研究者紹介システム
| 水谷 正治 |  |
| ミズタニ マサハル | |
| 大学院農学研究科 生命機能科学専攻 | |
| 准教授 | |
| 農芸化学関係 |
研究者情報
所属
【主配置】
大学院農学研究科 生命機能科学専攻【配置】
農学部 生命機能科学科
学位
授業科目
- 食と健康Ｂ, 国際教養教育院, 2022
- 卒業研究, 農学部, 2022
- 応用生命化学実験II, 農学部, 2022
- 植物代謝化学１, 農学部, 2022
- 分子生命農学入門, 農学部, 2022
- 分子生命農学入門１, 農学部, 2022
- 応用生命化学基礎英語, 農学部, 2022
- 植物代謝化学, 農学部, 2022
- 初年次セミナー（応用生命化学コース）, 農学部, 2022
- 植物代謝化学２, 農学部, 2022
- 分子生命農学入門２, 農学部, 2022
- 高度教養セミナー農学部応用生命化学入門, 農学部, 2022
- 高度教養セミナー農学部応用生命化学入門, 農学部, 2022
- 高度教養セミナー農学部応用生命化学, 農学部, 2022
- 高度教養セミナー農学部応用生命化学, 農学部, 2022
- 応用生命化学実験I, 農学部, 2022
- 応用生命化学実験III, 農学部, 2022
- 生命機能科学各論, 農学部, 2022
- 生理活性物質論, 大学院農学研究科, 2022
- 特定課題演習 I-1, 大学院農学研究科, 2022
- 課題開発演習, 大学院農学研究科, 2022
- 生命機能科学プレゼンテーション演習Ⅰ（オーラル・ペーパー）, 大学院農学研究科, 2022
- 特定課題演習 II-1, 大学院農学研究科, 2022
- 先端融合科学特論A（健康生命編）, 大学院農学研究科, 2022
- 植物酵素化学, 大学院農学研究科, 2022
- 特定課題演習 I-2, 大学院農学研究科, 2022
- 生命機能科学プレゼンテーション演習Ⅱ（オーラル・ペーパー）, 大学院農学研究科, 2022
- 特定課題演習 II-2, 大学院農学研究科, 2022
ジャンル
コメントテーマ
研究ニュース
研究活動
研究キーワード
研究分野
委員歴
- 2020年 - 現在, 日本植物生理学会, 欧文誌Plant and Cell Physiology、Regular Editor
- 2018年 - 現在, 日本植物バイオテクノロジー学会, 代議員
- 2016年 - 現在, 植物化学調節学会, 会計幹事
- 2013年 - 現在, 日本植物細胞分子生物学会, 学会誌Plant Biotechnology 編集委員
- 2012年 - 現在, 日本農芸化学会関西支部, 参与
- 2017年 - 2019年, 日本植物生理学会, 欧文誌Plant and Cell Physiology, Advisory Editorial Board
- 2017年, 日本農芸化学会2017年度大会（京都）, プログラム編成委員（植物）
- 2014年, 12th International Symposium on Cytochrome P450 Biodiversity and Biotechnology（Kyoto), Organizing Committee
- 2012年 - 2012年, 日本農芸化学会関西支部, 庶務幹事
- 2011年 - 2012年, 日本農芸化学会2011-12年度大会（京都大会）, プログラム編集委員（植物）
受賞
2021年09月 日本バイオテクノロジー学会, Plant Biotechnology誌論文賞, Hatching stimulation activity of steroidal glycoalkaloids toward the potato cyst nematode, Globodera rostochiensis.
2019年09月 日本植物細胞分子生物学会, Plant Biotechnology誌論文賞, Characterization of steroid 5a-reductase involved in a-tomatine biosynthesis in tomatoes.
2006年10月 植物化学調節学会, 植物化学調節学会奨励賞, アブシジン酸および植物ステロイドの代謝系に関わるシトクロムP450の酵素化学的研究
2006年03月 日本農芸化学会, B.B.B.論文賞, CYP724B2 and CYP90B3 function in the early C-22 hydroxylation steps of brassinosteroid biosynthetic pathway in tomato.
2001年03月 日本農芸化学会, B.B.B.論文賞, Substrate specificity of b-primeverosidase, a key enzyme in aroma formation during oolong tea and black tea manufacturing.
論文
Abstract Sterols are the essential components of the eukaryotic cell membranes. However, studies on sterol biosynthesis in bryophytes are limited. This study analyzed the sterol profiles in the bryophyte model plant Marchantia polymorpha L. The thalli contained typical phytosterols such as campesterol, sitosterol, and stigmasterol. BLASTX analysis of the M. polymorpha genome against the Arabidopsis thaliana sterol biosynthetic genes confirmed the presence of all of the enzymes responsible for sterol biosynthesis in M. polymorpha. In this study, we focused on characterizing two genes, MpDWF5A and MpDWF5B, which showed high homology with A. thaliana DWF5, encoding Δ5,Δ7-sterol Δ7-reductase. Functional analysis using a yeast expression system revealed that MpDWF5A converted 7-dehydrocholesterol to cholesterol, indicating that MpDWF5A is a Δ5,Δ7-sterol Δ7-reductase. Mpdwf5a-knockout lines (Mpdwf5a-ko) were constructed using CRISPR/Cas9 mediated genome editing. GC-MS analysis of Mpdwf5a-ko revealed that phytosterols such as campesterol, sitosterol, and stigmasterol disappeared, and instead, the corresponding Δ7-type sterols accumulated. The thalli of Mpdwf5a-ko grew smaller than those of the wild type, and excessive formation of apical meristem in the thalli was observed. In addition, the gemma cups of the Mpdwf5a-ko were incomplete, and only a limited number of gemma formations were observed. Treatment with 1 µM of castesterone or 6-deoxocastasterone, a bioactive brassinosteroid, partly restored some of these abnormal phenotypes, but far from complete recovery. These results indicate that MpDWF5A is essential for the normal growth and development of M. polymorpha and suggest that the dwarfism caused by the MpDWF5A defect is due to the deficiency of typical phytosterols and, in part, a brassinosteroid-like compound derived from phytosterols.
Oxford University Press (OUP), 2023年05月11日, Plant And Cell Physiology研究論文（学術雑誌）
The potato cyst nematode (PCN) causes extensive crop losses worldwide. Because the hatching of PCN requires host-derived molecules known as hatching factors (HFs), regulating HF production in host plants may help to control this harmful pest. Solanoeclepin A (SEA), isolated from potato, is the most active HF for PCN; however, its biosynthesis is completely unknown. We discovered a HF called solanoeclepin B (SEB) from potato and tomato root exudates and showed that SEB was biosynthesized in the plant and converted to SEA outside the plant by biotic agents. Moreover, we identified five SEB biosynthetic genes encoding three 2-oxoglutarate-dependent dioxygenases and two cytochrome P450 monooxygenases in tomato. Exudates from tomato hairy roots in which each of the genes was disrupted contained no SEB and had low hatch-stimulating activity for PCN. These findings will help to breed crops with a lower risk of PCN infection.
American Association for the Advancement of Science (AAAS), 2023年03月17日, Science Advances, 9 (11)研究論文（学術雑誌）
- 2023年03月, アグリバイオ, 7 (3), 20 - 23, 日本語ユニークな生理活性物質と合成生物学プラットフォーム
[招待有り]
Canonical strigolactones (SLs), such as orobanchol, consist of a tricyclic lactone ring (ABC-ring) connected to a methylbutenolide (D-ring). Tomato plants have been reported to produce not only orobanchol but also various canonical SLs related to the orobanchol structure, including orobanchyl acetate, 7-hydroxyorobanchol isomers, 7-oxoorobanchol, and solanacol. In addition to these, structurally unidentified SL-like compounds known as didehydroorobanchol isomers (DDHs), whose molecular mass is 2 Da smaller than that of orobanchol, have been found. Although the SL biosynthetic pathway in tomato is partially characterized, structural elucidation of DDHs is required for a better understanding of the entire biosynthetic pathway. In this study, three novel canonical SLs with the same molecular mass as DDHs were identified in tomato root exudates. The first was 6,7-didehydroorobanchol, while the other two were not in the DDH category. These two SLs were designated phelipanchol and epiphelipanchol because they induced the germination of Phelipanche ramosa, a noxious root parasitic weed of tomato. We also proposed a putative biosynthetic pathway incorporating these novel SLs from orobanchol to solanacol.
Frontiers Media SA, 2022年12月14日, Frontiers in Plant Science, 13研究論文（学術雑誌）
- 2022年10月, 植物の生長調節, 57 (2), 84 - 92, 日本語植物におけるステロイド生合成酵素の進化とそれらの代謝工学への利用
[査読有り][招待有り]
研究論文（学術雑誌）
Abstract Strigolactones (SLs) known as rhizosphere signaling molecules and plant hormones regulating shoot architecture, are classified into two distinct groups, canonical and non-canonical SLs based on their structures. Avenaol, a non-canonical SL found in the root exudates of black oat (Avena strigosa), has a characteristic bicyclo[4.1.0]heptane skeleton. Elucidating the biosynthetic mechanism of this peculiar structure is a challenge for further understanding the structural diversification of non-canonical SLs. In this study, a novel non-canonical SL, 6-epi-heliolactone in black oat root exudates was identified. Feeding experiments showed that 6-epi-heliolactone was a biosynthetic intermediate between methyl carlactonoate and avenaol. Inhibitor experiments proposed the involvement of 2-oxoglutarate-dependent dioxygenase in converting 6-epi-heliolactone to avenaol. These results provide new insights into the stereochemistry diversity of non-canonical SLs and a basis to explore the biosynthetic pathway causing avenaol.
Informa UK Limited, 2022年05月13日, Bioscience, Biotechnology, and Biochemistry研究論文（学術雑誌）
Cultivated tomato (Solanum lycopersicum) contains α-tomatine, a steroidal glycoalkaloid (SGA), which functions as a defense compound to protect against pathogens and herbivores; interestingly, wild species in the tomato clade biosynthesize a variety of SGAs. In cultivated tomato the metabolic detoxification of α-tomatine during tomato fruit ripening is an important trait which aided in its domestication, and two distinct 2-oxoglutarate-dependent dioxygenases (DOXs), a C-23 hydroxylase of α-tomatine (Sl23DOX) and a C-27 hydroxylase of lycoperoside C (Sl27DOX), are key to this process. There are tandemly duplicated DOX genes on tomato chromosome 1, with high levels of similarity to Sl23DOX. While these DOX genes are rarely expressed in cultivated tomato tissues, the recombinant enzymes of Solyc01g006580 and Solyc01g006610 metabolized α-tomatine to habrochaitoside A and (20R)-20-hydroxytomatine, and were therefore named as habrochaitoside A synthase (HAS) and α-tomatine 20-hydroxylase (20DOX), respectively. Furthermore, 20DOX and HAS exist in the genome of wild tomato S. habrochaites accession LA1777, which accumulates habrochaitoside A in its fruits, and their expression patterns were in agreement with the SGA profiles in LA1777. These results indicate that the functional divergence of α-tomatine-metabolizing DOX enzymes results from gene duplication and the neofunctionalization of catalytic activity and gene expression, and this contributes to the structural diversity of SGAs in the tomato clade.
2022年05月13日, Plant & cell physiology, 英語, 国内誌研究論文（学術雑誌）
- Japan Society for Bioscience, Biotechnology, and Agrochemistry, 2022年03月01日, 化学と生物, 60 (3), 107 - 109, 日本語
[招待有り]
研究論文（学術雑誌）
- 2022年02月, 化学, 77 (2), 12 - 16, 日本語ジャガイモの毒はトマトの苦味から進化した！
[招待有り]
- Wiley, 2022年01月03日, Journal of Phytopathology
研究論文（学術雑誌）
MAIN CONCLUSION: An Arabidopsis S-adenosyl-L-methionine-dependent methyltransferase belonging to the SABATH family catalyzes the specific carboxymethylation of (11R)-carlactonoic acid. Methyl carlactonoate (MeCLA), found in Arabidopsis (Arabidopsis thaliana) as a non-canonical strigolactone (SL), may be a biosynthetic intermediate of various non-canonical SLs and biologically active as a plant hormone. MeCLA is formed from carlactonoic acid (CLA), but the methyltransferases (MTs) converting CLA to MeCLA remain unclear. Previous studies have demonstrated that the carboxymethylation of acidic plant hormones is catalyzed by the same protein family, the SABATH family (Wang et al. in Evol Bioinform 15:117693431986086. https://doi.org/10.1177/1176934319860864 , 2019). In the present study, we focused on the At4g36470 gene, an Arabidopsis SABATH MT gene co-expressed with the MAX1 gene responsible for CLA formation for biochemical characterization. The recombinant At4g36470 protein expressed in Escherichia coli exhibited exclusive activity against naturally occurring (11R)-CLA among the substrates, including CLA enantiomers and a variety of acidic plant hormones. The apparent Km value for (11R)-CLA was 1.46 μM, which was relatively smaller than that of the other Arabidopsis SABATH MTs responsible for the carboxymethylation of acidic plant hormones. The strict substrate specificity and high affinity of At4g36470 suggested it is an (11R)-CLA MT. We also confirmed the function of the identified gene by reconstructing MeCLA biosynthesis using transient expression in Nicotiana benthamiana. Phylogenetic analysis demonstrated that At4g36470 and its orthologs in non-canonical SL-producing plants cluster together in an exclusive clade, suggesting that the SABATH MTs of this clade may be involved in the carboxymethylation of CLA and the biosynthesis of non-canonical SLs.
Springer Science and Business Media LLC, 2021年11月, Planta, 254 (5), 88 - 88, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Abstract Tomato (Solanum lycopersicum) contains α-tomatine, a steroidal glycoalkaloid that contributes to the plant defense against pathogens and herbivores through its bitter taste and toxicity. It accumulates at high levels in all the plant tissues, especially in leaves and immature green fruits, whereas it decreases during fruit ripening through metabolic conversion to the nontoxic esculeoside A, which accumulates in the mature red fruit. This study aimed to identify the gene encoding a C-27 hydroxylase that is a key enzyme in the metabolic conversion of α-tomatine to esculeoside A. The E8 gene, encoding a 2-oxoglutalate-dependent dioxygenase, is well known as an inducible gene in response to ethylene during fruit ripening. The recombinant E8 was found to catalyze the C-27 hydroxylation of lycoperoside C to produce prosapogenin A and is designated as Sl27DOX. The ripe fruit of E8/Sl27DOX-silenced transgenic tomato plants accumulated lycoperoside C and exhibited decreased esculeoside A levels compared with the wild-type (WT) plants. Furthermore, E8/Sl27DOX deletion in tomato accessions resulted in higher lycoperoside C levels in ripe fruits than in WT plants. Thus, E8/Sl27DOX functions as a C-27 hydroxylase of lycoperoside C in the metabolic detoxification of α-tomatine during tomato fruit ripening, and the efficient detoxification by E8/27DOX may provide an advantage in the domestication of cultivated tomatoes.Oxford University Press (OUP), 2021年10月01日, Plant and Cell Physiology, 62 (5), 775 - 783[査読有り]
研究論文（学術雑誌）
- 2021年09月, バイオサイエンスとインダストリー, 79 (5), 384 - 385, 日本語ジャガイモとトマトのアルカロイドの毒性を決定するジオキシゲナーゼの発見
[招待有り]
- Wiley, 2021年08月09日, The Plant Journal, 108 (1), 81 - 92
[査読有り]
研究論文（学術雑誌）
- 2021年08月, ファルマシア, 57 (8), 726 - 730, 日本語ジャガイモとトマトはおいしい野菜だが，毒がある？
[査読有り][招待有り]
- Elsevier BV, 2021年05月, Tetrahedron, 132194 - 132194
[査読有り]
研究論文（学術雑誌）
Plants produce ∼300 aromatic compounds enzymatically linked to prenyl side chains via C–O bonds. These
Proceedings of the National Academy of Sciences, 2021年04月27日, Proceedings of the National Academy of Sciences, 118 (17), e2022294118 - e2022294118, 英語, 国際誌O -prenylated aromatic compounds have been found in taxonomically distant plant taxa, with some of them being beneficial or detrimental to human health. Although theirO -prenyl moieties often play crucial roles in the biological activities of these compounds, no plant gene encoding an aromaticO -prenyltransferase (O -PT) has been isolated to date. This study describes the isolation of an aromaticO -PT gene,CpPT1 , belonging to the UbiA superfamily, from grapefruit (Citrus ×paradisi, Rutaceae). This gene was shown responsible for the biosynthesis ofO -prenylated coumarin derivatives that alter drug pharmacokinetics in the human body. Another coumarinO -PT gene encoding a protein of the same family was identified inAngelica keiskei , an apiaceous medicinal plant containing pharmaceutically activeO -prenylated coumarins. Phylogenetic analysis of theseO -PTs suggested that aromaticO -prenylation activity evolved independently from the same ancestral gene in these distant plant taxa. These findings shed light on understanding the evolution of plant secondary (specialized) metabolites via the UbiA superfamily.[査読有り]
研究論文（学術雑誌）
Abstract Strigolactones (SLs), first identified as germination stimulants for root parasitic weeds, act as endogenous phytohormones regulating shoot branching and as root-derived signal molecules mediating symbiotic communications in the rhizosphere. Canonical SLs typically have an ABCD ring system and can be classified into orobanchol- and strigol-type based on the C-ring stereochemistry. Their simplest structures are 4-deoxyorobanchol (4DO) and 5-deoxystrigol (5DS), respectively. Diverse canonical SLs are chemically modified with one or more hydroxy or acetoxy groups introduced into the A- and/or B-ring of these simplest structures, but the biochemical mechanisms behind this structural diversity remain largely unexplored. Sorgomol in sorghum (Sorghum bicolor [L.] Moench) is a strigol-type SL with a hydroxy group at C-9 of 5DS. In this study, we characterized sorgomol synthase. Microsomal fractions prepared from a high-sorgomol-producing cultivar of sorghum, Sudax, were shown to convert 5DS to sorgomol. A comparative transcriptome analysis identified SbCYP728B subfamily as candidate genes encoding sorgomol synthase. Recombinant SbCYP728B35 catalyzed the conversion of 5DS to sorgomol in vitro. Substrate specificity revealed that the C-8bS configuration in the C-ring of 5DS stereoisomers was essential for this reaction. The overexpression of SbCYP728B35 in Lotus japonicus hairy roots, which produce 5DS as an endogenous SL, also resulted in the conversion of 5DS to sorgomol. Furthermore, SbCYP728B35 expression was not detected in nonsorgomol-producing cultivar, Abu70, suggesting that this gene is responsible for sorgomol production in sorghum. Identification of the mechanism modifying parental 5DS of strigol-type SLs provides insights on how plants biosynthesize diverse SLs.Oxford University Press (OUP), 2021年04月02日, Plant Physiology, 185 (3), 902 - 913, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Abstract Potato (Solanum tuberosum ), a worldwide major food crop, produces the toxic, bitter tasting solanidane glycoalkaloids α-solanine and α-chaconine. Controlling levels of glycoalkaloids is an important focus on potato breeding. Tomato (Solanum lycopersicum ) contains a bitter spirosolane glycoalkaloid, α-tomatine. These glycoalkaloids are biosynthesized from cholesterol via a partly common pathway, although the mechanisms giving rise to the structural differences between solanidane and spirosolane remained elusive. Here we identify a 2-oxoglutarate dependent dioxygenase, designated as DPS (Dioxygenase for Potato Solanidane synthesis), that is a key enzyme for solanidane glycoalkaloid biosynthesis in potato. DPS catalyzes the ring-rearrangement from spirosolane to solanidane via C-16 hydroxylation. Evolutionary divergence of spirosolane-metabolizing dioxygenases contributes to the emergence of toxic solanidane glycoalkaloids in potato and the chemical diversity in Solanaceae.Springer Science and Business Media LLC, 2021年03月, Nature Communications, 12 (1)[査読有り]
研究論文（学術雑誌）
- Wiley, 2021年02月, The Plant Journal, 105 (4), 1026 - 1034
[査読有り]
研究論文（学術雑誌）
- Japan Society for Bioscience, Biotechnology, and Agrochemistry, 2020年11月01日, 化学と生物, 58 (11), 628 - 634, 日本語
[査読有り][招待有り]
研究論文（学術雑誌）
Heliolactone is a non-canonical strigolactone isolated from sunflower root exudates. We have previously demonstrated that exogenously administered carlactonoic acid (CLA) was converted to heliolactone in sunflower. The conversion of CLA to heliolactone requires the methyl esterification of the carboxylic acid at C-19. Also, the CLA conversion to its methyl ester, methyl carlactonoate (MeCLA), was demonstrated by feeding experiment in sunflower. However, the involvement of MeCLA in heliolactone biosynthesis remains unclear. We synthesised MeCLA in its racemic form and resolved it into its enantiomers. Feeding experiments revealed that (11R)-MeCLA was exclusively converted to heliolactone in sunflower. This result is an evidence that (11R)-MeCLA is the biosynthetic precursor of heliolactone. Further conversion of heliolactone to an unidentified metabolite with a molecular mass larger than heliolactone by 16 Da was confirmed. The conversion was inhibited by a cytochrome P450 inhibitor, suggesting the involvement of cytochrome P450-dependent monooxygenation.
Informa UK Limited, 2020年10月09日, Natural Product Research, 1 - 8, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
- Japanese Society for Plant Cell and Molecular Biology, 2020年09月25日, Plant Biotechnology, 37 (3), 319 - 325
[査読有り]
研究論文（学術雑誌）
Abstract The apple cultivar McIntosh Wijcik, which is a mutant of ‘McIntosh’, exhibits a columnar growth phenotype (short internodes, few lateral branches, many spurs, etc.) that is controlled by a dominant Co gene. The candidate gene (MdDOX-Co), encoding a 2-oxoglutarate-dependent dioxygenase, is located adjacent to an insertion mutation. Non-columnar apples express MdDOX-Co in the roots, whereas columnar apples express MdDOX-Co in the aerial parts as well as in the roots. However, the function of MdDOX-Co remains unknown. Here, we characterized tobacco plants overexpressing MdDOX-Co. The tobacco plants showed the typical dwarf phenotype, which was restored by application of gibberellin A3 (GA3). Moreover, the dwarf tobacco plants had low concentrations of endogenous bioactive gibberellin A1 (GA1) and gibberellin A4 (GA4). Similarly, ‘McIntosh Wijcik’ contained low endogenous GA4 concentration and its dwarf traits (short main shoot and internodes) were partially reversed by GA3 application. These results indicate that MdDOX-Co is associated with bioactive GA deficiency. Interestingly, GA3 application to apple trees also resulted in an increased number of lateral branches and a decrease in flower bud number, indicating that gibberellin (GA) plays important roles in regulating apple tree architecture by affecting both lateral branch formation (vegetative growth) and flower bud formation (reproductive growth). We propose that a deficiency of bioactive GA by ectopic expression of MdDOX-Co in the aerial parts of columnar apples not only induces dwarf phenotypes but also inhibits lateral branch development and promotes flower bud formation, and assembly of these multiple phenotypes constructs the columnar tree form.Oxford University Press (OUP), 2020年08月29日, Tree Physiology, 40 (9), 1205 - 1216, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
The shoot meristem, a stem-cell-containing tissue initiated during plant embryogenesis, is responsible for continuous shoot organ production in postembryonic development. Although key regulatory factors including KNOX genes are responsible for stem cell maintenance in the shoot meristem, how the onset of such factors is regulated during embryogenesis is elusive. Here, we present evidence that the two KNOX genes STM and KNAT6 together with the two other regulatory genes BLR and LAS are functionally important downstream genes of CUC1 and CUC2, which are a redundant pair of genes that specify the embryonic shoot organ boundary. Combined expression of STM with any of KNAT6, BLR, and LAS can efficiently rescue the defects of shoot meristem formation and/or separation of cotyledons in cuc1cuc2 double mutants. In addition, CUC1 and CUC2 are also required for the activation of KLU, a cytochrome P450-encoding gene known to restrict organ production, and KLU counteracts STM in the promotion of meristem activity, providing a possible balancing mechanism for shoot meristem maintenance. Together, these results establish the roles for CUC1 and CUC2 in coordinating the activation of two classes of genes with opposite effects on shoot meristem activity.
MDPI AG, 2020年08月15日, International Journal of Molecular Sciences, 21 (16), 5864 - 5864, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Genome editing using site-specific nucleases, such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9), is a powerful technology for crop breeding. For plant genome editing, the genome-editing reagents are usually expressed in plant cells from stably integrated transgenes within the genome. This requires crossing processes to remove foreign nucleotides from the genome to generate null segregants. However, in highly heterozygous plants such as potato, the progeny lines have different agronomic traits from the parent cultivar and do not necessarily become elite lines. Agrobacteria can transfer exogenous genes on T-DNA into plant cells. This has been used both to transform plants stably and to express the genes transiently in plant cells. Here, we infected potato, with Agrobacterium tumefaciens harboring TALEN-expression vector targeting sterol side chain reductase 2 (SSR2) gene and regenerated shoots without selection. We obtained regenerated lines with disrupted-SSR2 gene and without transgene of the TALEN gene, revealing that their disruption should be caused by transient gene expression. The strategy using transient gene expression by Agrobacterium that we call Agrobacterial mutagenesis, developed here should accelerate the use of genome-editing technology to modify heterozygous plant genomes.
Japanese Society for Plant Cell and Molecular Biology, 2020年06月25日, Plant Biotechnology, 37 (2), 205 - 211, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
- 2020年06月, 植物の生長調節, 56 (1), 35 - 41, 日本語ブラシのステロイド生合成における鍵酵素CYP90B1の結晶構造解析と実験上の工夫について
[査読有り][招待有り]
MAIN CONCLUSION: CYP722C from cotton, a homolog of the enzyme involved in orobanchol synthesis in cowpea and tomato, catalyzes the conversion of carlactonoic acid to 5-deoxystrigol. Strigolactones (SLs) are important phytohormones with roles in the regulation of plant growth and development. These compounds also function as signaling molecules in the rhizosphere by interacting with beneficial arbuscular mycorrhizal fungi and harmful root parasitic plants. Canonical SLs, such as 5-deoxystrigol (5DS), consist of a tricyclic lactone ring (ABC-ring) connected to a methylbutenolide (D-ring). Although it is known that 5DS biosynthesis begins with carlactonoic acid (CLA) derived from β-carotene, the enzyme that catalyzes the conversion of CLA remains elusive. Recently, we identified cytochrome P450 (CYP) CYP722C as the enzyme that catalyzes direct conversion of CLA to orobanchol in cowpea and tomato (Wakabayashi et al., Sci Adv 5:eaax9067, 2019). Orobanchol has a different C-ring configuration from that of 5DS. The present study aimed to characterize the homologous gene, designated GaCYP722C, from cotton (Gossypium arboreum) to examine whether this gene is involved in 5DS biosynthesis. Expression of GaCYP722C was upregulated under phosphate starvation, which is an SL-producing condition. Recombinant GaCYP722C was expressed in a baculovirus-insect cell expression system and was found to catalyze the conversion of CLA to 5DS but not to 4-deoxyorobanchol. These results strongly suggest that GaCYP722C from cotton is a 5DS synthase and that CYP722C is the crucial CYP subfamily involved in the generation of canonical SLs, irrespective of the different C-ring configurations.
Springer Science and Business Media LLC, 2020年05月, Planta, 251 (5), 97 - 97, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Abstract Tomato plants (Solanum lycopersicum) contain steroidal glycoalkaloid α-tomatine, which functions as a chemical barrier to pathogens and predators. α-Tomatine accumulates in all tissues and at particularly high levels in leaves and immature green fruits. The compound is toxic and causes a bitter taste, but its presence decreases through metabolic conversion to nontoxic esculeoside A during fruit ripening. This study identifies the gene encoding a 23-hydroxylase of α-tomatine, which is a key to this process. Some 2-oxoglutarate-dependent dioxygenases were selected as candidates for the metabolic enzyme, and Solyc02g062460, designated Sl23DOX, was found to encode α-tomatine 23-hydroxylase. Biochemical analysis of the recombinant Sl23DOX protein demonstrated that it catalyzes the 23-hydroxylation of α-tomatine and the product spontaneously isomerizes to neorickiioside B, which is an intermediate in α-tomatine metabolism that appears during ripening. Leaves of transgenic tomato plants overexpressing Sl23DOX accumulated not only neorickiioside B but also another intermediate, lycoperoside C (23-O-acetylated neorickiioside B). Furthermore, the ripe fruits of Sl23DOX-silenced transgenic tomato plants contained lower levels of esculeoside A but substantially accumulated α-tomatine. Thus, Sl23DOX functions as α-tomatine 23-hydroxylase during the metabolic processing of toxic α-tomatine in tomato fruit ripening and is a key enzyme in the domestication of cultivated tomatoes.Oxford University Press (OUP), 2020年01月01日, Plant and Cell Physiology, 61 (1), 21 - 28[査読有り]
研究論文（学術雑誌）
Strigolactones (SLs) are carotenoid-derived phytohormones and rhizosphere signaling molecules for arbuscular mycorrhizal fungi and root parasitic weeds. Why and how plants produce diverse SLs are unknown. Here, cytochrome P450 CYP722C is identified as a key enzyme that catalyzes the reaction of BC-ring closure leading to orobanchol, the most prevalent canonical SL. The direct conversion of carlactonoic acid to orobanchol without passing through 4-deoxyorobanchol is catalyzed by the recombinant enzyme. By knocking out the gene in tomato plants, orobanchol was undetectable in the root exudates, whereas the architecture of the knockout and wild-type plants was comparable. These findings add to our understanding of the function of the diverse SLs in plants and suggest the potential of these compounds to generate crops with greater resistance to infection by noxious root parasitic weeds.
American Association for the Advancement of Science (AAAS), 2019年12月18日, Science Advances, 5 (12), eaax9067 - eaax9067, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Potato (Solanum tuberosum) is one of the most important crops in the world. However, it is generally difficult to breed a new variety of potato crops because they are highly heterozygous tetraploid. Steroidal glycoalkaloids (SGAs) such as alpha-solanine and alpha-chaconine found in potato are antinutritional specialized metabolites. Because of their toxicity following intake, controlling the SGA levels in potato varieties is critical in breeding programs. Recently, genome-editing technologies using artificial site-specific nucleases such as TALEN and CRISPR-Cas9 have been developed and used in plant sciences. In the present study, we developed a highly active Platinum TALEN expression vector construction system, and applied to reduce the SGA contents in potato. Using Agrobacterium-mediated transformation, we obtained three independent transgenic potatoes harboring the TALEN expression cassette targeting SSR2 gene, which encodes a key enzyme for SGA biosynthesis. Sequencing analysis of the target sequence indicated that all the transformants could be SSR2-knockout mutants. Reduced SGA phenotype in the mutants was confirmed by metabolic analysis using LC-MS. In vitro grown SSR2knockout mutants exhibited no differences in morphological phenotype or yields when compared with control plants, indicating that the genome editing of SGA biosynthetic genes such as SSR2 could be a suitable strategy for controlling the levels of toxic metabolites in potato. Our simple and powerful plant genome-editing system, developed in the present study, provides an important step for future study in plant science.
Japanese Society for Plant Cell and Molecular Biology, 2019年09月25日, Plant Biotechnology, 36 (3), 167 - 173, 英語[査読有り]
研究論文（学術雑誌）
Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side-effects. Here, we report that UDP-glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP-glucuronic acid to glycyrrhetinic acid 3-O-monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin-deficient (83-555) strain of G. uralensis. The natural variant showed loss of specificity for UDP-glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83-555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP-glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP-glucuronic acid. The functional arginine residue and resultant specificity for UDP-glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP-sugar selectivity for the rational engineering of sweet triterpenoid saponins.
Wiley, 2019年09月, The Plant Journal, 99 (6), 1127 - 1143, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
- Informa UK Limited, 2019年07月03日, Plant Signaling & Behavior, 14 (7), 1605810 - 1605810
[査読有り]
研究論文（学術雑誌）
Brassinosteroids (BRs) are essential plant steroid hormones that regulate plant growth and development1. The most potent BR, brassinolide, is produced by addition of many oxygen atoms to campesterol by several cytochrome P450 monooxygenases (CYPs). CYP90B1 (also known as DWF4) catalyses the 22(S)-hydroxylation of campesterol and is the first and rate-limiting enzyme at the branch point of the biosynthetic pathway from sterols to BRs2. Here we show the crystal structure of Arabidopsis thaliana CYP90B1 complexed with cholesterol as a substrate. The substrate-binding conformation explains the stereoselective introduction of a hydroxy group at the 22S position, facilitating hydrogen bonding of brassinolide with the BR receptor3-5. We also determined the crystal structures of CYP90B1 complexed with uniconazole6,7 or brassinazole8, which inhibit BR biosynthesis. The two inhibitors are structurally similar; however, their binding conformations are unexpectedly different. The shape and volume of the active site pocket varies depending on which inhibitor or substrate is bound. These crystal structures of plant CYPs that function as membrane-anchored enzymes and exhibit structural plasticity can inform design of novel inhibitors targeting plant membrane-bound CYPs, including those involved in BR biosynthesis, which could then be used as plant growth regulators and agrochemicals.
Springer Science and Business Media LLC, 2019年06月, Nature Plants, 5 (6), 589 - 594, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
α-Tomatine and dehydrotomatine are major steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves and flowers of tomato (Solanum lycopersicum), and function as defensive compounds against bacteria, fungi, insects and animals. The aglycone of dehydrotomatine is dehydrotomatidine (5,6-dehydrogenated tomatidine, having the Δ5,6 double bond; the dehydro-type). The aglycone of α-tomatine is tomatidine (having a single bond between C5 and C6; the dihydro-type), which is believed to be derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5 reduction and C3 reduction; however, these conversion processes remain uncharacterized. In the present study, we demonstrate that a short-chain alcohol dehydrogenase/reductase designated Sl3βHSD is involved in the conversion of dehydrotomatidine to tomatidine in tomato. Sl3βHSD1 expression was observed to be high in the flowers, leaves and mature green fruits of tomato, in which high amounts of α-tomatine are accumulated. Biochemical analysis of the recombinant Sl3βHSD1 protein revealed that Sl3βHSD1 catalyzes the C3 oxidation of dehydrotomatidine to form tomatid-4-en-3-one and also catalyzes the NADH-dependent C3 reduction of a 3-ketosteroid (tomatid-3-one) to form tomatidine. Furthermore, during co-incubation of Sl3βHSD1 with SlS5αR1 (steroid 5α-reductase) the four reaction steps converting dehydrotomatidine to tomatidine were completed. Sl3βHSD1-silenced transgenic tomato plants accumulated dehydrotomatine, with corresponding decreases in α-tomatine content. Furthermore, the constitutive expression of Sl3βHSD1 in potato hairy roots resulted in the conversion of potato SGAs to the dihydro-type SGAs. These results demonstrate that Sl3βHSD1 is a key enzyme involved in the conversion processes from dehydrotomatidine to tomatidine in α-tomatine biosynthesis.
Oxford University Press (OUP), 2019年06月01日, Plant and Cell Physiology, 60 (6), 1304 - 1315, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
- Springer Science and Business Media LLC, 2019年03月, Nature Plants, 5 (3), 258 - 262
[査読有り]
研究論文（学術雑誌）
Plant specialized metabolites are often found as lineage-specific diastereomeric isomers. For example, Sesamum alatum accumulates the specialized metabolite (+)-2-episesalatin, a furofuran-type lignan with a characteristic diastereomeric configuration rarely found in other Sesamum spp. However, little is known regarding how diastereomeric specificity in lignan biosynthesis is implemented in planta. Here, we show that S. alatum CYP81Q3, a P450 orthologous to S. indicum CYP81Q1, specifically catalyzes methylenedioxy bridge (MDB) formation in (+)-epipinoresinol to produce (+)-pluviatilol. Both (+)-epipinoresinol and (+)-pluviatilol are putative intermediates of (+)-2-episesalatin based on their diastereomeric configurations. On the other hand, CYP81Q3 accepts neither (+)- nor (-)-pinoresinol as a substrate. This diastereomeric selectivity of CYP81Q3 is in clear contrast to that of CYP81Q1, which specifically converts (+)-pinoresinol to (+)-sesamin via (+)-piperitol by the sequential formation of two MDBs but does not accept (+)-epipinoresinol as a substrate. Moreover, (+)-pinoresinol does not interfere with the conversion of (+)-epipinoresinol to (+)-pluviatilol by CYP81Q3. Amino acid substitution and CO difference spectral analyses show that polymorphic residues between CYP81Q1 and CYP81Q3 proximal to their putative substrate pockets are crucial for the functional diversity and stability of these two enzymes. Our data provide clues to understanding how the lineage-specific functional differentiation of respective biosynthetic enzymes substantiates the stereoisomeric diversity of lignan structures.
2018年11月, Plant & cell physiology, 59 (11), 2278 - 2287, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
- Elsevier BV, 2018年10月, Plant Physiology and Biochemistry, 131, 70 - 77
[査読有り]
研究論文（学術雑誌）
Reductive metabolism of strigolactones (SLs) in several plants was investigated. Analysis of aquaculture filtrates of cowpea and sorghum each fed with four stereoisomers of GR24, the most widely used synthetic SL, revealed stereospecific reduction of the double bond at C-3' and C-4' in the butenolide D-ring with preference for an unnatural 2'S configuration. The cowpea metabolite converted from 2'-epi-GR24 and the sorghum metabolite converted from ent-GR24 had the methyl group at C-4' in the trans configuration with the substituent at C-2', different from the cis configuration of the synthetic H2-GR24 reduced with Pd/C catalyst. The plants also reduced the double bond in the D-ring of 5-deoxystrigol isomers with a similar preference. The metabolites and synthetic H2-GR24 stereoisomers were much less active than were the GR24 stereoisomers in inducing seed germination of the root parasitic weeds Striga hermonthica, Orobanche crenata, and O. minor. These results provide additional evidence of the importance of the D-ring for bioactivity of SLs.
