time reverse transcription-PCR (RT-PCR) and microarray analysis using wild-type plants and SL-deficient/insensitive mutants to understand the molecular mechanisms underlying SL biosynthesis and signaling. We found that there is responsiveness to SL in the gene expression of Arabidopsis seedlings, which includes feedback regulation of two carotenoid cleavage dioxygenase genes. Microarray analysis revealed that exogenously applied SL regulated the expression of several genes, including light signaling-related genes and auxin-inducible genes. We also found that MORE AXILLARY GROWTH (MAX)2 plays an important role in the expression of SL-regulated genes. Our data support previous studies indicating that SL might function at the seedling stage. Analysis of SL-responsive and MAX2 downstream gene candidates provides new opportunities to broaden our understanding of SL signaling.

DOI： 10.1271/bbb.90443

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

We analyzed global gene expression in Arabidopsis in response to various hormones and in related experiments as part of the AtGenExpress project. The experimental agents included seven basic phytohormones (auxin, cytokinin, gibberellin, brassinosteroid, abscisic acid, jasmonate and ethylene) and their inhibitors. In addition, gene expression was investigated in hormone-related mutants and during seed germination and sulfate starvation. Hormone-inducible genes were identified from the hormone response data. The effects of each hormone and the relevance of the gene lists were verified by comparing expression profiles for the hormone treatments and related experiments using Pearson's correlation coefficient. This approach was also used to analyze the relationships among expression profiles for hormone responses and those included in the AtGenExpress stress-response data set. The expected correlations were observed, indicating that this approach is useful to monitor the hormonal status in the stress-related samples. Global interactions among hormones-inducible genes were analyzed in a pairwise fashion, and several known and novel hormone interactions were detected. Genome-wide transcriptional gene-to-gene correlations, analyzed by hierarchical cluster analysis (HCA), indicated that our data set is useful for identification of clusters of co-expressed genes, and to predict the functions of unknown genes, even if a gene's function is not directly related to the experiments included in AtGenExpress. Our data are available online from AtGenExpressJapan; the results of genome-wide HCA are available from PRIMe. The data set presented here will be a versatile resource for future hormone studies, and constitutes a reference for genome-wide gene expression in Arabidopsis.

DOI： 10.1111/j.1365-313X.2008.03510.x

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PRIMe (http://prime.psc.riken.jp/), the Platform for RIKEN Metabolomics, is a Web site that has been designed and implemented to support research and analysis workflows ranging from metabolome to transcriptome analysis. The site provides access to a growing collection of standardized measurements of metabolites obtained by using NMR, GC-MS, LC-MS, and CE-MS, and metabolomics tools that support related analyses (SpinAssign for the identification of metabolites by means of NMR, KNApSAcK for searches within metabolite databases). In addition, the transcriptomics tools provide Correlated Gene Search, and Cluster Cutting for the analysis of mRNA expression. Use of the tools and database can contribute to the analysis of biological events at the levels of metabolites and gene expression, and we describe one example of such an analysis for Arabidopsis thaliana using the batch-learning self-organizing map (BL-SOM), which is provided via the Web site.

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Other Link： https://dblp.uni-trier.de/db/journals/isb/isb8.html#AkiyamaCYSTSHSKS08

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GAMYB is a component of gibberellin (GA) signaling in cereal aleurone cells, and has an important role in flower development. However, it is unclear how GAMYB function is regulated. We examined the involvement of a microRNA, miR159, in the regulation of GAMYB expression in cereal aleurone cells and flower development. In aleurone cells, no miR159 expression was observed with or without GA treatment, suggesting that miR159 is not involved in the regulation of GAMYB and GAMYB-like genes in this tissue. miR159 was expressed in tissues other than aleurone, and miR159 over-expressors showed similar but more severe phenotypes than the gamyb mutant. GAMYB and GAMYB-like genes are co-expressed with miR159 in anthers, and the mRNA levels for GAMYB and GAMYB-like genes are negatively correlated with miR159 levels during anther development. Thus, OsGAMYB and OsGAMYB-like genes are regulated by miR159 in flowers. A microarray analysis revealed that OsGAMYB and its upstream regulator SLR1 are involved in the regulation of almost all GA-mediated gene expression in rice aleurone cells. Moreover, different sets of genes are regulated by GAMYB in aleurone cells and anthers. GAMYB binds directly to promoter regions of its target genes in anthers as well as aleurone cells. Based on these observations, we suggest that the regulation of GAMYB expression and GAMYB function are different in aleurone cells and flowers in rice.

DOI： 10.1111/j.1365-313X.2006.02795.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

We examined whether auxin/indole-3-acetic acid (Aux/IAA) proteins, which are key players in auxin-signal transduction, are involved in brassinosteroid (BR) responses. iaa7/axr2-1 and iaa17/axr3-3 mutants showed aberrant BR sensitivity and aberrant BR-induced gene expression in an organ-dependent manner. Two auxin inhibitors were tested in terms of BR responses. Yokonolide B inhibited BR responses, whereas p-chlorophenoxyisobutyric acid did not inhibit BR responses. DNA microarray analysis revealed that 108 genes were up-regulated, while only eight genes were down-regulated in iaa7. Among the genes that were up- or down-regulated in axr2, 22% were brassinolide-inducible genes, 20% were auxin-inducible genes, and the majority were sensitive neither to BR nor to auxin. An inhibitor of BR biosynthesis, brassinazole, inhibited auxin induction of the DR5-GUS gene, which consists of a synthetic auxin-response element, a minimum promoter, and a beta-glucuronidase. These results suggest that Aux/IAA proteins function in auxin- and BR-signaling pathways, and that IAA proteins function as the signaling components modulating BR sensitivity in a manner dependent on organ type.

DOI： 10.1111/j.1365-313X.2005.02582.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Plant hormone abscisic acid (ABA) is an important factor for conferring drought stress resistance on plants. Therefore, small molecules that regulate ABA levels in plants can be useful both for investigating functions of ABA and for developing new plant growth regulators. Abscisic acid (ABA) catabolism in plants is primarily regulated by ABA 8'-hydroxylase, which is a cytochrome P450 (P450). We tested known P450 inhibitors containing a triazole group and found that uniconazole-P inhibited ABA catabolism in cultured tobacco Bright Yellow-2 cells. In a structure-activity study of uniconazole, we found a more effective ABA catabolic inhibitor (diniconazole) than uniconazole-P. Diniconazole, a fungicide, acted as a potent competitive inhibitor of recombinant Arabidopsis ABA 8'-hydroxylase, CYP707A3, in an in vitro assay. Diniconazole-treated plants retained a higher ABA content and higher transcription levels of ABA response genes during rehydration than did untreated plants and were more drought stress tolerant than untreated plants. These results strongly suggest that ABA catabolic inhibitors that target ABA 8'-hydroxylase can regulate the ABA content of plants and conferred drought stress resistance on plants. The optical resolution of diniconazole revealed that the S-form isomer, which is a weak fungicidal isomer, was more active as an ABA catabolic inhibitor than was the R-form isomer. (c) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2005.04.036

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Active brassinosteroids (BRs), such as brassinolide (BL) and castasterone (CS), are growth-promoting plant hormones. An Arabidopsis cytochrome P450 monooxygenase (CYP734A1, formerly CYP72B1), encoded by the BAS1 gene, inactivates BRs and modulates photomorphogenesis. BAS1 was identified as the overexpressed gene responsible for a dominant, BR-deficient mutant, bas1-D. This mutant was isolated in an activation-tagged screen designed to identify redundant genes that might not be identified in classic loss-of-function screens. Here we report the isolation of a second activation-tagged mutant with a BR-deficient phenotype. The mutant phenotype is caused by the overexpression of SOB7 (CYP72C1), a homolog of BAS1. We generated single and double null-mutants of BAS1 and SOB7 to test the hypothesis that these two genes act redundantly to modulate photomorphogenesis. BAS1 and SOB7 act redundantly with respect to light promotion of cotyledon expansion, repression of hypocotyl elongation and flowering time in addition to other phenotypes not regulated by light. We also provide biochemical evidence to suggest that BAS1 and SOB7 act redundantly to reduce the level of active BRs, but have unique mechanisms. Overexpression of SOB7 results in a dramatic reduction in endogenous CS levels, and although single null-mutants of BAS1 and SOB7 have the same level of CS as the wild type, the double null-mutant has twice the amount. Application of BL to overexpression lines of BAS1 or SOB7 results in enhanced metabolism of BL, though only BAS1 overexpression lines confer enhanced conversion to 26-OHBL, suggesting that SOB7 and BAS1 convert BL and CS into unique products.

DOI： 10.1111/j.1365-313X.2005.02358.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

To gain insight into the brassinosteroid (BR) signaling pathway, the expression of BR-regulated genes was analysed in the BR-signaling mutant br-insensitive 1 (bri1), and in the presence of a protein kinase inhibitor, staurosporin. BR-regulated genes were classified based on the results. This classification will perhaps prove useful in BR-signalling studies using BR-regulated genes as molecular markers.