2018年08月, Bioorganic & medicinal chemistry, 26 (14), 4225 - 4233, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Steroidal glycoalkaloids (SGAs) are specialized anti-nutritional metabolites that accumulate in Solanum lycopersicum (tomato) and Solanum tuberosum (potato). A series of SGA biosynthetic genes is known to be upregulated in Solanaceae species by jasmonate-responsive Ethylene Response Factor transcription factors, including JRE4 (otherwise known as GAME9), but the exact regulatory significance in planta of each factor has remained unaddressed. Here, via TILLING-based screening of an EMS-mutagenized tomato population, we isolated a JRE4 loss-of-function line that carries an amino acid residue missense change in a region of the protein important for DNA binding. In this jre4 mutant, we observed downregulated expression of SGA biosynthetic genes and decreased SGA accumulation. Moreover, JRE4 overexpression stimulated SGA production. Further characterization of jre4 plants revealed their increased susceptibility to the generalist herbivore Spodoptera litura larvae. This susceptibility illustrates that herbivory resistance is dependent on JRE4-mediated defense responses, which include SGA accumulation. Ethylene treatment attenuated the jasmonate-mediated JRE4 expression induction and downstream SGA biosynthesis in tomato leaves and hairy roots. Overall, this study indicated that JRE4 functions as a primary master regulator of SGA biosynthesis, and thereby contributes toward plant defense against chewing insects.
Wiley, 2018年06月, The Plant Journal, 94 (6), 975 - 990, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
The original version of of the Supplementary Information associated with this Article inadvertently omitted oligonucleotide primer sequences from Supplementary Table 3 and Supplementary Methods describing the molecular cloning of CYP92B14, CPR1 and CYP81Q cDNA fragments. The HTML has been updated to include a corrected version of the Supplementary Information.
2018年05月25日, Nature communications, 9 (1), 2140 - 2140, 英語, 国際誌研究論文（学術雑誌）
4-Coumaroyl-CoA ligase (4CL) is ubiquitous in the plant kingdom, and plays a central role in the biosynthesis of phenylpropanoids such as lignins, flavonoids, and coumarins. 4CL catalyzes the formation of the coenzyme A thioester of cinnamates such as 4-coumaric, caffeic, and ferulic acids, and the regulatory position of 4CL in the phenylpropanoid pathway renders the enzyme an attractive target that controls the composition of phenylpropanoids in plants. In this study, we designed and synthesized mechanism-based inhibitors for 4CL in order to develop useful tools for the investigation of physiological functions of 4CL and chemical agents that modulate plant growth with the ultimate goal to produce plant biomass that exhibits features that are beneficial to humans. The acylsulfamide backbone of the inhibitors in this study was adopted as a mimic of the acyladenylate intermediates in the catalytic reaction of 4CL. These acylsulfamide inhibitors and the important synthetic intermediates were fully characterized using two-dimensional NMR spectroscopy. Five 4CL proteins with distinct substrate specificity from four plant species, i.e., Arabidopsis thaliana, Glycine max (soybean), Populus trichocarpa (poplar), and Petunia hybrida (petunia), were used to evaluate the inhibitory activity, and the half-maximum inhibitory concentration (IC50) of each acylsulfamide in the presence of 4-coumaric acid (100 µM) was determined as an index of inhibitory activity. The synthetic acylsulfamides used in this study inhibited the 4CLs with IC50 values ranging from 0.10 to 722 µM, and the IC50 values of the most potent inhibitors for each 4CL were 0.10–2.4 µM. The structure–activity relationship observed in this study revealed that both the presence and the structure of the acyl group of the synthetic inhibitors strongly affect the inhibitory activity, and indicates that 4CL recognizes the acylsulfamide inhibitors as acyladenylate mimics.
Elsevier Ltd, 2018年05月15日, Bioorganic and Medicinal Chemistry, 26 (9), 2466 - 2474, 英語[査読有り]
研究論文（学術雑誌）
Potato (Solanum tuberosum) is a major food crop, while the most tissues of potato accumulates steroidal glycoalkaloids (SGAs) -solanine and -chaconine. Since SGAs confer a bitter taste on human and show the toxicity against various organisms, reducing the SGA content in the tubers is requisite for potato breeding. However, generation of SGA-free potato has not been achieved yet, although silencing of several SGA biosynthetic genes led a decrease in SGAs. Here, we show that the knockout of St16DOX encoding a steroid 16-hydroxylase in SGA biosynthesis causes the complete abolition of the SGA accumulation in potato hairy roots. Nine candidate guide RNA (gRNA) target sequences were selected from St16DOX by in silico analysis, and the two or three gRNAs were introduced into a CRISPR/Cas9 vector designated as pMgP237-2A-GFP that can express multiplex gRNAs based on the pre-tRNA processing system. To establish rapid screening of the candidate gRNAs that can efficiently mutate the St16DOX gene, we used a potato hairy root culture system for the introduction of the pMgP237 vectors. Among the transgenic hairy roots, two independent lines showed no detectable SGAs but accumulated the glycosides of 22,26-dihydroxycholesterol, which is the substrate of St16DOX. Analysis of the two lines with sequencing exhibited the mutated sequences of St16DOX with no wild-type sequences. Thus, generation of SGA-free hairy roots of tetraploid potato was achieved by the combination of the hairy root culture and the pMgP237-2A-GFP vector. This experimental system is useful to evaluate the efficacy of candidate gRNA target sequences in the short-term.
2018年04月, Plant Physiology and Biochemistry : PPB, Vol.131, 70 - 77, 英語[査読有り]
研究論文（学術雑誌）
Strigolactones (SLs), comprising compounds with diverse but related chemical structures, are determinant signals in elicitation of germination in root parasitic Orobanchaceae and in mycorrhization in plants. Further, SLs are a novel class of plant hormones that regulate root and shoot architecture. Dissecting common and divergent biosynthetic pathways of SLs may provide avenues for modulating their production in planta. Biosynthesis of SLs in various SL-producing plant species was inhibited by fluridone, a phytoene desaturase inhibitor. The plausible biosynthetic precursors of SLs were exogenously applied to plants, and their conversion to canonical and non-canonical SLs was analysed using liquid chromatography-tandem mass spectrometry. The conversion of carlactone (CL) to carlactonoic acid (CLA) was a common reaction in all investigated plants. Sorghum converted CLA to 5-deoxystrigol (5-DS) and sorgomol, and 5-DS to sorgomol. One sorgomol-producing cotton cultivar had the same SL profile as sorghum in the feeding experiments. Another cotton cultivar converted CLA to 5-DS, strigol, and strigyl acetate. Further, 5-DS was converted to strigol and strigyl acetate. Moonseed converted CLA to strigol, but not to 5-DS. The plant did not convert 5-DS to strigol, suggesting that 5-DS is not a precursor of strigol in moonseed. Similarly, 4-deoxyorobanchol was not a precursor of orobanchol in cowpea. Further, sunflower converted CLA to methyl carlactonoate and heliolactone. These results indicated that the biosynthetic pathways of hydroxy SLs do not necessarily involve their respective deoxy SL precursors.
2018年04月, Journal of experimental botany, 69 (9), 2305 - 2318, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
The bioconversion of 5-deoxystrigol (5DS) and 4-deoxyorobanchol (4DO), the simplest canonical strigolactones (SLs), into monohydroxylated SLs such as strigol, sorgomol and orobanchol was confirmed by administering of stable isotope-labeled substrates to hydroponically grown plants. Liquid chromatography-mass spectrometry analyses established that 5DS was stereoselectively converted into strigol and sorgomol by cotton (Gossypium hirsutum) and Chinese milk vetch (Astragalus sinicus), respectively. 4DO was converted into orobanchol by rice (Oryza sativa). However, the red bell pepper (Capsicum annuum), red clover (Trifolium pratense), and pea (Pisum sativum) negligibly converted 4DO into orobanchol. The red bell pepper converted ent-4DO into 2',8-bisepi-sorgomol. These results suggest that some plants generate orobanchol without passing through 4DO.
2018年, JOURNAL OF PESTICIDE SCIENCE, 43 (3-4), 198 - 206, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
(+)-Sesamin, (+)-sesamolin, and (+)-sesaminol glucosides are phenylpropanoid-derived specialized metabolites called lignans, and are rich in sesame (Sesamum indicum) seed. Despite their renowned anti-oxidative and health-promoting properties, the biosynthesis of (+)-sesamolin and (+)-sesaminol remained largely elusive. Here we show that (+)-sesamolin deficiency in sesame is genetically associated with the deletion of four C-terminal amino acids (Del4C) in a P450 enzyme CYP92B14 that constitutes a novel clade separate from sesamin synthase CYP81Q1. Recombinant CYP92B14 converts (+)-sesamin to (+)-sesamolin and, unexpectedly, (+)-sesaminol through an oxygenation scheme designated as oxidative rearrangement of α-oxy-substituted aryl groups (ORA). Intriguingly, CYP92B14 also generates (+)-sesaminol through direct oxygenation of the aromatic ring. The activity of CYP92B14 is enhanced when co-expressed with CYP81Q1, implying functional coordination of CYP81Q1 with CYP92B14. The discovery of CYP92B14 not only uncovers the last steps in sesame lignan biosynthesis but highlights the remarkable catalytic plasticity of P450s that contributes to metabolic diversity in nature.
2017年12月18日, NATURE COMMUNICATIONS, 8 (1), 2155 - 2155, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.
2017年10月05日, Cell, 171 (2), 287 - 304, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites that are found in the Solanaceae. Potato (Solanum tuberosum) contains the SGAs α-solanine and α-chaconine, while tomato (Solanum lycopersicum) contains α-tomatine, all of which are biosynthesized from cholesterol. However, although two cytochrome P450 monooxygenases that catalyze the 22- and 26-hydroxylation of cholesterol have been identified, the 16-hydroxylase remains unknown. Feeding with deuterium-labeled cholesterol indicated that the 16α- and 16β-hydrogen atoms of cholesterol were eliminated to form α-solanine and α-chaconine in potato, while only the 16α-hydrogen atom was eliminated in α-tomatine biosynthesis, suggesting that a single oxidation at C-16 takes place during tomato SGA biosynthesis while a two-step oxidation occurs in potato. Here, we show that a 2-oxoglutarate-dependent dioxygenase, designated as 16DOX, is involved in SGA biosynthesis. We found that the transcript of potato 16DOX (St16DOX) was expressed at high levels in the tuber sprouts, where large amounts of SGAs are accumulated. Biochemical analysis of the recombinant St16DOX protein revealed that St16DOX catalyzes the 16α-hydroxylation of hydroxycholesterols and that (22S)-22,26-dihydroxycholesterol was the best substrate among the nine compounds tested. St16DOX-silenced potato plants contained significantly lower levels of SGAs, and a detailed metabolite analysis revealed that they accumulated the glycosides of (22S)-22,26-dihydroxycholesterol. Analysis of the tomato 16DOX (Sl16DOX) gene gave essentially the same results. These findings clearly indicate that 16DOX is a steroid 16α-hydroxylase that functions in the SGA biosynthetic pathway. Furthermore, St16DOX silencing did not affect potato tuber yield, indicating that 16DOX may be a suitable target for controlling toxic SGA levels in potato.
Oxford University Press (OUP), 2017年09月, Plant Physiology, 175 (1), 120 - 133, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
In tomato, perception of jasmonates by a receptor complex, which includes the F-box protein CORONATINE INSENSITIVE 1 (COI1), elicits biosynthesis of defensive steroidal glycoalkaloids (SGAs) via a jasmonate-responsive ERF transcription factor, JRE4/GAME9. Although JRE4 is upregulated by jasmonate and induces the expression of many metabolic genes involved in SGA biosynthesis, it is not known whether JRE4 alone is sufficient for increased SGA biosynthesis upon activation of jasmonate signaling. Here, we show that application of methyl jasmonate induces the expression of JRE4 and SGA biosynthesis genes in leaves and hairy roots of wild-type tomato, but not in jasmonic acid insensitive I (jail), a loss-of-function mutant allele of the tomato COI1 gene. Induced overexpression of JRE4 increased the expression of SGA biosynthesis genes in transgenic hairy roots of both wild-type tomato and the jail mutant, suggesting that JRE4 is the primary transcription factor that functions downstream of the jasmonate signaling pathway. (C) 2017 Elsevier Inc. All rights reserved.
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2017年07月, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 489 (2), 206 - 210, 英語[査読有り]
研究論文（学術雑誌）
Steroidal glycoalkaloids （SGAs） are toxic specialized metabolites that are found in Solanaceae. Potato （Solanum tuberosum） contains the SGAs α-solanine and α-chaconine, which are biosynthesized from cholesterol. Several biosynthetic genes including SSR2 and two cytochrome P450 genes （CYP72A188 and CYP72A208） have been identified, and the transgenic potato plants silencing these biosynthetic genes showed SGA-reduced phenotypes. Here we summarize our recent results and strategy towards metabolic engineering of potato accumulating pharmaceutically useful compounds by genome editing. CYP88B1, which is involved in a later step of the SGA biosynthetic pathway with unknown catalytic function, is co-ordinately expressed with the SGA biosynthetic genes. We applied CRISPR/Cas9 system to knockout potato CYP88B1. The CYP88B1-knockout potatoes showed no accumulation of SGAs, and furthermore the corresponding amounts of steroidal saponins were accumulated in the knockout potatoes.
一般社団法人 植物化学調節学会, 2017年06月, 植物の生長調節, 52 (1), 92 - 98, 日本語[査読有り][招待有り]
- 2017年, Photosynthesis Research, 131, 41 - 50, 英語Essential role of the PSI-LHCII supercomplex in photosystem acclimation to light and/or heat conditions by state transitions.
[査読有り]
研究論文（学術雑誌）
Fifteen steroidal saponins 1–15, which include 4 furostanol glycosides 1–3 and 15, and 11 spirostanol glycosides 4–14, were isolated from the tubers and leaves of lesser yam (Dioscorea esculenta, Togedokoro). Their structures were identified by nuclear magnetic resonance and liquid chromatography mass spectroscopy. Four steroidal saponins 9, 11, 14, and 15 were found to be novel compounds.
Japan Society for Bioscience Biotechnology and Agrochemistry, 2017年, Bioscience, Biotechnology and Biochemistry, 81 (12), 2253 - 2260, 英語[査読有り]
研究論文（学術雑誌）
- Elsevier BV, 2016年11月, Bioorganic & Medicinal Chemistry, 24 (21), 5340 - 5352
[査読有り]
研究論文（学術雑誌）
2-オキソグルタル酸依存性ジオキシゲナーゼ（2OGD）は二価鉄を含む水溶性のジオキシゲナーゼであり，低分子化合物からタンパク質やDNAまで様々な生体分子に対して水酸化や脱メチル化など多彩な酸化反応を触媒する．2OGDは細菌から植物，動物まで広く存在しており，ヒトには約60個，各植物種のゲノムには0.5%を占める2OGD遺伝子が存在しているが，進化系統解析に基づく分類命名法は確立されていない．本解説では，生物界全体の2OGDを比較解析し，2OGDの進化と多様性，および代謝活性の有用性について考察する．
公益社団法人 日本農芸化学会, 2016年09月, 化学と生物, 54 (9), 640 - 649, 日本語[査読有り][招待有り]
研究論文（学術雑誌）
alpha-Solanine and alpha-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry.
AMER SOC PLANT BIOLOGISTS, 2016年08月, PLANT PHYSIOLOGY, 171 (4), 2458 - 2467, 英語[査読有り]
研究論文（学術雑誌）
The green odor of plants is characterized by green leaf volatiles (GLVs) composed of C6 compounds. GLVs are biosynthesized from polyunsaturated fatty acids in thylakoid membranes by a series of enzymes. A representative member of GLVs (E)-2-hexenal, known as the leaf aldehyde, has been assumed to be produced by isomerization from (Z)-3-hexenal in the biosynthesis pathway; however, the enzyme has not yet been identified. In this study, we purified the (Z)-3:(E)-2-hexenal isomerase (HI) from paprika fruits and showed that various plant species have homologous HIs. Purified HI is a homotrimeric protein of 110 kDa composed of 35-kDa subunits and shows high activity at acidic and neutral pH values. Phylogenetic analysis showed that HIs belong to the cupin superfamily, and at least three catalytic amino acids (His, Lys, Tyr) are conserved in HIs of various plant species. Enzymatic isomerization of (Z)-3-hexenal in the presence of deuterium oxide resulted in the introduction of deuterium at the C4 position of (E)-2-hexenal, and a suicide substrate 3-hexyn-1-al inhibited HI irreversibly, suggesting that the catalytic mode of HI is a keto-enol tautomerism reaction mode mediated by a catalytic His residue. The gene expression of HIs in Solanaceae plants was enhanced in specific developmental stages and by wounding treatment. Transgenic tomato plants overexpressing paprika HI accumulated (E)-2-hexenal in contrast to wild-type tomato plants mainly accumulating (Z)-3-hexenal, suggesting that HI plays a key role in the production of (E)-2-hexenal in planta.
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2016年07月, JOURNAL OF BIOLOGICAL CHEMISTRY, 291 (27), 14023 - 14033, 英語[査読有り]
研究論文（学術雑誌）
The green odor of plants is characterized by green leaf volatiles (GLVs) composed of C6 compounds. GLVs are biosynthesized from polyunsaturated fatty acids in thylakoid membranes by a series of enzymes. A representative member of GLVs (E)-2-hexenal, known as the leaf aldehyde, has been assumed to be produced by isomerization from (Z)-3-hexenal in the biosynthesis pathway; however, the enzyme has not yet been identified. In this study, we purified the (Z)-3:(E)-2-hexenal isomerase (HI) from paprika fruits and showed that various plant species have homologous HIs. Purified HI is a homotrimeric protein of 110 kDa composed of 35-kDa subunits and shows high activity at acidic and neutral pH values. Phylogenetic analysis showed that HIs belong to the cupin superfamily, and at least three catalytic amino acids (His, Lys, Tyr) are conserved in HIs of various plant species. Enzymatic isomerization of (Z)-3-hexenal in the presence of deuterium oxide resulted in the introduction of deuterium at the C4 position of (E)-2-hexenal, and a suicide substrate 3-hexyn-1-al inhibited HI irreversibly, suggesting that the catalytic mode of HI is a keto-enol tautomerism reaction mode mediated by a catalytic His residue. The gene expression of HIs in Solanaceae plants was enhanced in specific developmental stages and by wounding treatment. Transgenic tomato plants overexpressing paprika HI accumulated (E)-2-hexenal in contrast to wild-type tomato plants mainly accumulating (Z)-3-hexenal, suggesting that HI plays a key role in the production of (E)-2-hexenal in planta.
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2016年07月, JOURNAL OF BIOLOGICAL CHEMISTRY, 291 (27), 14023 - 14033, 英語[査読有り]
研究論文（学術雑誌）
Steroidal saponins are natural surfactants with various biological activities, and the tubers of Dioscorea, known as yam, contain a furostanol glycoside protodioscin and a spirostanol glycoside dioscin, which are valuable saponins required for semi-synthetic production of pharmaceutical steroidal drugs. Steroidal saponins are biosynthesized from cholesterol via several steps of oxygenation and transglycosylation, and a beta-glucosidase is involved in the hydrolytic conversion from furostanol glycosides to spirostanol glycosides. To investigate steroidal saponin biosynthesis in Dioscorea spps, comparative transcriptome analysis of high saponin producers, D. esculenta and D. cayenensis, and a low producer, D. alata, was performed using 454 pyrosequencing. In this study, we isolated and characterized a beta-glucosidase ( DeF26G1) from D. esculenta. The DeF26G1 cDNA encodes a family 1 glucosidase, and the DeF26G1 transcript was present at high levels in D. esculenta but not detected in D. alata. The recombinant DeF26G1 protein hydrolyzed the 26-O-glycosidic bond of protodioscin to form dioscin, indicating that the DeF26G1 gene encodes furostanol glycoside 26-O-beta-glucosidase. These results suggested that DeF26G1 is involved in the conversion of furostanol saponins to spirostanol saponins, which seems to be related to biological defense response in the leaves of Dioscorea plants.
The Japanese Society for Plant Cell and Molecular Biology, 2015年12月, Plant Biotechnology, 32 (4), 299 - U141, 英語[査読有り]
研究論文（学術雑誌）
Bioconversion of GR24, the most widely used synthetic strigolactone (SL), by hydroponically grown sorghum (Sorghum bicolor) and biological activities of hydroxylated GR24 stereoisomers were studied. Analysis of extracts and exudates of sorghum roots previously fed with a racemic and diastereomeric mixture of GR24, using liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (MRM), confirmed uptake of GR24 and suggested its conversion to mono-hydroxylated products. Two major GR24 metabolites, 7-hydroxy-GR24 and 8-hydroxy-GR24, were identified in the root extracts and exudates by direct comparison of chromatographic behavior with a series of synthetic mono-hydroxylated GR24 analogues. Separate feeding experiments with each of the GR24 stereoisomers revealed that the hydroxylated products were derived from 2'-epi-GR24, an evidence of sterical recognition of the GR24 molecule by sorghum. Trans-4-hydroxy-GR24 isomers derived from all GR24 stereoisomers were detected in the exudates as minor metabolites. The synthetic hydroxy-GR24 isomers induced germination of Striga hermonthica in decreasing order of C-8 > C-7 > C-6 > C-5 > C-4. In contrast the stereoisomers having the same configuration of orobanchol, irrespective of position of hydroxylation, induced germination of Striga gesnerioides. The results confirm previous reports on structural requirements of SLs and ascribe a critical role to hydroxylation, but not to the position of the hydroxyl group in the AB part of the molecule, in induction of S. gesnerioides seed germination. (c) 2015 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2015年09月, BIOORGANIC & MEDICINAL CHEMISTRY, 23 (18), 6100 - 6110, 英語[査読有り]
研究論文（学術雑誌）
Bioconversion of GR24, the most widely used synthetic strigolactone (SL), by hydroponically grown sorghum (Sorghum bicolor) and biological activities of hydroxylated GR24 stereoisomers were studied. Analysis of extracts and exudates of sorghum roots previously fed with a racemic and diastereomeric mixture of GR24, using liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (MRM), confirmed uptake of GR24 and suggested its conversion to mono-hydroxylated products. Two major GR24 metabolites, 7-hydroxy-GR24 and 8-hydroxy-GR24, were identified in the root extracts and exudates by direct comparison of chromatographic behavior with a series of synthetic mono-hydroxylated GR24 analogues. Separate feeding experiments with each of the GR24 stereoisomers revealed that the hydroxylated products were derived from 2'-epi-GR24, an evidence of sterical recognition of the GR24 molecule by sorghum. Trans-4-hydroxy-GR24 isomers derived from all GR24 stereoisomers were detected in the exudates as minor metabolites. The synthetic hydroxy-GR24 isomers induced germination of Striga hermonthica in decreasing order of C-8 > C-7 > C-6 > C-5 > C-4. In contrast the stereoisomers having the same configuration of orobanchol, irrespective of position of hydroxylation, induced germination of Striga gesnerioides. The results confirm previous reports on structural requirements of SLs and ascribe a critical role to hydroxylation, but not to the position of the hydroxyl group in the AB part of the molecule, in induction of S. gesnerioides seed germination. (c) 2015 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2015年09月, BIOORGANIC & MEDICINAL CHEMISTRY, 23 (18), 6100 - 6110, 英語[査読有り]
研究論文（学術雑誌）
Tea plants (Camellia sinensis) store volatile organic compounds (VOCs; monoterpene, aromatic, and aliphatic alcohols) in the leaves in the form of water-soluble diglycosides, primarily as beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides). These VOCs play a critical role in plant defenses and tea aroma quality, yet little is known about their biosynthesis and physiological roles in planta. Here, we identified two UDP-glycosyltransferases (UGTs) from C. sinensis, UGT85K11 (CsGT1) and UGT94P1 (CsGT2), converting VOCs into beta-primeverosides by sequential glucosylation and xylosylation, respectively. CsGT1 exhibits a broad substrate specificity toward monoterpene, aromatic, and aliphatic alcohols to produce the respective glucosides. On the other hand, CsGT2 specifically catalyzes the xylosylation of the 69-hydroxy group of the sugar moiety of geranyl beta-D-glucopyranoside, producing geranyl beta-primeveroside. Homology modeling, followed by site-directed mutagenesis of CsGT2, identified a unique isoleucine-141 residue playing a crucial role in sugar donor specificity toward UDP-xylose. The transcripts of both CsGTs were mainly expressed in young leaves, along with beta-PRIMEVEROSIDASE encoding a diglycoside-specific glycosidase. In conclusion, our findings reveal the mechanism of aroma beta-primeveroside biosynthesis in C. sinensis. This information can be used to preserve tea aroma better during the manufacturing process and to investigate the mechanism of plant chemical defenses.
AMER SOC PLANT BIOLOGISTS, 2015年06月, PLANT PHYSIOLOGY, 168 (2), 464 - 477, 英語[査読有り]
研究論文（学術雑誌）
Abiotic stresses cause serious damage to plants; therefore, plants undergo a complicated stress response through signal transduction originating from environmental stimuli. Here we show that a subset of short-chain leaf volatiles with an alpha, beta-unsaturated carbonyl bond in their structure (reactive short-chain leaf volatiles, RSLVs) like (E)-2-hexenal and (E)-2-butenal can act as signal chemicals that strongly induce the gene expression of abiotic-related transcription factors, such as heat stress-related transcription factors (HSFA2, MBF1c) and other abiotic stress-related transcription factors (DREB2A, ZATs). RSLV-induced expression of HSFA2 and MBF1c was eliminated in HSFA1s-, known as heat stress response master regulators, knockout mutant, whereas those of DREB2A and ZATs were not, suggesting that the RSLV signaling pathway is composed of HSFA1-dependent and -independent pathways. RSLV treatment induced production of chaperon proteins, and the RSLV-treated Arabidopsis thus demonstrated enhanced abiotic stress tolerance. Because oxidative stress treatment enhanced RSLV production, we concluded that commonly found RSLVs produced by environmental stresses are powerful inducer of abiotic stress-related gene expression as oxidative stress signals.
NATURE PUBLISHING GROUP, 2015年01月, SCIENTIFIC REPORTS, 5, 8030, 英語[査読有り]
研究論文（学術雑誌）
Abiotic stresses cause serious damage to plants; therefore, plants undergo a complicated stress response through signal transduction originating from environmental stimuli. Here we show that a subset of short-chain leaf volatiles with an alpha, beta-unsaturated carbonyl bond in their structure (reactive short-chain leaf volatiles, RSLVs) like (E)-2-hexenal and (E)-2-butenal can act as signal chemicals that strongly induce the gene expression of abiotic-related transcription factors, such as heat stress-related transcription factors (HSFA2, MBF1c) and other abiotic stress-related transcription factors (DREB2A, ZATs). RSLV-induced expression of HSFA2 and MBF1c was eliminated in HSFA1s-, known as heat stress response master regulators, knockout mutant, whereas those of DREB2A and ZATs were not, suggesting that the RSLV signaling pathway is composed of HSFA1-dependent and -independent pathways. RSLV treatment induced production of chaperon proteins, and the RSLV-treated Arabidopsis thus demonstrated enhanced abiotic stress tolerance. Because oxidative stress treatment enhanced RSLV production, we concluded that commonly found RSLVs produced by environmental stresses are powerful inducer of abiotic stress-related gene expression as oxidative stress signals.
NATURE PUBLISHING GROUP, 2015年01月, SCIENTIFIC REPORTS, 5, 8030, 英語[査読有り]
研究論文（学術雑誌）
Photosystems of higher plants alleviate heat-induced damage in the presence of light under moderate stressed conditions; however, in the absence of light (i.e., in the dark), the same plants are damaged more easily. (Yamauchi and Kimura, 2011) We demonstrate that regulating photochemical energy transfer in heat-treated wheat at 40 degrees C with light contributed to heat tolerance of the photosystem. Chlorophyll fluorescence analysis using heat-stressed wheat seedlings in light showed increased non-photochemical quenching (NPQ) of chlorophyll fluorescence, which was due to thermal dissipation that was increased by state 1 to state 2 transition. Transmission electron microscopy revealed structural changes in thylakoid membranes, including unstacking of grana regions under heat stress in light. It was accompanied by the phosphorylation of thylakoid proteins such as D1 and D2 proteins and the light harvesting complex II proteins Lhcb1 and Lhcb2. These results suggest that heat stress at 40 degrees C in light induces state 1 to state 2 transition for the preferential excitation of photosystem I (PSI) by phosphorylating thylakoid proteins more strongly. Structural changes of thylakoid membrane also assist the remodeling of photosystems and regulation of energy distribution by transition toward state 2 probably contributes to plastoquione oxidation; thus, light-driven electrons flowing through PSI play a protective role against PSII damage under heat stress.
MDPI AG, 2014年12月, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 15 (12), 23042 - 23058, 英語[査読有り]
研究論文（学術雑誌）
Root exudates of sunflower (Helianthus annuus L.) line 2607A induced germination of seeds of root parasitic weeds Striga hermonthica, Orobanche cumana, Orobanche minor, Orobanche crenata, and Phelipanche aegyptiaca. Bioassay-guided purification led to the isolation of a germination stimulant designated as heliolactone. FT-MS analysis indicated a molecular formula of C20H24O6. Detailed NMR spectroscopic studies established a methylfuranone group, a common structural component of strigolactones connected to a methyl ester of a C-14 carboxylic acid via an enol ether bridge. The cyclohexenone ring is identical to that of 3-oxo-alpha-ionol and the other part of the molecule corresponds to an oxidized carlactone at C-19. It is a carlactone-type molecule and functions as a germination stimulant for seeds of root parasitic weeds. Heliolactone induced seed germination of the above mentioned root parasitic weeds, while dehydrocostus lactone and costunolide, sesquiterpene lactones isolated from sunflower root exudates, were effective only on O. cumana and O. minor. Heliolactone production in aquacultures increased when sunflower seedlings were grown hydroponically in tap water and decreased on supplementation of the culture with either phosphorus or nitrogen. Costunolide, on the other hand, was detected at a higher concentration in well-nourished medium as opposed to nutrient-deficient media, thus suggesting a contrasting contribution of heliolactone and the sesquiterpene lactone to the germination of O. cumana under different soil fertility levels. (c) 2014 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2014年12月, PHYTOCHEMISTRY, 108, 122 - 128, 英語研究論文（学術雑誌）
(+)-Sesamin is a major furofran-class lignan in sesame seeds and harbors characteristic two methylenedioxy bridges (MDB) that are sequentially formed from (+)-pinoresinol via (+)-piperitol by a Sesamum indicum P450, CYP81Q1. However, the molecular basis for this unique catalytic activity of CYP81Q1 has been poorly understood. To elucidate MDB formation, we tested various natural and non-natural metabolites as substrates for CYP81Q1. A synthetic (+)-SC1mr and a naturally occurring (+)-kobusin showed inhibitory effect on the production of (+)-sesamin by CYP81Q1 unlike (+)-epipinoresinol and (-)-pinoresinol, indicating the strict diastereomer and enantiomer selectivity. Homology modeling followed by site-directed mutagenesis of CYP81Q1 showed that an amino acid residue crucial for MDB formation is a unique Ala residue (A308), located in I-helix proximal to the substrate pocket, responsible to the conserved distal-Thr residue. MDB by CYP81Q1 is produced possibly through the formation of a substrate-participated hydrogen-bonding network, since single replacement of the Ala by Thr severely and specifically lowered the MDB forming activity. This hypothesis is supported by a newly identified MDB-forming enzyme CYP81Q38 from Phryma leptostachya harboring an Ala responsible to Ala308 in CYP81Q1. An evolutional perspective of CYP81Q1 is discussed in relation to another MDB-forming CYP719As functionally conserved in Ranunculales.
JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY, 2014年12月, Plant Biotechnology, 31 (5), 493 - 503, 英語[査読有り]
研究論文（学術雑誌）
Lignans are a class of phenylpropanoids that are widely distributed in the plant kingdom and some lignans are known to be present as glycosides. In the model plant Arabidopsis thaliana, pinoresinol and lariciresinol as well as their glucosides are found to be accumulated in the roots, but the enzymes involved in the glucosylation of lignans remain to be characterized. UGT71C1 showed activity towards several phenylpropanoids in previous studies, although its activity towards lignans has not been investigated. In the present study, the involvement of UGT71C1 in lignan glucosylation was examined. Quantification of lignans in a T-DNA knockout line of the UGT71C1 gene, ugt71c1, by an ultra performance liquid chromatography-tandem mass spectrometry showed that the content of pinoresinol glucoside decreased in parallel with an increase of pinoresinol at the corresponding degree. Two major peaks corresponding to lariciresinol glucosides were detected in the mass chromatogram of the extract from the wild type and one of the peaks decreased in the ugt71c1 line suggesting that the amount of lariciersinol glucoside also decreased in the mutant. UGT71C1 expressed in Escherichia coli showed glucosyltransferase activity towards pinoresinol and lariciresinol. The present results suggest that UGT71C1 is involved in lignan glucosylation in A. thaliana.
JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY, 2014年12月, PLANT BIOTECHNOLOGY, 31 (5), 561 - 566, 英語[査読有り]
研究論文（学術雑誌）
Specialized metabolism in land plants produces the diverse array of compounds, which is important in interaction with the environments. Generally, specialized metabolism-related genes consist of large gene families (superfamily), including cytochrome P450 monooxygenases (CYPs), 2-oxoglutarate-dependent dioxygenases (DOXs), and family-1 UDP-sugar dependent glycosyltransferases (UGTs), especially in angiosperms and gymnosperms. We investigated the changes in the numbers of these superfamily genes during the evolution of angiosperms by inferring gain and loss events in ancestral lineages of 5 angiosperms and 1 lycophyte. We observed the clear difference in the changes in the gene number among ancestral lineages. Intriguingly, gene gain events were coordinately occurred among CYP, DOX and UGT in lineage-specific manner, and the gain events were in good accordance with ancient whole genome duplication (WGD) events. Thus, the WGD events in angiosperms would have an important role in the expansion and evolution of specialized metabolism by providing prerequisite genetic resources for subsequent lineage-specific local tandem duplication (LTD) of superfamily genes as well as functional differentiation of these superfamily genes.
JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY, 2014年12月, PLANT BIOTECHNOLOGY, 31 (5), 579 - 584, 英語[査読有り]
研究論文（学術雑誌）
The effects of an abscisic acid (ABA) 8'-hydroxylase inhibitor (ABAI) on ABA catabolism, stomatal aperture, and water potential were investigated in apple seedlings under dehydration conditions. In addition, 9-cis-epoxycarotenoid dioxygenase (MdNCED) and ABA 8'-hydroxylase (MdCYP707A) genes were isolated and their expressions were analyzed under dehydration conditions. The stomatal aperture decreased for 4 h after spraying with ABAI and the stomatal aperture in the ABAItreated leaves was lower than that in the untreated control- leaves during the dehydration conditions. The water potential was maintained at a higher level in the ABAItreated leaves during dehydration, however that in untreated control-leaves decreased with the progress of dehydration. In both the ABAI-treated and untreated controlleaves, endogenous ABA concentrations increased with dehydration, but the ABA levels in the ABAI-treated leaves were higher than those in the untreated controlleaves throughout dehydration. In contrast, the phaseic acid (PA) concentrations in the ABAI-treated leaves were lower than those in the untreated control-leaves during dehydration. Regardless of the higher endogenous ABA concentrations, the expressions of MdNCEDs in the ABAI-treated leaves were lower than those in the untreated control-leaves. In addition, the expressions of MdCYP707As in the ABAI-treated leaves were also lower than those in the untreated control-leaves. Higher 50% effective concentrations (EC50) were observed in the ABAI-treated leaves, which suggests that the oxidative damage under dehydration may be reduced by ABAI application. The results suggest that prompt stomata closure is required for survival under dehydration, and ABAI application may therefore be of practical use. The increase of endogenous ABA, which induced prompt stomata closure in ABAI-treated leaves, may depend on the retardation of the expression of MdCYP707As. Moreover, the results demonstrate a close relationship between MdNCEDs and MdCYP707As on ABA catabolism.
2014年07月20日, Acta Horticulturae, 1042, 151 - 157[査読有り]
研究論文（学術雑誌）
- Elsevier BV, 2014年06月, Journal of Biological Chemistry, 289 (24), 16826 - 16834
研究論文（学術雑誌）
The 2-oxoglutarate-dependent dioxygenase (2OGD) superfamily is the second largest enzyme family in the plant genome, and its members are involved in various oxygenation/hydroxylation reactions. Despite their biochemical significance in metabolism, a systematic analysis of plant 2OGDs remains to be accomplished. We present a phylogenetic classification of 479 2OGDs in six plant models, ranging from green algae to angiosperms. These were classified into three classes - DOXA, DOXB and DOXC - based on amino acid sequence similarity. The DOXA class includes plant homologs of Escherichia coliAlkB, which is a prototype of 2OGD involved in the oxidative demethylation of alkylated nucleic acids and histones. The DOXB class is conserved across all plant taxa and is involved in proline 4-hydroxylation in cell wall protein synthesis. The DOXC class is involved in specialized metabolism of various phytochemicals, including phytohormones and flavonoids. The vast majority of 2OGDs from land plants were classified into the DOXC class, but only seven from Chlamydomonas, suggesting that this class has diversified during land plant evolution. Phylogenetic analysis assigned DOXC-class 2OGDs to 57 phylogenetic clades. 2OGD genes involved in gibberellin biosynthesis were conserved among vascular plants, and those involved in flavonoid and ethylene biosynthesis were shared among seed plants. Several angiosperm-specific clades were found to be involved in various lineage-specific specialized metabolisms, but 31 of the 57 DOXC-class clades were only found in a single species. Therefore, the evolution and diversification of DOXC-class 2OGDs is partly responsible for the diversity and complexity of specialized metabolites in land plants.
WILEY, 2014年04月, PLANT JOURNAL, 78 (2), 328 - 343, 英語[査読有り]
研究論文（学術雑誌）
Key message: Structure-activity relationship studies of strigolactones and Striga gesnerioides seed germination revealed strict structural requirements for germination induction and a new function of the plant hormones as germination inhibitors. Stereoisomers of the naturally occurring strigolactones, strigol, sorgolactone, orobanchol, sorgomol and 5-deoxystrigol, 36 in total, were prepared and screened for the ability to induce and/or inhibit the germination of Striga hermonthica and Striga gesnerioides seeds collected from mature plants that parasitized on sorghum and cowpea, respectively. All of the compounds induced S. hermonthica seed germination, albeit displayed differential activities. On the other hand, only a limited number of the compounds induced significant germination in S. gesnerioides, thus indicating strict structural requirements. Strigolactones inducing high germination in S. gesnerioides induced low germination in S. hermonthica. Strigolactones with the same configuration at C3a, C8b and C2′ as that in 5-deoxystrigol (9a) induced high germination of S. hermonthica seeds, but most of them inhibited the germination of S. gesnerioides. The differential response of S. gesnerioides to strigolactones may play an important role in the survival of the species. However, the compounds could be used as means of control if mixed cropping of cowpea and sorghum is adopted. © 2013 Springer-Verlag Berlin Heidelberg.
2013年06月, Plant Cell Reports, 32 (6), 829 - 838, 英語研究論文（学術雑誌）
Strigolactones, important rhizosphere signalling molecules and a class of phytohormones that control shoot architecture, are apocarotenoids of plant origin. They have a structural core consisting of a tricyclic lactone connected to a butyrolactone group via an enol ether bridge. Deuterium-labelled 5-deoxystrigol stereoisomers were administered to aquacultures of a high sorgomol-producing sorghum cultivar, Sorghum bicolor (L.) Moench, and conversion of these substrates to sorgomol stereoisomers was investigated. Liquid chromatography-mass spectrometry analyses established that 5-deoxystrigol (5-DS) and ent-2′-epi-5-deoxystrigol were absorbed by sorghum roots, converted to sorgomol and ent-2′-epi-sorgomol, respectively, and exuded out of the roots. The conversion was inhibited by uniconazole-P, implying the involvement of cytochrome P450 in the hydroxylation. These results provide experimental evidence for the postulated biogenetic scheme for formation of strigolactones, in which hydroxylation at C-9 of 5-DS can generate sorgomol. © 2013 Elsevier B.V.
Elsevier Ltd, 2013年, Phytochemistry, 93, 41 - 48, 英語研究論文（学術雑誌）
Brassinosteroids are biosynthesized from campesterol via several cytochrome P450 (P450)-catalyzed oxidative reactions. We report the biochemical characterization of two brassinosteroid-biosynthetic P450s from rice: CYP90D2 and CYP90D3. A rice dwarf mutant, ebisu dwarf (d2), which contains a defective copy of CYP90D2, is known to be a brassinosteroid-deficient mutant, and CYP90D2 has been considered to act as a C-3 dehydrogenase. However, in vitro biochemical assays using baculovirus/insect cell-produced proteins revealed that both CYP9002 and CYP90D3 catalyze C-23 hydroxylation of various 22-hydroxylated brassinosteroids, but with markedly different catalytic efficiencies. Both enzymes preferentially convert (22S,24R)-22-hydroxyergost-4-en-3-one, (22S,24R)-22-hydroxy-5 alpha-ergostan-3-one, and 3-epi-6-deoxocathasterone to the corresponding 23-hydroxylated products, but are less active in the conversion of (22S)-22-hydroxycampesterol and 6-deoxocathasterone, in vitro. Consistently, the levels of 23-hydroxylated products of these intermediates, namely, 6-deoxoteasterone, 3-dehydro-6-deoxoteasterone, and 6-deoxotyphasterol were decreased in d2 mutants. These results indicate that CYP90D2 and CYP90D3 can act as brassinosteroid C-23 hydroxylases in rice. (C) 2012 Elsevier Masson SAS. All rights reserved.
ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER, 2012年09月, Plant Physiology and Biochemistry, 58, 220 - 226, 英語[査読有り]
研究論文（学術雑誌）
Brassinosteroids (BRs) are steroidal phytohormones that regulate plant growth and development. Whereas in Arabidopsis the network-like routes of BR biosynthesis have been elucidated in considerable detail, the roles of some of the biosynthetic enzymes and their participation in the different subpathways remained to be clarified. We investigated the function of the cytochrome P450 monooxygenase CYP90A1/CPD, which earlier had been proposed to act as a BR C-23 hydroxylase. Our GC-MS and genetic analyses demonstrated that the cpd mutation arrests BR synthesis upstream of the DET2-mediated 5 alpha reduction step and that overexpression of the C-23 hydroxylase CYP90C1 does not alleviate BR deficiency in the cpd mutant. In line with these results, we found that CYP90A1/CPD heterologously expressed in a baculovirus-insect cell system catalyzes C-3 oxidation of the early BR intermediates (22S)-22-hydroxycampesterol and (22R, 23R)-22,23-dihydroxycampesterol, as well as of 6-deoxocathasterone and 6-deoxoteasterone. Enzyme kinetic data of CYP90A1/CPD and DET2, together with those of the earlier studied CYP90B1, CYP90C1, and CYP90D1, suggest that BR biosynthesis proceeds mainly via the campestanol-independent pathway.
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012年09月, JOURNAL OF BIOLOGICAL CHEMISTRY, 287 (37), 31551 - 31560, 英語[査読有り]
研究論文（学術雑誌）
- 2012年08月, PLANTA, 236 (2), 753 - 761
[査読有り]
Cytochrome P450 monooxygenases (P450s) catalyze a wide variety of monooxygenation reactions in primary and secondary metabolism in plants. The share of P450 genes in each plant genome is estimated to be up to 1%. This implies that the diversification of P450 has made a significant contribution to the ability to acquire the emergence of new metabolic pathways during land plant evolution. The P450 families conserved universally in land plants contribute to their chemical defense mechanisms. Several P450s are involved in the biosynthesis and catabolism of plant hormones. Species-specific P450 families are essential for the biosynthetic pathways of phytochemicals such as terpenoids and alkaloids. Genome wide analysis of the gene clusters including P450 genes will provide a clue to defining the metabolic roles of orphan P450s. Metabolic engineering with plant P450s is an important technology for large-scale production of valuable phytochemicals such as medicines.
PHARMACEUTICAL SOC JAPAN, 2012年06月, BIOLOGICAL & PHARMACEUTICAL BULLETIN, 35 (6), 824 - 832, 英語[査読有り]
研究論文（学術雑誌）
The plant growth retardant uniconazole (UNI), which has been used as an effective inhibitor of ent-kaurene oxidase (CYP701A) involved in gibberellin biosynthesis, also strongly inhibits ABA 8'-hydroxylase (CYP707A), a key enzyme in abscisic acid catabolism. Azole P450 inhibitors bind to the P450 active site by both coordinating to the heme-iron atom via an sp(2) nitrogen and interacting with surrounding protein residues through a lipophilic region. We hypothesized that poor selectivity of UNI may result from its small molecular size and flexible conformation that allows it to fit into active sites differing in size and shape. To find a selective inhibitor of CYP701A based on this hypothesis, we examined inhibitory activities of three types of UNI analogues, which were conformationally constrained, enlarged in width, and enlarged in length, against recombinant rice CYP701A6 and Arabidopsis CYP707A3. Conformationally restricted analogues, UFAP2 and UFAP2N, inhibited CYP701A6 as strongly as UNI, whereas it inhibited CYP707A3 less than UNI. (C) 2012 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2012年05月, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 22 (9), 3240 - 3243, 英語[査読有り]
研究論文（学術雑誌）
We developed abscinazole-E2B (Abz-E2B), a practical and specific inhibitor of abscisic acid (ABA) 80hydroxylase (CYP707A), by structural modification of abscinazole-E1 (Abz-E1), another compound we developed. A butoxy group was introduced to Abz-E2B instead of the tosylate group of Abz-E1, in expectation of better water solubility, because the calculated log P value of Abz-E2B is 3.47, which is smaller than that of Abz-E1 (4.02). The water solubility of Abz-E2B was greater than 90% at a concentration of 100 mu M, at which the solubility of Abz-E1 was 20%. The enzyme specificity was improved significantly. In in vitro assays constructed using recombinant enzymes, (+/-)-Abz-E2B was a considerably weaker inhibitor than (+/-)-Abz-E1 for CYP701A, a GA biosynthetic enzyme, which is a target of S-uniconazole (S-UNI), a lead compound of Abz-E1. (+/-)-Abz-E2B application to plants resulted in improved desiccation tolerance and an increase in endogenous ABA, with little retardation of growth. We also prepared optically pure Abz-E2B and determined its absolute configuration. The R-enantiomer of Abz-E2B was the more potent inhibitor of CYP707A, unlike UNI, whereas both enantiomers were markedly less effective than S-UNI in inhibiting CYP701A. Because S-Abz-E2B arrested the growth of rice seedlings at 100 mu M, probably because of off-target effects, R-Abz-E2B should be used as a chemical tool for research focusing on CYP707A when 100 mu M or higher concentration is required, although (+/-)-Abz-E2B may be useful as an alternative option at a lower concentration. (C) 2012 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2012年05月, Bioorg Med Chem, 20 (10), 3162 - 3172, 英語[査読有り]
研究論文（学術雑誌）
Coumarins are important compounds that contribute to the adaptation of plants to biotic or abiotic stresses. Among coumarins, umbelliferone occupies a pivotal position in the plant phenylpropanoid network. Previous studies indicated that umbelliferone is derived from the ortho-hydroxylation of p-coumaric acid by an unknown biochemical step to yield 2,4-dihydroxycinnamic acid, which then undergoes spontaneous lactonization. Based on a recent report of a gene encoding a 2-oxoglutarate-dependent dioxygenase from Arabidopsis thaliana that exhibited feruloyl CoA 6-hydroxylase activity (Bourgaud, 2006), we combined a bioinformatic approach and a cDNA library screen to identify an orthologous ORF (Genbank accession number JF799117) from Ruta graveolens L. This ORF shares 59% amino acid identity with feruloyl CoA 6-hydroxylase, was functionally expressed in Escherichia coli, and converted feruloyl CoA into scopoletin and p-coumaroyl CoA into umbelliferone with equal activity. Its bi-functionality was further confirmed in planta: transient expression of JF799117 in Nicotiana benthamiana yielded plants with leaves containing high levels of umbelliferone and scopoletin when compared to control plants, which contained barely detectable traces of these compounds. The expression of JF799117 was also tightly correlated to the amount of umbelliferone that was found in UV-elicited R. graveolens leaves. Therefore, JF799117 encodes a p-coumaroyl CoA 2-hydroxylase in R. graveolens, which represents a previously uncharacterized step in the synthesis of umbelliferone in plants. Psoralen, which is an important furanocoumarin in R. graveolens, was found to be a competitive inhibitor of the enzyme, and it may exert this effect through negative feedback on the enzyme at an upstream position in the pathway. © 2011 Blackwell Publishing Ltd.
2012年05月, Plant Journal, 70 (3), 460 - 470, 英語[査読有り]
研究論文（学術雑誌）
Darjeeling teas are the highest grown teas in the world and preferred for its flavour, aroma and quality. Apart from the genetic makeup of the plant, earlier reports suggest that insect infestation, particularly jassids and thrips triggers the aroma and flavour formation in Darjeeling tea. The present work encompasses the identification of the genes/transcriptomes responsible for the typical flavour of Darjeeling tea, besides understanding the role of jassids and thrips in particular, in producing the best cup character and quality. The quantitative real time PCR analysis was based on a suppression subtractive hybridisation forward library of B157 (tea clone infested with thrips), providing us transcripts related to aroma and flavour formation. We observed the expression of genes like leucine zipper, ntd, nced, geraniol synthase, raffinose synthase, trehalose synthase, amylase, farnesyl transferase, catalase, methyl transferase, linalool synthase, peroxidases, elicitor responsive proteins, linamarase, nerolidol linalool synthase 2, 12-oxophytodienoate reductase, glucosidase, MYB transcription factor, and alcohol dehydrogenase, highly regulated due to insect infestation, manufacturing stresses and mechanical injury. The first report on gene expression dynamics in thrips infested Darjeeling tea leaves can be extrapolated with increase in volatiles which is responsible for enhancing the quality of Darjeeling tea, specially the flavour and aroma of the infusion. We hope to model these responses in order to understand the molecular changes that occur during Darjeeling tea flavour formation.
SPRINGER, 2012年04月, PLANT MOLECULAR BIOLOGY, 78 (6), 577 - 597, 英語[査読有り]
研究論文（学術雑誌）
Lipid peroxide-derived reactive carbonyls (RCs) can cause serious damage to plant functions. A chloroplastic NADPH-dependent alkenal/one oxidoreductase (AOR) detoxifies RCs, but its physiological significance remains unknown. In this study, we investigated the biological impacts of AOR using an AOR-knock out Arabidopsis line (aor). Methyl viologen treatment, mainly to enhance photosystem (PS) I-originated reactive oxygen species (ROS) production, caused more severe damage to aor than wild type (Col-0). In contrast, the high light treatment used to enhance PSII-originated ROS production resulted in no difference in PSII damage between Col-0 and aor. In conclusion, AOR can contribute to detoxify stromal RCs produced under oxidative stress. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
ELSEVIER SCIENCE BV, 2012年04月, FEBS LETTERS, 586 (8), 1208 - 1213, 英語[査読有り]
研究論文（学術雑誌）
Ortho-hydroxylation of cinnamates is a key step in coumarin biosynthesis in plants. Ortho-hydroxylated cinnamates undergo trans/cis isomerization of the side-chain and then lactonization to form coumarins. Sweet potato [Ipomoea batatas (L) Lam.] accumulates umbelliferone and scopoletin after biotic and abiotic stresses. To elucidate molecular aspects of ortho-hydroxylation involved in umbelliferone formation in sweet potato, isolation and characterization of cDNAs encoding 2-oxoglutarate-dependent dioxygenases (20GD) was performed from sweet potato tubers treated with a chitosan elicitor. Five cDNAs (designated as Ib) encoding a protein of 358 amino acid residues were cloned, and these were categorized into two groups, Ib1 and Ib2, based on their amino acid sequences. Whether the recombinant Ib proteins had any enzymatic activity toward cinnamates was examined. Ib1 proteins exhibited ortho-hydroxylation activity toward feruloyl coenzyme A (CoA) to form scopoletin (K-m = similar to 10 mu M, k(cat) = similar to 2.7 s(-1)). By contrast, Ib2 proteins catalyzed ortho-hydroxylation of feruloyl-CoA (K-m = 7.3-14.0 mu M, k(cat) = 0.28-0.55 s(-1)) and also of p-coumaroyl-CoA (K-m = 6.1-15.2 mu M, k(cat) = 0.28-0.64 s(-1)) to form scopoletin and umbelliferone, respectively. Fungal and chitosan treatments increased levels of umbelliferone and its glucoside (skimmin) in the tubers, and expression of the Ib2 gene was induced concomitantly. (C) 2011 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2012年02月, PHYTOCHEMISTRY, 74, 49 - 57, 英語[査読有り]
研究論文（学術雑誌）
The effects of an abscisic acid (ABA) 8'-hydroxylase inhibitor (Abz-F1) on ABA catabolism, stomatal aperture, and water potential were examined in apple seedlings under dehydration and rehydration conditions. In this study, 9-cis-epoxycarotenoid dioxigenase (MdNCED) and ABA 8'-hydroxylase (MdCYP707A) genes were isolated and their expressions were investigated under dehydration and rehydration conditions. The stomatal aperture decreased up to 4 h after spraying with Abz-F1 and the stomatal aperture in the Abz-F1-treated leaves was generally lower than that in the untreated control-leaves during the dehydration condition. Although the water potential in untreated control-leaves decreased with the progress of dehydration, it was maintained at a higher level in the Abz-F1 treated-leaves than in the untreated control-leaves during dehydration. Endogenous ABA concentrations increased with dehydration in both the Abz-F1 treated- and untreated-control-leaves, but the ABA levels in the Abz-F1 treated-leaves were higher than those in the untreated control-leaves throughout dehydration. In contrast, the phaseic acid (PA) concentrations in the Abz-F1 treated-leaves were lower than those in the untreated control-leaves during dehydration. The expressions of MdNCEDs in the Abz-F1 treated-leaves were lower than those in the untreated control-leaves regardless of the higher endogenous ABA concentrations. Moreover, the expressions of MdCYP707As in the Abz-F1 treated-leaves were also lower than those in the untreated control-leaves. Higher 50% effective concentrations (EC50) and ascorbic acid concentrations were observed in the Abz-F1 treated-leaves, which show that the oxidative damage under dehydration may be reduced by Abz-F1 application.These results suggest that prompt stomata closure is required for survival under dehydration, and Abz-F1 application may therefore be of practical use. The increase of endogenous ABA, which induced prompt stomata closure in Abz-F1 treated-leaves may depend on inhibition of the expression of MdCYP707As. Furthermore, the results showed the close relationship between MdNCEDs and MdCYP707As on ABA catabolism. (C) 2011 Published by Elsevier GmbH.
ELSEVIER GMBH, URBAN & FISCHER VERLAG, 2012年02月, JOURNAL OF PLANT PHYSIOLOGY, 169 (3), 234 - 241, 英語[査読有り]
研究論文（学術雑誌）
Triterpenoids are a diverse group of secondary metabolites that are associated with a variety of biological activities. Oleanolic acid, ursolic acid and betulinic acid are common triterpenoids in plants with diverse biological activities, including antifungal, antibacterial, anti-human immunodeficiency virus (HIV) and/or antitumor activities. In the present study, using the gene co-expression analysis tool of Medicago truncatula, we found a strong correlation between CYP716A12 and beta-amyrin synthase (bAS), which encodes the enzyme responsible for the initial cyclization of 2,3-oxidosqualene to beta-amyrin (the basic structural backbone of most triterpenoid saponins). Through an in vitro assay, we identified CYP716A12 as a beta-amyrin 28-oxidase able to modify beta-amyrin to oleanolic acid (through erythrodiol and, possibly, oleanolic aldehyde). We also confirmed its activity in vivo, by expressing CYP716A12 in transgenic yeast that endogenously produce beta-amyrin. In addition, CYP716A12 was evaluated for its potential alpha-amyrin- and lupeol-oxidizing activities. Interestingly, CYP716A12 was able to generate ursolic acid (through uvaol and, possibly, ursolic aldehyde) and betulinic acid (through betulin). Hence, CYP716A12 was characterized as a multifunctional enzyme with beta-amyrin 28-oxidase, alpha-amyrin 28-oxidase and lupeol 28-oxidase activities. We also identified homologs of CYP716A12 in grape (CYP716A15 and CYP716A17) that are involved in triterpenoid biosynthesis, which indicates the highly conserved functionality of the CYP716A subfamily among plants. These findings will be useful in the heterologous production of pharmacologically and industrially important triterpenoids, including oleanolic acid, ursolic acid and betulinic acid.
OXFORD UNIV PRESS, 2011年12月, PLANT AND CELL PHYSIOLOGY, 52 (12), 2050 - 2061, 英語[査読有り]
研究論文（学術雑誌）
Glycyrrhizin, a triterpenoid saponin derived from the underground parts of Glycyrrhiza plants (licorice), has several pharmacological activities and is also used worldwide as a natural sweetener. The biosynthesis of glycyrrhizin involves the initial cyclization of 2,3-oxidosqualene to the triterpene skeleton beta-amyrin, followed by a series of oxidative reactions at positions C-11 and C-30, and glycosyl transfers to the C-3 hydroxyl group. We previously reported the identification of a cytochrome P450 monooxygenase (P450) gene encoding beta-amyrin 11-oxidase (CYP88D6) as the initial P450 gene in glycyrrhizin biosynthesis. In this study, a second relevant P450 (CYP72A154) was identified and shown to be responsible for C-30 oxidation in the glycyrrhizin pathway. CYP72A154 expressed in an engineered yeast strain that endogenously produces 11-oxo-beta-amyrin (a possible biosynthetic intermediate between beta-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-beta-amyrin supplied in situ to produce glycyrrhetinic acid, a glycyrrhizin aglycone. Furthermore, CYP72A63 of Medicago truncatula, which has high sequence similarity to CYP72A154, was able to catalyze C-30 oxidation of beta-amyrin. These results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide a genetic tool for engineering the production of glycyrrhizin.
AMER SOC PLANT BIOLOGISTS, 2011年11月, PLANT CELL, 23 (11), 4112 - 4123, 英語[査読有り]
研究論文（学術雑誌）
Striga gesnerioides is a root parasitic weed of economic significance to cowpea ( Vigna unguiculata) crops in Western Africa. Seeds of the parasite germinate in response to cowpea root exudates. Germination stimulants for the seeds were isolated from the hydroponic culture filtrate of cowpea, and their structures were unambiguously determined as (-)-(3aR,412,8bR,2'R)-ent-2'-epi-orobanchol and (+)-(3aR,4R,8bR,2'R)-ent-2'-epi-orobanchyl acetate, on the basis of mass, CD, and (1)H NMR spectra; optical rotatory power; and chromatographic behavior on HPLC. The alcohol was first isolated and identified from the cowpea root exudates, and the acetate maybe the same compound that had been previously isolated from the exudates and designated as alectrol. Identity of the stimulants produced by cowpea to those produced by red clover (Trifolium pratense) was confirmed.
AMER CHEMICAL SOC, 2011年10月, JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 59 (19), 10485 - 10490, 英語研究論文（学術雑誌）
Strigolactones are highly potent germination stimulants for seeds of the parasitic weeds Striga and Orobanche spp., which severely reduce the yield of economically important crops in tropical and semitropical areas. GR24 (1) is a widely used synthetic strigolactone able to stimulate parasitic seed germination in the absence of a host plant to reduce soil seed levels. However, S. gesnerioides seeds do not respond to GR24 and only germinate by exposure to cowpea (Vigna unguiclata) root exudates or their active ingredient, the structure of which has not been established. Our present study provides substantial insight into the plant metabolism and structural requirements of strigolactones for host recognition by parasitic weeds. First, we clarified the structural and stereochemical requirements of synthetic strigolactones for seed germination of S. gesnerioides through bioassays of 4-hydroxy-GR24 (HO-GR24, 2) and 4-acetoxy-GR24 (AcO-GR24, 3) and their stereoisomers. These results suggest that both an oxygenated substituent at C-4 and the configuration of the tricyclic lactone and the D-ring are essential structural requirements for induction of S. gesnerioides seed germination. Furthermore, GR24 exhibited inhibitory activity against seed germination of S. gesnerioides at concentrations that induce seed germination of S. hermonthica and 0. minor. Second, we isolated the germination stimulants from hydroponic culture filtrate of cowpea, and their structures were unambiguously determined as (-)-(3aR,4R,8bR,2'R)-ent-2'-epi-orobanchol (11) and (+)-(3aR,4R,8bR,2'R)-ent-2'-epi-orobanchyl acetate (12), on the basis of mass, CD, and ^1H NMR spectra; optical rotator power; and chromatographic behavior on HPLC. These compounds are identical to products produced by red clover (Trifolium. pratense).
天然有機化合物討論会, 2011年09月02日, 天然有機化合物討論会講演要旨集, (53), 313 - 318, 日本語Strigolactones are highly potent germination stimulants for seeds of the parasitic weeds Striga and Orobanche spp. 4-Hydroxy-GR24 and 4-acetoxy-GR24 were prepared and their abilities to induce seed germination of Striga gesnerioides evaluated. Optically active (8bR,2'R)-isomers induced germination, although the racemic diastereomers were inactive. In contrast, the stereoisomer of GR24 with the same configuration induced negligible germination. Some stereoisomers of GR24 and its analogues acted as effective antagonists for induction of seed germination by cowpea root, exudates These results suggest that both an oxygenated substituent at C-4 and the configuration of the tricyclic lactone and the D-ring are essential structural requirements for induction of germination in S. gesnerioides seeds.
AMER CHEMICAL SOC, 2011年09月, JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 59 (17), 9226 - 9231, 英語[査読有り]
研究論文（学術雑誌）
Abstract Plants are often exposed to temperatures of around 40^o^C. These temperatures can cause serious damage to photosystems, yet plants can survive with minimum damage. Here, we show that plants switch photosystem to protect photosystem II (PSII) at 40^o^C. Using wheat and Arabidopsis seedlings, we investigated the mechanisms of heat-derived damage in the dark and avoidance of damage in the light. Heat treatment at 40^o^C in the dark caused serious damage to PSII: the maximum quantum yield of PSII (Fv/Fm) and oxygen evolution rapidly decreased. The damage was due to the degradation of the D1 protein (shown by immuno-chemical analysis) and the disturbance of energy transfer in PSII core chlorophyll-binding proteins CP43 and CP47 (shown by time-resolved fluorescence measurement). The damage to PSII might be due to enhanced introduction of electrons from the reducing power of the stroma into thylakoid membranes, causing subsequent electron backflow to PSII. Plants treated at 40^o^C in the light avoided PSII damage and showed preferential excitation of photosystem I (PSI), phosphorylation and migration of light-harvesting complex II (LHCII), which indicate state transition of the photosystem to enhance thermal dispersion and light-driven cyclic electron flow around PSI. These results suggest that heat damage to PSII is probably due to a backflow of reducing power from the stroma to PSII, and that light causes a state transition of photosystem, driving cyclic electron flow and thus protecting PSII from damage.Springer Science and Business Media LLC, 2011年08月01日, Nature Precedings研究論文（学術雑誌）
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.
WILEY-BLACKWELL, 2011年07月, PLANT JOURNAL, 67 (1), 1 - 12, 英語[査読有り]
研究論文（学術雑誌）
We prepared 19 amino acid conjugates of the plant hormone abscisic acid (ABA) and investigated their biological activity, enzymatic hydrolysis by a recombinant Arabidopsis amidohydrolases GST-ILR1 and GST-IAR3, and metabolic fate in rice seedlings. Different sets of ABA-amino acids induced ABA-like responses in different plants. Some ABA-amino acids, including some that were active in bioassays, were hydrolyzed by recombinant Arabidopsis GST-IAR3, although GST-ILR1 did not show hydrolysis activity for any of the ABA-amino acids. ABA-L-Ala, which was active in all the bioassays, an Arabidopsis seed germination, spinach seed germination, and rice seedling elongation assays, except in a lettuce seed germination assay and was hydrolyzed by GST-IAR3, was hydrolyzed to free ABA in rice seedlings. These findings suggest that some plant amidohydrolases hydrolyze some ABA-amino acid conjugates. Because our study indicates the possibility that different plants have hydrolyzing activity toward different ABA-amino acids, an ABA-amino acid may function as a species-selective pro-hormone of ABA. (C) 2011 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2011年03月, BIOORGANIC & MEDICINAL CHEMISTRY, 19 (5), 1743 - 1750, 英語[査読有り]
研究論文（学術雑誌）
Reactive carbonyls, especially alpha,beta-unsaturated carbonyls produced through lipid peroxidation, damage biomolecules such as proteins and nucleotides; elimination of these carbonyls is therefore essential for maintaining cellular homeostasis. In this study, we focused on an NADPH-dependent detoxification of reactive carbonyls in plants and explored the enzyme system involved in this detoxification process. Using acrolein (CH(2) = CHCHO) as a model alpha,beta-unsaturated carbonyl, we purified a predominant NADPH-dependent acrolein-reducing enzyme from cucumber leaves, and we identified the enzyme as an alkenal/one oxidoreductase (AOR) catalyzing reduction of an alpha,beta-unsaturated bond. Cloning of cDNA encoding AORs revealed that cucumber contains two distinct AORs, chloroplastic AOR and cytosolic AOR. Homologs of cucumber AORs were found among various plant species, including Arabidopsis, and we confirmed that a homolog of Arabidopsis (At1g23740) also had AOR activity. Phylogenetic analysis showed that these AORs belong to a novel class of AORs. They preferentially reduced alpha,beta-unsaturated ketones rather than alpha,beta-unsaturated aldehydes. Furthermore, we selected candidates of other classes of enzymes involved in NADPH-dependent reduction of carbonyls based on the bioinformatic information, and we found that an aldo-keto reductase (At2g37770) and aldehyde reductases (At1g54870 and At3g04000) were implicated in the reduction of an aldehyde group of saturated aldehydes and methylglyoxal as well as alpha,beta-unsaturated aldehydes in chloroplasts. These results suggest that different classes of NADPH-dependent reductases cooperatively contribute to the detoxification of reactive carbonyls.
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011年03月, JOURNAL OF BIOLOGICAL CHEMISTRY, 286 (9), 6999 - 7009, 英語[査読有り]
研究論文（学術雑誌）
We developed abscinazole-E1 (Abz-E1), a specific inhibitor of abscisic acid (ABA) 8'-hydroxylase (CYP707A). This inhibitor was designed and synthesized as an enlarged analogue of uniconazole (UNI), a well-known plant growth retardant, which inhibits a gibberellin biosynthetic enzyme (ent-kaurene oxidase, CYP701A) as well as CYP707A. Our results showed that Abz-E1 functions as a potent inhibitor of CYP707A and a poor inhibitor of CYP701A both in vitro and in vivo. Abz-E1 application to plants resulted in improved desiccation tolerance and an increase in endogenous ABA. (C) 2010 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2011年01月, BIOORGANIC & MEDICINAL CHEMISTRY, 19 (1), 406 - 413, 英語[査読有り]
研究論文（学術雑誌）
The plant growth-retardant uniconazole (UNI), a triazole inhibitor of gibberellin biosynthetic enzyme (CYP701A), inhibits multiple P450 enzymes including ABA 8'-hydroxylase (CYP707A), a key enzyme in ABA catabolism. Azole P450 inhibitors bind to a P450 active site by both coordinating to the heme-iron atom via sp(2) nitrogen and interacting with surrounding protein residues through a lipophilic region. We hypothesized that poor selectivity of UNI may result from adopting a distinct conformation and orientation for different active sites. Based on this hypothesis, we designed and synthesized novel UNI analogs with a disubstituted azole ring (DSI). These analogs were expected to have higher selectivity than UNI because the added functional group may interact with the active site to restrict orientation of the molecule in the active site. DSI-505ME and DSI-505MZ, which have an imidazolyl group with a methyl 5-acrylate, strongly inhibited recombinant CYP707A3, with no growth-retardant effect. (C) 2010 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2010年09月, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 20 (18), 5506 - 5509, 英語[査読有り]
研究論文（学術雑誌）
Serotonin is a well known neurotransmitter in mammals and plays an important role in various mental functions in humans. In plants, the serotonin biosynthesis pathway and its function are not well understood. The rice sekiguchi lesion (sl) mutants accumulate tryptamine, a candidate substrate for serotonin biosynthesis. We isolated the SL gene by map-based cloning and found that it encodes CYP71P1 in a cytochrome P450 monooxygenase family. A recombinant SL protein exhibited tryptamine 5-hydroxylase enzyme activity and catalyzed the conversion of tryptamine to serotonin. This pathway is novel and has not been reported in mammals. Expression of SL was induced by the N-acetylchitooligosaccharide (chitin) elicitor and by infection with Magnaporthe grisea, a causal agent for rice blast disease. Exogenously applied serotonin induced defense gene expression and cell death in rice suspension cultures and increased resistance to rice blast infection in plants. We also found that serotonin-induced defense gene expression is mediated by the RacGTPase pathway and by the G alpha subunit of the heterotrimeric G protein. These results suggest that serotonin plays an important role in rice innate immunity.
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010年04月, JOURNAL OF BIOLOGICAL CHEMISTRY, 285 (15), 11308 - 11313, 英語[査読有り]
研究論文（学術雑誌）
Numerous cytochrome P450 monooxygenases (P450s) have been known to be involved in the biosynthesis and metabolism of triterpenoids and steroids. This review will survey the oxidative reactions by such P450s and provide insights into the evolution of the steroid-biosynthetic P450 genes in the plant kingdom. Special emphasis is placed on brassinosteroids (BRs), plant steroid hormones, that play essential roles in the regulation of plant growth and development. Several P450s involved in BR biosynthesis and catabolism have recently been characterized by recombinant protein experiments, revealing a new route of the BR biosynthetic pathway. (C) 2009 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2009年12月, PHYTOCHEMISTRY, 70 (17-18), 1918 - 1929, 英語[査読有り]
研究論文（学術雑誌）
We enlarged the uniconazole (UNI) molecule to find a specific inhibitor of abscisic acid (ABA) 8'-hydroxylase, and synthesized various UNI derivatives that were substituted with hydrophilic and hydrophobic groups at the 4-chlorine of the phenyl group of UNI using click chemistry. Considering its potency in ABA 8'-hydroxylase inhibition, its small effect on seedling growth, and its ease of application, UT4, the UNI derivative containing the C-4 alkyltriazole, was the best candidate for a highly selective inhibitor of ABA 8'-hydroxylase. (C) 2009 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2009年10月, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 19 (19), 5782 - 5786, 英語[査読有り]
研究論文（学術雑誌）
To develop a specific inhibitor of abscisic acid (ABA) 8'-hydroxylase, a key enzyme in the catabolism of ABA, a plant hormone involved in stress tolerance, seed dormancy, and other various physiological events, we designed and synthesized conformationally restricted analogues of uniconazole (UNI), a well-known plant growth retardant, which inhibits a biosynthetic enzyme (ent-kaurene oxidase) of gibberellin as well as ABA 8'-hydroxylase. Although most of these analogues were less effective than UNI in inhibition of ABA 8'-hydroxylase and rice seedling growth, we found that a lactol-bridged analogue with an imidazole is a potent inhibitor of ABA 8'-hydroxylase but not of plant growth. This compound, abscinazole-F1, induced drought tolerance in apple seedlings upon spray treatment with a 10 mu M solution. (C) 2009 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2009年09月, BIOORGANIC & MEDICINAL CHEMISTRY, 17 (18), 6620 - 6630, 英語[査読有り]
研究論文（学術雑誌）
We have developed a highly sensitive and high-throughput method for the simultaneous analysis of 43 molecular species of cytokinins, auxins, ABA and gibberellins. This method consists of an automatic liquid handling system for solid phase extraction and ultra-performance liquid chromatography (UPLC) coupled with a tandem quadrupole mass spectrometer (qMS/MS) equipped with an electrospray interface (ESI; UPLC-ESI-qMS/MS). In order to improve the detection limit of negatively charged compounds, such as gibberellins, we chemically derivatized fractions containing auxin, ABA and gibberellins with bromocholine that has a quaternary ammonium functional group. This modification, that we call MS-probe, makes these hormone derivatives have a positive ion charge and permits all compounds to be measured in the positive ion mode with UPLC-ESI-qMS/MS in a single run. Consequently, quantification limits of gibberellins increased up to 50-fold. Our current method needs < 100mg (FW) of plant tissues to determine phytohormone profiles and enables us to analyze 180 plant samples simultaneously. Application of this method to plant hormone profiling enabled us to draw organ distribution maps of hormone species in rice and also to identify interactions among the four major hormones in the rice gibberellin signaling mutants, gid1-3, gid2-1 and slr1. Combining the results of hormone profiling data with transcriptome data in the gibberellin signaling mutants allows us to analyze relationships between changes in gene expression and hormone metabolism.
OXFORD UNIV PRESS, 2009年07月, PLANT AND CELL PHYSIOLOGY, 50 (7), 1201 - 1214, 英語[査読有り]
研究論文（学術雑誌）
Disaccharide-specific glycosidases (diglycosidases) are unique glycoside hydrolases, as their substrate specificities differ from those of monosaccharide-specific beta-glycosidases (monoglycosidases), in spite of similarities in their sequences and reaction mechanisms. Diglycosidases selectively hydrolyse the beta-glycosidic bond between glycone and aglycone of disaccharide glycosides, but do not cleave the bond between two saccharides, and barely hydrolyse monosaccharide glycosides. We analysed the substrate recognition mechanisms of diglycosidases by computational and experimental methods, using furcatin hydrolase (FH) (EC 3.2.1.161) derived from Viburnum furcatum. Amino acid sequence comparisons and model structure building revealed two residues, Ala419 and Ser504 of FH, as candidates determining the substrate specificity. These residues were specifically conserved in the diglycosidases. The model structure suggested that Ala419 is involved in the aglycone recognition, whereas Ser504 recognizes the external saccharide of the glycone. Mutations at these sites drastically decreased the diglycosidase activity. The mechanism by which the diglycosidases acquired their substrate specificity is discussed, based on these observations.
OXFORD UNIV PRESS, 2008年10月, JOURNAL OF BIOCHEMISTRY, 144 (4), 467 - 475, 英語[査読有り]
研究論文（学術雑誌）
Coumarins are derived via the phenylpropanoid pathway in plants. The 2H-1-benzopyran-2-one core structure of coumarins is formed via the ortho-hydroxylation of cinnamates, trans/cis isomerization of the side chain, and lactonization. Ortho-hydroxylation is a key step in coumarin biosynthesis as a branch point from lignin biosynthesis; however, ortho-hydroxylation of cinnamates is not yet fully understood. In this study, scopoletin biosynthesis was explored using Arabidopsis thaliana, which accumulates scopoletin and its beta-glucopyranoside scopolin in its roots. T-DNA insertion mutants of caffeoyl CoA O-methyltransferase 1 (CCoAOMT1) showed significant reduction in scopoletin and scopolin levels in the roots, and recombinant CCoAOMT1 exhibited 3'-O-methyltransferase activity on caffeoyl CoA to feruloyl CoA. These results suggest that feruloyl CoA is a key precursor in scopoletin biosynthesis. Ortho-hydroxylases of cinnamates were explored in the oxygenase families in A. thaliana, and one of the candidate genes in the Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2OGD) family was designated as F6'H1. T-DNA insertion mutants of F6'H1 showed severe reductions in scopoletin and scopolin levels in the roots. The pattern of F6'H1 expression is consistent with the patterns of scopoletin and scopolin accumulation. The recombinant F6'H1 protein exhibited ortho-hydroxylase activity for feruloyl CoA (K(m) = 36.0 +/- 4.27 mu M; k(cat) = 11.0 +/- 0.45 sec(-1)) to form 6'-hydroxyferuloyl CoA, but did not hydroxylate ferulic acid. These results indicate that Fe(II)- and 2-oxoglutarate-dependent dioxygenase is the pivotal enzyme in the ortho-hydroxylation of feruloyl CoA in scopoletin biosynthesis.
WILEY-BLACKWELL, 2008年09月, PLANT JOURNAL, 55 (6), 989 - 999, 英語[査読有り]
研究論文（学術雑誌）
Glycyrrhizin, a major bioactive compound derived from the underground parts of Glycyrrhiza (licorice) plants, is a triterpene saponin that possesses a wide range of pharmacological properties and is used worldwide as a natural sweetener. Because of its economic value, the biosynthesis of glycyrrhizin has received considerable attention. Glycyrrhizin is most likely derived from the triterpene beta-amyrin, an initial product of the cyclization of 2,3-oxidosqualene. The subsequent steps in glycyrrhizin biosynthesis are believed to involve a series of oxidative reactions at the C-11 and C-30 positions, followed by glycosyl transfers to the C-3 hydroxyl group; however, no genes encoding relevant oxidases or glycosyltransferases have been identified. Here we report the successful identification of CYP88D6, a cytochrome P450 monooxygenase (111450) gene, as a glycyrrhizin-biosynthetic gene, by transcript profiling-based selection from a collection of licorice expressed sequence tags (ESTs). CYP88D6 was characterized by in vitro enzymatic activity assays and shown to catalyze the sequential two-step oxidation of p-amyrin at C-11 to produce 11-oxo-beta-amyrin, a possible biosynthetic intermediate between P-amyrin and glycyrrhizin. CYP88D6 coexpressed with beta-amyrin synthase in yeast also catalyzed in vivo oxidation of beta-amyrin to 11-oxo-beta-amyrin. CYP88D6 expression was detected in the roots and stolons by RT-PCR; however, no amplification was observed in the leaves or stems, which is consistent with the accumulation pattern of glycyrrhizin in planta. These results suggest a role for CYP88D6 as a beta-amyrin 11-oxidase in the glycyrrhizin pathway.