DOI： 10.1271/bbb.68.1605

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We screened steroid derivatives and found that spironolactone, an inhibitor of both 17beta-hydroxy steroid dehydrogenase (17beta-HSD) and aldosterone receptor, is an inhibitor of phytohormone brassinosteroid (BR) action in plants. Under both dark and light growing conditions, spironolactone induced morphological changes in Arabidopsis, characteristic of brassinosteroid-deficient mutants. Spironolactone-treated plants were also nearly restored to the wild-type phenotype by treatment with additional BRs. In the spironolactone-treated Arabidopsis, the CPD gene in the BR biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR-deficiency. Spironolactone-treated tobacco plants grown in the dark showed expression of light-regulated genes as was observed in the deficient mutant. These data suggest that spironolactone inhibits brassinosteroid action probably due to the blockage of biosynthesis and exerts its activity against plants. Thus, spironolactone, in conjunction with brassinosteroid-deficient mutants, can be used to clarify the function of BRs in plants and characterize mutants. The spironolactone action site was also investigated by feeding BR biosynthesis intermediates to Arabidopsis grown in the dark, and the results are discussed. (C) 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jsbmb.2004.01.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

The transcriptional factor DREB/CBF (dehydration-responsive element/C-repeat-binding) specifically interacts with the dehydration-responsive element (DRE)/C-repeat (CRT) cis-acting element (A/GCCGAC) and controls the expression of many stress-inducible genes in Arabidopsis. Transgenic plants overexpressing DREB1A showed activated expression of many stress-inducible genes and improved tolerance to not only drought, salinity, and freezing but also growth retardation. We searched for downstream genes in transgenic plants overexpressing DREB1A using the full-length cDNA microarray and Affymetrix GeneChip array. We confirmed candidate genes selected by array analyses using RNA gel blot and identified 38 genes as the DREB1A downstream genes, including 20 unreported new downstream genes. Many of the products of these genes were proteins known to function against stress and were probably responsible for the stress tolerance of the transgenic plants. The downstream genes also included genes for protein factors involved in further regulation of signal transduction and gene expression in response to stress. The identified genes were classified into direct downstream genes of DREB1A and the others based on their expression patterns in response to cold stress. We also searched for conserved sequences in the promoter regions of the direct downstream genes and found A/GCCGACNT in their promoter regions from -51 to -450 as a consensus DRE. The recombinant DREB1A protein bound to A/GCCGACNT more efficiently than to A/GCCGACNA/G/C.

DOI： 10.1111/j.1365-313X.2004.02100.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Chloroplast division is mediated by the coordinated action of a prokaryote-derived division system(s) and a host eukaryote-derived membrane fission system(s). The evolutionary conserved prokaryote-derived system comprises several nucleus-encoded proteins, two of which are thought to control division site placement at the midpoint of the organelle: a stromal ATPase MinD and a topological specificity factor MinE. Here, we show that arc11, one of 12 recessive accumulation and replication of chloroplasts (arc) mutants in Arabidopsis, contains highly elongated and multiple-arrayed chloroplasts in developing green tissues. Genomic sequence analysis revealed that arc11 contains a missense mutation in alpha-helix 11 of the chloroplast-targeted AtMinD1 changing an Ala at position 296 to Gly (A296G). Introduction of wild-type AtMinD1 restores the chloroplast division defects of arc11 and quantitative RT-PCR analysis showed that the degree of complementation was highly dependent on transgene expression levels. Overexpression of the mutant ARC11/AtMinD1 in transgenic plants results in the inhibition of chloroplast division, showing that the mutant protein has retained its division inhibition activity. However, in contrast to the defined and punctate intraplastidic localization patterns of an AtMinD1-YFP fusion protein, the single A296G point mutation in ARC11/AtMinD1 results in aberrant localization patterns inside chloroplasts. We further show that AtMinD1 is capable of forming homodimers and that this dimerization capacity is abolished by the A296G mutation in ARC11/AtMinD1. Our data show that arc11 is a loss-of-function mutant of AtMinD1 and suggest that the formation of functional AtMinD1 homodimers is paramount for appropriate AtMinD1 localization, ultimately ensuring correct division machinery placement and chloroplast division in plants.

DOI： 10.1242/jcs.01092

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

When brassinosteroid (BR)-deficient mutant (det2) or wild-type (WT) seedlings were treated with brassinolide (BL), the most active BR, for 3 h, the abundance of PIN4 and PIN7 transcripts decreased, and there were fewer PIN4 and PIN7 transcripts in det2 than in the WT. This suggests that BL selectively regulates the PIN gene in a complex manner.

DOI： 10.1271/bbb.68.952

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Although numerous physiological studies have addressed the interactions between brassinosteroids and auxins, little is known about the underlying molecular mechanisms. Using an Affymetrix GeneChip representing approximately 8,300 Arabidopsis genes, we studied comprehensive transcript profiles over 24 h in response to indole-3-acetic acid (IAA) and brassinolide (BL). We identified 409 genes as BL inducible, 276 genes as IAA inducible, and 637 genes in total. These two hormones regulated only 48 genes in common, suggesting that most of the actions of each hormone are mediated by gene expression that is unique to each. IAA-up-regulated genes were enriched in genes regulated in common. They were induced quickly by IAA and more slowly by BL, suggesting divergent physiological roles. Many were early auxin-inducible genes and their homologs, namely SAUR, GH3, and IAA. The comprehensive comparison also identified IAA- and BL-specific genes, which should help to elucidate the specific actions of each hormone. The identified genes were classified using hierarchical clustering based on the similarity of their responses to the two hormones. Gene classification also allowed us to analyze the frequency of cis-elements. The TGTCTC element, a core element of the previously reported auxin response element, was not enriched in genes specifically regulated by IAA but was enriched in the 5'-flanking region of genes up-regulated by both IAA and BL. Such gene classification should be useful for predicting the functions of unknown genes, to understand the roles of these two hormones, and the promoter analysis should provide insight into the interaction of transcriptional regulation by the two hormones.

DOI： 10.1104/pp.103.034736

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DOI： 10.18978/jscrp.39.2_189

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DOI： 10.14841/jspp.2004.0.340.0

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

DOI： 10.18978/jscrp.39.1_17

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Despite numerous physiological studies addressing the interactions between brassinosteroids (BRs) and auxins, little is known about the underlying molecular mechanisms. We studied the expression of IAA5 and IAA19 in response to treatment with indole acetic acid (IAA) or brassinolide (BL), the most active BR. Exogenous IAA induced these genes quickly and transiently, whereas exogenous BL induced them gradually and continuously. We also found that a fusion of DR5, a synthetic auxin response element, with the GUS (beta-glucuronidase) gene was induced with similar kinetics to those of the IAA5 and IAA19 genes in response to both IAA and BL treatment of transgenic plants. These results suggest that the IAA genes are induced by BL, at least in part, via the activation of the auxin response element. Endogenous IAA levels per gram fresh weight did not increase when seedlings of Arabidopsis wild type (WT) or the BR-deficient mutant det2 were treated with BL. Furthermore, the levels of IAA transcripts were lower in the det2 mutant than in the WT, even though endogenous IAA levels per gram fresh weight were higher in the det2 mutant than in the WT. In conclusion, the lack of evidence for auxin-mediated activation of early auxin-inducible genes in response to BL suggests that the BR and auxin signaling pathways independently activate the transcriptional system of the IAA and DR5-GUS genes.

DOI： 10.1104/pp.103.030031

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Active brassinosteroids, such as brassinolide (BL) and castasterone, are growth promoting plant hormones. An Arabidopsis cytochrome P450 monooxygenase encoded by CYP72B1 has been implicated in brassinosteroid catabolism as well as photomorphogenesis. We expressed CYP72B1 in yeast, coupled with brassinosteroid feeding, and established the biochemical function to be the hydroxylation of BL and castasterone, to give 26-hydroxybrassinolide and 26-hydroxycastasterone, respectively. Brassinosteroid feeding experiments with wild-type Arabidopsis, a CYP72B1 null mutant, and a CYP72B1 overexpression line demonstrated that carbon 26 hydroxylation of active brassinosteroids is an endogenous function of CYP72B1. Seedling growth assays demonstrated that 26-hydroxybrassinolide is an inactive brassinosteroid. Genetic and physiological analysis of the hypocotyl response to exogenous BL and varying intensities of white and monochromatic light suggested that CYP72B1 modulates photomorphogenesis primarily through far-red light and to a lesser extent through blue-and red-light pathways. CYP72B1 transcript accumulation in dark-grown seedlings was organ specific and down-regulated after 1 h of illumination in dim white, red, and blue light, but not far-red light. CYP72B1 translational fusions with the beta-glucuronidase reporter gene demonstrated that protein levels increased in the hypocotyl elongation zone when shifted from the dark to far-red light, but not blue or red light. We propose a model in which Arabidopsis seedling development switches from dark-grown development (skotomorphogenesis) to light-grown development (photomorphogenesis). in part by rapid modulation of brassinosteroid sensitivity and levels. CYP72B1 provides an intersection between the light and brassinosteroid pathways mainly by far-red-light-dependent modulation of brassinosteroid levels.

DOI： 10.1104/pp.103.030882

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

When exogenous chemicals allow rapid, conditional, reversible, selective, and dose-dependent control of biological functions, they act like conditional mutations, either inducing or suppressing the formation of a specific phenotype of interest. Exploration of the small molecules that induce the brassinosteroid (BR) deficient-like phenotype in Arabidopsis led us to identify brassinazole as the first candidate for a BR biosynthesis inhibitor. Brassinazole treatment reduced BR content in plant cells. Investigation of target site(s) of brassinazole revealed that the compound directly binds to the DWF4 protein, a cytochrome P450 monooxygenase that catalyzes 22-hydroxylation of the side chain of BRs. These results suggest that brassinazole is a BR biosynthesis inhibitor. There are currently at least two BR biosynthesis inhibitors that act like conditional mutations in BR biosynthesis. They allow the investigation of the functions of BRs in a variety of plant species. Application of BR biosynthesis inhibitors to a standard genetic screen to identify mutants that confer resistance to these inhibitors allowed the identification of new components working in BR signal transduction. This method has advantages over mutant screens using BR-deficient mutants as a background. Development of chemicals that induce phenotypes of interest is now emerging as a useful way to study biological systems in plants and this would be a complement to classical biochemical and genetic methods.