NATL ACAD SCIENCES, 2008年09月, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 105 (37), 14204 - 14209, 英語[査読有り]
研究論文（学術雑誌）
- Biosynthetic Origin of the 1-Oxygen of Umbelliferone in the Root Tissue of Sweet Potato
Oxidation of p-coumarate at the ortho-position is a key step to form umbelliferone. A tracer analysis using (18)O(2) was performed in order to take information about the formation of umbelliferone in the root tissue of sweet potato. Mass fragmentation experiments revealed incorporation of an (18)O atom into the 1-position of umbelliferone. This result indicated that lactone of umbelliferone is formed via ortho-hydroxylation of the p-coumarate unit using O(2).
VERLAG Z NATURFORSCH, 2008年09月, ZEITSCHRIFT FUR NATURFORSCHUNG SECTION C-A JOURNAL OF BIOSCIENCES, 63 (9-10), 687 - 690, 英語[査読有り]
研究論文（学術雑誌）
The function of an Arabidopsis thaliana gene, At5g54160 annotated as a caffeic acid O-methyltransferase CAOMT gene was characterized. The recombinant enzyme of this gene (AtOMT1) catalyzed the O-methylation of phenylpropanoid and flavonoid substrates. The specificity constants (k (cat)/K (m)) for 5-hydroxyconiferaldehyde (5-HCAld) and quercetin were both 0.11 mu M(-1).min(-1). On the other hand, lignins of At5g54160-knockout Arabidopsis mutants lacked syringyl units. In addition, we showed that the gene silencing also resulted in significant accumulation of caffeyl alcohol (CaAlc). These results strongly suggested that At5g54160 gene is involved in syringyl lignin synthesis for the methylation of both 5-hydroxyconiferaldehyde and 3,4-dihydroxyphenyl compound(s).
SPRINGER TOKYO, 2008年08月, JOURNAL OF WOOD SCIENCE, 54 (4), 312 - 317, 英語[査読有り]
研究論文（学術雑誌）
A lignan, lariciresinol, was isolated from Arabidopsis thaliana, the most widely used model plant in plant bioscience sectors, for the first time. In the A. thaliana genome database, there are two genes (At1g32100 and At4g13660) that are annotated as pinoresinol/lariciresinol reductase (PLR). The recombinant AtPLRs showed strict substrate preference toward pinoresinol but only weak or no activity toward lariciresinol, which is in sharp contrast to conventional PLRs of other plants that can reduce both pinoresinol and lariciresinol efficiently to lariciresinol and secoisolariciresinol, respectively. Therefore, we renamed AtPLRs as A. thaliana pinoresinol reductases (AtPrRs). The recombinant AtPrR2 encoded by At4g13660 reduced only (-)-pinoresinol to (-)-lariciresinol and not (+)-pinoresinol in the presence of NADPH. This enantiomeric selectivity accords with that of other PLRs of other plants so far reported, which can reduce one of the enantiomers selectively, whatever the preferential enantiomer. In sharp contrast, AtPrR1 encoded by At1g32100 reduced both (+)-and (-)-pinoresinols to (+)-and (-)-lariciresinols efficiently with comparative k(cat)/K-m values. Analysis of lignans and spatio-temporal expression of AtPrR1 and AtPrR2 in their functionally deficient A. thaliana mutants and wild type indicated that both genes are involved in lariciresinol biosynthesis. In addition, the analysis of the enantiomeric compositions of lariciresinol isolated from the mutants and wild type showed that PrRs together with a dirigent protein(s) are involved in the enantiomeric control in lignan biosynthesis. Furthermore, it was demonstrated conclusively for the first time that differential expression of PrR isoforms that have distinct selectivities of substrate enantiomers can determine enantiomeric compositions of the product, lariciresinol.
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2008年06月, JOURNAL OF BIOLOGICAL CHEMISTRY, 283 (23), 15550 - 15557, 英語[査読有り]
研究論文（学術雑誌）
The plant growth retardant S-(+)-uniconazole (UNI-OH) is a strong inhibitor of abscisic acid (ABA) 8'-hydroxylase, a key enzyme in the catabolism of ABA, a plant hormone involved in stress tolerance, stomata] closure, flowering, seed dormancy, and other physiological events. In the present study, we focused on the two polar sites of UNI-OH and synthesized 3- and 2 ''-modified analogs. Conformational analysis and an in vitro enzyme inhibition assay yielded new findings on the structure-activity relationship of UNI-OH: (1) by substituting imidazole for triazole, which increases affinity to heme iron, we identified a more potent compound, IMI-OH; (2) the polar group at the 3-position increases affinity for the active site by electrostatic or hydrogen-bonding interactions; (3) the conformer preference for a polar environment partially contributes to affinity for the active site. These findings should be useful for designing potent azole-containing specific inhibitors of ABA 8'-hydroxylase. (C) 2007 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2008年03月, BIOORGANIC & MEDICINAL CHEMISTRY, 16 (6), 3141 - 3152, 英語[査読有り]
研究論文（学術雑誌）
beta-Primeverosidase (PD) is a family 1 glycosidase catalyzing the hydrolysis of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to release a disaccharide primeverose. To investigate how PD recognizes the disaccharide moiety of beta-primeverosides, the recombinant PD was expressed by a baculovirus-insect cell system. The recombinant PD was secreted from High Five cells and was properly modified with N-glycosylation and correct cleavage at the N-terminal signal peptide. The recombinant PD exhibited high substrate specificity to beta-primeverosides in terms of the glycone moiety, consistently with the substrate specificity of native PD from Camellia sinensis. Next, beta-glycosylamidines were synthesized as substrate analog inhibitors. beta-Primeverosylamidine strongly inhibited PD activity, but beta-glucosylamidine did not. Hence beta-primeverosylamidine is an ideal chemical tool for probing disaccharide recognition in the active site of PD. An affinity adsorbent for PD was prepared using beta-primeverosylamidine as a ligand. Affinity chromatography gave large amounts of PD with high purity, permitting crystallographic study.
TAYLOR & FRANCIS LTD, 2008年02月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 72 (2), 376 - 383, 英語[査読有り]
研究論文（学術雑誌）
Arabidopsis thaliana (Arabidopsis) treated with the four stereoisomers of Brz220 (2RS, 4RS-1-[4-propyl-2-(4-trifluoromethylphenyl)-1, 3-dioxane-2-ylmethyl]-1H-1, 2, 4-triazole) showed a dwarf phenotype like brassinosteroid (BR) biosynthesis mutants that were rescued by treatment of BRs. The target sites of each Brz220 stereoisomer were investigated by treatment of Arabidopsis with BRs in the dark. The results suggest that the stereoisomers block the 22-hydroxylation step in BR biosynthesis. This step is catalyzed by DWF4, an Arabidopsis cytochrome P450 identified as a steroid 22-hydroxylase. The enzyme was expressed in E. coli, and the binding affinity of the stereoisomers to recombinant DWF4 was analyzed. The results indicate that in these stereoisomers there exists a positive correlation between binding affinity to DWF4 and inhibition of Arabidopsis hypocotyl growth. In this context, we concluded that DWF4 is the target site of Brz220 in Arabidopsis.
TAYLOR & FRANCIS LTD, 2008年01月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 72 (1), 7 - 12, 英語[査読有り]
研究論文（学術雑誌）
To examine the effect of the minor abscisic acid (ABA) metabolite 7'-hydroxy-ABA on Arabidopsis ABA W-hydroxylase (CYP707A3), we developed a novel and facile, four-step synthesis of 7'-hydroxy-ABA from alpha-ionone. Structural analogues of Thydroxy-ABA, 1'-deoxy-7'-hydroxy-ABA, and 7'-oxo-ABA were also synthesized to evaluate the role of the T-hydroxyl group on binding to the enzyme. The result of enzyme inhibition assay suggests that the local polarity at C-T, neither steric bulkiness nor overall molecular hydrophilicity, would be the major reason why (+)-7'-hydroxy-ABA is not a potent inhibitor of CYP707A3. (C) 2007 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2007年09月, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 17 (17), 4977 - 4981, 英語[査読有り]
研究論文（学術雑誌）
\Abscisic acid (ABA), a plant stress hormone, has a chiral center (CV) in its molecule, yielding the enantiomers (1'S)-(+)ABA and (1'R)-(-)-ABA during chemical synthesis. ABA 8'-hydroxylase (CYP707A), which is the major and key P450 enzyme in ABA catabolism in plants, catalyzes naturally occurring (VS)-(+)-enantiomer, whereas it does not recognize naturally not occurring (1'R)-(-)-enantiomer as either a substrate or an inhibitor. Here we report a structural ABA analogue (AHI1), whose both enantiomers bind to recombinant Arabidopsis CYP707A3, in spite of stereo-structural similarity to ABA. The difference of AHI1 from ABA is the absence of the side-chain rnethyl group (C6) and lack of the alpha,beta-tinsaturated carbonyl (C2'=C3'-C4'=O) in the six-membered ring. To explore which moiety is responsible for asymmetrical binding by CYP707A3, we synthesized and tested ABA analogues that lacked each moiety. Competitive inhibition was observed for the (PR) enantiomers of these analogues in the potency order of (1'R,2'R)-(-)-2',3'-dihydro-4'-deoxo-ABA (K(1) = 0.45 mu M) > (VR)-(-)-4-oxo-ABA (K(1) = 27 mu M) > (1'R)-(-)-6-nor-ABA and (1'R,2'R)-(-)-2',3'-dihydro-ABA (no inhibition). In contrast to the (1'R)-enantiomers, the inhibition potency of the (US)-analogues declined with the saturation of the C2',C3'-double bond or with the elimination of the C4'-oxo moiety. These findings suggest that the C4'-oxo moiety coupled with the C2',C3'-double bond is the significant key functional group by which ABA K-hydroxylase distinguishes (1'S)-(+)-ABA from (1'R)-(-)-ABA. (c) 2007 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2007年09月, BIOORGANIC & MEDICINAL CHEMISTRY, 15 (18), 6311 - 6322, 英語[査読有り]
研究論文（学術雑誌）
The cyanogenic disaccharide glycoside, vicianin [ mandelonitrile beta- vicianoside ( 6- O- alpha- L- arabinopyranosyl- beta- D- glucopyranoside)], is accumulated in seeds of Vicia angustifolia var. segetalis. Vicianin hydrolase ( VH) catalyzes the hydrolysis of vicianin into mandelonitrile and a disaccharide vicianose. VH was purified from the seeds using DEAE-, CM- and Con A- Sepharose chromatography, and the molecular mass of the purified VH was estimated to be 56 kDa on SDS - PAGE. The N- terminal amino acid sequence of the purified VH was determined, and a cDNA encoding VH was obtained. The deduced VH protein consists of a 509 amino acid polypeptide containing a putative secretion signal peptide. It shares about 50% identity with various kinds of plant beta- glycosidases including tea leaf beta- primeverosidase and furcatin hydrolase, and is classified in family 1 of the glycosyl hydrolases. The VH transcript was detected abundantly in seeds and moderately in flowers, but only slightly in leaves, stems and roots, indicating that the organ distribution of VH expression is similar to that of the substrate vicianin. The recombinant VH was produced in insect cells with a baculovirus system, and was compared with the native VH in terms of substrate specificity. Both enzymes hydrolyzed vicianin to release vicianose, demonstrating that VH is a disaccharide- specific beta- glycosidase. VH also hydrolyzed the mandelonitrile beta- glucoside prunasin to some extent but did not hydrolyze the gentiobioside amygdalin, both of which contain the same aglycone as vicianin. Thus, VH is a unique cyanogenic glycosidase showing high glycone specificity for the disaccharide vicianoside.
OXFORD UNIV PRESS, 2007年07月, PLANT AND CELL PHYSIOLOGY, 48 (7), 938 - 947, 英語[査読有り]
研究論文（学術雑誌）
Oriental Beauty, which is made from tea leaves infested by the tea green leafhopper (Jacobiasca formosana) in Taiwan, has a unique aroma like ripe fruits and honey. To determine what occurs in the tea leaves during the oolong tea manufacturing process, the gene expression profiles and the chemical profiles were investigated. Tea samples were prepared from Camellia sinensis var. sinensis cv. Chin-shin Dah-pang while the tea leaves were attacked by the insect. The main volatile compounds, such as linalool-oxides, benzyl alcohol, 2-phenylethanol, and 2,6-dimethylocta-3,7-diene-2,6-diol, increased during manufacture. The gene expression profiles during manufacture were analyzed by differential screening between fresh leaves and tea leaves of the first turn over. Many up-regulated transcripts were found to encode various proteins homologous to stress response proteins. Accordingly, the endogenous contents of abscisic acid and raffinose increased during manufacture. Thus the traditional manufacturing method is a unique process that utilizes plant defense responses to elevate the production of volatile compounds and other metabolites.
TAYLOR & FRANCIS LTD, 2007年06月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 71 (6), 1476 - 1486, 英語[査読有り]
研究論文（学術雑誌）
- 日本森林学会, 2007年, 日本森林学会大会発表データベース, 118, 119 - 119, 日本語
カンペステロール (CR) からブラシノライドに至るブラシノステロイド生合成経路では、カンペスタノール (CN) を経由する経路がこれまで提唱されてきた。しかしC-22位水酸化酵素CYP90B1および、C-23位水酸化酵素CYP90C1, 90D1の基質特異性の解析から、早期にC-22位が水酸化される経路、すなわちCNを経由しない新規経路の存在が示唆された。5α還元酵素であるDET2が (24R)-ergost-4-en-3-one (4-en-3-one) を基質とすることは既に報告されているが、今回新たに22-OH-4-en-3-oneと22,23-diOH-4-en-3-oneも基質となることを明らかにした。また、我々はCYP90A1がC-3位酸化/異性化反応を触媒することを明らかにした。CYP90A1はCRを基質としないのに対し、22-OH-CRと22,23-diOH-CRは基質となり、基質特異性を調べた結果、22-OH-CRは22,23-diOH-CRに比べkcat/Km値が13倍高いことがわかった。CYP90B1の基質特異性と考え合わせると、CRから22-OH-CRを経て22-OH-4-en-3-oneへと反応する経路が主流であることが強く示唆された。DET2の基質特異性の解析、CYP90A1とは異なるCRのC-3位酸化/異性化酵素についても報告する予定である。
日本植物生理学会, 2007年, 日本植物生理学会年会およびシンポジウム 講演要旨集, 2007 (0), 409 - 409カンペスタノールからブラシノライドに至るブラシノステロイド (BR) 生合成経路の多くの酸化反応はシトクロムP450 (P450) 酵素により触媒されることが示されている。1996年、シロイヌナズナのBR欠損突然変異体constitutive photomorphogenesis and dwarfism (cpd) が単離され、CPD遺伝子がCYP90A1をコードしていることが明らかになった。またBR生合成中間体投与によるcpdの表現型回復実験の結果、CYP90A1がC-23位水酸化酵素であると報告された。しかし、CYP90A1についての生化学的証明はなされていない。昨年度の本大会で我々はシロイヌナズナCYP90C1およびCYP90D1がC-23位水酸化酵素であることを酵素学的解析により明らかにした。
日本植物生理学会, 2007年, 日本植物生理学会年会およびシンポジウム 講演要旨集, 2007 (0), 733 - 733
今回、CYP90A1の機能を生化学的に証明するために、CYP90A1の酵素化学的解析を行った。バキュロウィルス-昆虫細胞発現系を用いてCYP90A1を発現させ、酵素活性実験を行った。その結果、CYP90A1はC-23位水酸化酵素ではなく、C-3位酸化/異性化酵素であることを明らかにした。またcpd突然変異株の内生BR量の分析およびBR生合成中間体の投与による表現型回復実験の結果から、カンペスタノールを経由しないBR新規生合成経路の存在を明らかにした。Sterols are isoprenoid-derived lipids that are produced via the mevalonate pathway and are involved in various cellular functions in eukaryotes such as maintenance of membrane integrity and biosynthetic precursors of steroid hormones. Among cellular sterols, Delta(22)-sterols containing a double bond at C-22 in the sterol side chain specifically occur in fungi (ergosterol) and plants (stigmasterol and brassicasterol), and several lines of experimental evidence have suggested specific physiological roles of Delta(22)-sterols in plants. Fungal cytochrome P450 (P450), CYP61, has been established as the sterol C-22 desaturase functioning at the penultimate step in the ergosterol biosynthetic pathway. On the other hand, no particular sequence has been assigned as to the enzyme responsible for the introduction of the double bond into the sterol side chain in plants. in this review, we summarize our recent findings demonstrating that CYF710A P450 family genes encode the plant sterol C-22 desaturases to produce stigmasterol and brassicasterol/crinosterol from beta-sitosterol and 24-epi-campesterol respectively.
PORTLAND PRESS LTD, 2006年12月, BIOCHEMICAL SOCIETY TRANSACTIONS, 34, 1202 - 1205, 英語[査読有り]
研究論文（学術雑誌）
Brassinosteroids (BRs) are biosynthesized from campesterol via several cytochrome P450 (P450)-catalyzed oxidative reactions. We report the functional characterization of two BR-biosynthetic P450s from Arabidopsis thaliana: CYP90C1/ROTUNDIFOLIA3 and CYP90D1. The cyp90c1 cyp90d1 double mutant exhibits the characteristic BR-deficient dwarf phenotype, although the individual mutants do not display this phenotype. These data suggest redundant roles for these P450s. In vitro biochemical assays using insect cell-expressed proteins revealed that both CYP90C1 and CYP90D1 catalyze C-23 hydroxylation of various 22-hydroxylated BRs with markedly different catalytic efficiencies. Both enzymes preferentially convert 3-epi-6-deoxocathasterone, (22S,24R)-22-hydroxy-5 alpha-ergostan-3-one, and (22S,24R)-22-hydroxyergost-4-en-3-one to 23-hydroxylated products, whereas they are less active on 6-deoxocathasterone. Likewise, cyp90c1 cyp90d1 plants were deficient in 23-hydroxylated BRs, and in feeding experiments using exogenously supplied intermediates, only 23-hydroxylated BRs rescued the growth deficiency of the cyp90c1 cyp90d1 mutant. Thus, CYP90C1 and CYP90D1 are redundant BR C-23 hydroxylases. Moreover, their preferential substrates are present in the endogenous Arabidopsis BR pool. Based on these results, we propose C-23 hydroxylation shortcuts that bypass campestanol, 6-deoxocathasterone, and 6-deoxoteasterone and lead directly from (22S,24R)-22-hydroxy-5 alpha-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol.
AMER SOC PLANT BIOLOGISTS, 2006年11月, PLANT CELL, 18 (11), 3275 - 3288, 英語[査読有り]
研究論文（学術雑誌）
Several cytochrome P450 monooxygenases (P450s) catalyze essential oxidative reactions in brassinosteroid (BR) biosynthesis as well as in BR catabolism; however, only limited information exists on the P450s involved in the BR catabolic pathway. Here, we report the characterization of two P450 mRNAs, CYP734A7 and CYP734A8, from Lycopersicon esculentum. These P450s show high homology with Arabidopsis CYP734A1/BAS1 (formerly CYP72B1), which inactivates BRs via C-26 hydroxylation. Transgenic tobacco plants that constitutively overexpressed CYP734A7 showed an extreme dwarf phenotype similar to BR deficiency. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in the transgenic plants showed that the levels of castasterone and 6-deoxocastasterone significantly decreased in comparison with those in wild-type plants. By measuring the Type I substrate-binding spectra using recombinant CYP734A7, the dissociation constants for castasterone, brassinolide, and 6-deoxocastasterone were determined to be 6.7, 12, and 12 mu M, respectively. In an in vitro assay, CYP734A7 was confirmed to metabolize castasterone to 26-hydroxycastasterone. In addition, 28-norcastasterone and brassinolide were converted to the hydroxylated products. The expression of CYP734A7 and CYP734A8 genes in tomato seedlings was upregulated by exogenous application of bioactive BRs. These results indicated that CYP734A7 is a C-26 hydroxylase of BRs and is likely involved in BR catabolism in tomato. The presence of the CYP734A subfamily in various plant species suggests that oxidative inactivation of BRs by these proteins is a widespread phenomenon in plants. (c) 2006 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2006年09月, PHYTOCHEMISTRY, 67 (17), 1895 - 1906, 英語[査読有り]
研究論文（学術雑誌）
We characterized a new cytochrome P450 monooxygenase (P450), CYP724B2, from tomato (Lycopersicon esculentum). CYP724B2 showed 42% and 62% amino acid sequence identity with Arabidopsis DWARF4/CYP90B1 and rice DWARF11/CYP724B1 respectively. Functional assay of CYP724B2 heterologously expressed in insect cells revealed that CYP724B2 catalyzes C-22 hydroxylation of campesterol, indicating that CYP724B2 is a C-22 hydroxylase. We also isolated a tomato CYP90B homolog (CYP90B3) and found that CYP90B3 is a C-22 hydroxylase as well. CYP724B2 and CYP90B3 showed substrate specificities similar to each other toward the biosynthetic intermediate compounds from campesterol to campestanol. Campesterol was the best substrate, and (24R)-ergost-4-en-3-one was also metabolized to the C-22 hydroxylated product to some extent. On the other hand, the P450s catalyzed C-22 hydroxylation of (24R)-5 alpha-ergostan-3-one and campestanol at a trace level, indicating that the compounds after C-5 alpha reduction are poor substrates of CYP724B2 and CYP90B3. In addition, cholesterol (C-27 sterol) and sitosterol (C-29 sterol) were also converted to C-22 hydroxylated products by the P450s. Furthermore, CYP724B2 and CYP90B3 genes were ubiquitously expressed, and their transcript levels were down-regulated by the exogenous application of brassinolide. These findings strongly suggest that CYP724B2 and CYP90B3 function in the early C-22 hydroxylation steps of brassinosteroid biosynthetic pathway in tomato.
TAYLOR & FRANCIS LTD, 2006年09月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 70 (9), 2071 - 2080, 英語[査読有り]
研究論文（学術雑誌）
- 天然有機化合物討論会, 2006年07月23日, International Symposium on the Chemistry of Natural Products, 2006, "P - 357", 英語
Plant growth retardants (PGRs) reduce the shoot growth of plants by inhibiting gibberellin biosynthesis. In this study, we performed detailed analyses of the inhibitory effects of PGRs on Arabidopsis abscisic acid (ABA) 8-hydroxylase, a major ABA catabolic enzyme, recently identified as CYP707As. In an in vitro assay with CYP707A3 microsomes expressed in insect cells, uniconazole-P inhibited CYP707A3 activity more effectively than paclobutrazol or tetcyclacis, whereas the other PGRs tested did not inhibit it significantly. Uniconazole-P was found to be a strong competitive inhibitor (K-i = 8.0 nm) of ABA 8'-hydroxylase. Uniconazole-P-treated Arabidopsis plants showed enhanced drought tolerance. In uniconazole-P-treated plants, endogenous ABA levels increased 2-fold as compared with the control, and co-application of GA(4) did not suppress the effects, indicating that the effects were not due to gibberellin deficiency. Thus uniconazole-P effectively inhibits ABA catabolism in Arabidopsis plants. We also discuss the structure-activity relationship of the azole-type compounds on ABA 8'-hydroxylase inhibitory activity.
TAYLOR & FRANCIS LTD, 2006年07月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 70 (7), 1731 - 1739, 英語[査読有り]
研究論文（学術雑誌）
We designed and synthesized AHI4 that has an axial hydroxyl group instead of geminal methyl groups at C-6' of AHI1, previously reported as a lead compound for the development of non-azole inhibitors of ABA 8'-hydroxylase. (+)-AHI4 competitively inhibited 8'-hydroxylation of ABA by recombinant CYP707A3. The K-I value was found to be 0.14 mu M, 10-fold less than that of (+)-AHI1, suggesting that enzyme affinity increased by a factor of 10 due to substitution of the hydroxyl group by the geminal methyls at C-6'. This finding should assist in the design of more effective, non-azole ABA 8'-hydroxylase inhibitors. (c) 2006 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2006年06月, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 16 (12), 3302 - 3305, 英語[査読有り]
研究論文（学術雑誌）
Abscisic acid (ABA) regulates many important processes in normal growth and development as well as in adaptive responses to environmental stresses. For correct and accurate actions, physiologically active ABA level is controlled through fine-tuning of de novo biosynthesis and catabolism. Hydroxylation at the 8′-position of ABA is the key step in the oxidative catabolism of ABA, and this reaction is catalyzed by ABA 8′-hydroxylase, a cytochrome P450 (P450). Recently, the CYP707A family of Arabidopsis has been identified as ABA 8′-hydroxylase through genomic and biochemical approaches. The CYP707A family is present in a wide range of plant kingdom and functions in ABA catabolism in plants. CYP707A is the pivotal enzyme controlling the endogenous ABA levels by its transcriptional regulation and plays a key role in ABA-mediated physiological processes such as seed dormancy and stress response. Specific inhibitors of ABA catabolism can manipulate ABA homeostasis in plants and are potentially very useful tools for cellular and molecular investigations in the field of plant physiology as well as for potential agricultural chemicals. Identification of the ABA catabolic genes gives us new insight into the development of chemical inhibitors specific to ABA 8′-hydroxylase. © 2006 Springer Science+Business Media B.V.
2006年06月, Phytochemistry Reviews, 5 (2-3), 385 - 404, 英語[査読有り]
研究論文（学術雑誌）
Delta 22-Unsaturated sterols, containing a double bond at the C-22 position in the side chain, occur specifically in fungi and plants. Here, we describe the identification and characterization of cytochrome P450s belonging to the CYP710A family as the plant C-22 desaturase. Recombinant proteins of CYP710A1 and CYP710A2 from Arabidopsis thaliana and CYP710A11 from tomato (Lycopersicon esculentum) were expressed using a baculovirus/insect system. The Arabidopsis CYP710A1 and tomato CYP710A11 proteins exhibited C-22 desaturase activity with beta-sitosterol to produce stigmasterol (CYP710A1, K(m) = 1.0 mu M and kinetic constant [k(cat)] 0.53 min(-1); CYP710A11, K(m) = 3.7 mu M and k(cat) 10 min(-1)). In Arabidopsis transgenic lines with CYP710A1 and CYP710A11 overexpression, stigmasterol levels increased by 6- to 32-fold. Arabidopsis CYP710A2 was able to produce brassicasterol and stigmasterol from 24-epi-campesterol and beta-sitosterol, respectively. Sterol profiling analyses for CYP710A2 overexpression and a T-DNA insertion event into CYP710A2 clearly demonstrated in planta that CYP710A2 was responsible for both brassicasterol and stigmasterol production. Semiquantitative PCR analyses and promoter: beta-glucuronidase transgenic approaches indicated strict tissue/organ-specific regulation for each CYP710A gene, implicating differential tissue distributions of the Delta(22)-unsaturated sterols in Arabidopsis. Our results support the possibility that the CYP710 family may encode P450s of sterol C-22 desaturases in different organisms.
AMER SOC PLANT BIOLOGISTS, 2006年04月, PLANT CELL, 18 (4), 1008 - 1022, 英語[査読有り]
研究論文（学術雑誌）
Arabidopsis dwf4 is a brassinosteroid (BR)-deficient mutant, and the DWF4 gene encodes a cytochrome P450, CYP90B1. We report the catalytic activity and substrate specificity of CYP90B1. Recombinant CYP90B1 was produced in Escherichia coli, and CYP90B1 activity was measured in an in vitro assay reconstituted with NADPH-cytochrome P450 reductase. CYP90B1 converted campestanol (CN) to 6-deoxocathasterone, confirming that CYP90B1 is a steroid C-22 hydroxylase. The substrate specificity of CYP90B1 indicated that sterols with a double bond at positions C-5 and C-6 are preferred substrates compared with stanols, which have no double bond at the position. In particular, the catalytic efficiency (k(cat)/K-m) of CYP90B1 for campesterol (CR) was 325 times greater than that for CN. As CR is more abundant than CN in planta, the results suggest that C-22 hydroxylation of CR before C-5 alpha reduction is the main route of BR biosynthetic pathway, which contrasts with the generally accepted route via CN. In addition, CYP90B1 showed C-22 hydroxylation activity toward various C27-29 sterols. Cholesterol (C-27 sterol) is the best substrate, followed by CR (C-28 sterol), whereas sitosterol (C-29 sterol) is a poor substrate, suggesting that the substrate preference of CYP90B1 may explain the discrepancy between the in planta abundance of C-27/C-28/C-29 sterols and C-27/C-28/C-29 BRs.
BLACKWELL PUBLISHING, 2006年03月, PLANT JOURNAL, 45 (5), 765 - 774, 英語[査読有り]
研究論文（学術雑誌）
The biosynthesis of coumarins in plants is not well understood, although these metabolic pathways are often found in the plant kingdom. We report here the occurrence of coumarins in Arabidopsis thaliana ecotype Columbia. Considerably high levels of scopoletin and its P-D-glucopyranoside, scopolin, were found in the wild-type roots. The scopolin level in the roots was similar to 1200 nmol/gFW, which was similar to 180-fold of that in the aerial parts. Calli accumulated scopolin at a level of 70 nmol/gFW. Scopoletin and scopolin formation were induced in shoots after treatment with either 2,4-dichlorophenoxyacetic acid (at 100 mu M) or a bud-cell suspension of Fusarium oxysporum. In order to gain insight into the biosynthetic pathway of coumarins in A. thaliana, we analyzed coumarins in the Mutants obtained from the SALK Institute collection that carried a T-DNA insertion within the gene encoding the cytochrome P450, CYP98A3, which catalyzes 3'-hydroxylation of p-coumarate units in the phenylpropanoid pathway. The content of scopoletin and scopolin in the mutant roots greatly decreased to similar to 3% of that in the wild-type roots. This observation suggests that scopoletin and scopolin biosynthesis in A. thaliana are strongly dependent on the 3'-hydroxylation of p-cournarate units catalyzed by CYP98A3. We also found that the level of skimmin, a beta-(D)-glucopyranoside of umbelliferone, was slightly increased in the mutant roots. (c) 2005 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2006年02月, PHYTOCHEMISTRY, 67 (4), 379 - 386, 英語[査読有り]
研究論文（学術雑誌）
- OXFORD UNIV PRESS, 2006年, PLANT AND CELL PHYSIOLOGY, 47, S195 - S195, 英語Identification and enzymatic characterization of the plant sterol C-22 desaturase
[査読有り]
- OXFORD UNIV PRESS, 2006年, PLANT AND CELL PHYSIOLOGY, 47, S139 - S139, 英語Cytochrome P450CYP710A encodes the sterol C-22 desaturase in plants
[査読有り]
New cultivars with very erect leaves, which increase light capture for photosynthesis and nitrogen storage for grain filling, may have increased grain yields(1). Here we show that the erect leaf phenotype of a rice brassinosteroid-deficient mutant, osdwarf4-1, is associated with enhanced grain yields under conditions of dense planting, even without extra fertilizer. Molecular and biochemical studies reveal that two different cytochrome P450s, CYP90B2/OsDWARF4 and CYP724B1/D11, function redundantly in C-22 hydroxylation, the rate-limiting step of brassinosteroid biosynthesis. Therefore, despite the central role of brassinosteroids in plant growth and development, mutation of OsDWARF4 alone causes only limited defects in brassinosteroid biosynthesis and plant morphology. These results suggest that regulated genetic modulation of brassinosteroid biosynthesis can improve crops without the negative environmental effects of fertilizers.
NATURE PUBLISHING GROUP, 2006年01月, NATURE BIOTECHNOLOGY, 24 (1), 105 - 109, 英語[査読有り]
研究論文（学術雑誌）
(1'S*,2'S*)-(+/-)-6-Nor-2',3'-dihydro-4'-deoxo-ABA (2) was designed and synthesized as a candidate lead compound for developing a potent and specific inhibitor of ABA 8'-hydroxylase. This compound acted as an effective competitive inhibitor of the enzyme, with a K-I value of 0.40 mu M, without exhibiting ABA activity. However, compound 2 also functioned as an enzyme substrate, making it a short-lived inhibitor. The 8'-difluorinated derivative of 2 (4) was synthesized as a long-lasting alternative. Compound 4 resisted 8'-hydroxylation, but inhibited ABA 8'-hydroxylation as effectively as 2. These results suggest that compound 2 is a useful lead compound for the future design and development of an ideal ABA 8'-hydroxylase inhibitor. (c) 2005 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2005年12月, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 15 (23), 5226 - 5229, 英語[査読有り]
研究論文（学術雑誌）
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.
PERGAMON-ELSEVIER SCIENCE LTD, 2005年07月, BIOORGANIC & MEDICINAL CHEMISTRY, 13 (14), 4491 - 4498, 英語[査読有り]
研究論文（学術雑誌）
A major catabolic enzyme of the plant hormone abscisic acid (ABA) is the cytochrome P450 monooxygenase ABA 8 '-hydroxylase. For designing a specific inhibitor of this enzyme, the substrate specificity and inhibition of CYP707A3, an ABA 8 '-hydroxylase from Arabidopsis thaliana, was investigated using 45 structural analogues of ABA and compared to the structural requirements for ABA activity. Substrate recognition by the enzyme strictly required the 6 '-methyl groups (C-8 ' and C-9 ') which were unnecessary for ABA activity, whereas elimination of the 3-methyl (C-6) and V-hydroxyl groups, which significantly affected ABA activity, had little effect on the ability of analogues to competitively inhibit the enzyme. Fluorination at C-8 ' and C-9 ' resulted in resistance to 8 '-hydroxylation and competitive inhibition of the enzyme. In particular, 8 ',8 '-difluoro-ABA and 9 ',9 '-difluoro-ABA yielded no enzyme reaction products and strongly inhibited the enzyme (K-1 = 0.16 and 0.25 mu M, respectively). (c) 2005 Elsevier Ltd. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2005年05月, BIOORGANIC & MEDICINAL CHEMISTRY, 13 (10), 3359 - 3370, 英語[査読有り]
研究論文（学術雑誌）
植物の細胞伸長、細胞分化、屈曲、暗所の形態形成には、植物ホルモンであるブラシノステロイドが深く関与している。Activation T-DNA taggingにより単離されたシロイヌナズナchibi2変異体は、ブラシノステロイド欠損により極めて矮性を示した。chibi2においてシトクロムP450 (CYP72C1) が過剰発現してしたことから、本P450酵素が内生ブラシノステロイドの代謝 (不活性化） を行うことが示唆された。
日本植物生理学会, 2005年, 日本植物生理学会年会およびシンポジウム 講演要旨集, 2005 (0), 764 - 764
そこで本研究では、CHIBI2 (CYP72C1) の生化学的解析を行った。昆虫細胞発現系を用いてCHIBI2を発現させ、NADPH-P450還元酵素との再構成系を構築してCHIBI2の酵素活性を測定した。その結果、CHIBI2により6-デオキソカスタステロンが代謝されることをLC-MS分析により確認した。現在、その代謝物の詳細な構造解析を行っている。ブラシノステロイドは、植物の細胞伸長、細胞分化、屈曲、暗所の形態形成など重要な生理作用を担う植物ホルモンである。カンペステロールからブラシノライドに至るブラシノステロイド生合成経路には複数の酸化反応があり、C-22、C-23水酸化やC-6酸化を行うシトクロムP450遺伝子が同定されている。
日本植物生理学会, 2005年, 日本植物生理学会年会およびシンポジウム 講演要旨集, 2005 (0), 767 - 767
シロイヌナズナ、トマト、イネなどの植物のシトクロムP450遺伝子を網羅的に系統樹解析した結果、ブラシノステロイド生合成に関与する遺伝子群はクラスターを形成することがわかった。
今回、我々はそのクラスターに属するトマト由来の新規シトクロムP450遺伝子であるCYP724B2に注目した。昆虫細胞発現系を用いてCYP724B2を発現させ、NADPH-シトクロムP450還元酵素との再構成系を構築した。基質としてブラシノステロイド生合成中間体を用いることにより、CYP724B2の酵素活性の同定を行った。
その結果、CYP724B2がC-22位水酸化反応を触媒することがGC-MS分析により確認された。発表では、CYP724B2の詳細な酵素学的特徴についても報告する。A β-primeverosidase from tea plants (Camellia sinensis) is a unique disaccharide-specific diglycosidase, which hydrolyses aroma precursors of β-primeverosides (6-O-β-D-xylopyranosyl-β-D-glucopyranosides) to liberate a primeverose unit and various aroma compounds. Recently we reported the purification and cloning of β-primeverosidase from tea plants. β-Primeverosidase is classified in glycosyl hydrolase family 1, in which many β-glucosidases from various plants are also present. In addition to β-primeverosidase, various kinds of disaccharide-specific diglycosidases such as β-acuminosidase, β-vicianase, β-rutinosidase, etc. are thought to be present in a wide range of plant kingdom. The purpose of this study is to investigate these diglycosidases from chemical, biochemical as well as physiological points of view. The cDNA coding for another diglycosidase (furcatin hydrolase), which hydrolyzes p-allylphenyl β-acuminoside, has been isolated from Viburnum fitrcatum. Furthermore, we have succeeded in the purification and cloning of the third diglycosidase (vicianin hydrolase) from immature seeds of Vicia angustifolia. These diglycosidases cluster together with β-primeverosidase in the phylogenetic tree of family 1 of plant β-glucosidases, suggesting that the disaccharide specific glycosidases form a new subfamily of family 1 glycosidase. The physiological functions of these diglycosidases in defense mechanism in plants as well as the evolution of the diglycosidases in relation to the presence of disaccharide glycosides in some plants are discussed.
天然有機化合物討論会, 2004年10月01日, 天然有機化合物討論会講演要旨集, (46), 665 - 670, 日本語- Redundancy or flexibility: Molecular diversity of the electron transfer components for P450 monooxygenases in higher plants
Specific metabolic roles of P450-dependent monooxygenase systems are determined by enzymatic properties and substrate specificity of P450s, the terminal enzymes of the electron transfer chain. On the other hand, molecular diversity has also been reported for NADPH-cytochrome P450 reductase, cytochrome b(5), and cytochrome b5 reductase in plants. Several lines of evidence indicate that the electron transfer components for plant P450 reactions have specific physiological roles. In this review, we describe the current status of knowledge of the biochemistry, molecular biology, gene regulation, and molecular diversity of plant P450-related electron transfer components and summarize possible individual physiological roles of the diversified P450 electron transfer systems in plants.
FRONTIERS IN BIOSCIENCE INC, 2004年05月, FRONTIERS IN BIOSCIENCE-LANDMARK, 9, 1587 - 1597, 英語[査読有り]
研究論文（学術雑誌）
Furcatin hydrolase (FH) is a unique disaccharide-specific acuminosidase, which hydrolyzes furcatin(p-allylphenyl 6-O-beta-D-apiofuranosyl-beta-D-glucopyranoside (acuminoside)) into p-allylphenol and the disaccharide acuminose. We have isolated a cDNA coding for FH from Viburnum furcatum leaves. The open reading frame in the cDNA encoded a 538-amino acid polypeptide including a putative chloroplast transit peptide. The deduced protein showed 64% identity with tea leaf beta-primeverosidase, which is another disaccharide glycosidase specific to beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides). The deduced FH also shared greater than 50% identity with various plant beta-glucosidases in glycosyl hydrolase family 1. The recombinant FH expressed in Escherichia coli exhibited the highest level of activity toward furcatin with a K-m value of 2.2 mM and specifically hydrolyzed the beta-glycosidic bond between p-allylphenol and acuminose, confirming FH as a disaccharide glycosidase. The FH also hydrolyzed beta-primeverosides and beta-vicianoside (6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside) but poorly hydrolyzed beta-gentiobiosides (6-O-beta-D-glucopyranosyl-beta-D-glucopyranosides), indicating high substrate specificity for the disaccharide glycone moiety. The FH exhibited activity toward p-allylphenyl beta-D-glucopyranoside containing the same aglycone as furcatin but little activity toward the other beta-D-glucopyranosides. Stereochemical analysis using H-1 NMR spectroscopy revealed that FH is a retaining glycosidase. The subcellular localization of FH was analyzed using green fluorescent protein fused with the putative N-terminal signal peptide, indicating that FH is localized to the chloroplast. Phylogenetic analysis of plant beta-glucosidases revealed that FH clusters with beta-primeverosidase, and this suggests that the disaccharide glycosidases will form a new subfamily in glycosyl hydrolase family 1.
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2004年05月, JOURNAL OF BIOLOGICAL CHEMISTRY, 279 (22), 23405 - 23414, 英語[査読有り]
研究論文（学術雑誌）
Abscisic acid (ABA) is involved in a number of critical processes in normal growth and development as well as in adaptive responses to environmental stresses. For correct and accurate actions, a physiologically active ABA level is controlled through fine-tuning of de novo biosynthesis and catabolism. The hydroxylation at the 8'-position of ABA is known as the key step of ABA catabolism, and this reaction is catalyzed by ABA 8'-hydroxylase, a cytochrome P450. Here, we demonstrate CYP707As as the P450 responsible for the 8'-hydroxylation of (+)-ABA. First, all four CYP707A cDNAs were cloned from Arabidopsis and used for the production of the recombinant proteins in insect cells using a baculovirus system. The insect cells expressing CYP707A3 efficiently metabolized (+)-ABA to yield phaseic acid, the isomerized form of 8'-hydroxy-ABA. The microsomes from the insect cello exhibited very strong activity of 8'-hydroxylation of (+)-ABA (K(m) = 1.3 muM and k(cat) = 15 min(-1)). The solubilized CYP707A3 protein bound (+)-ABA with the binding constant K(s) = 3.5 muM, but did not bind (-)-ABA. Detailed analyses of the reaction products confirmed that CYP707A3 does not have the isomerization activity of 8'-hydroxy-ABA to phaseic acid. Further experiments revealed that Arabidopsis CYP707A1 and CYP707A4 also encode ABA 8'-hydroxylase. The transcripts of the CYP707A genes increased in response to salt, osmotic, and dehydration stresses as well as ABA. These results establish that the CYP707A family plays a key role in regulating the ABA level through the 8'-hydroxylation of (+)-ABA.
AMER SOC PLANT BIOLOGISTS, 2004年04月, PLANT PHYSIOLOGY, 134 (4), 1439 - 1449, 英語[査読有り]
研究論文（学術雑誌）
- OXFORD UNIV PRESS, 2004年, PLANT AND CELL PHYSIOLOGY, 45, S76 - S76, 英語Characterization of cytochromes P450 involved in ABA catabolism
[査読有り]
Triazole compounds like paclobutrazol and uniconazole-P are used as growth retardant. The action mechanism of these is inhibition of the cytochrome P-450. It is widely known that paclobutrazol and uniconazole-P not only inhibit vegetative growth, but give environmental stress tolerance to various plants in recent years. As a result of our paying attention to this feature, we found that the eucalyptus seedling processed by Paclobutrazol or Uniconazole-P showed the tolerance against various environmental stress including drought, high temperature, and low temperature with strong wind. Abscisic acid (ABA) 8'-hydroxylase which is the first step of ABA catabolism to phaseic acid (PA) is a member of cytochrome P-450. When plants have some water stress, ABA accumulate especially in leaves, and in recent years it came to be known that ABA is participating deeply in expression of the environmental stress responsive genes. Moreover, it was proved that the plant overexpressed with ABA synthesized gene become drought stress tolerance. Therefore, we predict that P-450 inhibitors, such as uniconazole-P, inhibits the action of ABA 8'-hydroxylase, and then the increase in the amount of ABA, its precursors and/or its glycosyl ester (ABA-GE) occurs, and stress tolerance may increase by that cause. Then, the quantity ant the ratio of free ABA, ABA-GE, and PA in the eucalyptus seedling adapted to the environmental stress by UNI-P processing was investigated by ELISA and the GC-MS analysis. We report here a possibility that controlling the metabolism of ABA by the cytochrome P-450 inhibitors would be concerned with stress tolerance.
一般社団法人 植物化学調節学会, 2004年, 植物化学調節学会 研究発表記録集, 39, 44 - 44, 日本語- 2004年, Internat. J. Tea Sci, 3, 167 - 173, 英語Improvement of Flavour Quality of CTC Black Tea By Glycosidases in Tea Leaves
[査読有り]
研究論文（学術雑誌）
- Japan Society for Bioscience, Biotechnology, and Agrochemistry, 2003年07月01日, 日本農芸化学会誌, 77 (7), 666 - 667, 日本語
We have identified and characterized novel types of ferredoxin and ferredoxin reductase from Arabidopsis. Among a number of potential ferredoxin reductase genes in the Arabidopsis genome, AtMFDR was identified to encode a homologue of mitochondrial ferredoxin reductase, and AtMFDX1 and AtMFDX2 were predicted to code for proteins similar to mitochondrial ferredoxin. First, we isolated cDNAs for these proteins and expressed them in heterologous systems of insect cells and Escherichia coli, respectively. The recombinant AtMFDX1 and AtMFDR proteins exhibited spectral properties characteristic of ferredoxin and ferredoxin reductase, respectively, and a pair of recombinant AtMFDX1 and AtMFDR proteins was sufficient to transfer electrons from NAD(P)H to cytochrome c in vitro. Subcellular fractionation analyses suggested membrane association of AtMFDR protein, and protein-gel blot analyses and transient expression studies of green fluorescence protein fusions indicated mitochondrial localization of AtMFDX1 and AtMFDR. RNA-gel blot analyses revealed that the accumulation levels of AtMFDXs and AtMFDR gene transcripts were specifically high in flowers, while protein-gel blot analysis demonstrated substantial accumulation of AtMFDR protein in leaf, stem, and flower. Possible physiological roles of these mitochondrial electron transfer components are discussed in relation to redox dependent metabolic pathways in plants.
KLUWER ACADEMIC PUBL, 2003年07月, PLANT MOLECULAR BIOLOGY, 52 (4), 817 - 830, 英語[査読有り]
研究論文（学術雑誌）
An affinity adsorbent for beta-glycosidases has been prepared by using beta-glycosylamidine as a ligand. beta-Glucosylamidine and beta-galactosylamidine, highly potent and selective inhibitors of beta-glucosidases and beta-galactosidases, respectively, were immobilized by a novel one-pot procedure involving the addition of a beta-glycosylamine and 2-iminothiolane . HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give an affinity adsorbent for beta-glucosidases and beta-galactosidases, respectively. This one-pot procedure enables various beta-glycosylamidine ligands to be formed and immobilized conveniently according to the glycon substrate specificities of the enzymes. A crude enzyme extract from tea leaves (Camellia sinensis) and a beta-galactosidase from Penicillium multicolor were chromatographed directly on each affinity adsorbent to give a beta-glucosidase and a beta-galactosidase to apparent homogeneity in one step by eluting the column with glucose or by a gradient NaCl elution, respectively. The beta-glucosidase and beta-galactosidase were inhibited competitively by a soluble form of the corresponding beta-glycosylamidine ligand with an inhibition constant (K-i) of 2.1 and 0.80 muM, respectively. Neither enzyme was bound to the adsorbent with a mismatched ligand, indicating that the binding of the glycosidases was of specific nature that corresponds to the glycon substrate specificity of the enzymes. The ease of preparation and the selective nature of the affinity adsorbent should promise a large-scale preparation of the affinity adsorbent for the purification and removal of specific glycosidases according to their glycon substrate specificities. (C) 2003 Elsevier Science Ltd. All rights reserved.
ELSEVIER SCI LTD, 2003年07月, CARBOHYDRATE RESEARCH, 338 (14), 1477 - 1490, 英語[査読有り]
研究論文（学術雑誌）
Triadimefon (Bayleton((R))), a widely used triazole-type fungicide, affects gibberellin (GA) biosynthesis and 14alpha-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 phenotype 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 triardimefon 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.
PORTLAND PRESS, 2003年01月, BIOCHEMICAL JOURNAL, 369 (1), 71 - 76, 英語[査読有り]
研究論文（学術雑誌）
A beta-primeverosidase from tea (Camellia sinensis) plants is a unique disaccharide-specific glycosidase, which hydrolyzes aroma precursors of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to liberate various aroma compounds, and the enzyme is deeply concerned with the floral aroma formation in oolong tea and black tea during the manufacturing process. The beta-primeverosidase was purified from fresh leaves of a cultivar for green tea (C. sinensis var sinensis cv Yabukita), and its partial amino acid sequences were determined. The beta-primeverosidase cDNA has been isolated from a cDNA library of cv Yabukita using degenerate oligonucleotide primers. The cDNA insert encodes a polypeptide consisting of an N-terminal signal peptide of 28 amino acid residues and a 479-amino acid mature protein. The beta-primeverosidase protein sequence was 50% to 60% identical to beta-glucosidases from various plants and was classified in a family 1 glycosyl hydrolase. The mature form of the beta-primeverosidase expressed in Escherichia coli was able to hydrolyze beta-primeverosides to liberate a primeverose unit and aglycons, but did not act on 2-phenylethyl beta-D-glucopyranoside. These results indicate that the beta-primeverosidase selectively recognizes the beta-primeverosides as substrates and specifically hydrolyzes the beta-glycosidic bond between the disaccharide and the aglycons. The stereochemistry for enzymatic hydrolysis of 2-phenylethyl beta-primeveroside by the beta-primeverosidase was followed by H-1-nuclear magnetic resonance spectroscopy, revealing that the enzyme hydrolyzes the beta-primeveroside by a retaining mechanism. The roles of the beta-primeverosidase in the defense mechanism in tea plants and the floral aroma formation during tea manufacturing process are also discussed.
AMER SOC PLANT BIOLOGISTS, 2002年12月, Plant Physiol, 130 (4), 2164 - 2176, 英語[査読有り]
研究論文（学術雑誌）
Brain-derived neurotrophic factor (BDNF) binds to and activates the TrkB tyrosine kinase receptor to regulate cell differentiation, survival, and neural plasticity in the nervous system. However, the identities of the downstream signaling proteins involved in this process remain unclear. Using a yeast two-hybrid screen with the intracellular domain (ICD-TrkB) of the TrkB BDNF receptor, we identified the Nck2 adaptor protein as a novel interaction partner of the active form of TrkB. Additionally, we identified three tyrosines in ICD-TrkB (Y694, Y695, and Y771) that are crucial for this interaction. Similar results were obtained for Nck1, an Nck2 homolog. We also found that TrkB could be co-precipitated with GST-Nck2 recombinant protein or anti-Nck antibody in BDNF-activated cortical neurons. These results suggest that BDNF stimulation promotes interaction of Ncks with TrkB in cortical neurons. (C) 2002 Elsevier Science (USA). All rights reserved.
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2002年06月, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 294 (5), 1087 - 1092, 英語[査読有り]
研究論文（学術雑誌）
- OXFORD UNIV PRESS, 2002年, PLANT AND CELL PHYSIOLOGY, 43, S96 - S96, 英語Identification and characterization of a mitochondrial electron transfer chain in plants comprising of an adrenocortical ferredoxin homologue and its oxidoreductase
[査読有り]
- OXFORD UNIV PRESS, 2002年, PLANT AND CELL PHYSIOLOGY, 43, S184 - S184, 英語Functional analysis of tomato CYP72B involved in brassinosteroid catabolism
[査読有り]
- 一般社団法人植物化学調節学会, 2002年, 植物の生長調節, 37 (2), 227 - 228, 日本語
- Association of SH2-B to phosphorylated tyrosine residues in the activation loop of TRKB
Neurotrophins are essential for the survival and differentiation of neurons in the central and peripheral nervous systems. The binding of neurotrophins to their Trk receptors induces autophosphorylation of tyrosine residues and activation of several signaling components. However, the downstream signaling cascades remain to be fully elucidated. Here we describe molecular cloning of human SH2-Bα, PH and SH2-domain-containing adaptor protein, as a TrkB binding protein, and how SH2-Bα associate with the cytoplasmic domain of TrkB at phosphorylated tyrosine residues in the kinase activation loop. There was no distinct inhibitory or inducing effect on kinase activity detected by either a full-length or an SH2 domain of SH2-Bα in vitro, even though the regulation mechanism of the activation loop on tyrosine kinase activity has been described. In addition to SH2-Bα, the expression of three SH2-B alternative splice variants, SH2-Bβ, γ and δ, was detected in human cell lines. These splicing variants have unique carboxyl-terminal amino acid sequences due to insertion sequences as well as reading frameshifts.
2002年, Research Communications in Molecular Pathology and Pharmacology, 111 (1-4), 27 - 39, 英語[査読有り]
研究論文（学術雑誌）
We synthesized nine kinds of diglycosides and a monoglycoside of 2-phenylethanol to investigate the substrate specificity of the purified beta -primeverosidase from fresh leaves of a tea cultivar (Camellia sinensis var. sinensis cv. Yabukita) in comparison with the apparent substrate specificity of the crude enzyme extract from tea leaves. The crude enzyme extract mainly showed beta -primeverosidase activity, although monoglycosidases activity was present to some extent. The purified beta -primeverosidase showed very narrow substrate specificity with respect to the glycon moiety, and especially prominent specificity for the beta -primeverosyl (6-O-beta -D-xylopyranosyl-beta -D-glueopyranosyl) moiety. The enzymes hydrolyzed naturally occurring diglycosides such as beta -primeveroside, beta -vicianoside, beta -acuminoside, beta -gentiobioside and 6-O-alpha -L-arabinofuranosyl-beta -D-glucopyranoside, but were unable to hydrolyze synthetic unnatural diglycosides. The purified enzyme was inactive toward 2-phenylethyl beta -D-glucopyranoside. The enzyme hydrolyzed each of the diglycosides into the corresponding disaccharide and 2-phenylethanol. These results indicate the beta -primeverosidase, a diglycosidase, to be a key enzyme involved in aroma formation during the tea manufacturing process.
TAYLOR & FRANCIS LTD, 2001年12月, Biosci. Biotechnol. Biochem., 65 (12), 2719 - 2729, 英語[査読有り]
研究論文（学術雑誌）
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.
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2001年07月, JOURNAL OF BIOLOGICAL CHEMISTRY, 276 (28), 25687 - 25691, 英語[査読有り]
研究論文（学術雑誌）
- Analysis of tyrosine phosphorylation-dependent protein-protein interactions in TrkB-mediated intracellular signaling using modified yeast two-hybrid system
Activated receptor tyrosine kinases induce a large number of tyrosine phosphorylation-dependent protein-protein interactions through which they mediate their various ligand-exerted functions including regulation of proliferation, differentiation and survival. TrkB receptor tyrosine kinase activated by binding of brain-derived neurotrophic factor (BDNF) also stimulates various protein interactions in a tyrosine phosphorylation-dependent manner in neuronal cells. To examine tyrosine phosphorylation-dependent interactions stimulated by active TrkB, we developed a modified yeast two-hybrid system, which we call the yeast two-and-a-half-hybrid system. In this system, yeast was engineered to express a tyrosine kinase domain of TrkB as an effector, in addition to two fusion proteins with GAL4 DNA-binding and GAL4 activation domains as bait and prey proteins, respectively. Using this system with Shp2 as the bait, we demonstrated that Shp2 interacts directly with BPT/SHPS-1 (also called SIRP) and Grb2 depending on tyrosine phosphorylation mediated by TrkB, Furthermore, we screened an adult human brain cDNA library with the yeast two-and-a-half-hybrid system in order to identify other Shp2-binding proteins in TrkB-stimulated tyrosine phosphorylation signaling. We found that fibroblast growth factor receptor substrate 2 beta (FRS2 beta), also called SNT2, interacts with Shp2 dependently on TrkB-mediated tyrosine phosphorylation of FRS2 beta /SNT2, Therefore, we show that the two-and-a-half-hybrid system is a powerful tool for studying tyrosine phosphorylation-dependent protein-protein interactions in intracellular signaling pathways stimulated by TrkB receptor tyrosine kinase.
JAPANESE BIOCHEMICAL SOC, 2001年07月, JOURNAL OF BIOCHEMISTRY, 130 (1), 157 - 165, 英語[査読有り]
研究論文（学術雑誌）
- Molecular cloning and characterization of ATP-phosphoribosyl transferase from Arabidopsis, a key enzyme in the histidine biosynthetic pathway
We have characterized two isoforms of ATP-phosphoribosyl transferase (ATP-PRT) from Arabidopsis (AtATP-PRT1 [accession no. AB025251] and AtATP-PRT2), catalyzing the first step of the pathway of hisidine (His) biosynthesis. The primary structures deduced from AtATP-PRT1 and AtATP-PRT2 cDNAs share an overall amino acid identity of 74.6% and contain N-terminal chloroplast transit peptide sequences. DNA-blot analyses indicated that the ATP-PRTs in Arabidopsis are encoded by two separate genes with a closely similar gene structural organization. Both gene transcripts were detected throughout development, and protein-blot analysis revealed predominant accumulation of the AtATP-PRT proteins in Arabidopsis leaves. The His auxotrophy of a his1 mutant of Saccharomyces cerevisiae was suppressed by the transformation with AtATP-PRT1 and AtATP-PRT2, cDNAs, indicating that both isoforms are functionally active ATP-PRT enzymes. The K-m values for ATP and phosphoribosyl pyrophosphate of the recombinant AtATP-PRT proteins were comparable to those of the native ATP-PRTs from higher plants and bacteria. It was demonstrated that the recombinant AtATP-PRTs were inhibited by L-His (50% inhibition of initial activity = 40-320 mu M), suggesting that His biosynthesis was regulated in plants through feedback inhibition by L-His.
AMER SOC PLANT PHYSIOLOGISTS, 2000年03月, PLANT PHYSIOLOGY, 122 (3), 907 - 914, 英語[査読有り]
研究論文（学術雑誌）
AtCBR, a cDNA encoding NADH-cytochrome (Cyt) b(5) reductase, and AtB5-A and AtB5-B, two cDNAs encoding Cyt b(5), were isolated from Arabidopsis. The primary structure-deduced from the AtCBR cDNA was 40% identical to those of the NADH-Cyt b(5) reductases of yeast and mammals. A recombinant AtCBR protein prepared using a baculovirus system exhibited typical spectral properties of NADH-Cyt b(5) reductase and was used to study its electron-transfer activity. The recombinant NADH-Cyt b(5) reductase was functionally active and displayed strict specificity to NADH for the reduction of ;a recombinant Cyt b(5) (AtB5-A), whereas no Cyt b(5) reduction was observed when NADPH was used as the electron donor. Conversely, a recombinant NADPH-Cyt P450 reductase of Arabidopsis was able to reduce Cyt b(5) with NADPH but not with NADH. To our knowledge, this is the first evidence in higher plants that both NADH-Cyt b(5) reductase and NADPH-Cyt P450 reductase can reduce Cyt b(5) and have clear specificities in terms of the electron donor, NADH or NADPH, respectively. This substrate specificity of the two reductases is discussed in relation to the NADH- and NADPH-dependent activities of microsomal fatty acid desaturases.
AMER SOC PLANT PHYSIOLOGISTS, 1999年01月, PLANT PHYSIOLOGY, 119 (1), 353 - 361, 英語[査読有り]
研究論文（学術雑誌）
We have isolated multiple cDNAs encoding cytochromes P450 (P450s) from Arabidopsis thaliana employing a PCR strategy. Degenerate oligonucleotide primers were designed from amino acid sequences conserved between two plant P450s, CYP71A1 and CYP73A2, including the heme-binding site and the proline-rich motif found in the N-terminal region, and 11 putative P450 fragments were amplified from first-strand cDNA from 7-day-old Arabidopsis as a template. With these PCR fragments as hybridization probes, 13 full-length and 3 partial cDNAs encoding different P450s have been isolated from an Arabidopsis cDNA library. These P450s have been assigned to either one of the established subfamilies: CYP71B, CYP73A, and CYP83A; or novel subfamilies: CYP76C, CYP83B, and CYP91A. The primary protein structures predicted from the cDNA sequences revealed that the regions around both the heme-binding site and the proline-rich motif were highly conserved among all these P450s. The N-terminal structures of the predicted P450 proteins suggested that these Arabidopsis P450s were located at the endoplasmic reticulum membrane. The loci of four P450 genes were determined by RFLP mapping. One of the clones, CYP71B2, was located at a position very close to the ga4 and gai mutations. RNA blot analysis showed expression patterns unique to each of the P450s in terms of tissue specificity and responsiveness to wounding and light/dark cycle, implicating involvement of these P450s in diverse metabolic processes.
KLUWER ACADEMIC PUBL, 1998年05月, PLANT MOLECULAR BIOLOGY, 37 (1), 39 - 52, 英語[査読有り]
研究論文（学術雑誌）
We have investigated two NADPH-cytochrome (Cyt) P450 reductase isoforms encoded by separate genes (ARI and AR2) in Arabidopsis thaliana. We isolated AR1 and AR2 cDNAs using a mung bean (Phaseolus aureus L.) NADPH-Cyt P450 reductase cDNA as a probe. The recombinant AR1 and AR2 proteins produced using a baculovirus expression system showed similar K-m values for Cyt c and NADPH, respectively. In the reconstitution system with a recombinant cinnamate 4-hydroxylase (CYP73A5), the recombinant AR1 and AR2 proteins gave the same level of cinnamate 4-hydroxylase activity (about 70 nmol min(-1) nmol(-1) P450). The AR2 gene expression was transiently induced by 4- and 3-fold within 1 h of wounding and light treatments, respectively, and the induction time course preceded those of CYP73A5 and a phenylalanine ammonia-lyase (PALI) gene. On the contrary, the ARI expression level did not change during the treatments. Analysis of the AR1 and AR2 gene structure revealed that only the AR2 promoter contained three putative sequence motifs (boxes P, A, and L), which are involved in the coordinated expression of CYP73A5 and other phenylpropanoid pathway genes. These results suggest the possibility that AR2 transcription may be functionally linked to the induced levels of phenylpropanoid pathway enzymes.
AMER SOC PLANT PHYSIOLOGISTS, 1998年01月, PLANT PHYSIOLOGY, 116 (1), 357 - 367, 英語[査読有り]
研究論文（学術雑誌）
We have isolated a cDNA for a cytochrome P450, cinnamate 4-hydroxylase (C4H), of Arabidopsis thaliana using a C4H cDNA from mung bean as a hybridization probe. The deduced amino acid sequence is 84.7% identical to that of mung bean C4H and therefore was designated CYP73A5. The CYP73A5 protein was expressed in insect cells using the baculovirus expression system and when reconstituted with lipid and NADPH-cytochrome P450 reductase resulted in C4H activity with a specific activity of 68 nmol min(-1) nmol(-1) P450. Southern blot analysis revealed that CYP73A5 is a single-copy gene in Arabidopsis. C4H (CYP73A5) expression was apparently coordinated in Arabidopsis with both PAL1 and 4CL in response to light and wounding. Although the light induction of CHS followed a time course similar to that observed with C4H, no induction of CHS was detected upon wounding. On the other hand, the C4H expression patterns exhibited no significant coordination with those of PAL2 and PAL3. A C4H promoter region of 907 bp contained all of the three cis-acting elements (boxes P, A, and L) conserved among the PAL and 4CL genes so far reported as controlling expression.
AMER SOC PLANT PHYSIOLOGISTS, 1997年03月, PLANT PHYSIOLOGY, 113 (3), 755 - 763, 英語[査読有り]
研究論文（学術雑誌）
- PURIFICATION AND CHARACTERIZATION OF A CYTOCHROME-P450 (TRANS-CINNAMIC ACID 4-HYDROXYLASE) FROM ETIOLATED MUNG BEAN SEEDLINGS
A Cyt P450 (P450C4H) PoSsessing trans-cinnamate 4-hydroxylase (C4H) activity was purified to apparent homogeneity from microsomes of etiolated mung bean seedlings. Upon SDS-polyacrylamide gel electrophoresis, the purified preparation gave a single protein band with a molecular mass of 58-kDa. Its specific P450 content was 12.6 nmol (mg protein)-1. Using NADPH as electron donor, purified P450C4H aerobically converted trans-cinnamic acid to p-coumaric acid with a specific activity of 68 nmol min-1 nmol-1 P450 in a reconstituted system containing NADPH-Cyt P450 reductase purified from the seedlings or rabbit liver microsomes, dilauroyl phosphatidylcholine, and cholate. This specific activity is by far the highest for reconstituted C4H systems so far reported and provides direct evidence that C4H activity is actually associated with a P450 protein. In the oxidized state P450C4H showed a typical low-spin type absorption spectrum with a Soret peak at 419 nm. A partial spectral shift to the high spin state was observed when trans-cinnamic acid was added to oxidized P450C4H. By spectral titration, the dissociation constant of the cinnamic acid-P450C4H complex was determined to be 2.8 muM. This value is similar to the K(m) value (1.8 muM) for trans-cinnamic acid determined in the reconstituted system.
JAPANESE SOC PLANT PHYSIOLOGISTS, 1993年04月, PLANT AND CELL PHYSIOLOGY, 34 (3), 481 - 488, 英語[査読有り]
研究論文（学術雑誌）
- ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 1993年02月, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 190 (3), 875 - 880, 英語
[査読有り]
研究論文（学術雑誌）
- 1993年, Jpn. J. Soil Sci. Plant Nutr., 64, 71 - 73, 英語A Sensitive and Microscale Method for Boron Determination of Plant Materials
[査読有り]
研究論文（学術雑誌）
- BORON NUTRITION OF CULTURED TOBACCO BY-2 CELLS .1. REQUIREMENT FOR AND INTRACELLULAR-LOCALIZATION OF BORON AND SELECTION OF CELLS THAT TOLERATE LOW-LEVELS OF BORON
Cultured cells of tobacco (Nicotiana tabacum L. cv. Bright Yellow 2) grown under the standard culture conditions (1 mg boron liter-1 medium as boric acid) contained boron at a concentration of 2.26 mg boron kg-1 oven-dried cells and the protoplast contained 1.26% of the boron in the cells. The cells required boron for growth and the half-maximum growth rate was obtained with 0.056 mg of boron liter-1 medium. Subculturing the cells in media with lower concentrations of boron allowed selection of cells that can grow even in the presence of 1 mug boron liter-1 medium. Cell walls of the selected cells seemed to be thicker than those of the control cells and Golgi bodies were accompanied by more secretory vesicles than those in the control cells.
JAPANESE SOC PLANT PHYSIOLOGISTS, 1992年12月, PLANT AND CELL PHYSIOLOGY, 33 (8), 1135 - 1141, 英語[査読有り]
研究論文（学術雑誌）
MISC
- 2022年, 日本植物生理学会年会(Web), 63rdステロイドグリコアルカロイド生合成に関わる2-オキソグルタル酸依存性ジオキシゲナーゼ
- 2021年, 日本農薬学会大会講演要旨集, 46th (CD-ROM)活性型ジベレリンの欠乏がリンゴの樹形を円柱状にする
- 2021年, 日本農芸化学会大会講演要旨集(Web), 2021円柱状のリンゴ樹形はジベレリンの欠乏が原因だった
- 2020年, 農研機構果樹茶業研究部門成果情報(Web), 2020リンゴのカラムナー性は,活性型ジベレリンの欠乏により生じる
- 2020年, 日本農芸化学会大会講演要旨集(Web), 2020典型的ストリゴラクトンの生合成経路の解明
α-tomatine and dehydrotomatine are steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves, and flowers of tomatoes (Solanum lycopersicum) and function as defensive compounds against pathogens and predators. The aglycones of α-tomatine and dehydrotomatine are tomatidine and dehydrotomatidine (5,6-dehydrogenated tomatidine), and tomatidine is derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5α reduction, and C3 reduction. Our previous studies (Lee et al. 2019) revealed that Sl3βHSD is involved in the three reactions except for C5α reduction, and in the present study, we aimed to elucidate the gene responsible for the C5α reduction step in the conversion of dehydrotomatidine to tomatidine. We characterized the two genes, SlS5αR1 and SlS5αR2, which show high homology with DET2, a brassinosteroid 5α reductase of Arabidopsis thaliana. The expression pattern of SlS5αR2 is similar to those of SGA biosynthetic genes, while SlS5αR1 is ubiquitously expressed, suggesting the involvement of SlS5αR2 in SGA biosynthesis. Biochemical analysis of the recombinant proteins revealed that both of SlS5αR1 and SlS5αR2 catalyze the reduction of tomatid-4-en-3-one at C5α to yield tomatid-3-one. Then, SlS5αR1- or SlS5αR2-knockout hairy roots were constructed using CRISPR/Cas9 mediated genome editing. In the SlS5αR2-knockout hairy roots, the α-tomatine level was significantly decreased and dehydrotomatine was accumulated. On the other hand, no change in the amount of α-tomatine was observed in the SlS5αR1-knockout hairy root. These results indicate that SlS5αR2 is responsible for the C5α reduction in α-tomatine biosynthesis and that SlS5αR1 does not significantly contribute to α-tomatine biosynthesis.
2019年12月01日, Plant biotechnology (Tokyo, Japan), 36 (4), 253 - 263, 英語, 国内誌- 2019年, 日本農芸化学会関西支部講演会講演要旨集, 510thジャガイモのソラニン生合成系のソラニダン形成に関わる還元酵素の解析
- 2019年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 37thジャガイモのソラニン生合成におけるソラニダン骨格形成機構の解析
- 2019年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 37thジャガイモシストセンチュウ孵化促進物質の生合成に関与する酸化酵素の探索
- 2018年05月, 10th World Potato Congress, Cusco, Peru, 英語Metabolic Engineering of Glycoalkaloid-Free Potatoes Accumulating Useful Steroidal Saponins by Genome Editing
研究発表ペーパー・要旨（国際会議）
- 2018年03月16日, 日本農芸化学会2018年度大会，名古屋, 2018, 日本語栽培種および野生種トマトにおけるα-トマチン代謝酵素遺伝子の機能解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2018年03月05日, 日本農芸化学会大会講演要旨集(Web), 2018, ROMBUNNO.2A25p10 (WEB ONLY), 日本語ゴマリグナン新規酸化酵素の同定
- 2018年03月05日, 日本農芸化学会大会講演要旨集(Web), 2018, ROMBUNNO.2A27p07 (WEB ONLY), 日本語ジャガイモのステロイドグリコアルカロイド生合成に関わる糖転移酵素の機能解析
- 2018年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 36thトマトにおけるα-トマチン生合成に関わる5位還元酵素の解析
- 2018年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 36thナス属植物におけるステロイドグリコアルカロイドの構造多様性の進化
- 2018年, 日本農芸化学会関西支部講演会講演要旨集, 506thゲノム編集技術を用いた16DOX遺伝子の破壊による有用形質ジャガイモの作出
- 2018年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 36thジャガイモのα-ソラニン生合成遺伝子と共発現する還元酵素の解析
- 2018年, 日本農芸化学会大会講演要旨集(Web), 2018ゲノム編集によるジャガイモCYP88B1遺伝子の破壊
- 2018年, 日本植物生理学会年会(Web), 59th, ROMBUNNO.3aJ06, 日本語新規ゴマリグナン生合成酵素CYP92B14はセサミンの酸化を介してセサモリンとセサミノールを同時生成する
- 公益社団法人 日本農芸化学会, 2018年, 化学と生物, 56 (8), 566 - 71, 日本語
[査読有り]
記事・総説・解説・論説等（学術雑誌）
- 2017年10月28日, 植物化学調節学会第52回大会，鹿児島, 52 (Supplement), 日本語シロイヌナズナ由来短鎖型脱水素酵素/還元酵素 (AtSDR2, 6) の酵素学的解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2017年08月31日, 第35回日本植物細胞分子生物学会大会，さいたま, 35th, 日本語野生種トマトのステロイドグリコアルカロイドの多様性を担う酵素遺伝子の機能解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2017年08月29日, 第35回日本植物細胞分子生物学会大会，さいたま, 35th, 日本語ジャガイモα-ソラニン生合成遺伝子St16DOXのゲノム編集
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2017年03月05日, 日本農芸化学会大会講演要旨集(Web), 2017, ROMBUNNO.2J31p06 (WEB ONLY), 日本語CRISPR/Cas9によるジャガイモα‐ソラニン生合成遺伝子のゲノム編集
- 2017年, 植物の生長調節, 52 (Supplement)ヤマノイモ属ステロイドサポニン生合成に関わる配糖化酵素の同定および機能解析
- 2017年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 35thトマトのステロイドアルカロイド生合成のジャスモン酸応答性はCOI1受容体とJRE4転写因子に依存する
- 2017年, 日本農芸化学会大会講演要旨集(Web), 2017ヤマノイモ属におけるステロイドサポニン生合成の配糖化段階に関わる糖転移酵素の探索
- 2017年, 日本農芸化学会大会講演要旨集(Web), 2017トマトにおけるα-トマチン生合成に関わるA環酸化還元酵素の解析
- 2017年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 35thヤマノイモ属トゲドコロのステロイドサポニン生合成に関わる糖転移酵素の機能解析
- 2017年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 35thTALENによるジャガイモCYP88B1遺伝子の破壊
- 2017年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 35thゲノム編集によるジャガイモSSR2破壊とヌルセグリガント獲得に向けて
- 2017年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 35th大腸菌発現系を用いた植物オキシドスクアレン環化酵素の機能解析
- 2017年, 天然有機化合物討論会講演要旨集(Web), 59thセサモリン合成酵素の同定と反応機構
Steroidal glycoalkaloids （SGAs） are toxic specialized metabolites that are found in Solanaceae. Potato （Solanum tuberosum） contains the SGAs α-solanine and α-chaconine, which are biosynthesized from cholesterol. Several biosynthetic genes including SSR2 and two cytochrome P450 genes （CYP72A188 and CYP72A208） have been identified, and the transgenic potato plants silencing these biosynthetic genes showed SGA-reduced phenotypes. Here we summarize our recent results and strategy towards metabolic engineering of potato accumulating pharmaceutically useful compounds by genome editing. CYP88B1, which is involved in a later step of the SGA biosynthetic pathway with unknown catalytic function, is co-ordinately expressed with the SGA biosynthetic genes. We applied CRISPR/Cas9 system to knockout potato CYP88B1. The CYP88B1-knockout potatoes showed no accumulation of SGAs, and furthermore the corresponding amounts of steroidal saponins were accumulated in the knockout potatoes.