DOI： 10.1007/s00344-003-0065-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Limited information is available concerning the interactions between the brassinosteroid (BR) and auxin signaling pathways. The expression pattern of the SAUR-AC1 gene, an early auxin-inducible gene in Arabidopsis, was studied in response to brassinolide (BL), in the presence of a BR-biosynthesis inhibitor, in a BR-deficient mutant, and in combination with auxin. The results suggested that the SAUR-AC1 gene is regulated by BRs independently of auxin levels, and that it is important in BR-mediated elongation. The axr1 (auxin insensitive 1) mutant was less sensitive to BL-induced elongation and BL-induced SAUR-AC1 expression, suggesting that a ubiquitin ligase-mediated system is involved in BR-mediated elongation. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/S0014-5793(03)00945-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Plant Growth Regulation Society of America  

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Triadimefon (Bayleton®), a widely used triazole-type fungicide, affects gibberellin (GA) biosynthesis and 14α-demethylase in sterol biosynthesis. The present study revealed that the phenotype of Arabidopsis treated with triadimefon resembled that of a brassinosteroid (BR)-biosynthesis mutant, and that the pheno-type was rescued by brassinolide (BL), the most active BR, partly rescued by GA, and fully rescued by the co-application of BL and GA, suggesting that triadimefon affects both BR and GA biosynthesis. The target sites of triadimefon were investigated using a rescue experiment, feeding triadimefon-treated Arabidopsis BR-biosynthesis intermediates, and a binding assay to expressed DWF4 protein, which is reported to be involved in the BR-biosynthesis pathway. The binding assay indicated that the dissociation constant for triadimefon was in good agreement with the activity in an in planta assay. In the triadimefon-treated Arabidopsis cells, the CPD gene in the BR-biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR deficiency. These results strongly suggest that triadimefon inhibits the reaction catalysed by DWF4 protein and induces BR deficiency in plants. As triadimefon treatment has proved to be beneficial to plants, this result suggests that BR-biosynthesis inhibitors can be applied to crops.

DOI： 10.1042/BJ20020835

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Blue flowers generally contain 3',5'-hydroxylated anthocyanins (delphinidin derivatives) as pigments, which are formed only in the presence of flavonoid 3',5'hydroxylases (F3'5'H). Heterologous expression of a F3'5'H gene therefore provides an opportunity to produce novel blue flowers for a number of ornamental plants missing blue flowering varieties. However, our previous study indicated difficulties in obtaining good accumulation of delphinidin derivatives in plants expressing F3'5'H. Here we report the isolation of a putative F3'5'H cDNA (Ka1) from canterbury bells (Campanula medium) and its expression in tobacco. Surprisingly, compared with other F3'5'H cDNAs, Kal encoded a protein with a unique primary structure that conferred high competence in the accumulation of delphinidin derivatives (up to 99% of total anthocyanins) and produced novel purple flowers. These results suggest that, among F3'5'H cDNAs, Ka1 is the best genetic resource for the creation of fine blue flowers by genetic engineering.

DOI： 10.1271/bbb.67.161

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. There is only limited information on where BRs are synthesized and used. We studied the organ specificity of BR biosynthesis in Arabidopsis, using two different approaches: We analyzed the expression of BR-related genes using real-time quantitative reverse transcriptase-polymerase chain reaction, and analyzed endogenous BRs using gas chromatography-mass spectrometry. Before starting this study, we cloned the second BR-6-oxidase (BR6ox2) gene from Arabidopsis and found that the encoded enzyme has the same substrate specificity as the enzyme encoded by the previously isolated 6-oxidase gene (BR6ox1) of Arabidopsis. Endogenous BRs and the expression of BR-related genes were detected in all organs tested. The highest level of endogenous BRs and the highest expression of the BR6ox1, BR6ox2, and DWF4 genes were observed in apical shoots, which contain actively developing tissues. These genes are important in BR biosynthesis because they encode the rate-limiting or farthest downstream enzyme in the BR biosynthesis pathway. The second highest level of endogenous BRs and expression of BR6ox1 and DWF4 were observed in siliques, which contains actively developing embryos and seeds. These findings indicate that BRs are synthesized in all organs tested, but are most actively synthesized in young, actively developing organs. In contrast, synthesis was limited in mature organs. Our observations are consistent with the idea that BRs function as the growth-promoting hormone in plants.

DOI： 10.1104/pp.013029

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

The plant hormone, auxin, regulates many aspects of growth and development. Despite its importance, the molecular mechanisms underlying the action of auxin are largely unknown. To gain a more comprehensive understanding of the primary responses to auxin, we analyzed the expression of genes in Arabidopsis seedlings treated with indole-3-acetic acid (IAA) for 15 min. We identified a single gene that is downregulated early, and 29 genes that are upregulated early. Several types of typical transcription factors are identified as early upregulated genes, suggesting that auxin signals are mediated by a master set of diverse transcriptional regulators. Of the genes that responded to auxin, the expression of the homeobox gene, HAT2 , was induced rapidly. Furthermore, we show that the expression of HAT2 is induced by auxin, but not by other phytohormones. To analyze the function of HAT2 in the plant's response to auxin, we generated 35S::HAT2 transgenic plants. These produced long hypocotyls, epinastic cotyledons, long petioles, and small leaves, which are characteristic of the phenotypes of the auxin-overproducing mutants, superroot1 (sur1) and superroot2 (sur2) . On the other hand, 35S::HAT2 plants showed reduced lateral root elongation, and reduced auxin sensitivity compared to wild-type plants. Together with the results of RNA blotting and biochemical analyses, these findings suggest that HAT2 plays opposite roles in the shoot and root tissues in regulating auxin-mediated morphogenesis.

DOI： 10.1046/j.1365-313X.2002.01488.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. Although insights into the functions of BRs have been provided by recent studies of biosynthesis and sensitivity mutants, the mode of action of BRs is poorly understood. With the use of DNA microarray analysis, we identified BR-regulated genes in the wild type (WT; Columbia) of Arabidopsis and in the BR-deficient mutant, det2. BR-regulated genes generally responded more potently in the det2 mutant than in the WT, and they showed only limited response in a BR-insensitive mutant, bri1. A small group of genes showed stronger responses in the WT than in the det2. Exposure of plants to brassinolide and brassinazole, which is a specific inhibitor of BR biosynthesis, elicited opposite effects on gene expression of the identified genes. The list of BR-regulated genes is constituted of transcription factor genes including the phytochrome-interacting factor 3, auxin-related genes, P450 genes, and genes implicated in cell elongation and cell wall organization. The results presented here provide comprehensive view of the physiological functions of BRs using BR-regulated genes as molecular markers. The list of BR-regulated genes will be useful in the characterization of new mutants and new growth-regulating compounds that are associated with BR function.

DOI： 10.1104/pp.011254

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Molecular genetic and physiological studies on brassinosteroid (BR)-related mutants of dicot plants have revealed that BRs play important roles in normal plant growth and development. However, little is known about the function of BR in monocots (grasses), except for the phenotypic analysis of a rice mutant partially insensitive to BR signaling. To investigate the function of BR in monocots, we identified and characterized BR-deficient mutants of rice, BR-deficient dwarf1 (brd1). The brd1 mutants showed a range of abnormalities in organ development and growth, the most striking of which were defects in the elongation of the stem and leaves. Light microscopic observations revealed that this abnormality was primarily owing to a failure in the organization and polar elongation of the leaf and stem cells. The accumulation profile of BR compounds in the brd1 mutants suggested that these plants may be deficient in the activity of BR C-6 oxidase. Therefore, we cloned a rice gene, OsDWARF, which has a high sequence similarity to the tomato C-6 oxidase gene, DWARF. Introduction of the wild-type OsDWARF gene into brd1 rescued the abnormal phenotype of the mutants. The OsDWARF gene was expressed at a low level in all of the examined tissues, with preferential expression in the leaf sheath, and the expression was negatively regulated by brassinolide treatment. On the basis of these findings, we discuss the biological function of BRs in rice plants.

DOI： 10.1046/j.1365-313X.2002.01438.x

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

DOI： 10.18978/jscrp.37.2_227_5

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

DOI： 10.18978/jscrp.37.2_227_3

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

DOI： 10.18978/jscrp.37.2_229_4

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

DOI： 10.18978/jscrp.37.2_178

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Brassinazole, a synthetic chemical developed in our laboratory, is a triazole-type brassinosteroid biosynthesis inhibitor that induces dwarfism in various plant species. The target sites of brassinazole were investigated by chemical analyses of endogenous brassinosteroids (BRs) in brassinazole-treated Catharanthus roseus cells. The levels of castasterone and brassinolide in brassinazole-treated plant cells were less than 6% of the levels in untreated cells. In contrast, campestanol and 6-oxocampestanol levels were increased, and levels of BR intermediates with hydroxy groups on the side chains were reduced, suggesting that brassinazole treatment reduced BR levels by inhibiting the hydroxylation of the C-22 position. DWF4, which is an Arabidopsis thaliana cytochrome P450 isolated as a putative steroid 22-hydroxylase, was expressed in Escherichia coli, and the binding affinity of brassinazole and its derivatives to the recombinant DWF4 were analyzed. Among several triazole derivatives, brassinazole had both the highest binding affinity to DWF4 and the highest growth inhibitory activity. The binding affinity and the activity for inhibiting hypocotyl growth were well correlated among the derivatives. In brassinazole-treated A. thaliana, the CPD gene involved in BR biosynthesis was induced within 3 h, most likely because of feedback activation caused by the reduced levels of active BRs. These results indicate that brassinazole inhibits the hydroxylation of the C-22 position of the side chain in BRs by direct binding to DWF4 and that DWF4 catalyzes this hydroxylation reaction.

DOI： 10.1074/jbc.M103524200

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Flavonoid-3',5'-hydroxylase (F3'5'H) is the key enzyme in the synthesis of 3',5'-hydroxylated anthocyanins, which are generally required for the expression of blue or purple flower color. It has been predicted that the introduction of this enzyme into a plant species that lacks it would enable the production of blue or purple flowers by altering the anthocyanin composition. We present here the results of the genetic engineering of petunia flower color, pigmentation patterns and anthocyanin composition with sense or antisense constructs of the F3'5'H gene under the control of the CaMV 35S promoter. When sense constructs were introduced into pink flower varieties that are deficient in the enzyme, transgenic plants showed flower color changes from pink to magenta along with changes in anthocyanin composition. Some transgenic plants showed novel pigmentation patterns, e.g. a star-shaped pattern. When sense constructs were introduced into blue flower petunia varieties, the flower color of the transgenic plants changed from deep blue to pale blue or even pale pink. Pigment composition analysis of the transgenic plants suggested that the F3'5'H transgene not only created or inhibited the biosynthetic pathway to 3',5'-hydroxylated anthocyanins but switched the pathway to 3',5'-hydroxylated or 3'-hydroxylated anthocyanins.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

DOI： 10.18978/jscrp.36.2_231_3

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Inhibitors of phytohormone have proven to be useful tools for understanding hormonal function. Recently, brassinolide has been designated as a new class of phytohormone based on the physiological responses of brassinolide-deficient mutants. However, information on other roles of this hormone is limited because studies have been confined to mutants in a limited number of plant species. Therefore, specific inhibitors of brassinosteroid biosynthesis would be valuable tools for investigating their roles at various stages of plant development, such as germination, leaf expansion and flowering. Recent advances in developing brassinosteroid biosynthesis inhibitor, brassinazole (Brz), have shown the importance of brassinosteroids in broad aspects of plant growth and development. This inhibitor induced drastic morphological changes in treated plants, almost identical to those found in brassinosteroid-deficient mutants. The normal phenotype of inhibitor-treated plants could be recovered by the addition of brassinolide. This result suggests that brassinosteroids are essential for plant growth, and that specific brassinosteroid biosynthesis inhibitors can be used to clarify the functions of brassinosteroids in plants when used as a complement to brassinosteroid-deficient mutants. The action site of brassinazole was an oxidative processes from 6-oxo-campestanol to teasterone.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Flavonoid-3',5'-hydroxylase (F3'5'H), a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3',5'-hydroxylated anthocyanins, which are generally required for blue or purple flowers. A full-length cDNA, TG1, was isolated from prairie gentian by heterologous hybridization with a petunia cDNA, AK14, which encodes F3'5'H, To investigate the in vivo function of TG1 and AK14, they were subcloned into a plant expression vector and expressed under the control of the CaMV35S promoter in transgenic tobacco or petunia, both of which originally lack the enzyme, Transgenic petunia plants had a dramatic change in flower color from pink to magenta with a high content of 3',5'-hydroxylated anthocyanins. In contrast, transgenic tobacco plants had minimal color change with at most 35% 3',5'-hydroxylated anthocyanin content. These results indicate that the products of TG1 and AK14 have F3'5'H activity in planta and that interspecific gene transfer alters anthocyanin pigment synthesis. The difference in apparent F3'5'H activity between tobacco and petunia is discussed. (C) 1999 Federation of European Biochemical Societies.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TRANSACTION PERIOD CONSORTIUM  

We present a method to identify cDNA clones of a cytochrome P450 enzyme. Flavonoid-3', 5'-Hydroxylase (F3',5'H), the key enzyme for the expression of blue Or purple color in flowers, was cloned as an example. We have made a catalog of cDNA fragments encoding conserved regions of P450s for petunia (Petunia hybrida Vilm.) petals. Single specific primers were designed for these cDNA sequences and RT-PCRs were performed with cDNA templates. The amplified bands were tested for linkage to the delphinidin producing phenotype using a backcrossed population that had been prepared to have a genetic background of cyanidin-type petunia but segregated for the hydroxylation at the B-ring of anthocyanin. We were successful in amplifying a cDNA fragment that has close linkage to the F3',5'H gene. A full length cDNA clone of the F3',5'H gene was isolated using the amplified fragment as a probe.

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Language：English   Publisher：Japanese Society for Plant Cell and Molecular Biology  

DOI： 10.5511/plantbiotechnology.14.175

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Other Link： https://jlc.jst.go.jp/DN/JALC/00084371171?from=CiNii

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The relationship between the accumulation of Chl and the apoproteins of the light-harvesting Chl a/b-protein complex of PS II (LHCII) during the greening of cucumber cotyledons was studied. LHCII apoproteins were not detected in etiolated cotyledons. Upon illumination, Chl a was formed as a result of photoconversion of protochlorophyllide (Pchlide) which had accumulated in the dark. During the lag period that preceded the accumulation of Chl, a small amount of LHCII apoproteins appeared. The amount of LHCII apoproteins increased with increases in levels of Chl b, though somewhat more rapidly during the first 10 h of greening. Treatment with benzyladenine (BA) or levulinic acid (LA) was used to vary the supply of Chl a for apoproteins by promoting or inhibiting the synthesis of Chl a, respectively. LA decreased but BA increased the rate of accumulation of Chl b and LHCII apoproteins. Only small amounts of Chl b and LHCII apoproteins were formed under intermittent illumination. However, in the presence of chloramphenicol (CAP), which inhibits the synthesis of plastome-coded proteins including apoproteins of the P700-Chl a-protein complex (CP1) and a Chl a-protein complex of PS II (CPa), we observed the accumulation of Chl b and LHCII apoproteins, both of which are of nuclear origin. During incubation in the dark after intermittent exposure to light, CAP alone allowed neither destruction nor accumulation of Chl b and LHCII apoproteins, but it did enhance the effect of CaCl2 in inducing both Chl b and these apoproteins. These results can be explained by assuming that apoproteins of CP1 and CPa have a higher affinity for Chl a than do LHCII apoproteins. When the availability of Chl a is limited, these apoproteins compete with one another for Chl a, with the resultant preferential formation of CP1 and CPa. However, when the supply of Chl a becomes large enough for saturation of apoproteins of CP1 and CPa, some of the Chl a is incorporated into LHCII apoproteins either directly or after conversion to Chl b. Thus, the formation of different Chl-protein complexes (CPs) is regulated by the relative rates of synthesis of Chl a and apoproteins and by differential affinities of the apoproteins for Chl a. © 1990. The Japanese Society of Plant Physiologists (JSPP).

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Language：Japanese   Publisher：Pesticide Science Society of Japan  

DOI： 10.1584/jpestics.w19-19

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Other Link： https://www.jstage.jst.go.jp/article/jjpestics/44/1/44_W19-19/_pdf

Language：Japanese  

DOI： 10.1584/jpestics.w19-07

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Language：Japanese   Publisher：日本農芸化学会 ; 1962-  

DOI： 10.1271/kagakutoseibutsu.54.408

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Language：Japanese   Publisher：一般社団法人 日本土壌肥料学会  

DOI： 10.20710/dohikouen.62.0_281_2

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Language：Japanese   Publisher：一般社団法人 日本土壌肥料学会  

DOI： 10.20710/dohikouen.62.0_44_2

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Language：Japanese   Publisher：一般社団法人 植物化学調節学会  

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Language：Japanese   Publisher：The Janapese Society for Chemical Regulation of Plants  

DOI： 10.18978/jscrpanb.50.Supplement_42

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Language：Japanese   Publisher：一般社団法人 植物化学調節学会  

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Recently we reported the identification of the auxin-biosynthesis inhibitor, L-aminooxyphenylpropionic acid (AOPP), which blocks L-Tryptophan aminotransferase (PCP (2010) 51 (4): 524-536). We designed and synthesized novel AOPP analogues by modifying functional groups of AOPP and finally identified KOK1169 as more selectivity inhibitor than AOPP by several screening. Thus we examined the effect of KOK1169 treatment on auxin biosynthesis in Arabidopsis and Rice in vivo. The profile of endogenous auxin-biosynthesis related compounds will be reported. 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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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Indole-3-acetic acid (IAA), the most important member of the auxin family, has been reported to play an important role in plant development and growth. TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1) is involved in IAA biosynthesis. We reported that L-aminooxy-phenylpropionic acid (AOPP) is an inhibitor of TAA1. However AOPP has side effects, such as inhibition of phenylalanine ammonia-lyase and 1-Aminocyclopropane-l-carboxylic acid synthase (ACS). Here, we developed more stable and specific auxin-biosynthesis inhibitor, KOK1169, using AOPP as a lead compound. We examined the inhibitor activity of KOK1169 for AtACS8. The K, value of KOK1169 is 246 nM, and it was lower than that of AOPP (847 nM). While, the K, value of KOK1169 is higher than that of L-α-(2-Aminoethoxyvinyl)glyeine (AVG), which is a typical inhibitor of the ACS (37 nM). Although KOK1169 showed an inhibitor activity for ACS in enzyme assay, ethylene biosynthesis of Arabidopsis seedling was scarcely affected by KOK1169. Furthermore, the K_i value of KOK1169 for TAA1 is 76 nM, and it was superior to that of KOK1169 for AtACS8. Therefore, we concluded that KOK1169 has the higher specificity for IAA biosynthesis than AOPP. 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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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Plant hormone indole-3-acetic acid (IAA) has various physiological function, but IAA biosynthetic and signal pathway is still unclear. Also the components mediating the crosstalk between IAA and other plant hormones are little known. As IAA is essential for plant growth, 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. We can raise the next point as an overall merit of using IAA biosynthesis inhibitor for isolating IAA mutants. That is, we could isolate double or triple mutant-like mutants by screening mutants with the inhibitor.