一般社団法人 植物化学調節学会, 2017年, 植物の化学調節, 52 (2), 92 - 98, 日本語[査読有り][招待有り]
記事・総説・解説・論説等（学術雑誌）
- 2016年12月01日, 日本農芸化学会関西支部講演会講演要旨集, 497th, 10, 日本語ジャガイモCYP88B1のゲノム編集による有毒α‐ソラニンから有用サポニンへの代謝変換
- 2016年09月02日, 第34回日本植物細胞分子生物学会大会，上田, 34th, 日本語野生種トマトにおけるα-トマチン代謝酵素遺伝子の機能解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2016年09月01日, 日本植物学会大会研究発表記録, 80th, 178, 日本語ナス科作物のステロイドグリコアルカロイド生合成遺伝子を標的としたゲノム編集植物の解析
- 2016年08月20日, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 34th, 72, 日本語ステロイドグリコアルカロイド生合成遺伝子CYP88B1をターゲットとしたゲノム編集ジャガイモの解析
- 2016年03月29日, 日本農芸化学会2016年度大会，札幌, 2016, 日本語ナス科植物の有毒ステロイドグリコアルカロイド生合成に関わるアミノトランスフェラーゼの機能解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2016年03月19日, 第57回日本植物生理学会年会，岩手, 日本語ナス科植物ステロイドグリコアルカロイドのF環形成に関わる26位アミノ基転移酵素の機能解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2016年03月05日, 日本農芸化学会大会講演要旨集(Web), 2016, 2H043 (WEB ONLY), 日本語ステロイドグリコアルカロイド生合成遺伝子SGA3をターゲットとしたゲノム編集トマトの解析
- 2016年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 34thジャガイモのα-ソラニン生合成遺伝子と共発現する機能未知遺伝子の解析
- 2016年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 34thサポニン生合成遺伝子過剰発現によるトマトのトマチン生産能の増強
- 2016年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 34thヤマノイモ属トゲドコロのステロイドサポニン生合成に関わる糖転移酵素の探索
- 2016年, 日本植物学会大会研究発表記録, 80th沖縄特産トゲドコロのステロイドサポニン生合成の解析
- 2016年, 日本農芸化学会大会講演要旨集(Web), 2016トマトにおけるα-トマチン生合成に関わるステロイド3位酸化異性化酵素の解析
- 2016年, 日本植物学会大会研究発表記録, 80thトマトにおけるα-トマチン生合成に関わるA環酸化還元酵素の機能解析
- 2016年, 日本農芸化学会大会講演要旨集(Web), 2016ヤマノイモ属のステロイドサポニン生合成に関わるCYP90B遺伝子の機能解析
- 2016年, 日本農芸化学会関西支部講演会講演要旨集, 497thシロイヌナズナの酸化ストレス情報伝達系に関わるシグナル受容体の探索
- 2016年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 34thプラチナTALENによる効果的なジャガイモ4倍体のゲノム編集
- 植物化学調節学会, 2015年10月01日, 植物化学調節学会研究発表記録集, 50, 117 - 117, 日本語P099 アブシジン酸に対するストライガの発芽と気孔の応答(ポスター発表,植物化学調節学会第50回大会)
- 植物化学調節学会, 2015年10月01日, 植物化学調節学会研究発表記録集, 50, 107 - 107, 日本語P089 トマトにおけるα-トマチン生合成に関わるステロイド3位酸化異性化酵素の解明(ポスター発表,植物化学調節学会第50回大会)
- 植物化学調節学会, 2015年10月01日, 植物化学調節学会研究発表記録集, 50, 55 - 55, 日本語P037 ササゲにおけるorobanchol生合成遺伝子の解明に向けたRNA-seq解析(ポスター発表,植物化学調節学会第50回大会)
- 植物化学調節学会, 2015年10月01日, 植物化学調節学会研究発表記録集, 50, 54 - 54, 日本語P036 様々な植物におけるストリゴラクトン生合成の多様性の解析(ポスター発表,植物化学調節学会第50回大会)
- 植物化学調節学会, 2015年10月01日, 植物化学調節学会研究発表記録集, 50, 53 - 53, 日本語P035 ソルガムにおけるSorgomol合成酵素遺伝子の同定及び機能解析(ポスター発表,植物化学調節学会第50回大会)
- 植物化学調節学会, 2015年10月01日, 植物化学調節学会研究発表記録集, 50, 52 - 52, 日本語P034 植物による5-deoxystrigol立体異性体の水酸化反応とストリゴラクトン前駆体の探索(ポスター発表,植物化学調節学会第50回大会)
- 植物化学調節学会, 2015年10月01日, 植物化学調節学会研究発表記録集, 50, 97 - 97, 日本語P079 傷害時に放出される2-ヘキセナールを生合成するヘキセナールイソメラーゼの同定と機能解析(ポスター発表,植物化学調節学会第50回大会)
2-オキソグルタル酸依存性ジオキシゲナーゼ（2OGD）は二価鉄を含む水溶性のジオキシゲナーゼであり，低分子化合物からタンパク質やDNAまで様々な生体分子に対して水酸化や脱メチル化など多彩な酸化反応を触媒する．2OGDは細菌から植物，動物まで広く存在しており，ヒトには約60個，各植物種のゲノムには0.5%を占める2OGD遺伝子が存在しているが，進化系統解析に基づく分類命名法は確立されていない．本解説では，生物界全体の2OGDを比較解析し，2OGDの進化と多様性，および代謝活性の有用性について考察する．
日本農芸化学会, 2015年09月, 化学と生物, 54 (9), 640 - 649, 日本語[査読有り][招待有り]
記事・総説・解説・論説等（学術雑誌）
- 2015年08月11日, 第33回日本植物細胞分子生物学会大会，東京, 33rd, 日本語ナス科植物ステロイドグリコアルカロイド生合成に関わるアミノ基転移酵素の機能解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2015年03月27日, 日本農芸化学会2014年度大会，岡山, 2015, 日本語ナス科植物ステロイドグリコアルカロイドの構造多様性に関わる生合成酵素の機能解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2015年, 日本植物生理学会年会要旨集, 56thナス科植物ステロイドサポニンのF環の構造多様性に関わるコレステロール26位水酸化酵素の機能解析
- 2015年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 33rdソラニン・チャコニンの他のグリコアルカロイドを含むジャガイモ系統
- 2015年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 33rdα-トマチン生合成に関わる3位酸化異性化酵素の解析
- 2015年, 植物の生長調節, 50 (Supplement)トマトにおけるα-トマチン生合成に関わるステロイド3位酸化異性化酵素の解明
- 2014年10月18日, 植物化学調節学会第49回大会，京都, 日本語ブラシノステロイド不活性化に関わるシトクロムP450酵素CYP72C1の酵素機能解明
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 植物化学調節学会, 2014年10月01日, 植物化学調節学会研究発表記録集, 49, 85 - 85, 日本語67. ヒマワリの生産する非セスキテルペンラクトン型根寄生雑草発芽刺激物質heliolactoneの同定(口頭発表,植物化学調節学会第49回大会)
- 植物化学調節学会, 2014年10月01日, 植物化学調節学会研究発表記録集, 49, 87 - 87, 日本語69. ソルガムにおけるGR24水酸化反応の立体特異性と位置選択性(口頭発表,植物化学調節学会第49回大会)
- 植物化学調節学会, 2014年10月01日, 植物化学調節学会研究発表記録集, 49, 65 - 65, 日本語47. チャにおける加害応答性モノテルペンアルコール生合成酵素の同定(口頭発表,植物化学調節学会第49回大会)
- 植物化学調節学会, 2014年10月01日, 植物化学調節学会研究発表記録集, 49, 68 - 68, 日本語50. 防御シグナル因子ピペコリン酸の生合成に関わるALD1の機能解析(口頭発表,植物化学調節学会第49回大会)
- 2014年09月20日, 2014年度日本農芸化学会関西支部大会, 486th, 日本語ナス科植物ステロイドサポニンの構造多様性に関わるステロイド26位水酸化酵素の機能解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2014年03月29日, 日本農芸化学会2014年度大会，東京, 2014, 日本語ジャガイモの有毒ソラニン生合成に関わる新規ジオキシゲナーゼの機能解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2014年03月18日, 第55回日本植物生理学会，富山, 55th, 日本語ナス科植物ステロイドグリコアルカロイド生合成に関わる16位水酸化を触媒するジオキシゲナーゼの機能解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2014年03月, 日本農芸化学会2014年度大会，東京, 2014, 日本語ヤマノイモ属トゲドコロに蓄積するステロイドサポニンの単離精製および定量解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2014年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 32ndトマト果実におけるα-トマチン代謝酵素の探索
- 2014年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 32ndナス科植物ステロイドグリコアルカロイド生合成に関わるジオキシゲナーゼの機能解析
- 2014年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 32ndジャガイモにおける有用サポニン高生産系の構築
- 2014年, 日本農薬学会大会講演要旨集, 39th根寄生雑草ヤセウツボの発芽時に発現するβ-マンノシダーゼの役割
- 2014年, 日本農芸化学会大会講演要旨集(Web), 2014ヤセウツボ発芽種子で発現する糖加水分解酵素の機能解析
- 植物化学調節学会, 2013年10月04日, 植物化学調節学会研究発表記録集, 48, 71 - 71, 日本語56. S. gesnerioides D14-like遺伝子の探索とストライゴラクトン加水分解活性の評価(口頭発表,植物化学調節学会第48回大会)
- 植物化学調節学会, 2013年10月04日, 植物化学調節学会研究発表記録集, 48, 65 - 65, 日本語50. 植物における5-デオキシストリゴールからモノヒドロキシストリゴラクトンへの変換反応(口頭発表,植物化学調節学会第48回大会)
- 植物化学調節学会, 2013年10月04日, 植物化学調節学会研究発表記録集, 48, 44 - 44, 日本語29. ブラシノステロイド不活性化酵素CYP72C1酵素学的解析(口頭発表,植物化学調節学会第48回大会)
- 植物化学調節学会, 2013年10月04日, 植物化学調節学会研究発表記録集, 48, 29 - 29, 日本語14. ジベレリン生合成・代謝酵素CYP714ファミリーのリガンド認識機構(口頭発表,植物化学調節学会第48回大会)
- 植物化学調節学会, 2013年10月04日, 植物化学調節学会研究発表記録集, 48, 33 - 33, 日本語18. ファゼイン酸還元酵素の阻害剤を指向したフッ素化基質アナログの合成(口頭発表,植物化学調節学会第48回大会)
- 植物化学調節学会, 2013年10月04日, 植物化学調節学会研究発表記録集, 48, 60 - 60, 日本語45. ヒマワリが分泌する根寄生雑草発芽刺激物質の解析(口頭発表,植物化学調節学会第48回大会)
- 植物化学調節学会, 2013年10月04日, 植物化学調節学会研究発表記録集, 48, 84 - 84, 日本語69. ヤマノイモ属トゲドコロのステロイドサポニン生合成に関わるステロール22位水酸化酵素の同定(口頭発表,植物化学調節学会第48回大会)
- 植物化学調節学会, 2013年10月04日, 植物化学調節学会研究発表記録集, 48, 64 - 64, 日本語49. β-カロテン生産菌を用いたストライゴラクトン生合成中間体カルラクトン酵素合成の試み(口頭発表,植物化学調節学会第48回大会)
- 2013年10月, 植物化学調節学会第48回大会，新潟, 48 (Supplement), 日本語ヤマノイモ属トゲドコロにおけるステロイドサポニン生合成 16 位水酸化酵素の同定
研究発表ペーパー・要旨（全国大会，その他学術会議）
- (一社)日本生薬学会, 2013年08月, 日本生薬学会年会講演要旨集, 60回, 141 - 141, 日本語薬用植物カンゾウのグリチルリチン生合成に関わる糖転移酵素遺伝子の単離と機能解析
- 2013年06月, TERPNET 2013, Crete, Greece, 31st, 英語Identification and Quantification of Steroidal Saponins in Dioscorea esculenta
研究発表ペーパー・要旨（国際会議）
- 2013年, 植物の生長調節, 48 (Supplement)ヤマノイモ属トゲドコロのステロイドサポニン生合成に関わるステロール22位水酸化酵素の同定
- 2013年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 31stヤマノイモ属トゲドコロ由来のフロスタノール配糖体を加水分解するβ-グルコシダーゼの同定
- 2013年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 31stトマトにおけるステロイドアルカロイド生合成に関わる配糖化酵素の探索
- 2013年, 日本農芸化学会大会講演要旨集(Web), 2013ヤマノイモ属における有用ステロイドサポニン生合成に関わるβ-グルコシダーゼの同定
- 2013年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 31stヤマノイモ属トゲドコロ由来NADPH-P450還元酵素遺伝子の単離同定
- 2013年, 植物の生長調節, 48 (Supplement)ナス科植物ステロイドグリコアルカロイド生合成に関わる新規2-オキソグルタル酸依存性ジオキシゲナーゼ:16位水酸化酵素の同定と機能解析
- 2013年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 31stトマト果実におけるα-トマチン代謝酵素遺伝子の探索
- 2013年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 31stヤマノイモ属トゲドコロにおけるステロイドサポニン生合成P450遺伝子の探索
- 2013年, 日本農芸化学会大会講演要旨集(Web), 2013ヤセウツボ発芽種子で発現する糖加水分解酵素の機能解析
- 2013年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 31stシロイヌナズナの配糖化酵素UGT71Cサブファミリーのリグナン生合成への関与
- 2013年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 31stグリコアルカロイド生合成遺伝子群の同定について
- 日本生物工学会, 2013年, 日本生物工学会大会講演要旨集, 65, 243 - 243, 日本語3P-221 薬用植物カンゾウのグリチルリチン生合成に関わる糖転移酵素遺伝子の単離と機能解析(植物細胞工学,組織培養,育種工学,一般講演)
- 一般社団法人 植物化学調節学会, 2013年, 植物の生長調節, 48 (0), 86 - 86, 日本語
The C22-unsaturated sterols are primarily found in fungi and plants. The C22-desaturation reaction is catalyzed by independent cytochrome P450 family proteins, CYP61 in fungi, and CYP710 in plants. We describe our extensive characterization studies of plant CYP710 family proteins and discuss possible evolutional relationshipsof C22-desaturation reactions among eukaryotic organisms. We also discuss possible research directions toward understanding physiological implications of sterols in unidentifiedbrassinosteroid-independent growth/developmental processes.
Springer New York, 2013年01月01日, Isoprenoid Synthesis in Plants and Microorganisms: New Concepts and Experimental Approaches, 381 - 391, 英語[査読有り]
記事・総説・解説・論説等（商業誌、新聞、ウェブメディア）
- 2012年10月27日, 植物化学調節学会第47回大会，鶴岡, 47 (Supplement), 日本語ナス科植物におけるステロイドグリコアルカロイド生合成に関わるP450の酵素解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2012年10月27日, 植物化学調節学会第47回大会，鶴岡, 日本語シトクロムP450酵素CYP90A1はブラシノステロイドC-3位酸化酵素である
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 植物化学調節学会, 2012年10月05日, 植物化学調節学会研究発表記録集, 47, 92 - 92, 日本語75. ファゼイン酸還元酵素の基質特異性(口頭発表,植物化学調節学会第47回大会)
- 植物化学調節学会, 2012年10月05日, 植物化学調節学会研究発表記録集, 47, 68 - 68, 日本語51. ヒマワリが生産するストライゴラクトンの探索(口頭発表,植物化学調節学会第47回大会)
- 植物化学調節学会, 2012年10月05日, 植物化学調節学会研究発表記録集, 47, 64 - 64, 日本語47. Strigolactone の生理活性における最小構造(口頭発表,植物化学調節学会第47回大会)
- 植物化学調節学会, 2012年10月05日, 植物化学調節学会研究発表記録集, 47, 79 - 79, 日本語62. ソルガムによる5-deoxystrigol からsorgomol への変換(口頭発表,植物化学調節学会第47回大会)
- 植物化学調節学会, 2012年10月05日, 植物化学調節学会研究発表記録集, 47, 66 - 66, 日本語49. Striga gesnerioides 種子に対する発芽阻害物質の探索(口頭発表,植物化学調節学会第47回大会)
- 植物化学調節学会, 2012年10月05日, 植物化学調節学会研究発表記録集, 47, 65 - 65, 日本語48. 根寄生植物 Striga gesnerioides 種子のストライゴラクトンに対する構造要求性の解明
- 植物化学調節学会, 2012年10月05日, 植物化学調節学会研究発表記録集, 47, 80 - 80, 日本語63. 様々な植物における5-deoxystrigol の酸化的代謝(口頭発表,植物化学調節学会第47回大会)
- 2012年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 30thヤマノイモ属における有用ステロイドサポニン生合成に関わるβ-グルコシダーゼの解析
- 2012年, 日本農芸化学会大会講演要旨集(Web), 2012ヤマノイモ属トゲドコロにおけるプロトジオシンを加水分解するβ-グルコシダーゼの解析
- 2012年, 日本植物生理学会年会要旨集, 53rdCYP716Aサブファミリーメンバーはトリテルペンサポニン生合成においてマルチファンクショナルP450として機能する
- 2012年, 日本植物生理学会年会要旨集, 53rdブドウゲノム上でオペロン様遺伝子クラスターを作るCYP716AはトリテルペンC28位酸化酵素である。
- 2012年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 30thグリコアルカロイドを非常に低下させたジャガイモ
- 2012年, 平成22-23年度研究成果報告書，京都大学生存基盤科学研究ユニット, 47 - 51, 日本語アオモリヒバの代謝産物および遺伝子発現プロファイリング
その他
- 53. 植物によるGR24変換反応の解析(口頭発表,植物化学調節学会第46回大会)
Strigolactones (SLs) have been identified from plant root exudates as important bioactive compounds. Based on their structural similarity, 5-deoxystrigol can be a common precursor of SLs. In this study, a synthetic SL analog GR24 was fed to Menispermum dauricum (Md) roots, Lotus japonicas (Lj) roots and sorghum (Sorghum bicolor) seedlings to study incorporation and metabolic conversion. GR24 was detected in Md roots, Lj roots and sorghum seedlings incorporated GR24. Md roots converted GR24 to a metabolite with M.W.314. Sorghum seedlings converted GR24 into three metabolites with M.W.314. On the other hand, Lj roots did not convert GR24. A hydroxylated product of GR24 by sorghum exhibited identical behavior to 4-hydroxy-GR24 on HPLC. The other two hydroxylated products of sorghum and a hydroxylated product of Md exhibited similar but not identical chromatographic behavior. These three hydroxylated product of GR24 afforded fragment ion at m/z 97, indicating that hydroxylation occurred in the A ring of GR24. These results suggest that plants which produce hydroxylated SLs have the ability to hydroxylate GR24. Structural analysis of the hydroxylated products of GR24 is now in progress.
植物化学調節学会, 2011年10月03日, 植物化学調節学会研究発表記録集, (46), 69 - 69, 日本語 - 54. 水酸化GR24の合成と構造解析(口頭発表,植物化学調節学会第46回大会)
To identify hydroxylated products of GR24, the synthesis of 5-, 6-, 7- and 8-hydroxy GR24 was undertaken. First, 5- and 6-hydroxy-1-indanone 1, 2 were protected with a MOM group, gave MOM protected indanone 3, 4, respectively. Indanone 3 and 4 reacted with dimethyl carbonate in the presence of sodium hydride, then ethyl bromoacetate gave compounds 5 and 6. Acid catalyzed hydrolysis and subsequent decarboxylation gave carboxylic acids 7 and 8. Then, sodium borohydride reduction, followed by acid treatment gave tricyclic lactones 9 and 10, respectively. Formylation of ABC-ring and coupling with D-ring are ongoing.
植物化学調節学会, 2011年10月03日, 植物化学調節学会研究発表記録集, (46), 70 - 70, 日本語 - 5. ABA8'-水酸化酵素選択的阻害剤abscinazole-E2Bの光学活性体(口頭発表,植物化学調節学会第46回大会)
S-Uniconazole (UNI), which was developed as an inhibitor of GA biosynthetic enzyme (CYP701A), is a low selective P450 inhibitor that inhibits multiple P450 enzymes including ABA 8'-hydroxylase (CYP707A). Based on our speculation that the low selectivity of S-UNI may be resulted from its small molecule, we developed enlarged UNI analogues to find a selective inhibitor of CYP707A. Finally we found a practical inhibitor of CYP707A, (±)-abscinazole-E2B (Abz-E2B). (±)-Abz-E2B showed strong inhibitory activity (inhibition constant, K_1=36 nM) against CYP707A, which was equivalent to that of S-UNI (K_1=10 nM). The (±)-Abz-E2B-sprayed plants exhibited drought tolerance, stomatal closure, and an increase in the amount of ABA. On the other hand, against CYP701A, Abz-E2B was a poorer inhibitor, and it did not inhibit the growth of rice seedlings, contrary to UNI. In this study, we optically resolved (±)-Abz-E2B and determined the absolute configuration. The K_1 values of S-(-)- and R-(+)-Abz-E2B for CYP707A were 28 nM and 360 nM, respectively. The similar tendency is observed in UNI whose S-enantiomer is more potent than the R-enantiomer. Similarly, in bioassays, Abz-E2B enhanced the effect of ABA in the order of potency: the S-enantiomer, racemic form, and R-enantiomer. Because no side effects were observed by administration of R-enantiomer, the use of the racemic form may be a reasonable alternative option in the field experiments using a large amount of chemicals.
植物化学調節学会, 2011年10月03日, 植物化学調節学会研究発表記録集, (46), 21 - 21, 日本語 - 3. ジベレリン生合成酵素CYP701A選択的阻害剤(口頭発表,植物化学調節学会第46回大会)
CYP701A, a gibberellin (GA) biosynthetic P450 enzyme, catalyzes the three step oxidation of ent-kaurene to ent-kaurenoic acid. A selective inhibitor of this enzyme is a promising research tool for chemical genetic studies of GA deficiency. Uniconazole (UNI), paclobutrazol (PAC), inabenfide (IBF) and ancymidol (ANC) have been known as inhibitors of CYP701A. Although their selectivity has little been investigated biochemically, recent researches suggest that UNI, PAC, and ANC are not specific to CYP701A; UNI and PAC act as an inhibitor of CYP707A, a catabolic enzyme of abscisic acid, and ANC inhibits cellulose synthesis. Hence, we launched the development of a novel selective inhibitor of CYP701A. Recently we developed a selective and potent inhibitor of CYP707A, abscinazoles, by screening our library of UNI analogues whose structures are enlarged or conformationally restricted to eliminate structural factors that may cause the low enzyme selectivity. This library may contain a selective inhibitor of CYP701A. We examined inhibitory activities of compounds in the library against recombinant rice CYP701A6 and Arabidopsis CYP707A3 enzymes. Finally we found a conformationally restricted UNI analogue, UFAP2, that strongly inhibited CYP701A6 without exhibiting considerable inhibitory effect on CYP707A3. Here we present the enzyme selectivity, biological activity and water solubility of UFAP2 in comparison with those of other known CYP701A inhibitors.
植物化学調節学会, 2011年10月03日, 植物化学調節学会研究発表記録集, (46), 19 - 19, 日本語 - 56. ストライゴラクトンの枝分かれ抑制活性における構造要求性(口頭発表,植物化学調節学会第46回大会)
Strigolactones (SLs) act as a phytohormone which inhibit shoot branching. Naturally occurring SLs such as 5-deoxystrigol consist of tricyclic ABC-ring and D-ring connected with enol ether bridge. Synthetic SL analogues such as GR24 were known to exhibit similar activity as natural SLs. However, it is still unclear whether SLs themselves are the active form or precursors of active principles. To get insight into the structure essential for inhibiting shoot branching, information about SL structure-activity relationship is necessary. In this study, we employed over 20 ABC-ring analogues of SL and tested their activity toward rice d10 mutant grown in hydroponic system. Based on the branching bioassay, D-ring and enol ether bridge structure were found to be critical but the AB-ring moiety is less important on SL activity. Furthermore, compounds having phenyl ether as a substitute for enol ether exhibited high activity, but the replacement of enol ether by benzyl ether resulted in the loss of activity. These results imply that D-ring and π-conjugated system are required for shoot branching inhibition.
植物化学調節学会, 2011年10月03日, 植物化学調節学会研究発表記録集, (46), 72 - 72, 日本語 - 57. 根寄生雑草Striga gesnerioides種子の発芽を誘導するストライゴラクトンの構造要求性(口頭発表,植物化学調節学会第46回大会)
Strigolactones are highly potent germination stimulants for seeds of the parasitic weeds Striga and Orobanche spp. A synthetic strigolactone GR24 induces germination of seeds of several root parasitic plants including O. minor and S. hermonthica, but not S. gesnerioides. Germination of the latter is only elicited by cowpea root exudates or alectrol, isolated from its root exudates. The recent proposal that the true structure of alectrol is the same as that of orobanchyl acetate, suggests that a modification in the B-ring of the strigolactones is important for germination induction of S. gesnerioides seeds. In the present study, 4-hydroxy-GR24 (HO-GR24) and 4-acetoxy-GR24 (AcO-GR24) were prepared and evaluated for ability to induce germination of parasitic plant seeds. Racemic mixtures of these compounds induced seed germination of S. hermonthica and O. minor. However, S. gesnerioides seeds did not respond to any of the compounds. The seeds responded to the (8bR,2'R)-isomer of HO-GR24. The stereoisomer of GR24 with the same configuration induced negligible germination. Some stereoisomers of GR24 and its analogs acted as effective inhibitor for induction of seed germination by cowpea root exudates. The results suggested that both an oxidized substituent at C-4 and the configuration of the tricyclic lactone and the D ring are essential structural requirements for induction of germination in S. gesnerioides seeds.
植物化学調節学会, 2011年10月03日, 植物化学調節学会研究発表記録集, (46), 73 - 73, 日本語 - 55. ソルガムにおける5-deoxystrigolの酸化的代謝(口頭発表,植物化学調節学会第46回大会)
Strigolactones have been isolated from root exudates of various plants. A series of strigolactones may be derived from 5-deoxystrigol by oxidation, acetylation, methylation and demethylation. We found that some plants can convert GR24 to its hydroxylated products. In this study, 5-deoxystrigol was applied to hydroponically grown sorghum (Sorghum bicolor), and the conversion of the probable common precursor of other strigolactones to hydroxylated strigolactones was analyzed by LC-MS/MS. A sorghum variety referred to as NM24 was selected as the highest producer of sorgomol (9-hydroxylated 5-deoxystrigol). NM24 converted [6'-D]-5-deoxystrigol and [6'-D]-2'-epi-5-deoxystrigol to [6'-D]-sorgomol and its 2'-epimer, respectively. Accordingly, the ability to hydroxylate 5-deoxystrigol at C-9 in sorghum was confirmed.
植物化学調節学会, 2011年10月03日, 植物化学調節学会研究発表記録集, (46), 71 - 71, 日本語 - 58. 根寄生雑草Striga gesnerioides種子発芽を誘導するササゲ由来ストライゴラクトンの単離同定(口頭発表,植物化学調節学会第46回大会)
Alectrol was originally isolated from root exudates of cowpea (Vigna unguiculata) as a germination stimulant for Striga gesnerioides and Alectra vogelii. The strigolactone was also isolated from red clover (Trifolium pretense) as a stimulant for Orobanche minor and identified most probably as (+)-orobanchyl acetate. However, authentic (+)-orobanchyl acetate induced negligible S. gesnerioides seed germination. In this study, the germination stimulants for seeds of S. gesnerioides produced by cowpea were reinvestigated. Root exudates of cowpea were collected and fractionated. Two germination stimulants were detected. Based on chromatographic behavior on HPLC and physicochemical properties including CD and NMR spectra, structures of the isolated stimulants were determined as ent-2'-epi-orobanchol and ent-2'-epi-orobanchyl acetate. The same strigolactones were identified in root exudates of red clover.
植物化学調節学会, 2011年10月03日, 植物化学調節学会研究発表記録集, (46), 74 - 74, 日本語 - 4. ジベレリン生合成における13位水酸化反応を阻害する物質(口頭発表,植物化学調節学会第46回大会)
The 13-hydroxylation in gibberellin (GA) biosynthesis-the substrate, the enzyme, and the biological significance are still poorly understood. Although recent studies suggest that some CYP714 enzymes catalyze the 13-hydroxylation of ent-kaurenoic acid (KA) or GA_<12>, the biological significance of the 13-hydroxylation remains unclear. The GA receptor, GID1 in Arabidopsis and rice, prefers GA_4 with no hydroxy group at C13 rather than GA_1, the 13-hydroxylated GA. Nevertheless, GA_1 is the major active GA in rice. Why does the 13-hydroxylation exist in GA biosynthesis? Our goal in this study is the development of a selective inhibitor of the 13-hydroxylases in GA biosynthesis as a chemical tool for probing the biological significance of the 13-hydroxylation. We screened inhibitors for Arabidopsis KA 13-hydroxylase, CYP714A, from commercial available azole compounds, and found imazalil (IMZ), which was 30-fold stronger than uniconazole in inhibition of CYP714A. Because IMZ is a fungicide that inhibits fungal CYP51, it is not excluded the possibility that this compound inhibits plant CYP51. Hence, we are constructing the assay system of plant and fungal CYP51 to test the selectivity of IMZ. We are also modifying the structure of IMZ to raise the selectivity.
植物化学調節学会, 2011年10月03日, 植物化学調節学会研究発表記録集, (46), 20 - 20, 日本語 - 98. ヤマノイモ属トゲドコロにおけるステロイドサポニン生合成に関与する遺伝子の探索(口頭発表,植物化学調節学会第46回大会)
Steroidal saponins have various biological activities. Steroidal saponins are often found in monocotyledons such as plants of the families Dioscoreaceae, Agavaceae, and Liliaceae. In particular, the rhizomes of Dioscorea, known as yam, contain furostane and spirostane glycosides such as protodioscin and dioscin, respectively. These steroid saponins are derived from choresterol by sequential modification with oxygenation and transglycosylation reaction as shown in Figure 1. Namely, several P450s are likely involved in oxygenations at the C-16, C-22, and C-26 positions, and UGTs will function in transglycosylation at C-3 and C-26. But little is known about enzyme and genes for dioscin biosynthesis. To investigate steroidal saponin biosynthesis in Dioscorea spp., we performed comparative transcriptome analysis of the rhizomes of Dioscorea spp. We will utilize these datasets to identify key genes for cholesterol and dioscin biosynthesis, and will apply the results to plant metabolic engineering of steroidal saponins.
植物化学調節学会, 2011年10月03日, 植物化学調節学会研究発表記録集, (46), 114 - 114, 日本語 Plant cytochromes P450 (P450s) participate in a variety of biochemical pathways to produce a vast diversity of plant natural products. The number of P450 genes in plant genomes is estimated to be up to 1% of the total gene annotations of each plant species, implying that plants are huge sources for various P450-dependent reactions. Plant P450s catalyze a wide variety of monooxygenation/hydroxylation reactions in secondary metabolism, and some of them are involved in unusual reactions such as methylenedioxy-bridge formation, phenol coupling reactions, oxidative rearrangement of carbon skeletons, and oxidative C-C bond cleavage. Here, we summarize unusual P450 reactions in various plant secondary metabolisms, and describe their proposed reaction mechanisms. (C) 2010 Elsevier Inc. All rights reserved.
ELSEVIER SCIENCE INC, 2011年03月, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 507 (1), 194 - 203, 英語[査読有り]
書評論文,書評,文献紹介等
- 2011年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 29thヤマノイモ属トゲドコロにおけるステロイドサポニン生合成酵素の探索
- 2011年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 29thトマトにおけるα-トマチン生合成酵素の探索
- 2011年, 植物の生長調節, 46 (Supplement)ヤマノイモ属トゲドコロにおけるステロイドサポニン生合成に関与する遺伝子の探索
- 2011年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 29thトマト果実におけるα-トマチンの代謝酵素の探索
- 80.キュウリ葉からのα,β-不飽和カルボニル化合物還元酵素の精製と遺伝子解析(口頭発表)
Polyunsaturated fatty acids in biomembranes are oxidized easily, and result in various low-molecular weight compounds. These compounds include α,β-unsaturated carbonyls like acrolein and methylvinylketone, which are highly reactive. The toxicity of these reactive compounds are based on their ability to form the Michael adducts with thiols and amino groups in proteins and nucleic acids. Therefore, it is important to scavenge α,β-unsaturated carbonyls for maintaining homeostasis in plants. In this study, we purifed the acrolein-reducing enzyme from cucumber leaves and determined its partial amino acid sequences. Based on RT-PCR with degenerate primers and RACE strategies, two cDNA sequences contained fully functional region of the enzyme were obtained. One has chloroplast-targeted transit sequence at N-terminal region, and the other has no. These results indicate that cucumber leaves have α,β-unsaturated carbonyl-detoxifying enzymes localizing in chloroplast and cytosol.
植物化学調節学会, 2010年10月01日, 植物化学調節学会研究発表記録集, (45), 97 - 97, 日本語 - 42.ABAアミノ酸複合体の合成と生物活性(口頭発表)
We prepared 19 amino acid conjugates of the plant hormone abscisic acid (ABA) and investigated their biological activity, enzymatic hydrolysis by recombinant Arabidopsis amidohydrolases, and metabolic fate in rice seedlings. Different sets of ABA-amino acids induced ABA-like responses in different plants. Some ABA-amino acids, including some that were active in bioassays, were hydrolyzed by recombinant Arabidopsis GST IAR3. ABA-L-Ala, which was active except in a lettuce bioassay and was hydrolyzed by GST-IAR3, was hydrolyzed to free ABA in rice seedlings. These findings suggest that some plant amidohydrolases hydrolyze some ABA-amino acid conjugates. Because our study indicates the possibility that different plants have hydrolyzing activity toward different ABA-amino acids, an ABA-amino acid may function as a species-selective mimic of ABA.
植物化学調節学会, 2010年10月01日, 植物化学調節学会研究発表記録集, (45), 59 - 59, 日本語 - 83.植物におけるクマリン類生合成の鍵酵素である桂皮酸類オルト位水酸化酵素の解析(口頭発表)
Coumarins are ubiquitously found as the plant secondary metabolites in plants and are biosynthesized via ortho-hydroxylation of cinnamates by cinnamates 2'-hydroxylase (C2'H), which is a key enzyme in coumarins biosynthesis. We investigated C2'Hs from Arabidopsis and sweet potato (Ipomoea batatas), which accumulate the glucosides of scopoletin and umbelliferone, respectively. Previous studies showed that C2'H is a 2-oxoglutarate-dependent dioxygenase and catalyzes ortho-hydroxylation of the CoA-ester of cinnamates to yield coumarins. The results suggest that the chemical composition of coumarins in plant species is determined by the substrate specificity of C2'H in each of the plants. To understand coumarins biosynthesis in various plants, we have isolated a C2'H (LjC2'H) cDNA from Lotus japonicus, in which coumarins have not yet been found so far. We found that LjC2'H catalyzes ortho-hydroxylation of feruloyl-CoA and cafeoyl-CoA to form scopoletin and esculetin, respectively. We have also isolated a C2'H cDNA from grapefruit (Citrus x paradisi). Grapefruit accumulates furanocoumarins, which is derived from umbelliferone, and characterization of grapefruit C2'H is in progress. In addition, we have established bacterial biosynthesis of coumarins. Plant C2'Hs were co-expressed with 4-coumaroyl CoA:ligase in E. coli, and the feeding of cinnamates resulted in the accumulation of coumarins in the culture medium.
植物化学調節学会, 2010年10月01日, 植物化学調節学会研究発表記録集, (45), 100 - 100, 日本語 - 34.メスキート葉に由来する根寄生植物の種子発芽抑制物質の探索(口頭発表)
Mesquite (Prosopis juliflora) is the leguminous shrubs growing in the tropical or subtropical regions. In this study, we are searching for biologically active substances produced by mesquite leaves toward root parasitic plants. Dried mesquite leaves (250g) were extracted with methanol. After partitioning, the extract (48.6g) yielded crude alkaloids (3.9g), which exhibited the potent inhibitory activity for seed germination and radicle elongation of root parasitic plants Striga hermonthica and Orobanche minor. Bioassay-guided separation of the crude alkaloids by silica gel column chromatography, TLC and HPLC gave two active principles. Structural analysis of the compounds is in progress.
植物化学調節学会, 2010年10月01日, 植物化学調節学会研究発表記録集, (45), 51 - 51, 日本語 - 32.根寄生植物オロバンキの成長過程における脱分化・分化の制御機構の解明(口頭発表)
Orobanche minor seedlings were attached onto red clover (Trifolium pretense) roots grown in rhizotrons. Approximately 30% of the O. minor seedlings formed tubercles on red clover roots 2 weeks after attachment. Root primordia were appeared on the surface of the tubercles, and crown roots were developed 19 days after attachement. A shoot bud was differentiated from the crown roots 5 weeks after attachment. O. minor parasitism induced little morphological changes in red clover roots during the development process from tubercle formation (de-differentiation) to shoot bud formation (re-differentiation). These observations suggest that O. minor produces growth regulators in its own metabolism to control its differentiation. In addition, the crown roots and the shoot bud were differentiated without the seed embryo of O. minor, suggesting that the developmental process seems to be the process of adventitious root and shoot formation. To investigate the involvement of phytohormone homeostasis in the differentiation process, the endogenous levels of various plant hormones during the developmental process from seed germination to flowering were measured. Furthermore, gene expression involved in the developmental process of O. minor has been extensively studied with the next-generation sequencing platform.
植物化学調節学会, 2010年10月01日, 植物化学調節学会研究発表記録集, (45), 49 - 49, 日本語 - 44.タンパク質二量体誘導化合物(CID)を利用した新規植物化学調節剤の創出(口頭発表)
A dimer of a ligand for a target protein functions in some cases as a chemical inducer of dimerization (CID) that induces dimerization of the target protein. The CID can bind strongly to the target protein because of protein-protein interactions to function as a stronger ligand (an enzyme inhibitor or an antagonist) than the corresponding monomer. However, there have been a few reports (especially no reports for plant enzymes) of CID, probably because chemical modifications for dimerization have a negatively affect on the affinity to the target. Abscinazole-E1 (Abz-E1), which is a uniconazole (UNI) derivative, inhibits CYP707A, a key P450 enzyme of ABA catabolism, as strongly as UNI, in spite of possessing a long diethylene glycol chain. Therefore, a UNI dimer linked by oligoethylene glycol chain may function as a CID for CYP707A. Base on this expectation, we designed and synthesized five UNI dimmers, UTD1-En, and examined their inhibitory activity against CYP707A3 and plant growth. Although UTD1-En inhibited CYP707A as strongly as UNI, we cannot have confirmed in our present assay system whether or not UTD1-En functioned as CID. UTD1-En promoted and inhibited the root elongation of rice seedlings at 30 and 100μM, respectively. This effect depended on the number of ethylene glycol unit, n. Because UNI and Abz-E1 had no effect on the root elongation, this activity may be caused by that two UNI units is linked with an appropriate distance.
植物化学調節学会, 2010年10月01日, 植物化学調節学会研究発表記録集, (45), 61 - 61, 日本語 - 45.ジベレリン生合成酵素選択的P450阻害剤のスクリーニング(口頭発表)
Gibberellins (GAs) are plant hormone involved in stem elongation, seed germination, induction of flowering and other physiological events. Uniconazole (UNI), which is well known as a triazole-containing inhibitor of GA biosynthetic enzyme (CYP701A), functions as an inhibitor of multiple P450 enzymes including ABA 8'-hydroxylase (CYP707A), which is a key enzyme for ABA catabolism. We speculated that low selectivity of UNI may be resulted from its small and flexible conformation adjustable for variety of substrate pockets. Based on this speculation, we developed UNI analogues which is conformationally restricted (Abscinazole-F1), enlarged (-E1), disubstituted with azole (-D1), as specific inhibitors of CYP707A. We examined their inhibitory activity against recombinant CYP707A and the growth of rice seedlings, which is regulated by GA. However, because the growth of rice seedlings is also regulated by other plant hormones, the inhibitory activity is not fully equivalent to inhibitory activity against GA biosynthetic P450s. Thus we constructed an assay system of CYP701A inhibition using recombinant rice CYP701 As. We are performing in vitro screening of azole compound libraries using this system to find a selective inhibitor for CYP701A.
植物化学調節学会, 2010年10月01日, 植物化学調節学会研究発表記録集, (45), 62 - 62, 日本語 - 43.ABA 8'-水酸化酵素に対する実用性の高い選択的阻害剤アブシナゾールE2B(口頭発表)
Uniconazole (UNI), which was developed as an inhibitor of GA biosynthetic enzyme (CYP701A), is a low selective P450 inhibitor that inhibits multiple P450 enzymes including abscisic acid (ABA) 8'-hydroxylase (CYP707A). Based on our speculation that the low selectivity of UNI may be resulted from its small molecule, we developed enlarged UNI analogues (UT) that have a 1,2,3-triazolyl alkyl chain. Although UT compounds showed the strong CYP707A inhibition, the biological activity of most of UT compounds was not tested because of their water-insolubility. Because the introduction of protic functional groups in the alkyl chain diminished the inhibitory activity, we developed Abz-E1 that has a diethylene glycol chain with a terminal tosylate. Abz-E1 showed good water solubility and strong inhibitory activity against CYP707A in vitro and in vivo. Because Abz-E1 has a terminal tosylate, it is expected to be useful precursor for multifunctional chemical probes. Because of the same reason, however, Abz-E1 seems to be unstable in vivo. Thus we synthesized four compounds from Abz-E1 to examine the requirement of the tosylate for CYP707A inhibition and to generate a new practical CYP707A inhibitor. The results showed that the tosylate is not necessary for the inhibition, whereas the hydrophobicity is more significant for it than the molecular length. Finally we found a more practical inhibitor of CYP707A, abscinazole-E2B (Abz-E2B), with no tosylate and with good water solubility.
植物化学調節学会, 2010年10月01日, 植物化学調節学会研究発表記録集, (45), 60 - 60, 日本語 Plant cytochromes P450 (P450s) catalyze a wide variety of monooxygenation/hydroxylation reactions in primary and secondary metabolism. The number of P450 genes in plant genomes is estimated to be up to 1% of total gene annotations of each plant species. This implies that diversification within P450 gene superfamilies has led to the emergence of new metabolic pathways throughout land plant evolution. The conserved P450 families contribute to chemical defense mechanisms under terrestrial conditions and several are involved in hormone biosynthesis and catabolism. Species-specific P450 families are essential for the biosynthetic pathways of species-specialized metabolites. Future genome-wide analyses of P450 gene clusters and coexpression networks should help both in identifying the functions of many orphan P450s and in understanding the evolution of this versatile group of enzymes.
ANNUAL REVIEWS, 2010年, ANNUAL REVIEW OF PLANT BIOLOGY, VOL 61, 61, 291 - 315, 英語[査読有り]
記事・総説・解説・論説等（学術雑誌）
- 38.メスキートが生産する有用二次代謝産物の検索(口頭発表)
Mesquite (Prosopis juliflora) is the leguminous shrubs growing in the tropical or subtropical regions. In this study, we conduct screening of biologically active substances produced by mesquite. Dried mesquite leaves (250g) were extracted with methanol. The extract (48.6g) was partitioned into the acidic and neutral fraction (22.2g) and the basic fraction (3.9g). The acidic and neutral fraction exhibited the inhibitory activity for root and shoot growth of lettuce seedlings. Furthermore, the basic fraction and the acidic and neutral fraction inhibited seed germination of Striga hermonthica, S. gesnerioides and Orobanche minor. The purification of these bioactive substances is in progress.
植物化学調節学会, 2009年10月06日, 植物化学調節学会研究発表記録集, (44), 52 - 52, 日本語 - 41.柑橘およびマメ科植物におけるクマリン化合物生合成酵素の探索(口頭発表)
Coumarins are ubiquitously found as the plant secondary metabolites in plants. They are thought to play important roles in plant defense due to their antimicrobial and antioxidative activities. Coumarins are biosynthesized via ortho-hydroxylations of cinnamates (C2'H). This is a key step in the lactone ring formation in coumarin biosynthesis, and C2'H is classified into the 2-oxoglutarate-dependent dioxygenase (2OGD) family. Arabidopsis roots accumulate scopoletin β-glucoside, and Arabidopsis C2'H shows the high substrate specificity to feruloyl-CoA but does not accept ferulic acid as a substrate. Sweet potato (Ipomoea batatas L.) tubers accumulate the β-glucosides of scopoletin and umbelliferone, and C2'H of sweet potato can accept feruloyl-CoA and p-coumaroyl-CoA as its substrates to form scopoletin and umberiferone, respectively. These results suggest that the substrate specificity of C2'H determines the accumulation patterns of coumarins in each plant species. In this study, we have found that there are C2'H homologs in the EST database of citrus and legume (Medicago truncatula and Lotus japonicus). Citrus fruits are known to accumulate furanocoumarins, but little is known about coumarin synthesis in M. truncatula and L. japonicus. In order to explore coumarin biosynthesis in citrus and the legume plants, we have performed the functional analysis of the C2'H homologs of these plants.
植物化学調節学会, 2009年10月06日, 植物化学調節学会研究発表記録集, (44), 55 - 55, 日本語 - 42.イソキノリンアルカロイド生合成に関わるシトクロムP450 cDNAの単離および機能解析(口頭発表)
In the biosynthesis of isoquinoline alkaloids, several unusual monooxygenation reactions such as the intermolecular C-O phenol-coupling and the intramolecular C-C phenol coupling reactions are involved, and cytochromes P450 such as CYP80A1 and CYP80G2 have been shown to catalyze these reactions. Root cultures of Stephania cephatantha produce several isoquinoline alkaloids, and, in this study, we have isolated five P450 cDNAs from the root cultures by RT-PCR with the degenerated primers specific to CYP80A1 and CYP80G2. The isolated P450s showed high amino-acid sequence identities with the CYP80 family members, suggesting their involvement in isoquinoline biosynthesis in S. cepharantha. Further analyses to determine the function of these P450 cDNAs with heterologous expression are now in progress.
植物化学調節学会, 2009年10月06日, 植物化学調節学会研究発表記録集, (44), 56 - 56, 日本語 - 57.根寄生植物ヤセウツボの寄生成立後の成長過程に関する形態学的観察(口頭発表)
Orobanche minor seedlings were attached onto red clover (Trifolium pretense) roots grown in rhizotrons. Approximately 30% of the O. minor seedlings formed tubercles on red clover roots 2 weeks after attachment. Root primordia were appeared on the surface of the tubercles, and crown roots were developed 19 days after attachment. A shoot bud differentiated from crown roots 5 weeks after attachment with or without the cotyledon of O. minor. O. minor parasitism induced little morphological changes in red clover roots during the development process from tubercle formation to shoot differentiation. These observations suggest that O. minor produces growth regulators in its own metabolism to control its differentiation. In addition, the crown roots and the shoot bud differentiated without the cotyledon of O. minor, suggesting the developmental process seems to be the process of adventitious root and shoot formation.
植物化学調節学会, 2009年10月06日, 植物化学調節学会研究発表記録集, (44), 71 - 71, 日本語 - 47.ブラシノステロイド生合成に関与すると推定されるコケおよびシダ植物P450の機能解析(口頭発表)
Several cytochromes P450 (P450s) are involved in the biosynthesis of phtohormones in higher plants. Genome sequencing projects of Physcomitrella patents and Selaginella moellendorffii have revealed that some orthologous genes to phytohormone-related P450s from the flowering plants may be present in bryophyte and pteridophyte. We think that the comparative analysis of plant P450s conserved across the evolutionary stages can give us a clue to study the evolutionary aspects of phytohormone homeostasis. In this study, we performed functional analysis of putative brassinosteroid-biosynthetic P450s from P. patents and S. moellendorffii. We have found that CYP763A1 and CYP763B1/B2 from P. patents and CYP90E1/E2/E3 and CYP90F1 from S. moellendorffii may be the orthologs of brassinosteroid-biosynthetic P450s from the flowering plants. We have isolated the full-length cDNAs of the P450s except for CYP90F1 by RT-PCR. The recombinant P450 proteins were expressed in E. coli and with a baculovirusinsect cell system in order to analyze their catalytic activities in vitro. Now, we are looking for their proper substrates among the BR-biosynthesis intermediates and try to reveal the enzymatic reactions catalyzed by them.
植物化学調節学会, 2009年10月06日, 植物化学調節学会研究発表記録集, (44), 61 - 61, 日本語 - 71.アブシジン酸8'-水酸化酵素CYP707A特異的阻害剤アブシナゾールEの開発(口頭発表)
Uniconazole (UNI), which is well known as a plant growth regulator, is a triazole-containing inhibitor of GA biosynthetic enzyme (CYP701A). UNI is a low selective P450 inhibitor that inhibits multiple P450 enzymes including abscisic acid (ABA) 8'-hydroxylase (CYP707A), which is a key enzyme for ABA catabolism. Based on our speculation that the low selectivity of UNI may be resulted from its small molecule, we designed and synthesized enlarged UNI analogues of UNI (UT) that have a 1,2,3-triazolyl alkyl chain at C4 of chlorophenyl group of UNI. Although UT compounds showed the strong inhibitory activity against CYP707A, the biological activity of most of UT compounds, especially those with a long alkyl chain (>C4), was not tested because of their water-insolubility. On the other hand, the introduction of protic functional groups in the alkyl chain diminished the inhibitory activity against CYP707A. Thus we designed and synthesized UT1-E2Ts that has a diethylene glycol chain as a novel, water-soluble UT analogue, and examined its water solubility, inhibitory activity against CYP707A, and biological activity. UT1-E2Ts showed strong inhibitory activity against CYP707A and good water-solubility, equivalent to UNI, whereas it did not inhibit the growth of rice seedlings, contrary to UNI.
植物化学調節学会, 2009年10月06日, 植物化学調節学会研究発表記録集, (44), 85 - 85, 日本語 - 73.ABA代謝物質ファゼイン酸の還元酵素の精製(口頭発表)
A plant hormone abscisic acid (ABA) protects plants from water stress, so suppression of its metabolic inactivation could increase resistance of plants against water stress. ABA is metabolized to 8'-hydroxy-ABA by ABA 8'-hydroxylase, which spontaneously isomerizes to phaseic acid (PA). PA is converted to dihydrophaseic acid (DPA) and epi-DPA by PA 4'-reductase. 8'-Hydroxy-ABA and PA still have biological activity although it is low, but DPAs are almost inactive. The gene of ABA 8'-hydroxylase was identified in Arabidopsis, and its inhibitors have been developed. However, research on PA 4'-reductase has not progressed. The objective of this research is identification of PA 4'-reductase gene, and development of its inhibitor. We have already found that the extract of immature garden pea (Pisum sativum) showed the PA 4'-reductase activity. After centrifugation of the extract, the supernatant was purified with an anion exchange resin. The activity was eluted with a buffer of pH 8.0 containing 50mM NaCl. The active fraction was purified on a 2'5'ADP-Sepharose 4B. The activity was eluted by a buffer of pH 8.0 containing 1mM NADP^+. The specific activity was increased to 137-fold. The fraction was purified on Sephadex G-75. The SDS-PAGE analysis of the active fraction showed several bands after silver staining. Further purification of PA 4'-reductase is under progress.
植物化学調節学会, 2009年10月06日, 植物化学調節学会研究発表記録集, (44), 87 - 87, 日本語 - 60.根寄生植物の種子発芽刺激活性に対するストライゴラクトンB環の修飾の影響について(口頭発表)
The seeds of root parasitic plants Orobanche spp. and Striga spp. only germinate when they are exposed to stimulant molecules, collectively referred to as strigolactones. In this study, strigolactones and their synthetic analogs with modification at C-4 were evaluated for their potency in inducing seed germination of S. hermonthica, S. gesnerioides, and O. minor. We prepared three synthetic analogs, GR24, OH-GR24 having a hydroxy group at position C-4, and AcO-GR24 having an acetoxy group at C-4, and these compounds were used for the seed germination assay. In the S. hermonthica germination assay, the activity was GR24>OH-GR24 and AcO-GR24. In the O. minor germination assay, the activity was OH-GR24>GR24 and AcO-GR24. These results suggest that the modification at C-4 is an important factor to determine the activity and the specifity of strigolactones for the parasites. In contrast, S. gesnerioides seeds did not respond to any of the synthetic strigolactones while the seeds were responsive to cowpea root exudates that contain orobanchyl acetate as a stimulant. Although it is known that the CD-ring structure of strigolactones is essential for their bioactivity, the results obtained in this study suggested the importance of the A-ring structure for the bioactivity toward S. gesnerioides.
植物化学調節学会, 2009年10月06日, 植物化学調節学会研究発表記録集, (44), 74 - 74, 日本語 - 58.根粒共生がオロバンキの寄生に及ぼす影響(口頭発表)
Effects of rhizobial symbiosis on Orobanche aegyptiaca parasitism to Lotus japonicus were studied. Approximately 30% of the parasite attached onto the host root formed tubercles, and root primordia developed from the tubercles. Concurrent inoculation of Mesorhizobium loti doubled the number of tubercles, suggesting some positive effects of rhizobial symbiosis on the parasite invasion. Inoculation of the rhizobium three weeks before attachment of the parasite did not affect the number of tubercles. However, the tubercles turned brown, and root development of the parasite delayed significantly, suggesting the negative effects of the symbiosis on the parasite growth.
植物化学調節学会, 2009年10月06日, 植物化学調節学会研究発表記録集, (44), 72 - 72, 日本語 - 72.アゾール環修飾による高選択性アゾール系植物P450阻害剤の創製(口頭発表)
Uniconazole (UNI), which is well known as a triazole-containing inhibitor of GA biosynthetic enzyme (CYP701A), functions as inhibitors of multiple P450 enzymes including ABA 8'-hydroxylase (CYP707A), which is a key enzyme for ABA catabolism. Azole-containing P450 inhibitors bind to a P450 active site by both coordinating to the heme-iron atom via sp2 nitrogen and interacting with surrounding protein residues through the lipophilic region. We speculated that poor selectivity of UNI may be resulted from its small and flexible conformation adjustable for a variety of substrate pockets. Based on this speculation, we designed and synthesized novel UNI analogues with a disubstituted azole ring (DSI). These analogues were expected to have higher selectivity than UNI because the added functional group may have an interaction with the active site to increase a rigidity of the molecule in the active site. DSI-505M, which has an imidazolyl group with a methyl 5-acrylate at C2, strongly inhibited recombinant CYP707A3, whereas it did not inhibit the growth of rice seedlings.
植物化学調節学会, 2009年10月06日, 植物化学調節学会研究発表記録集, (44), 86 - 86, 日本語 - 日本農薬学会, 2009年02月27日, 講演要旨集, (34), 126 - 126, 日本語9B25 植物に乾燥耐性を付与するAHI1(アブシジン酸8'-水酸化酵素阻害剤)の構造基盤(生物活性・検定法,一般講演要旨)
- 2009年, 園芸学研究 別冊, 8 (1)アブシシン酸8’水酸化酵素制御とリンゴの乾燥耐性
- 2008年10月30日, 植物化学調節学会 第 43 回大会 ,つくば都市振興財団 つくばカピオ, 日本語オーキシン不活性化を阻害する安定な化学的ツール ーIAA-アミノ酸複合体合成酵素 (GH3) 阻害剤の阻害活性ー
- 54.配座固定とアゾール環修飾による高選択性アゾール系植物P450阻害剤の創製(口頭発表)
Uniconazole (UNI), which is well known as a triazole-containing inhibitor of ent-kaurene oxidase, functions as a very strong inhibitor of ABA 8'-hydroxylase, CYP707A. Azole-containing P450 inhibitors bind to a P450 active site by both coordinating to the heme-iron atom via sp2 nitrogen and interactioning with surrounding protein residues through the lipophilic region. We speculated that poor selectivity of UNI may be resulted from its flexibile conformation adjustable for a variety of substrate pockets. Based on this speculation, we designed and synthesized conformationally restricted UNI analogues, which are expected to have higher selectivity than UNI. The rigid analogues were examined the selectivity in the enzyme and plant growth assays. We also examined synthetic intermediate whose azole ring was modified. UFAP1 and IFAP2, which were restricted differently, showed contrasting results. UFAP1 did not inhibit CYP707A, but inhibited growth of rice. On the other hand, IFAP2 strongly inhibited CYP707A, but did not inhibit growth of rice. This suggests that the conformational restriction may improve the enzyme specificity of UNI. We are examining other conformationally-rigid and azole-modified analogues.
植物化学調節学会, 2008年10月06日, 植物化学調節学会研究発表記録集, (43), 68 - 68, 日本語 - 55.クリック反応を利用した高選択性アゾール系P450阻害剤の創製と探索(口頭発表)
Uniconazole (UNI), which is well known as a plant growth regulator, is a triazole-containing inhibitor of GA biosynthetic enzyme (CYP701A). UNI is also an inhibitor for ABA metabolic enzyme (CYP707A) and BR biosynthetic enzyme (CYP90B). The substrate specificity of P450 depends on the distal helical region which is located far from the heme. Because UNI binds to the heme by coordinating the nitrogen atom to the heme-iron, the 4-chlorophenyl group may interact with this region. Therefore, the alteration of the 4-chlorophenyl group of UNI may cause an increase in the enzyme selectivity. We synthesized UNI analogues modified at the 4-chlorophenyl group by 'click' reaction, and tested their enzyme specificity. UNI analogues with the 4-azidophenyl group (UNI-azide), which was prepared for click reaction, showed equivalent activity to UNI in the recombinant CYP707A inhibition assay, whereas the inhibitory activity for rice seedling elongation was much lower than that of UNI. This suggests that the alteration of the 4-chlorophenyl group of UNI enhances the enzyme selectivity. Eight UNI analogues were synthesized by click reaction between UNI-azide and acetylene compounds in presence of Cu(I). We are examining their inhibitory activity for P450 enzymes and plant growth.
植物化学調節学会, 2008年10月06日, 植物化学調節学会研究発表記録集, (43), 69 - 69, 日本語 - 52.ABAアミノ酸複合体の生物活性(口頭発表)
ABA is a phytohormone critical for plant growth, development and adaption to environmental stress conditions. Although the amino acid conjugates of IAA and JA (IAA-aa and JA-aa) exist in plants, ABA amino acid conjugates (ABA-aa) have not been yet found and chemically synthesized. We synthesized ABA-aa and examined their inhibitory activities. We synthesized 19 species of ABA-aa in a yield of 10-74%, and examined their inhibitory activity for rice seedling elongation and lettuce seed germination, ABA-Asp and ABA-Glu showed strong activity in lettuce seed germination assay, and ABA-Ala, -Met, -Ser, -Asn and -Gly showed strong activity in rice seedling elongation assay. Because the biologically active C1-modified ABA has not been found, the activity of ABA-aa may depend on free ABA released by hydrolysis. If ABA-aa is an endogenous metabolite, there may be a hydrolase specific to ABA-aa. If not so, ABA-aa may be hydrolysed by an enzyme not specific for ABA-aa, for example, IAA-aa hydrolase. We are examining that whether ABA-aa is a substrate of IAA-aa hydrolase or not.
植物化学調節学会, 2008年10月06日, 植物化学調節学会研究発表記録集, (43), 66 - 66, 日本語 - 53.ABA代謝物質ファゼイン酸の還元酵素の性質(口頭発表)
Abscisic acid (ABA) is inactivated through metabolism. First, ABA is hydroxylated by 8'-hydroxylase to give 8'-hydroxy-ABA. 8'-Hydroxy-ABA is spontaneously isomerized to phaseic acid (PA). PA is converted to dihydrophaseic acid (DPA) and epi-DPA by PA 4'-reductase. 8'-Hydroxy-ABA and PA still have biological activity although the activity is low, but DPAs are almost inactive. The gene of ABA 8'-hydroxylase was identified in Arabidopsis and other plants, and its inhibitors were developed to protect plants from water stress. However, the research on PA 4'-reductase has not progressed. We have started the research on the enzyme for regulating inactivation of ABA. Several plants were screened for detection of the PA 4'-reductase activity. The supernatants of the extracts after centrifugation were used to detect the PA 4'-reductase activity. The enzyme activity was found in the extract of immature garden pea (Pisum sativum). The reaction product was DPA, and epi-DPA was not detected. The optimum pH is 8.0, and NADPH is a better coenzyme than NADH. The extract kept 80% of activity after stored 9 days at -80℃. Purification of the enzyme is under progress.
植物化学調節学会, 2008年10月06日, 植物化学調節学会研究発表記録集, (43), 67 - 67, 日本語 - 40.サツマイモにおけるクマリン化合物の生合成と桂皮酸類オルト位水酸化酵素遺伝子のクローニング(口頭発表)
Coumarins are often found as the plant secondary metabolites in plants. They are thought to play important roles in plant defense due to their antimicrobial and antioxidative activity. We have identified the feruloyl-CoA ortho-(6'-)hydroxylase (AtF6'H1) in Arabidopsis, which catalyzes the key step of scopoletin biosynthesis. AtF6'H1 is classified into 2-oxoglutarate-dependent dioxygenase (2OGD) and exhibits the high substrate specificity for feruloyl-CoA. Arabidopsis mainly accumulates scopoletin. These results indicate that the substrate specificity of AtF6'H1 determines the accumulation patterns of the coumarins in Arabidopsis. In sweet potato (Ipomoea batatas L.) roots, both umbelliferone and scopoletin are accumulated in response to various stresses such as wounding and microbial infection. In order to identify the ortho-hydroxylase involved in formation of umbelliferone and scopoletin in sweet potato, we performed cloning and functional analysis of the AtF6'H1 homologs of sweet potato (Kokei No.14). The total sequences of the homologs were obtained by RT-PCR and RACE methods using cDNA from sweet potato roots as a template. The primers for cloning were designed according to the EST sequences of sweet potato, which are homologous to AtF6'H1.
植物化学調節学会, 2008年10月06日, 植物化学調節学会研究発表記録集, (43), 54 - 54, 日本語 - 39.イネ由来ブラシノステロイド不活性化酵素の酵素化学的解析(口頭発表)
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.
植物化学調節学会, 2008年10月06日, 植物化学調節学会研究発表記録集, (43), 53 - 53, 日本語 - Japan Society for Bioscience, Biotechnology, and Agrochemistry, 2008年08月, KAGAKU TO SEIBUTSU, 46 (8), 518 - 520, 日本語
- 2008年03月28日, 日本農芸化学会 2008 年度（平成 20 年度）大会 ,名古屋（名城大学）, 日本語植物由来アシル活性化酵素のケミカルバイオロジー（その２）ーオーキシン-アミノ酸合成酵素 (GH3) の新たな阻害剤の設計と阻害活性ーー
- 2008年03月28日, 日本農芸化学会 2008 年度（平成 20 年度）大会 ,名古屋（名城大学）, 日本語植物由来アシル活性化酵素のケミカルバイオロジー（その１）ーアッセイ方法の確立と酵素学的解析ー
- 2008年03月20日, 第 49 回 日本植物生理学会年会 ,札幌コンベンションセンター, 日本語
- 2008年, 日本生薬学会年会講演要旨集, 55th甘草グリチルリチン生合成遺伝子の同定(2)酵母発現系を用いた遺伝子機能同定
- 2008年, 日本生薬学会年会講演要旨集, 55th甘草グリチルリチン生合成遺伝子の同定(1)ESTライブラリーの構築と遺伝子マイニング
- 2008年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 26th薬用植物カンゾウのグリチルリチン生合成に関わるP450遺伝子のクローニング(2)
- 2008年, 園芸学研究 別冊, 7 (1)果樹におけるアブシシン酸8’水酸化酵素阻害による乾燥耐性付与
- 2008年, 第26回植物細胞分子生物学会大阪大会・シンポジウム講演要旨集，p. 154，2008．ピノレジノールレダクターゼによるリグナンのエナンチオマー組成の制御
- 2008年, 第58回日本木材学会大会講演要旨集，p. 70，2008．Arabidopsis thalianaのリグナン生合成酵素遺伝子の機能解析
- 2008年, Proceedings of Phytochemical Society of North America 2008 Annual Meeting, p. 47Arabidopsis thaliana pinoresinol reductases control enantiomeric compositions of lariciresinol
[査読有り]
- オーキシン不活性化を阻害する化学的ツール ?IAA-アミノ酸複合体合成酵素（GH3）阻害剤の設計とin vivo活性?
The formation and degradation of IAA-amino acid conjugates (IAA-aa) play an important role in auxin homeostasis. GH3s encode IAA-aa synthetases, but their functional redundancy has hampered the genetic approach to study auxin homeostasis. GH3s belong to the acyl-activating enzyme superfamily that activates IAA by adenylation. According to this catalytic mechanism, we designed and synthesized N-sulfamoyladenosine (SA) derivatives of auxins (IAA-SA, PAA-SA, NAA-SA and 2,4-D-SA) as intermediate analog inhibitors of GH3s. These compounds not only served as potent in vitro inhibitors of GH3s with IC_<50> values of 3 to 45 μM, but also exhibited in vivo activities. In the excised Arabidopsis leaves treated with exogenous IAA, the endogenous levels of IAA-Asp, IAA-Glu and IAA-Gln greatly increased. When the SA derivatives were applied together with IAA, the increase was significantly repressed. Thus, the SA-derivatives of auxins were adsorbed in plant tissues and inhibited GH3s involved in lAA-aa synthesis, thereby serving as highly effective chemical tools to probe otherwise inaccessible auxin homeostasis. The cell permeability was suggested to be an important factor that affected the in vivo activities of SA derivatives. We therefore designed and synthesized a series of N-acylsulfamide derivatives that are chemically stable and more hydrophobic than SA derivatives. The results on N-acylsulfamide derivatives will also be presented.
植物化学調節学会, 2007年10月29日, 植物化学調節学会 第 42 回大会 ,静岡県男女共同参画センター「あざれあ」, 42, 26 - 26, 日本語 - アブシジン酸8'-水酸化酵素リガンドの基本構造
ABA 8'-hydroxylase (recombinant CYP707A3) active site specifically binds (S)-(+)-ABA, naturally occurring ABA, but not its enantiomer (R)-(-)-ABA. On the other hand, enone-lacking (-)-ABA analogs (e.g. (-)-4'-deoxo-ABA and (-)-AHI1) bind to the enzyme, indicating that the ketone in the ring of (-)-ABA prevents binding to the enzyme due to decrease of the lipophilicity or unfavorable electrostatic interaction with the active site. This hypothesis has led us to speculate that an ABA analog with the same side chain as that of ABA can bind to the CYP707A3 active site independent of its C1'-configuration if its six-membered ring has no oxygenated functional group except for the C1'-hydroxyl group. Therefore, we tested the epimer of AHI1 (epi-ARI1), AHI1 analogs which are lacking the methyl group(s) in the ring (C8' and C9'), β-ionylideneacetic acid and phenylpentadienoic acid. These compounds except for phenylpentadienoic acid inhibited the enzyme reaction, thus supporting our speculation. Notably, 6-hydroxy-6-isopropyl-7-methyloctadienoic acid, a ring-opened compound, also exhibited enzyme inhibitory activity, whereas 7,7,7-trifluoro-6-hydroxy-6-methylheptadienoic acid cannot bind to the active site. These results suggest that 2Z,4E-pentadienoic acid can bind to ABA 8'-hydroxylase if it has a branched C7 hydrocarbon, which is accommodated by the hydrophobic binding pocket, at the C-5 position.
植物化学調節学会, 2007年10月05日, 植物の生長調節 = Regulation of plant growth & development, 42, 57 - 57, 日本語 - 2007年10月, 植物化学調節学会第42回大会，静岡, 日本語Uniconazoleの立体配座とABA 8'-hydroxylase阻害活性の関係
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2007年03月28日, 第48回日本植物生理学会年会 ,愛媛大学, 日本語
- 2007年03月28日, 第48回日本植物生理学会年会 ,愛媛大学, 日本語オーキシン不活性化を制御する化学的ツール ーIAA-アミノ酸複合体合成酵素（GH3）阻害剤のin vivo阻害活性の検討ー
- 2007年03月25日, 日本農芸化学会 2007 年度大会 ,東京農業大学, 日本語オーキシン恒常性を制御する化学ツール ーIAA-amino acid synthetase (GH3) 阻害剤と in vivo 活性ー
ジグリコシダーゼは二糖配糖体を二糖とアグリコンに加水分解する酵素である。ジグリコシダーゼはグリコシダーゼファミリー 1 に属しており、植物のモノグルコシダーゼである β-グルコシダーゼもこのファミリー1 に分類される。植物の β-グルコシダーゼは単糖配糖体を加水分解し、生体防御など様々な生理活性を持つアグリコンを放出する。グルコースの六位水酸基にさらに単糖が結合した二糖配糖体も多くの植物から報告されており、ジグリコシダーゼも植物界に広く分布していると考えられる。ジグリコシダーゼは β-グルコシダーゼと 50 % 以上の高い相同性を示すことから、β-グルコシダーゼから進化したと考えられる。その進化過程において、酵素のどのような構造変化が二糖特異性の獲得につながったのかを X 線結晶構造解析によって明らかにした。ジグリコシダーゼの一種である β-プリメベロシダーゼ (チャ樹由来) に基質アナログ阻害剤が結合した結晶を調整し 1.8 Å の分解能で構造解析した。その結果、活性残基とグルコース認識残基の構造は β-グルコシダーゼのものと完全に一致した。一方、 β-プリメベロシダーゼには二糖に水素結合するための特別な二つのアミノ酸 (Ser473、Gln477) が存在した。ジグリコシダーゼは基質結合部位の僅かな構造の変化によって二糖配糖体に対する加水分解活性を獲得したと考えられる。
日本植物生理学会, 2007年03月15日, 日本植物生理学会年会要旨集, 48th (0), 298 - 814, 日本語- 2007年03月05日, 日本農芸化学会大会講演要旨集, 2007, 205, 日本語チャ樹由来β‐プリメペロシダーゼの二糖認識機構の解明
- 2007年, 日本植物細胞分子生物学会大会・シンポジウム講演要旨集, 25th薬用植物カンゾウのグリチルリチン生合成に関わるP450遺伝子のクローニング
- OXFORD UNIV PRESS, 2007年, PLANT AND CELL PHYSIOLOGY, 48, S220 - S220, 英語Structural basis for evolution of diglycosidases from monoglucosidases in plants
研究発表ペーパー・要旨（国際会議）
- OXFORD UNIV PRESS, 2007年, PLANT AND CELL PHYSIOLOGY, 48, S64 - S64, 英語Identification of ortho-hydroxylase of cinnamates involved in coumarin biosynthesis in Arabidopsis
研究発表ペーパー・要旨（国際会議）
- 共立出版, 2007年, 蛋白質・核酸・酵素, 52 (12), 1454 - 1464, 日本語シトクロムP450の分子進化と植物の化学的多様性の獲得
記事・総説・解説・論説等（学術雑誌）
Cytochrome P450 monoxygenases (P450s) are heme-containing membrane proteins and catalyze the monooxygenation reactions in a wide range of metabolic pathways of various bioactive substances. In this study, several plant P450s belonging to 85-clan and 710-clan were investigated by biochemical approaches. (1) CYP707As have been identified as a major ABA catabolic enzyme, ABA 8'-hydroxylase. A triazole-type growth retardant, uniconazole-P, was found to be a potent inhibitor of ABA 8'-hydroxylase, and the inhibitor treated plants showed enhanced drought tolerance because of ABA accumulation. (2) Enzymatic functions of CYP90A1, 90B1, 90C1, 90D1, and 724B1 involved in BR biosynthesis were clarified by using an in vitro assay reconstituted with the recombinant P450s expressed either in a baculovirus-insect cell system or in a bacterial-expression system, and these studies revealed that BRs are biosynthesized via the campestanol-independent pathway. (3) CYP710As were found to encode plant sterol C-22 desaturases. Arabidopsis CYP710A1/A3/A4 catalyze C-22 desaturation of β-sitosterol to produce stigmasterol, while CYP710A2 specifically C-22 desaturate 24-epi-campesterol to produce brassicasterol, which is a unique sterol in Brassica species.
一般社団法人植物化学調節学会, 2007年, 植物の生長調節, 42 (1), 260 - 268, 日本語記事・総説・解説・論説等（学術雑誌）
- 植物化学調節学会, 2006年10月04日, 植物の生長調節 = Regulation of plant growth & development, 41, 15 - 15, 日本語アブシジン酸および植物ステロイドの代謝系に関わるシトクロムP450の酵素化学的研究
- 植物化学調節学会, 2006年10月04日, 植物の生長調節 = Regulation of plant growth & development, 41, 30 - 30, 日本語7'-Hydroxy-ABA は ABA 8'-hydroxylase を阻害しない
- 植物化学調節学会, 2006年10月04日, 植物の生長調節 = Regulation of plant growth & development, 41, 29 - 29, 日本語強力な ABA 8'-hyrdoxylase 阻害剤 uniconazole の構造活性研究
- 植物化学調節学会, 2006年10月04日, 植物の生長調節 = Regulation of plant growth & development, 41, 39 - 39, 日本語ブラシノステロイドはカンペスタノールを経由しない経路で生合成される
- 2006年10月01日, 日本農芸化学会 2006 年度（平成 18 年度）関西支部大会 ,京都工芸繊維大学, 日本語カラスノエンドウ由来 vicianin hydrolase の疑似二糖配糖体加水分解活性
- 2006年08月08日, 11th IUPAC International Congress of Pesticide Chemistry ,Kobe, Japan, 英語Inhibitors of IAA-Amino Acid Conjugate Synthetases and Hydrolases as Chemical Probes to Study IAA Homeostasis
- 2006年08月, 日本木材学会Arabidopsis thaliana CAldOMTの機能について
- 2006年07月, 8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology ,Swansea, Wales, 英語CYP724Bs and CYP90Bs are Redundant C-22 Hydroxylases in the Early C-22 Hydroxylation Routes of Brassinosteroid Biosynthesis
研究発表ペーパー・要旨（国際会議）
- 2006年07月, 8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology, Swansea, Wales, 英語Functional Characterization of Arabidopsis CYP90C1 and CYP90D1 Encode Brassinosteroids C-23 Hydroxylazes
研究発表ペーパー・要旨（国際会議）
- 2006年07月, ICOB-5 & ISCNP-25 IUPAC International Conference on Biodiversity and Natural ProductsFUNCTIONS OF ARABIDOPSIS CAldOMT
- 2006年03月20日, 第 47 回日本植物生理学会年会 ,筑波大学, 日本語IAA ホメオスタシスの解明を目指した化学的ツールの開発
- 2006年03月05日, 日本農芸化学会大会講演要旨集, 2006, 306, 日本語植物由来ジグリコシダーゼのX線結晶構造解析
- バイオインダストリー協会, 2006年03月01日, バイオサイエンスとインダストリー = Bioscience & industry, 64 (3), 145 - 150, 日本語チャ葉のジグリコシダーゼによる香気生成と食品への応用
ブラシノステロイド (BR) 生合成経路の多くの酸化反応はシトクロムP450 (P450) 酵素により触媒されることが示されている。これまでに様々な植物種から多くのBR欠損矮性変異体が単離され、その原因遺伝子として多数のP450分子種（CYP85A, 90A, 90B, 90C, 90D, 724B）が同定されている。それら変異体の内生BR量の分析およびBR中間体処理による回復実験の結果から各P450遺伝子の酵素機能が推定されているが、生化学的証明はC-6位酸化反応 (CYP85A) とC-22位水酸化反応 (CYP90B1) のみである。今回、我々はBR生合成酵素と推定されているシロイヌナズナ由来CYP90C1およびCYP90D1に注目し、その機能解析を行ったので報告する。各P450を昆虫細胞-バキュロウィルス発現系により発現させ、様々なBR中間体を基質として酵素アッセイを行い、酵素反応生成物をGC-MSによって分析した。CYP90C1および CYP90D1は6-deoxocathasteroneから6-deoxoteasteroneを生成し、両P450は共にC-23位水酸化反応を触媒した。一方、これまでC23位水酸化はCYP90A1が触媒すると推定されてきたが、CYP90A1はC23位水酸化活性を示さなかった。発表では、C-23位水酸化酵素の基質特異性などの酵素化学的詳細とcyp90c1cyp90d1二重変異株の内生BR量の分析結果およびBR中間体処理による回復実験の結果についても報告する。本研究によりCYP90C1とCYP90D1が共にC-23位水酸化酵素であることを明らかにした。
日本植物生理学会, 2006年03月, 第47回日本植物生理学会，つくば, 2006 (0), 485 - 485, 日本語研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2006年03月, 第47回日本植物生理学会，つくば, 日本語植物ステロール C-22 位不飽和化酵素 (CYP710A) の同定と酵素学的解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2006年03月, 第47回日本植物生理学会，つくば, 日本語
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2006年03月, 日本農芸化学会2006年度大会，京都, 日本語代謝プロファイリングによる植物ステロール側鎖不飽和化酵素の同定と機能解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- OXFORD UNIV PRESS, 2006年, PLANT AND CELL PHYSIOLOGY, 47, S199 - S199, 英語Methyltransferase related to biosynthesis of coumarins
研究発表ペーパー・要旨（国際会議）
- OXFORD UNIV PRESS, 2006年, PLANT AND CELL PHYSIOLOGY, 47, S116 - S116, 英語Chemical tools for probing into IAA homeostasis-synthesis of IAA-amino acid synthetase inhibitors
研究発表ペーパー・要旨（国際会議）
- OXFORD UNIV PRESS, 2006年, PLANT AND CELL PHYSIOLOGY, 47, S198 - S198, 英語Glucosylation of scopoletin in Arabidopsis roots
研究発表ペーパー・要旨（国際会議）
- 2005年12月20日, 根の研究 = Root research, 14 (4), 183 - 183, 日本語シロイヌナズナの根を用いたクマリン類縁体生合成経路の解析
- 56. ABA 8'-水酸化酵素のnon-azole阻害剤
Although some azole compounds inhibit ABA 8'-hydroxylase, their enzyme specificity is difficult to be estimated. An inhibitor having similar structure to ABA should be a specific inhibitor of ABA 8'-hydroxylase. Therefore, we designed and synthesized (1'S^*,2'S^*)-(±)-6-nor-2',3'-dihydro-4'-deoxo-ABA as a lead compound for the development specific inhibitor of the enzyme. The lead and ABA compound has similar affinity for the enzyme, although the lead has no ABA bioactivity. In the present study, we have tried to develop the stronger inhibitor than the lead by modification of C-8'. The new non-azole inhibitors, (1'S^*,2'S^*,6'S^*)-(±)-6-nor-2',3'-dihydro-4'-deoxo-8',8'-difluoro-ABA [(±)-1], (1'S^*,2'S^*,6'S^*)-(±)-6-nor-2',3'-dihydro-4'-deoxo-6'-hydroxy-8',9'-dinor-ABA [(±)-2] and (1'S^*,2'S^*)-(±)-6-nor-2',3'-dihydro-4'-deoxo-8',9'-cyolo-ABA [(±)-3] exhibited similar inhibition activity to the lead. Moreover, we found that (-)-1, witch is the unnatural type, is the stronger inhibitor than the natural type, (+)-1.