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Publisher：The Japanese Society of Plant Physiologists  

Glycerolipids are major components of membrane-lipids. Although glycerolipids can solely form lipid bilayer membrane, other type lipids, sterols and sphingolipids, also exist in cellular membrane. These lipid diversities are considered to be important for the function of cellular membrane. We have shown the developmental function of sterols by molecular genetics. So what is the function of sterols for the membrane lipids environment? We here report that the potential regulatory mechanisms of plastidial lipids from the analyses of sterol and glycerolipids profiles of sterol biosynthetic mutants. <br>LC-MS analyses demonstrated no difference of phospholipids profile on 19 mutants of sterol biosynthetic pathway. Although phospholipids as well as sterols are components of cellular membrane, alteration of sterol profile did not affect phospholipids profile. On the contrary, the profile of galactolipids, major components of plastidial lipids, was altered in the mutant of HMGR, the key enzyme of the sterol biosynthesis. Since the level of plastidial phospholipids was not affected in this mutant, HMGR may contribute to the regulatory mechanisms of the galactolipids biosynthesis in plastids.

DOI： 10.14841/jspp.2011.0.0099.0

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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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Publisher：日本植物生理学会  

Sterols are isoprenoid-type lipids that are biosynthesized &lt;I&gt;via&lt;/I&gt; the cytosolic mevalonate pathway. Through the study of several sterol biosynthetic genes, we have identified that sterols play important roles on cell elongation, senescence, male gametophyte development and chloroplast differentiation. To understand total physiological function of sterols, we have comprehensively collected and characterized Arabidopsis mutants lacking sterol biosynthetic genes.&lt;br&gt; Mutants on 19 loci were obtained as homozygote genotype. The sterol accumulation and profiles of these 19 mutants were quantitatively analyzed. The gene expression profiles of these mutants were examined by microarray analyses and the data were analyzed statistically. The results of sterol analyses and transcriptomics suggest that the magnitude of influence of sterol deficiency is compared with that of brassinosteroid deficiency. Although the alteration of sterol composition also affects gene expression profiles, the magnitude of influence was less than that that of sterol and brassinosteroid deficiency.

DOI： 10.14841/jspp.2010.0.0225.0

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Publisher：日本植物生理学会  

Phytohormones are key players in regulation of plant growth, development and defense response. In this study, we conducted comprehensive analysis of phytohormones regulating metabolic pathways using integrated omics data collected from Arabidopsis mutants of basic phytohormones, Auxin, Cytokinin, Gibberellin, Abscisic acid, Ethylene, Jasmonic acid and Brassinosteroid. &lt;br&gt; We used 28 hormone mutants and 4 wild types to collect gene expression profiles and metabolome profiles. The seven-day-old seedling grown in 1/2 MS medium were analyzed by DNA microarray (Affymetrix ATH1 GeneChip) and by LC-Q-TOF/MS, GC-TOF/MS, LCMS-IT-TOF/MS and CE-TOF/MS. Omics profile of each mutant was characterized by Ontology analysis. Furthermore, gene expression responses and metabolite accumulations related to specific hormone mutants were identified. We also analyzed co-expression pattern between genes and metabolites to extract metabolites accumulated in hormone-related pathways. We will present relationships between phytohormone functions and metabolic pathways.

DOI： 10.14841/jspp.2010.0.0190.0

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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. Analyses of SL-responsive and MAX2 downstream gene candidates provide a clue to broaden our understanding of SL signaling. We previously identified some SL-responsive genes by using real-time RT-PCR and microarray analyses. We also showed MAX2 plays an important role in the expression of those genes and the feedback regulation of MAX3 and MAX4. In this study, we over-expressed SL-responsive genes in Arabidopsis and analyze those mutants phenotype. We also performed yeast two-hybrid screening to identify the target of F-box protein, MAX2. We isolated some proteins that seem to interact with MAX2 Leu rich repeat.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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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Language：Japanese   Publisher：Pesticide Science Society of Japan  

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Language：Japanese   Publisher：Pesticide Science Society of Japan  

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Publisher：日本植物生理学会  

In order to investigate an inter-tissue diversity of plant secondary metabolisms, the metabolic profiles of 32 tissues of &lt;i&gt;Arabidopsis thaliana&lt;/i&gt; (Col-0 ecotype) were acquired by using the LC-Q-TOF/MS method with considering growth conditions of the public transcriptome data such as AtGenExpress Development. The obtained data matrix (called AtMetExpress Development LC/MS) contained 2522 peaks with annotations of about 100 metabolites. A detected metabolite (m/z 138, Rt 2.06 min) specifically accumulated in flower buds and apical meristem was identified to be tyramine from the annotation data A comparison of the accumulation profiles with that of gene expression data revealed that one homolog of putative aromatic amino acid decarboxylase gene is likely to be responsible for the tyramine biosynthsis in A. thaliana due to those identical accumulation and expression patterns.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Novel Oxylipin, 9-hydroxy-10-oxo-(Z), 15(Z)-octadecadienoic acid (KODA) is one of signal compounds, which is synthesized from the linolenic acid. It was reported that KODA has a function to enhance stress tolerance and to activate inflorescence development in some species. It has been suggested that KODA biosynthesis involve two kinds of enzymes (LOX, AOS) as in the case of Jasmonate (JA). It has also suggested that there are some differences in substrate specificity of enzyme activity between biosynthesis of KODA and JA. Namely 9-specific oxidation enzymes were necessary in KODA biosynthesis, while 13-specific enzymes in JA. According to a recent study, 9-specific enzyme accumulates tissue-specifically in vascular bundle of Solanum lycopersicom, and Solanum tuberosum. However there was little information about 9-specific enzyme in detail. In Arabidopsis thaliana the expression of one AOS and 6 LOXs were detected, but their involvement in KODA biosynthesis remained to be elucidated. To clarify 9-specific enzyme activity for KODA production, we have established that loss of function or overexpression mutants of six LOXs. Differences were observed in leaf enlargement and inflorescence elongation. To clear whether the levels of endogenous KODA were regulated by the expression of LOXs or not, we established GC/MS analysis method. Preliminary analysis to detect the levels of endogenous oxylipins in Arabidopsis were performed using by this method.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Brassinosteroids (BRs) are inactivated by cytochrome P450 monooxygenases in plants. It is known that Arabidopsis CYP734A1 (BAS1) and tomato CYP734A7 inactivate BRs by C26 hydroxylation of castasterone and brassinolide. Rice (Oriza sativa) has four CYP734A homologues (CYP734A2, A4, A5, and A6). Transgenic rice plants overexpressing these CYP734As, except for CYP734A5, showed a severe dwarf phenotype, and their endogenous BR levels were decreased. In order to investigate BR inactivation mechanism in rice, we have prepared the recombinant CYP734As expressed using a baculovirus insect cells system, and performed the enzyme assays with various BR-biosynthetic intermediates as a substrate. Rice CYP734As showed wide substrate specificities for BRs and exhibited high activity for BR intermediates located at the upstream of BR biosynthetic pathway. Futhermore, CYP734As catalyzed a three-step-oxidation of BR intermediates to the corresponding alcohols, aldehydes, and carboxylic acids. These results suggest that BR inactivation mechanism in rice is different from that in Arabidopsis and tomato.

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Language：Japanese  

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Gravitropic curvature occurs in stem along the whole elongation zone in various plant species. The mechanism to define the elongation zone and the gravitropic responding zone is unknown. Here, we used Arabidopsis and Mung bean to study mechanisms regulating elongation and the gravitropic response. Brassinosteroid (BR) biosynthesis genes, BR6ox1/CYP85A1 and BR6ox2/CYP85A2 are expressed in elongating zone in Arabidopsis. Mung bean CYP85A is also expressed higher in elongating zone. Analysis of endogenous BRs in Mung bean hypocotyls revealed that level of Castasterone was higher in elongating zone than in non-elongation zone. Brassinolide promoted gravitropic response, whereas Brassinazole inhibited it. These results suggested that BR is a key factor regulating stem elongation and gravitropic response.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

To understand hormonal status and their functions, we have been using hormone-responsive-marker genes or endogenous hormone levels. There is no conclusive way to monitor hormonal status in vivo including plant's sensitivities to hormones in a comprehensive way. Here we tested a novel method to monitor phytohormonal status using transcriptome data. We have been providing data for hormonal response of Arabidopsis seedlings using Affymetrix GeneChip as a part of AtGenExpress project. The data set was used to extract hormone-specifically responsive marker genes (threshold: ANOVA P < 0.001, fold change 2 or 4) and their expression patterns. The data for each hormone was used to calculate Pearson correlation coefficients (PCCs) with various data sets from the AtGenExpress project. Hormone inhibitors, such as, brassinazole, prohexandion, or aminoethoxyvinylglycine were also used to justify and evaluate results of this analysis.

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：Japanese   Publisher：The Janapese Society for Chemical Regulation of Plants  

To understand hormonal status and their functions, we have been using hormone-responsive-marker genes or endogenous hormone levels. There is no conclusive way to monitor hormonal status in vivo including plant's sensitivities to hormones in a comprehensive way. Here we tested a novel method to monitor phytohormonal status using transcriptome data. We have been providing data for hormonal response of Arabidopsis seedlings using Affymetrix GeneChip as a part of AtGenExpress project. The data set was used to extract hormone-specifically responsive marker genes (threshold: ANOVA P < 0.001, fold change 2 or 4) and their expression patterns. The data for each hormone was used to calculate Pearson correlation coefficients (PCCs) with various data sets from the AtGenExpress project. Hormone inhibitors, such as, brassinazole, prohexandion, or aminoethoxyvinylglycine were also used to justify and evaluate results of this analysis.

DOI： 10.18978/jscrpanb.42.0_90

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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At GenExpress is a multinational project to collect and distribute a transcriptome-data set of a multicellular-model organism, Arabidopsis thalinana. In the spring of 2003, the AtGenExpress consorcium has establised and agreed to generate a freely available transcriptome resource, using a hightly standerlized platform Affymetrix GeneChip, ATH1, which covers most Arabidopsis genes in the genome (about 23,000 probe sets corresponding to about 25,000 genes). It consists of Max Planck Institute from Gemany, Nottingham Arabidopsis Stock Center (NASC) and the Genomic Arabidopsis Resource Network (GARNet) from UK, The Arabidopsis Information Resource (TAIR) from U.S.A., RIKEN Plant Science Center from Japan, and etc. The consorcium collected data of developmental series, organ specificity series, light response series, stress response series, pathogen response series, hormone response series, nutrition response series, and etc. The data consists of >1,000 GeneChips (hybridizations). More than 20,000 genes were detected to be expressed significantly in any tissues or growth condisions. This is one of the largest number of gene expression reported in an organism. All collected data are integrated, displayed, distributed for public access at TAIR, http://www.arabidopsis.org. RIKEN-data set is also available at http://pfg.psc.riken.jp/AtGenExpress/index.html. Voluntary data submission from individual researchers is also accepted by TAIR. Starting form the core data set, the repsitory will expand by additonal data from the entire Arabidpisis community. The resource will be useful not only for plant basic scieces but also for plant applied siences as well as for other basic siences, such as bioinformatics and systems biology.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

The microarray technique is a powerful tool for otaining an overview of plant hormone actions using inducible genes as molecular markers. We have already studied comprehensive transcript profiles of auxin-regulated and brassinosteroid-regulated genes in arabidopsis. In this presentation, microarray analyses of other phytohormone (cytokinin, gibberellin, abscisic acid, jasmonic acid and ethylene and plant growth regulator including biosynthesis inhibitor are presented. Comparison of phytohormone's actions between triazol compound's actions, indicating the specificity of Brz220 for brassinosteroid's action.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Abscisic acid (ABA) catabolism in plant is predominantly regulated by ABA 8'-hydroxylase, which is a cytochrome P450. Among the known cytochrome P450 inhibitors with triazole group we tested, uniconazole-P inhibited ABA catabolism in cultured tobacco bright yellow-2 cells. On the basis of a structure-activity relationship study of uniconazole, we found that diniconazole, which is known as a fungicide, was more effective ABA catabolic inhibitor than uniconazole-P. Diniconazole showed the potent inhibitory activity to CYP707A3, Arabidopsis ABA 8'-hydroxylase, in in vitro assay. Diniconazole-treated plants kept higher ABA contents and higher transcription levels of ABA response genes during rehydration than those of non-treatmented. These results strongly suggest that ABA catabolic inhibitors targeting ABA 8'-hydroxylase can regulated the ABA content in plants. Moreover, we performed optical resolution of diniconazole and showed that S-form of diniconazole, a less active isomer as fungicide, was more active isomer as ABA catabolic inhibitor than R-form.

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Publisher：The Japanese Society of Plant Physiologists  

Plants produce a wide variety of triterpenes. Squalene, a linear triterpene synthesized via the cytosolic mevalonate pathway, is metabolized to steroids and triterpenoids. HMG-CoA reductase (HMGR), the key enzyme of the mevalonate pathway, is encoded by two genes, <I>HMG1</I> and <I>HMG2</I>, in Arabidopsis. We have been isolated T-DNA insertion mutants for these genes and identified that triterpenes are involved in the cell elongation, the inhibition of senescence and the pollen development.<br>The gene expression profiles of wild-type plants treated with inhibitors of HMGR, squalene synthase, squalene epoxydase, C-14 reductase (FACKEL) and CYP90B1 (DWF4) were compared of that of <I>hmg1</I> using a microarray technique. It was suggested that squalene is involved in the inhibition of senescence. The finding corresponded to the gene expression analyses in the various mutants downstream of <I>hmg1</I> in the MVA pathway. We consider that squalene may play a role for the inhibition of senescence as an anti-oxidant compound.

DOI： 10.14841/jspp.2005.0.667.0

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Publisher：The Japanese Society of Plant Physiologists  

AtGenExpress is a multinational project to collect and distribute transcriptome-data sets of Arabidopsis thalinana. using a highly standardized platform Affymetrix GeneChip, ATH1, which covers most Arabidopsis genes in the genome (about 23,000 probe sets corresponding to about 25,000 genes). The consortium collected data of developmental series, organ specificity series, light response series, stress response series, pathogen response series, hormone response series, nutrition response series, and etc. The data consists of >1,000 GeneChips (hybridizations). More than 20,000 genes were detected to be expressed significantly in any of the experiments. This is one of the largest numbers of gene expression detected in an organism. All collected data are integrated, displayed, distributed for public access at TAIR, http://www.arabidopsis.org. RIKEN-data set is also available at http://pfg.psc.riken.jp/AtGenExpress/index.html. In this presentation, we will mainly focus on how to use the data and results from large-scale clustering analyses.

DOI： 10.14841/jspp.2005.0.467.0

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Publisher：The Japanese Society of Plant Physiologists  

For plants, sun light is one and the only source of energy for photosynthesis and thus indispensable for growth of plants. On the other hand, intense and excess light environment causes the evolution of oxygen radicals in chloroplasts and has the potential to damage them. However, plants are able to respond to the light stress and protect the chloroplasts by various means, including transcriptional regulation at the nucleus. Activation of the light stress-responsive genes is mediated via hydrogen peroxide-dependent and -independent pathways. In this report, we will introduce our expression analysis for classification of light stress responses using Affimetrix GeneChip.

DOI： 10.14841/jspp.2004.0.779.0

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

There has been limited information how BR biosynthesis is regulated and where BRs are synthesized and used. To address this issue, we isolated two BR-6-oxidase genes (BR6ox) from Arabidopsis and analyzed their gene expression with distribution of endogenous BRs. The results suggested that BRs are synthesized in all of the organs tested, but most actively synthesized in young actively developing organs. To further gain insight into the site of BR biosynthesis, transgenic Arabidopsis carrying reporter, BR6ox-GUS gene were produced. Using these lines, the site of biosynthesis and action is now under investigation at the cellular level. Although recent studies of biosynthesis and sensitivity mutants have provided insights into the functions of BRs, the mode of action of BRs is poorly understood. Using microarray analysis, we identified BR-regulated genes. The list of BR-regulated genes includes transcription factor genes, auxin-related genes, P450 genes, and genes implicated in cell expansion and cell wall organization. These results provide a comprehensive view of the actions of BRs, using BR-regulated genes as molecular markers. Despite numerous physiological studies addressing the interactions between BRs and auxin, little is known about the underlying molecular mechanisms. Using auxin-reporter gene system and auxin-related mutants, signaling interaction between auxin and BR have been investigated.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

We investigated CYP72B1,an Arabidopsis cytochrome P450,to determine the biochemical and physiological functions of this enzyme. Using a yeast functional assay, we have demonstrated that CYP72B1 is a steroid C-26 hydroxylase that converts brassinolide (BL) to 26-hydroxybrassinolide (26-OHBL) and castasterone (CS) to 26-hydroxycastasterone (26-OHCS). We tested the ability of an Arabidopsis CYP72B1-null mutant, the wild type, and a CYP72B1 over-expressor to metabolize BL or CS. Reduced levels of 26-OHBL and 26-OHCS were detected in the null mutant, and increased levels were detected in the over-expressor, demonstrating that 26-hydroxylation of BRs is an endogenous biochemical function of CYP72B1. Bioassays with BL and 26-OHBL have provided evidence that 26-hydroxylation is an inactivation step. We also showed that CYP72B1-mediated brassinosteroid inactivation provides positive modulation of photomorphogenesis.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Abamine (abscisic acid biosynthesis inhibitor with amine moiety) is abscisic acid (ABA) biosynthesis inhibitor targeting the oxidative cleavage reaction of 9-cis-epoxycarotenoids, which is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). To examine the effects of abamine during developmental stage, we compared the phenotype of abamine-treated Arabidopsis or cress seedlings grown in the dark with those of non-treated plants. In abamine-treated seedlings, hook formation was inhibited. It has been reported that this inhibition of hook formation is observed in ABA deficient mutant, aba2-2. These results suggest that the inhibition of hook formation induced by abamine should be due to ABA deficiency.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

In dicotyledonous plants including Arabidopsis, brassinosteroids deficiency induces morphological features of light-grown plants in the dark, i. e. short hypocotyls, expanded cotyledons, and true leaves, in a dose-dependent manner. While how brassinsteroid deficiency does affect the growth of monocotyledonous plants in the dark is still not clear. In order to investigate the effect brassinosteroid deficiency in monocotyledonous plants, a brassinosteroid biosynthesis inhibitor was applied to rice in the dark and the morphological change was intensively observed and the expression of genes induced under light condition was examined.

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Language：Japanese   Publisher：日本農芸化学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Brassinosteroid (BR) has been suggested to interact closely with auxin to regulate plant growth and development. We have demonstrated that many auxin-inducible genes were induced by BR. In this work, we chose SAUR-AC1 gene for further analysis. Arabidopsis BR deficient mutant, det2,was grown under continuous light or darkness for 7 days, then treated with 10 nM brassinolide (BL). The mRNA of SAUR-AC1 was accumulated within 30 min both in light and dark. The data suggest that SAUR-AC1 is the earliest induced gene by BL. Wild type plants (Col-0) was also treated by 10 nM, 100 nM and 1 μM BL or IAA. The induction kinetics by BL was different from auxin. The induction of SAUR-AC1 mRNA treated by BL and IAA was larger than BL of IAA alone. The result indicated that the effect of BL and auxin was synergistic. Transgenic arabidopsis seedlings containing SAUR-AC1 : : GUS fusion gene were treated with BL, and then stained for GUS activity. In light-grown plants, GUS activity was detected in cotyledons and hypocotyl. Similar staining pattern was observed in auxin treatment. GUS staining was reduced by BR-biosynthesis inhibitor, Brz. From these results, it is suggested that the SAUR-AC1 is a shared signaling component in BR- and auxin-signaling pathways, and it functions as an activator to regulate cell elongation in both signaling pathways.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Recent studies of the brassinosteroid-deficient mutants have revealed that brassinosteroid plays essential roles to control plant growth and development, such as cell elongation, division and photomorphogenesis. However, its regulatory mechanisms and signal transduction pathway are still under unclear. To provide insight into the signaling pathway responsible for the induction of gene expression by Brassinolide, we examined the effects of Brassiolide on Brassinosteroid-inducible genes using the protein kinase inhibitor staurosporin or Brassinoseroid-Insensitive 1 mutant. These studies revealed that Brassinosteroid inducible-gene expression was mediated by at least two distinct pathways, one of that inhibited by protein kinase inhibitor staurosporin or the other is not.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. There is only limited information on where BRs are synthesized and used. We studied the organ-specificity of BR biosynthesis in Arabidopsis, using two different approaches : we analyzed the expression of BR-related genes using real-time quantitative RT-PCR, and analyzed endogenous BRs using gas chromatography-mass spectrometry. Endogenous BRs and the expression of BR-related genes were detected in all organs tested. The highest level of endogenous BRs and the highest expression of the BR6ox1,the BR6ox2,and the DWF4 genes were observed in flower buds, which contain actively developing organs, such as the shoot apical meristem, young stems, and young cauline leaves. These genes are important in BR biosynthesis, since they encode the rate-limiting or farthest downstream enzyme in the BR biosynthesis pathway. The second highest level of endogenous BRs and expression of BR6ox1 and DWF4 were observed in silique, which contains actively developing embryos and seeds. These findings indicate that BRs are synthesized in all organs tested, but are most actively synthesized in young actively developing organs. In contrast, synthesis was limited in mature organs. Our observations are consistent with the idea that BRs function as the growth-promoting hormone in plants.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Triadimefon (Bayleton〓), a widely used triazole-type fungicide, affects gibberellin biosynthesis and 14α-demethylase in sterol biosynthesis. This study revealed that the phenotype of Arabidopsis treated with triadimefon resembled that of a brassinosteroid (BR) biosynthesis mutant, and that the phenotype was rescued by BL, the most active BR, partly rescued by GA, and fully rescued by the co-application of BL and GA, suggesting that triadimefon affects both BR and GA biosynthesis. The target sites of triadimefon were investigated using a rescue experiment, feeding triadimefon-treated Arabidopsis BR biosynthesis intermediates, and a binding assay to expressed DWF4 protein, which is reported to be involved in the BR biosynthesis pathway. The binding assay indicated that the dissociation constant for triadimefon was in good agreement with the activity in an in planta assay. In the triadimefon-treated Arabidopsis, the CPD gene in the BR biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR-deficiency. These results strongly suggest that triadimefon inhibits the reaction catalyzed by DWF4 protein and induces BR deficiency in plants. As triadimefon treatment has proved beneficial to plants, this result suggests that brassinosteroid biosynthesis inhibitors can be applied to crops.

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Language：Japanese   Publisher：Pesticide Science Society of Japan  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Recent studies of the brassinosteroid-deficient mutants have revealed that brassinosteroid plays essential roles to control plant growth and development, such as cell elongation, division and photomorphogenesis. However, its regulatory mechanisms and signal transduction pathway are still under unclear. We used DNA microarray (Gene Chip, Affymetfix) to analyze BR-regulated genes comprehensively. BL inhibited the expression of the gene for phytochrome-.interacting factor 3, indicating that BRs modulate the light signaling pathway rather than function as signaling molecules downstream of light. After a lag period, BL also increased the expression of early auxin-inducible genes, suggesting that BR signaling overlaps with the auxin signaling pathway to controlcellular processes.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Light has been suggested to be a negative regulator of brassinosteroid biosynthesis, but there have been no direct evidences. We analyzed light regulation of brassinoseroid biosynthesis both from biochemical and molecular biological approach. We isolated brassinosteroid-6-oxidase II gene (BR6oxII) from Arabidopsis. Total RNAs were extracted from Arabidopsis seedlings grown in light or darkness for 5 to 7 days. They were then subjected to real-time monitoring RT-PCR. Transcript levels of BR biosynthetic genes were organ specific.Some of them were up-regulated upon transfer from darkness to light. We conclude, together with endogenous BR levels determined by GC-SIM, that BR biosynthesis is activated during photomorphogenesis.

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Language：English   Publisher：AMER SOC PLANT PHYSIOLOGISTS  

Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. It has been proposed that BRs are synthesized via two parallel pathways, the early and late C-6 oxidation pathways according to the C-6 oxidation status. The tomato (Lycopersicon esculent um) Dwarf gene encodes a cytochrome P450 that has be en shown to catalyze the C-6 oxidation of 6-deoxocastasterone to castasterone. We isolated an Arabidopsis ortholog (AtBX6ox gene) of the tomato Dwarf gene. The encoded polypeptide has characteristics of P450s and is classified into the CYP85 family. The AtBR6ox and tomato Dwarf gene were expressed in yeast and the ability of the transformed yeast cells to metabolize 6-deoxo-BRs was tested. Metabolites were analyzed by gas chromatography-mass spectrometry. Both Enzymes catalyze multiple steps in BR biosynthesis: 6-deoxoteasterone to teasterone, 3-dehydro-6-deoxoteasterone to 3-dehydroteasterone, 6-deoxotyphasterol to typhasterol, and 6-deoxocastasterone to castasterone. Our results indicate that the AtBR6ox gene and the tomato Dwarf gene encode steroid-6-oxidases and that these enzymes have a broad substrate specificity. This suggests that the BR biosynthetic pathway consists of a metabolic grid rather than two separate parallel pathways.

DOI： 10.1104/pp.126.2.770

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Language：Japanese   Publisher：Pesticide Science Society of Japan  

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Language：Japanese   Publisher：The Janapese Society for Chemical Regulation of Plants  

Brassinazole (Brz) is a specific inhibitor of brassinosteroid biosynthesis. The dark-grown Brz-treated Arabidopsis and non-Brz-treated der2, which is a brassinosteroid biosynthesis-deficient Arabidopsis mutant, show characteristics of de-etiolated plants, i.e., short hypocotyls, expanded cotyledons and development of true leaves, when grown in darkness. In this context, brassinosteroids seem to function in the suppression of photomorphogenesis in the dark. In general, the application of cytokinin reduces hypocotyls length and induces development of true leaves in the dark. Here, we examined that brassinosteroid-deficiency and cytokinin-excess exhibit whether common effects or different effects in photomorphogenesis in the dark. In development of true leaves in the dark, Brz induced expanding of leaves mainly and a little promote of proliferation of shoot apex. In contrast, Arabidopsis treated by benzyladenine (BA) exhibited many, slender and wind true leaves. Treatment of Brz plus BA induced many true leaves which is alike to normal leaves in light. Both of brassinosteroid-deficiency and cytokinin-excess may be necessary to shape normal leaves. The Brz--treated and BA-treated plants exhibited fast greening after transferring from dark to light. Then, we examined content of protochlorophylide (Pchlide) and protochlorophyll (Pchl) of Brz-treated and BA-treated Arabidopsis. The results showed that Brz induces accumulation of high contents of Pchl. However, BA induced accumulation of Pchlide rather than Pchl. Thus, brassinosteroid-deficiency and cytokinin-excess exhibited different effects in photomorphogenesis in the dark.

DOI： 10.18978/jscrpanb.36.0_27

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

DOI： 10.18978/jscrp.36.2_231_4

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Language：English   Publisher：Japanese Society of Plant Physiologists  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184812

Language：English   Publisher：Japanese Society of Plant Physiologists  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184778

Language：English   Publisher：Japanese Society of Plant Physiologists  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184762

Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

DOI： 10.18978/jscrpi.35.2_138

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Brassinosteroids are phytohormones that play important roles in plant growth, development and chloroplast differentiation. Recently, Brz (brassinazole) was synthesized as a first specific inhibitor of brassinosteroid biosynthesis. For analyzing the detail of mechanism of brassinosteroid biosynthesis and signal transduction, we started to screen for mutations that show resistance to the Brz actions. In genetic screening of Arabidopsis 140,000 seeds mutagenized by EMS and fast neutron, some mutants that were significantly taller than wild type when grown in the dark with Brz were screened. At least four mutants of dark grown with 3 μM Brz showed long hypocotyl as tall as wild type of dark grown in Brz-free condition. These are named as bil (B__-rz-i__-nsensitive-l__-ong hypocotyl). Another three mutants, that showed shorten hypocotyl but could not caused cotyledon-opening when grown in the dark with 3 μM Brz, were also screened. These are named as bih (B__-rz-i__-nsensitive-h__-ooked hypocotyl).

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Light has been suggested to be a negative regulator of brassinosteroid biosynthesis, but there have been no direct evidences. We analyzed light regulation of brassinosteroid biosynthesis both from biochemical and molecular biological approach. We first isolated brassinosteroid-6-oxidase gene (B6OX) from Arabidopsis. Total RNAs were extracted from Arabidopsis seedlings grown in light or darkness for 5 to 7 days. They were then subjected to realtime monitoring RT-PCR. Transcript level of B6OX gene was higher in plants grown in darkness than in light. We also performed feeding experiment of [^2H_6]6-deoxoCS using light- and dark-grown cucumber and Arabidopsis seedlings. Although we successfully detected [^2H_6]CS from both cucumber and Arabidopsis, significant differences were not observed between light- and dark-grown seedlings.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Previously, microscopical observations of brassinosteroid biosynthesis-deficient mutants have reported few cytologicalchanges other than inhibition of cell elongation. Here, we examinedin detail cytological changes in cells from a different perspective than that of previous studies and could postulate that a decrease in the amount of endogenous brassinosteroid in plants may induce initial differentiationfrom etioplastto chloroplast and quicken the pace of greening. Brassinazole (Brz) is a specific inhibitor of brassinosteroid biosynthesis. The cotyledons of dark-grown Br-treated Arabidopsis and non-Brz-treated det2, which is a brassinosteroid biosynthesis-deficient Arabidopsis mutant, were observed using DAPI-staining. We revealed that condensation and scattering of plastid nucleoids is advanced in the cotyledons of them. In general, these changes in the distribution of plastid nucleoids occur early on when a plant is changed from dark-grown to light-grown, and also when cells change from undifferentiated to differentiated, before the development of thylakoid membranes. In cucumber (Cucumis sativus) treated with Brz, The scattering of nucleoids in cotyledons was putted great emphasis on those in the Brz-treated Arabidopsis and det2. the Brz-treated cucumber exhibited fast greening after transferring from dark to light. We also found that bil (B__-rzi__-nsensitive-l__-ong hypocotyl) mutant, witch have long hypocotyl in the dark with Brz as tall as will type grown in the dark without Brz, exhibits late greening after transferring from dark to light. These results indicate that brassinosteroid may involve in differentiation from etioplastto chloroplast.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Recent studies of the brassinosteroid-deficient mutants have revealed that brassinosteroid plays essential roles to control plant growth and development, such as cell elongation, division and photomorphogenesis. However, its regulatory mechanisms and signal transduction pathway are still under unclear. We performed DNA microarray analysis (Gene Chip, Affymetrix) to analyze BR-regulated genes comprehensively. The known BR-regulated gene, TCH4 gene that encodes xylogucan (endotransglycosylase) XET was hightly induced by BR treatment. The expression of genes, which involved in BR biosynthesis, such as CPD, were repressed by BR treatment. Novel BR-regulated genes were also found.

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Language：English   Publisher：Japanese Society of Plant Physiologists  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184259

Language：English   Publisher：Japanese Society of Plant Physiologists  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184258

Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

CiNii Books

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

CiNii Books

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

CiNii Books

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

When the brassinosteroid biosynthesis inhibitor (Brz) was exogenously applied to 1/2 MS medium at a concentration of 0.1-2μM, Arabidopsis seedlings in the dark exhibited photomorphogenesis, such as inhibition of cell elongation in hypocotyls, expansion of cotyledons, development of true leaves. At 40 days after sowing in the dark of 2μM Brz, significant expansion of several rosette leaves was observed. For the analysis of DNA synthesis, BrdU (an analogue of thymidine) was applied to seedlings in the dark at 8-10 days after sowing. Many nuclei in the shoot apical meristem in the dark were labeled with BrdU in Brz-treated seedling, while less nuclei were labeled in non-treated seedling. The activation of DNA synthesis n shoot apical meristems was shown by treatment with Brz. We examined plastid morphology in cotyledons in dark grown seedlings by electron microscopy. They were typical etioplast, that is absence of thylakoid membranes, in both Brz-treated and non-treated cotyledons. In Brz-treatment, the lack of diggerentiation of etiplasts to chloroplasts was suggested. However, Immunoblot analyses and immunostaining revealed that the accumulated levels of Rubisco protein in Brz-treated seedlings were much higher that non-treated seedlings. These results suggested that Brz exhibits photomorphogenesis in the dark to some extent.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

Brz was selected through the screening for brassinosteroid biosynthesis inhibitors. In dark grown Arabidopsis, Brz-induced morphological changes were nearly restored to those of wild type by the additional treatment of brassinolide. The structure of Brz is similar to pacrobutrazol, a gibberellin biosynthesis inhibitor, the assays on cress plants, Brz-treated plants do not show recovery by the addition of gibberellin, but show good recovery by the addition of brassinolide. Brz-treated cress also showed dwarfism with altered leaf morphology, including the downward curling and dark-green color typical of Arabidopsis brassinosteroid deficient mutants, and this dwrfism was reversed by the application of 10 nM brassinolide. This result suggests that brassinosteroids are essential for plant growth and Brz can be used to clarify the function of brassinosteroids in plants as a complement to brassinosteroid-deficient mutants. The Brz action site was also investigated by feeding brassinosteroid biosynthesis intermediates to cress grown in the light. The results suggested that Brz blocks at least one step upstream of teasterone formation.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

In order to find new brassinosteroid biosynthesis inhibitors, we investigate the effect of human 3β-hydroxysteroid dehydorgenase inhibitors including spironolactone and progesterone on cress and Arabidopsis. Among the tested compounds, only spironolactone induced photomorphorogical changes in the dark, and dwarfisim and early development of leaves in the light. These morphological changes were restored by the coapplication of brassinolide, which suggests that spironolcatone should interfer with biosynthesis of brassinosteroids.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

We studied hormonal regulation of photomorphogenesis using cucumber seedlings with brassinosteroid-biosynthesis-specfic inhibitor, Brassinazole(Brz). When seedlings were germinated in the dark in half MS agar medium containing Brz, cotyledon opening was observed with inhibited hypocotylelongation. However, when they were germinated in distilled water, agar or soil containing Brz in the dark, neigher hook unfolding nor cotyledon opening was observed. In contrast, cytokinin treatment caused both of them in any of these growth conditions. We also analyzed endogenous BRs for light-and dark-grown seedlings by GC-MS. Light-grown plants accumulated more castasterone than dark-grown ones. Based upon these observations, we will discuss on the hormonal regulation of photomorphgenesis

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Language：English  

CiNii Books

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Language：English   Publisher：Japanese Society of Plant Physiologists  

In an attempt to isolate cDNA clones for dark-inducible chloroplast proteins, we screened a cDNA library which was prepared from radish cotyledons by a two-step method. The source plants were grown under continuous light for 14 d and kept in darkness for 24 h. One of the selected clones, S2D12, corresponded to the din1 gene which we previously reported as a dark-inducible, senescence-associated gene [Azumi and Watanabe (1991) Plant PPhysiol.95: 577]. A 22 kDa polypeptide was produced from the cDNA in an in vitro expression system in the presence of [^<35>S]methionine. This polypeptide was capable of being imported by isolated chloroplasts, processed to a smaller mature form and localized in the stromal fraction. As the amino acid sequence of the putative mature protein has no homology to any known chloroplast protein, din1 was suggested to be the first gene for a chloroplast protein which is negatively controlled by light. The putative mature protein has similarity to sulfide dehydrogenase from Wolinella succinogenes and other small stress proteins; glpE and pspE from Escherichia coli and hsp67B2 from Drosophila melanogaster.

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Other Link： http://dl.ndl.go.jp/info:ndljp/pid/10772360

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CiNii Research

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CiNii Books

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Language：Japanese   Publisher：日本植物生理学会  

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Applicant：公立大学法人横浜市立大学, 学校法人神奈川大学

Application no：特願2019-009335  Date applied：2019.1

Announcement no：特開2019-131540  Date announced：2019.8

Patent/Registration no：特許第7169584号  Date registered：2022.11 

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Applicant：国立研究開発法人理化学研究所, 国立研究開発法人農業・食品産業技術総合研究機構, 公立大学法人横浜市立大学

Application no：特願2016-092258  Date applied：2016.5

Announcement no：特開2016-169221  Date announced：2016.9

Publication no：WO2012-118216  Date published：2012.9

Patent/Registration no：特許第6164761号  Date registered：2017.6 

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Applicant：独立行政法人理化学研究所

Application no：特願2013-000207  Date applied：2013.1

Announcement no：特開2013-067656  Date announced：2013.4

Publication no：WO2008-150031  Date published：2008.12

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Applicant：公立大学法人横浜市立大学, 国立研究開発法人農業・食品産業技術総合研究機構

Application no：特願2013-008344  Date applied：2013

Announcement no：特開2014-138562  Date announced：2014.7

Patent/Registration no：特許第6078351号  Date registered：2017.1 

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Applicant：国立研究開発法人農業・食品産業技術総合研究機構, 公立大学法人横浜市立大学

Application no：特願2013-042031  Date applied：2013

Announcement no：特開2014-169246  Date announced：2014.9

Patent/Registration no：特許第6120272号  Date registered：2017.4 

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Applicant：独立行政法人理化学研究所, 独立行政法人農業・食品産業技術総合研究機構, 公立大学法人横浜市立大学

Application no：JP2012055498  Date applied：2012.2

Publication no：WO2012-118216  Date published：2012.9

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Applicant：協和醗酵工業株式会社

Application no：特願平5-515520  Date applied：1992.11

Patent/Registration no：特許第3403196号  Date registered：2003.2 

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