植物化学調節学会, 2005年10月13日, 植物化学調節学会研究発表記録集, (40), 75 - 75, 日本語 - 27. シロイヌナズナを用いたクマリン類縁体生合成経路の解析
Coumarins are often found as plant secondary metabolites in the plant kingdom. Their exact roles in plants are unclear. However, they are thought to play some roles in plant defense due to the induction of their biosynthesis following various stresses as well as their antimicrobial activities. We detected the coumarins, scopoletin and its glucoside, scopolin, in Arabidopsis thaliana roots. Details of coumarin biosynthesis pathway in plants are also unclear, although the coumarins are known to be biosynthesized via phenylpropanoid pathway. We quantified the coumarins in the T-DNA insertion mutants of p-coumaroylshikimate/quinate 3'-hydroxylase (C3'H) and caffeoyl CoA O-methyltransferase (CCoAOMT) of A. thaliana to collect information on the coumarin biosynthesis. Analysis of the coumarins in the T-DNA insertion mutants was performed by HPLC and LC-ESI/MS/MS. Mutation of C3'H and CCoAOMT1 resulted in change of the profiles of the coumarins. The level of skimmin, a glucoside of umbelliferone, rose to be detected in the c3'h roots. The levels of scopoletin and scopolin in the c3'h roots and in the ccoaomt1 roots were 〜5% and 〜15% of those in the wild type, respectively. These results suggest that scopoletin and scopolin are biosynthesized via C3'H and CCoAOMT in A. thaliana.
植物化学調節学会, 2005年10月13日, 植物化学調節学会研究発表記録集, (40), 46 - 46, 日本語 - 55. ABA 8'-水酸化酵素におけるABA不斉認識に必要な官能基
The plant hormone (1'S)-(+)-ABA is inactivated through 8'-hydroxylation by ABA 8'-hydroxylase (Arabidopsis CYP707A3), whereas (1'R)-(-)-ABA does not bind to the enzyme. (1'S,2'S)-(+)-6-Nor-2',3'-dihydro-4'-deoxo-ABA [(+)-1], which we developed as a lead compound for inhibitors of ABA 8'-hydroxylase, functioned as an enzyme substrate. The lead (-)-1 was not a substrate of the enzyme, however, the inhibitory activity of (-)-1 on 8'-hydroxylation of ABA was higher than that of (+)-1. The result suggests that the enone of cyclohexenone ring is the key to chiral recognition by ABA 8'-hydroxylase. Thus, we planned to synthesize 2',3'-dihydro-ABA and 4'-deoxo-ABA and test these compounds for the inhibition in the ABA 8'-hydroxylase reaction to investigate the phenomena in detail. (1'S,2'S)-(+)-2',3'-dihydro-ABA slightly inhibited the CYP707A3 reaction, whereas (1'R,2'R)-(-)-2',3'-dihydro-ABA did not bind to the enzyme as well as (-)-ABA. The observation indicates the importance of the carbonyl group at C-4' for chiral recognition by ABA 8'-hydroxylase. We are preparing 4'-deoxo-ABA and measuring its inhibitory activity on ABA 8'-hydroxylase reaction at present.
植物化学調節学会, 2005年10月13日, 植物化学調節学会研究発表記録集, (40), 74 - 74, 日本語 - 2005年10月, 植物調節化学会第40回大会，東京, 日本語ブラシノステロイドC-23位水酸化酵素の酵素学的解析
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2005年09月, The Second Yamada Symposium on Key Natural Molecules in Biological Systems, Hyogo, Japan, 英語Biosynthesis and Catabolism of Brassinosteroids by Cytochrome P450 in Plants.
研究発表ペーパー・要旨（国際会議）
- ABA 8'-水酸化酵素の基質特異性
The substrate specificity of ABA 8'-hydroxylase been examined using ABA analogs. More than 35 ABA analogs were reacted with CYP707A3 microsomes as a substrate and were tested for their ability to act as an inhibitor. The results are summarized in Table. The present findings are useful for developing the specific inhibitors of ABA 8'-hydroxylase.
植物化学調節学会, 2005年05月28日, 植物の生長調節 = Regulation of plant growth & development, 39, 43 - 43, 日本語 - アブシジン酸代謝阻害剤の探索
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.
植物化学調節学会, 2005年05月28日, 植物の生長調節 = Regulation of plant growth & development, 39, 52 - 52, 日本語 - 2005年03月30日, 日本農芸化学会 2005 年度（平成 17 年度）大会 ,札幌コンベンションセンター, 日本語植物および微生物由来の二糖配糖体加水分解酵素における基質認識の比較
- 2005年03月26日, 第 46 回 日本植物生理学会年会 ,新潟コンベンションセンター朱鷺メッセ, 日本語
- 2005年03月, 日本農芸化学会2005年度大会，札幌, 日本語アブシジン酸8'-水酸化酵素(CYP707A)阻害剤の探索，合成および活性
研究発表ペーパー・要旨（全国大会，その他学術会議）
- OXFORD UNIV PRESS, 2005年, PLANT AND CELL PHYSIOLOGY, 46, S50 - S50, 英語Structural requirements for substrate recognition of ABA 8 '-hydroxylase
研究発表ペーパー・要旨（国際会議）
- OXFORD UNIV PRESS, 2005年, PLANT AND CELL PHYSIOLOGY, 46, S215 - S215, 英語Uniconazole-P is a potent inhibitor for ABA catabolism
研究発表ペーパー・要旨（国際会議）
- OXFORD UNIV PRESS, 2005年, PLANT AND CELL PHYSIOLOGY, 46, S240 - S240, 英語Analysis of coumarin biosynthesis pathway in Arabidopsis thaliana
研究発表ペーパー・要旨（国際会議）
- OXFORD UNIV PRESS, 2005年, PLANT AND CELL PHYSIOLOGY, 46, S207 - S207, 英語Chemical knockout for probing into IAA homeostasis - Synthesis of the inhibitors of IAA-amino acid amidohydrolases
研究発表ペーパー・要旨（国際会議）
- OXFORD UNIV PRESS, 2005年, PLANT AND CELL PHYSIOLOGY, 46, S208 - S208, 英語Chemical knockout for probing into IAA homeostasis - Synthesis of IAA-amino acid synthetase inhibitor
研究発表ペーパー・要旨（国際会議）
- 日本農芸化学会, 2005年, 化学と生物, 43 (10), 628 - 630, 日本語シトクロムP450阻害剤による植物の乾燥ストレス耐性の向上—ジベレリン生合成阻害剤はアブシジン酸代謝阻害剤である？！—
記事・総説・解説・論説等（学術雑誌）
- 一般社団法人植物化学調節学会, 2005年, 植物の生長調節, 40 (1), 67 - 82, 日本語
記事・総説・解説・論説等（学術雑誌）
- 2004年10月, 植物化学調節学会第39回大会，秋田, 日本語シロイヌナズナCYP90B1はC27, C28, C29ブラシノステロイドのC-22位を水酸化する
研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2004年09月, 18th International Conference on Plant Growth Substances, Canberra, Australia, 英語Arabidopsis CYP90B1 Catalyzes the C-22 Hydroxylation of C27-, C28-, and C29-Brassinosteroids
研究発表ペーパー・要旨（国際会議）
- 日本農芸化学会, 2004年, 化学と生物, 42(12) (12), 774 - 776, 日本語植物の二糖配糖体特異的グリコシダーゼ：基質との共進化は生体防御の策だった？
記事・総説・解説・論説等（学術雑誌）
- 26. ABA 8'-位水酸化を触媒するシトクロム P450 の同定と機能解析
Catabolic inactivation of (+)-abscisic acid (ABA) proceeds via hydroxylation at the 8' position to form the unstable intermediate, 8'-hydroxy ABA (8'-OH ABA), which subsequently cyclizes spontaneously to form (-)-phaseic acid (PA). ABA 8'-hydroxylase has been known as P450,but not isolated. Arabidopsis genome contains 246 cytochrome P450 (CYP or P450) genes, which are classified in two main clades : A-type and non-A-type. Several P450 genes involved in brassinosteroids and gibberellins biosynthesis are clustered in the 85 clan of non-A-type. CYP707A family is also in the 85 clan, but its function has not been identified yet. In this study, we have characterized biochemical properties of CYP707A. RT-PCR analysis of expression profiles in response to the phytohormone treatment revealed that expressions of CYP707A genes significantly increased in response to ABA. In addition, expressions of CYP707A genes also induced by various stress conditions such as high salinity, osmotic and drought stresses. These results suggest the involvement of CYP707A in ABA catabolism. To confirm the hypothesis, CYP707As were expressed in insect cells. (+)-ABA was added to the suspension-cultured cells. An EtOAc extract was analyzed by HPLC. PA and 8'-OH ABA were detected. For biochemical characterization, the microsomal fractions were prepared from the insect cells expressing CYP707A3. The recombinant CYP707A3 catalyzed the hydroxylation of ABA to form PA in the presence of NADPH, but not NADH. The recombinant CYP707A3 was solubilized and partially purified by column chromatography. Type I spectra were detected by adding (+)-ABA to the purified CYP707A3 in a dose-dependent manner. Thus, these results demonstrate that CYP707A genes encode ABA 8'-hydoxylase.
植物化学調節学会, 2003年10月10日, 植物化学調節学会研究発表記録集, (38), 43 - 43, 日本語 - FFIジャーナル編集委員会, 2003年, 食品・食品添加物研究誌, 208 (12), 991 - 1003, 日本語茶の香気生成とβ-プリメベロシダーゼ
- ACADEMIC PRESS INC, 2003年, RECOGNITION OF CARBOHYDRATES IN BIOLOGICAL SYSTEMS, PART B: SPECIFIC APPLICATIONS, 363, 444 - 459, 英語
[査読有り]
書評論文,書評,文献紹介等
- 94 殺菌剤トリアジメフォンはブラシノステロイドとジベレリンの生合成を同時に阻害する
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.
植物化学調節学会, 2002年10月31日, 植物化学調節学会研究発表記録集, (37), 197 - 198, 日本語 - Institute for Chemical Research, Kyoto University, 2002年03月, ICR annual report, 8, 44 - 45, 英語Design, Synthesis and Applications of Selective β-Glycosidase Inhibitors, β-Glycosylamidines (MOLECULAR BIOFUNCTION - Chemistry of Molecular Biocatalysts)
- 14 トマト子房で特異的に発現しているチトクローム P450 の機能解析
Several cytochromes P450 are involved in gibberellin (GA) biosynthesis. We have isolated. two cDNAs from tomato, to88B1 and to707A5, specifically expressed in ovary. The cDNAs showed high homology to CYP88A (ent-kaurenoic acid oxidase in GA biosynthesis) in phylogenetic analysis, suggesting that both P450s may be in GA biosynthesiS. Because of only 40% identity to CYP88A, to88B1 may have a different function from CYP88A. Furthermore, the Thr residue conserved in most P450s, which plays an important role to stabilize the ferrous P450-O_2 intermediate, is substituted for Pro in to88B 1. GA biosynthetic pathway has a side branch that lead ent-kaurenoic acid to kaurenolides by C6-desaturation, and to88B1 may be involved in the pathway, together with to707A5. To confirm this, the P450s were expressed in insect cells and the measurement of their activities is in progress.
植物化学調節学会, 2001年10月09日, 植物化学調節学会研究発表記録集, (36), 39 - 40, 日本語 - 植物化学調節学会, 2001年10月09日, 植物化学調節学会研究発表記録集, (36), 19 - 20, 日本語4 ブラシノステロイド生合成阻害剤の作用部位
- 3 ブラシノステロイドの代謝に関わるトマト由来シトクロームP450の機能解析
Brassinosteroid (BR) am now recognized as a major hormone controlling plant development and growth. Recently several cytochrome P450 genes have been isolated from the mutants deficient in BR (Arabidopsis cpd, dwf4, bas1 and tomato dwarf). The physiological function of these P450s has been proposed from analysis of the corresponding routants in chemical levels. But enzymatic characters of these P450s are not yet clear. Recently, Arabidopsis BAS1 (CYP72B1) has been isolated and suggested to catalyze the C26-hydroxylation of brassinolide. To investigate regulation of BR metabolism in tomato, we have obtained P450 cDNA belonging to CYP72 family from tomato. The tomato cDNA tomato CYP72B, showed 60% identity to BAS 1. in this study, we produced recombinant proteins, which was over-expression of tomato CYP72B in the hetelogous expression system of baculovirus-insect cell. The tomato CYP72B over-expressed protein has typical character of P450 enzyme. Secondary, we cultivated a recombinant tobacco transformed by tomato CYP72B. The transgenic tobacco had typical brassinosteroids deftdent phenotype.
植物化学調節学会, 2001年10月09日, 植物化学調節学会研究発表記録集, (36), 17 - 18, 日本語 - 2001年10月03日, 第４３回天然有機化合物討論会 ,大阪, 日本語茶の香気生成酵素 β-プリメベロシダーゼの基質の合成および酵素学的特性の解明
- 2001年03月25日, 日本農芸化学会 2001 年度大会 ,京都, 日本語ジベレリン生合成に関わるチトクロム P450 cDNA の単離と機能解析
- 日本農薬学会, 2001年02月22日, 講演要旨集, 26, 107 - 107, 日本語ブラシノステロイド生合成阻害剤の作用部位と利用の可能性
- 植物化学調節学会, 2000年12月25日, 植物の化学調節, 35 (2), 229 - 229, 日本語28. トマト由来のプラシノステロイド生合成・代謝に関わるチトクロムP450のクローニングと機能解析(第35回大会研究発表抄録)
- 植物化学調節学会, 2000年12月25日, 植物の化学調節, 35 (2), 236 - 237, 日本語51. オーキシン受容体のクローニングに向けた酵母three-hybrid法の改良(第35回大会研究発表抄録)
- オーキシン受容体のクローニングに向けた酵母three-hybrid法の改良
Our objective is cloning of an auxin receptor cDNA with a yeast three-hybrid system which can detect small ligand-protein interaction in yeast cells and enables us to isolate a cDNA encoding a ligand-binding protein directly. In the original system, the interaction between a rat glucocorticoid receptor (GR) and dexamethasone (Dex) has been used as a hook. To improve the sensitivity of the system, we applied the combination of His-tag and Ni-NTA which interacts stronger than that of GR and Dex. In the hook plasmid 6 × His-tag was fused at the C-terminus of GAL4 DNA binding domain, and on the other hand, Dex binding domain for rat GR was fused to the transcriptional activation domain of GAL4 in a fish construct. As a bait ligand, the NTA was covalently crosslinked to Dex, and the carboxyl group of NTA was also protected as acetoxymethylester, which confers higher membrane permeability and is easily hydrolyzed in yeast cells. Yeast cells (strain Ah109) were transfomed with the two plasmids and were plated on medium containing Ni^<2+> and the synthesized bait ligands.
植物化学調節学会, 2000年11月02日, 植物化学調節学会研究発表記録集, (35), 109 - 110, 日本語 - 28 トマト由来のブラシノステロイド生合成・代謝に関わるチトクロムP450のクローニングと機能解析
Cytochromes P450 are membrane-bound heme proteins catalyzing the monooxygenation of lipophilic substances. Recently several P450 genes have been isolated in the course of cloning of the genes from the mutants deficient in brassinosteroid (BR) (Arabidopsis cpd, dwf4, bas1, and tomato dwarf) and gibberellin (GA) (Arabidopsis ga3, and maize dwafr3). Interestingly, most of the P450s so far isolated from these mutants belong to 85 clan in phylogenetic tree of plant P450s. Recently Arabidopsis BAS1 (CYP72B1) has been isolated and suggested to catalyze the C26-hydroxylation of brassinolide. In this study, to investigate regulation of BR biosynthesis and metabolism in tomato, we have obtained several P450 cDNAs belonging to 85 clan and CYP72 family from tomato. The tomato cDNA, to90AH, showed 74% identity to CPD, suggesting that to90AH catalyzes the C23-hydroxylation. The other tomato clone, to72BH showed 60% identity to BAS1 and 35〜45% to CHIBI2 and CYP72A. Thus to 72BH may catalyze the C25 or C26 hydroxylation of BR to inacrivate BR. molecules. Functional analysis of these P450s by expression in heterologous cells and in Arabidopsis is in progress.
植物化学調節学会, 2000年11月02日, 植物化学調節学会研究発表記録集, (35), 63 - 64, 日本語 - Japanese Society of Plant Physiologists, 2000年, Plant and cell physiology, 41, s167, 英語INTRACELLULAR LOCALIZATION STUDY OF TWO NADPH-CYT P450 REDUCTASE PROTEINS IN ARABIDOPSIS :
- Japanese Society of Plant Physiologists, 2000年, Plant and cell physiology, 41, s201, 英語CHARACTERIZATION OF THE DWF4 PROTEIN :
- 植物化学調節学会, 1999年12月25日, 植物の化学調節, 34 (2), 349 - 349, 日本語53. トリアゾール環を有するブラシノステロイド生合成阻害剤の構造活性相関と作用部位
- 植物化学調節学会, 1999年12月25日, 植物の化学調節, 34 (2), 336 - 336, 日本語15. 酵母Three-hybrid法によるオーキシン受容体のクローニング
- 15 酵母Tree-hybrid法によるオーキシン受容体のクローニング
In order to clarify the signal transduction mechanisms by which plant hormones control various physiological events in plans, it is very important to isolate the receptors of plant hormones. Our objective in this study is the cloning of an auxin receptor cDNA with a yeast three-hybrid system, which was adapted from the yeast two-hybrid system to detect small ligand-protein interactions. As a third hybird ligand, a synthetic auxin, 1-naphtalene acetic acid (1-NAA), was covalently linked to dexamethasone (Dex). The yeast expression plasmids for the three-hybrid system were constructed as followed; the plasmid to express the fusion protein of the GAL4 DNA binding domain and the rat glucocorticoid receptor (GR), and the plasmids in which an Arabidopsis cDNA library was fused to the GAL4 transcriptional activation domain. Yeast cells transformed with both the plasmids were plated on medium containing the synthesized hybrid ligand, and the screening of an auxin receptor is now in progress. Next, the combination of His-tag and Ni-NTA, which form a stronger interaction than that of GR and Dex, was adapted to improve the three-hybrid system. The Ni-NTA was covalently corsslinked to Dex. The yeast cells expressing the His-tagged GAL4 DNA-binding domain and the fusion protein of the GAL4 activation domain and GR activate reporter genes when plated on medium containing the synthesized heterodimer of Ni-NTA and Dex. This new method is now under construction.
植物化学調節学会, 1999年10月01日, 植物化学調節学会研究発表記録集, (34), 34 - 35, 日本語 - 53 トリアゾール環を有するブラシノステロイド生合成阻害剤の構造活性相関と作用部位
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.
植物化学調節学会, 1999年10月01日, 植物化学調節学会研究発表記録集, (34), 107 - 108, 日本語 - 1999年03月05日, 日本農芸化学会誌, 73, 474 - 474, 日本語植物のP450をめぐる話題 : アルカロイド生合成系とミクロソームにおける電子伝達系
- 1999年03月, Plant and cell physiology, 40, s125 - s125, 英語Cloning and expression of a β-primeverosidase concerned with the alcoholic aroma formation from the tea plant : Camellia sinensis var. sinensis cv. Yabukita
- 1997年03月, Plant and cell physiology, 38, s67, 英語CHARACTERIZATION OF TWO ISOFORMS OF NADPH-P450 REDUCTASE FROM Arabidopsis thaliana
- 1995年03月, Plant and cell physiology, 36, S105, 英語ISOLATION AND CHARACTERIZATION OF NOVELP450S IN ARABIDOPSIS
書籍等出版物
- 共編者(共編著者), 羊土社, 2019年01月, 日本語, 植物天然物化学・植物生化学の教科書, ISBN: 9784758120906基礎から学ぶ植物代謝生化学
教科書・概説・概論
- その他, 共立出版植物のシグナル伝達（柿本辰男、高山誠司、福田裕穂、松岡信編）、, 2010年, 日本語第１章 植物ホルモン合成とシグナル分子受容の新発見：シトクロムP450の酵素化学から導き出された新規ブラシノステロイド生合成経路
学術書
- その他, 文永堂植物栄養学（間藤徹、馬建鋒、藤原徹編）、, 2010年, 日本語第2章 植物栄養学を理解するために： 植物ホルモン
教科書・概説・概論
- その他, 廣川書店廣川タンパク質化学 第４巻 酵素 4.3P.232-240, 2004年, 日本語配糖体や少糖に作用する酵素 β-プリメベロシダーゼ（EC 3.2.2.149）
学術書
講演・口頭発表等
- 日本農芸化学会大会2014年度東京大会, 2014年03月, 日本語, 国内会議ソルガムにおける5-deoxystrigolからsorgomolへの変換反応
[招待有り]
口頭発表（招待・特別）
- 10th JSOL International Symposium on Solanaceae Genomics, 2013年11月, 英語, 国際会議Steroidal Glycoalkaloid biosynthesis in Solanaceae
[招待有り]
口頭発表（招待・特別）
- 奈良先端未来開拓コロキウム, 2013年11月, 英語, 国際会議Diversity and evolution of steroidal saponin biosynthesis in plants
[招待有り]
口頭発表（招待・特別）
- 第48回大会植物化学調節学会, 2013年10月, 日本語, 国際会議植物における5-デオキシストリゴールからモノヒドロキシストリゴラクトンへの変換
口頭発表（一般）
- 第48回大会植物化学調節学会, 2013年10月, 日本語, 国内会議β－カロテン生産菌を用いたストライゴラクトン生合成中間体カルラクトン酵素合成の試み
口頭発表（一般）
- 第48回植物化学調節学会, 2013年10月, 日本語, 国内会議ヤマノイモ属トゲドコロのステロイドサポニン生合成に関わるステロール22位水酸化酵素の同定
ポスター発表
- 植物化学調節学会第48回大会, 2013年10月, 日本語, 国内会議ヤマノイモ属トゲドコロにおけるステロイドサポニン生合成16位水酸化酵素の同定
口頭発表（一般）
- 植物化学調節学会第48回大会, 2013年10月, 日本語, 国内会議ナス科植物ステロイドグリコアルカロイド生合成に関わる新規2-オキソグルタル酸依存性ジオキシゲナーゼ：16位水酸化酵素の同定と機能解析
口頭発表（一般）
- INRA_Colmerセミナー, 2013年10月, 英語, 国際会議Diversity and evolution of steroidal saponin biosynthesis in plants
口頭発表（招待・特別）
- ボルドー大学セミナー, 2013年10月, 英語, 国際会議Diversity and Evolution of Steroidal Saponin Biosynthesis in Plants
口頭発表（招待・特別）
- 日本生薬学会第60回年会, 2013年09月, 日本語, 国内会議薬用植物カンゾウのグリチルリチン生合成に関わる糖転移酵素遺伝子の単離と機能解析
口頭発表（一般）
- 第65回日本生物工学会大会, 2013年09月, 日本語, 国内会議薬用植物カンゾウのグリチルリチン生合成に関わる糖転移酵素遺伝子の単離と機能解析
口頭発表（一般）
- 第31回植物細胞分子生物学会, 2013年09月, 日本語, 国内会議薬用植物カンゾウのグリチルリチン生合成に関わる糖転移酵素遺伝子の単離と機能解析
口頭発表（一般）
- 第31回植物細胞分子生物学会, 2013年09月, 日本語, 国内会議ヤマノイモ属トゲドコロ由来のフロスタノール配糖体を加水分解するβ-グルコシダーゼの同定
口頭発表（一般）
- 第31回植物細胞分子生物学会, 2013年09月, 日本語, 国内会議ヤマノイモ属トゲドコロにおけるステロイドサポニン生合成P-450遺伝子の探索
口頭発表（一般）
- 第31回植物細胞分子生物学会, 2013年09月, 日本語, 国内会議トマト果実におけるα-トマチン代謝酵素遺伝子の探索
ポスター発表
- 第31回植物細胞分子生物学会, 2013年09月, 日本語, 国内会議トマトにおけるステロイドアルカロイド生合成に関わる配糖化酵素の探索
ポスター発表
- 第31回植物細胞分子生物学会, 2013年09月, 日本語, 国内会議グリコアルカロイド生合成遺伝子群の同定について
口頭発表（一般）
- 第31回植物細胞分子生物学会, 2013年09月, 日本語, 国内会議アグロインフィルトレーション法による植物発現系を用いた植物代謝機能の解明およびその応用性の探索
ポスター発表
- World Congress of Parasitic Plants, 2013年07月, 英語, 国際会議The bioconversion of 5-deoxystrigol to monohydroxylated strigolactones by plants
口頭発表（一般）
- 12th World Congress of Parasitic Plants, 2013年07月, 英語, 国際会議Structural requirements of strigolactones for germination induction and inhibition of Striga gesnerioides seeds
口頭発表（一般）
- 蛋白質科学会, 2013年06月, 日本語, 国内会議植物における2−オキソグルタル酸依存性ジオキシゲナーゼスーパーファミリーの進化と多様性
ポスター発表
- TERPNET2013, 2013年06月, 英語, 国際会議Transgenic potatoes remarkably decreasing content of glycoalkaloids
[招待有り]
口頭発表（招待・特別）
- TERPNET2013, 2013年06月, 英語, 国際会議Identification and quantification of steroidal saponins in Dioscorea esculenta
ポスター発表
- TERPNET2013, 2013年06月, 英語, 国際会議Evolution and diversity of the 2-oxoglutarate-dependent dioxygenase superfamily in plants
ポスター発表
- TERPNET2013, 2013年06月, 英語, 国際会議Characterization of furostanol glycoside 26-O-β-glucosidase involved in hydrolysis of protodioscin from Dioscorea esculenta,
口頭発表（一般）
- TERPNET2013, 2013年06月, 英語, 国際会議Characterization of furostanol glycoside 26-O-β-glucosidase involved in hydrolysis of protodioscin from Dioscorea esculenta
口頭発表（一般）
- TERPNET2013, 2013年06月, 英語, 国際会議Characterization of De90B, cholesterol 22-hydroxylase involved in steroidal saponin biosynthesis in the tubers of Dioscorea esculenta
口頭発表（一般）
- 岩手生物工学研究所セミナー, 2013年04月, 日本語, 国内会議ヤマノイモ属トゲドコロの有用ステロイドサポニン生合成に関わる酵素遺伝子の解析
口頭発表（招待・特別）
- 次世代創薬プロジェクトワークショップ, 2013年02月, 日本語, 国内会議植物二次代謝の多様性とシトクロムP450の分子進化
[招待有り]
口頭発表（招待・特別）
- The 6th Asia and Oceania Conference on Photobiology, 2013年, 英語, 国際会議Remodeling of photosystems to induce thermal dissipation and state transition accompanied by structural changes of thylakoid membranes in heat-stressed wheat
ポスター発表
- P450発見50周年記念シンポジウム, 2012年12月, 日本語, 国内会議Cytochromes P450 in Steroid Glycoalkaloids Biosynthesis in Solanum Plants
ポスター発表
- 第28回ユーグレナ研究会, 2012年11月, 日本語, 国内会議植物二次代謝（適応代謝）の多様性とP450の分子進化
[招待有り]
口頭発表（招待・特別）
- 10th International Congress on Plant Molecular Biology, 2012年10月, 英語, 国際会議Oxidative Bioconversion of a Synthetic Strigolactone Analog GR24 in the Root Cultures of Menispermum dauricum
ポスター発表
- 10th International Congress on Plant Molecular Biology, 2012年10月, 英語, 国際会議Oxidation of 5-deoxystrigol in sorghum; Sorghum bicolor (L.) Moech
ポスター発表
- 10th International Congress on Plant Molecular Biology, 2012年10月, 英語, 国際会議D1 protein is degraded in heat-stressed plants in the dark; presumably due to electron inflow of the stromal reducing power into the plastoquinone
ポスター発表
- 第１回植物二次代謝フロンティア, 2012年09月, 日本語, 国内会議植物ステロイドサポニン生合成の解明
[招待有り]
口頭発表（招待・特別）
- 第30回日本植物細胞分子学会, 2012年08月, 日本語, 国内会議ヤマノイモ属における有用ステロイドサポニン生合成に関わるβ-グルコシダーゼの解析
口頭発表（一般）
- 第30回日本植物細胞分子学会, 2012年08月, 日本語, 国内会議コウモリカズラにおける含塩素イソキノリンアルカロイド 生合成中間体の同定
口頭発表（一般）
- 11th International Symposium on Cytochrome P450, 2012年06月, 英語, 国際会議Isolation and characterization of furostanol 26-O-glucoside β-glucosidase involved in hydrolysis of protodioscin; furostanol saponin from Dioscorea esculenta; based on transcriptome analysis of the rhizomes
ポスター発表
- 日本農芸化学会大会2012年度大会, 2012年03月, 日本語, 国内会議根寄生雑草Striga gesnerioides種子発芽誘導物質alectrolの単離と構造決定
口頭発表（一般）
- 日本農芸化学会大会2012年度大会, 2012年03月, 日本語, 国内会議合成ストライゴラクトンGR24 の植物による代謝と生成物の同定
口頭発表（一般）
- 第52回日本植物生理学会, 2012年03月, 日本語, 国内会議高温ストレスを受けた植物がステート遷移によりPSIIを保護するメカニズムの解析
口頭発表（一般）
- 日本農芸化学会大会2012年度大会, 2012年03月, 日本語, 国内会議ヤマノイモ属トゲドコロにおけるプロトジオシンを加水分解するβ－グルコシダーゼの解析
口頭発表（一般）
- 日本農芸化学会大会2012年度大会, 2012年03月, 日本語, 国内会議ソルガムにおける5-deoxystrigolの酸化的代謝の解析
口頭発表（一般）
- 日本農芸化学会2012年大会, 2012年03月, 日本語, 国内会議LC-MS/MSによる新奇ストリゴラクトン探索法の確立
口頭発表（一般）
- 第53回日本植物生理学会年会, 2012年, 日本語, 国内会議高温ストレスを受けた植物がステート遷移によりPSIIを保護するメカニズムの解析
口頭発表（一般）
- 植物化学シンポジウム, 2011年11月, 日本語, 国内会議植物の酸素添加酵素の多彩な機能と代謝工学への展開
[招待有り]
口頭発表（招待・特別）
- 植物化学調節学会第46回大会, 2011年11月, 日本語, 国内会議根寄生雑草Striga gesnerioides 種子の発芽を誘導するストライゴラクトンの構造要求性
口頭発表（一般）
- 天然有機化合物討論会, 2011年09月, 日本語, 国内会議根寄生雑草Striga gesnerioides種子発芽刺激物質の立体化学
口頭発表（一般）
- 第29回植物細胞分子生物学会, 2011年09月, 日本語, 国内会議ヤマノイモ属トゲドコロにおけるステロイドサポニン生合成酵素の探索
口頭発表（一般）
- 第29回植物細胞分子生物学会, 2011年09月, 日本語, 国内会議トマト果実におけるα-トマチンの代謝酵素の探索
口頭発表（一般）
- 第29回植物細胞分子生物学会, 2011年09月, 日本語, 国内会議トマトにおけるα-トマチン生合成酵素の探索
口頭発表（一般）
- 第29回植物細胞分子生物学会, 2011年09月, 日本語, 国内会議クマリン類生合成の鍵酵素である桂皮酸オルト位水酸化酵素の解析
口頭発表（一般）
- 11th World Congress on Parasitic Plants, 2011年09月, 英語, 国際会議Structural requirements of strigolactones for induction of germination in root parasitic plants
ポスター発表
- 日本農芸化学会大会2011年度大会, 2011年03月, 日本語, 国内会議キュウリ葉のα;β-不飽和カルボニル化合物還元酵素遺伝子の解析
口頭発表（一般）
- 日本農芸化学会関西・中部支部合同大会, 2011年, 日本語, 国内会議植物が光化学系の高温障害を回避するメカニズムの解析
口頭発表（一般）
- 第53回天然有機化合物討論会, 2011年, 日本語, 大阪, 国内会議根寄生雑草Striga gesnerioides種子発芽刺激物質の立体化学
ポスター発表
- Terpnet2011, 2011年, 英語, 国際会議Transcriptome analysis of the rhizomes of Dioscorea spp. to investigate steroidal saponin biosynthesis
ポスター発表
- 植物化学調節学会第45回大会, 2010年10月, 日本語, 国内会議植物におけるクマリン類生合成の鍵酵素である桂皮酸オルト位水酸化酵素の解析
ポスター発表
- 植物化学調節学会第45回大会, 2010年10月, 日本語, 国内会議タンパク質二量体誘導体化合物(CID)を利用した新規植物化学調節剤の創出
ポスター発表
- 植物化学調節学会第45回大会, 2010年10月, 日本語, 国内会議ジベレリン生合成酵素選択的P450阻害剤のスクリーニング
ポスター発表
- 植物化学調節学会第45回大会, 2010年10月, 日本語, 国内会議キュウリ緑葉からのα;β-不飽和カルボニル化合物還元酵素の精製と遺伝子解析
ポスター発表
- P450 Biodiversity and Biotechnology; 10th International Symposium, 2010年10月, 英語, 国際会議Exploring P450s involved in ABA catabolism from Bryophyte and Pteridophyte
ポスター発表
- 植物化学調節学会第45回大会, 2010年10月, 日本語, 国内会議ABAアミノ酸複合体の合成と生物活性
ポスター発表
- 植物化学調節学会第45回大会, 2010年10月, 日本語, 国内会議ABA 8’-水酸化酵素に対する実用性の高い選択性阻害剤アブシナゾールE2B
ポスター発表
- SATREPS-JSPS AA Science Platform Program Joint Seminar on Striga spp.; the food security scourge in Africa, 2010年07月, 英語, 国際会議Qualitative and quantitative analysis of strigolactones using LC-MS/MS
口頭発表（一般）
- 日本農芸化学会大会2010年度大会, 2010年03月, 日本語, 国内会議根寄生植物ヤセウツボの成長過程の形態学的観察および植物ホルモン分析
口頭発表（一般）
- 第51回日本植物生理学会年会, 2010年03月, 日本語, 国内会議コケおよびシダ植物におけるABA不活性化P450の探索
口頭発表（一般）
- 日本農芸化学会大会2010年度大会, 2010年03月, 日本語, 国内会議キュウリ緑葉由来NADPH 依存性Alkenal/one oxidoreductase の精製と遺伝子解析
口頭発表（一般）
- 日本農芸化学会大会2010年度大会, 2010年03月, 日本語, 国内会議ABAアミノ酸複合体の生理作用発現機構
口頭発表（一般）
- 日本農芸化学会大会2010年度大会, 2010年03月, 日本語, 国内会議ABA 8’-水酸化酵素CYP707A特異的阻害剤アブシナゾールEの開発
口頭発表（一般）
- 日本農芸化学会大会2010年度大会, 2010年03月, 日本語, 国内会議ABA 8’-水酸化酵素CYP707Aの特異的阻害剤アブシナゾールDの創出
口頭発表（一般）
- 日本農芸化学会関西支部例会, 2009年12月, 日本語, 国内会議キュウリ緑葉由来NADPH 依存性Alkenal/one oxidoreductase の精製と遺伝子解析
口頭発表（一般）
- 植物化学調節学会第44回大会, 2009年10月, 日本語, 国内会議根粒共生がオロバンキの寄生に及ぼす影響
ポスター発表
- 植物化学調節学会第44回大会, 2009年10月, 日本語, 国内会議根寄生植物ヤセウツボの寄生成立後の生長過程に関する形態学的観察
ポスター発表
- 植物化学調節学会第44回大会, 2009年10月, 日本語, 国内会議根寄生植物の種子発芽刺激活性に対するストライゴラクトンB環の修飾の影響について
ポスター発表
- 植物化学調節学会第44回大会, 2009年10月, 日本語, 国内会議柑橘およびマメ科植物におけるクマリン生合成酵素の探索
ポスター発表
- 植物化学調節学会第44回大会, 2009年10月, 日本語, 国内会議メスキートが生産する有用二次代謝産物の検索
ポスター発表
- 農芸化学会合同支部会, 2009年10月, 日本語, 国内会議ベンジルイソキノリンアルカロイド生合成に関わるシトクロムP450遺伝子の単離
口頭発表（一般）
- 植物化学調節学会第44回大会, 2009年10月, 日本語, 国内会議ブラシノステロイド生合成に関与すると推定されるコケおよびシダ植物P-450の機能解析
ポスター発表
- 植物化学調節学会第44回大会, 2009年10月, 日本語, 国内会議イソキノリンアルカロイド生合成に関わるシトクロムP-450 cDNAの単離および機能解析
ポスター発表
- 植物化学調節学会第44回大会, 2009年10月, 日本語, 国内会議アブシジン酸8'-位水酸化酵素CYP707A特異的阻害剤アブシナゾールEの開発
ポスター発表
- 植物化学調節学会第44回大会, 2009年10月, 日本語, 国内会議アゾール環修飾による高選択性アゾール系植物P450阻害剤の創製
ポスター発表
- 植物化学調節学会第44回大会, 2009年10月, 日本語, 国内会議ABA代謝物質ファゼイン酸の還元酵素の精製
ポスター発表
- 25th Naito Conference, 2009年09月, 英語, 国際会議Unravelling coumarin biosynthesis in plants and in vivo synthesis in E. coli
口頭発表（一般）
- Terpnet 2009, 2009年06月, 英語, 国際会議Rice CYP734As are multifunctional P450s in brassinosteroid catabolism.
[招待有り]
口頭発表（招待・特別）
- 農芸化学会2009年度大会, 2009年03月, 日本語, 国内会議東方美人茶における香気成分hortrienol前駆体の単離と精製
口頭発表（一般）
- 第50回植物生理学会, 2009年03月, 日本語, 国内会議新規ブラシノステロイド不活性化経路の解明
口頭発表（一般）
- 第50回植物生理学会, 2009年03月, 日本語, 国内会議植物免疫信号伝達系におけるセロトニンの機能解析
口頭発表（一般）
- 農芸化学会2009年度大会, 2009年03月, 日本語, 国内会議サツマイモにおけるクマリン生合成段階を触媒する桂皮酸類のオルト位水酸化酵素
口頭発表（一般）
- 第50回植物生理学会, 2009年03月, 日本語, 国内会議サツマイモにおけるクマリン生合成段階を触媒する桂皮酸類のオルト位水酸化酵素
口頭発表（一般）
- 農芸化学会2009年度大会, 2009年03月, 日本語, 国内会議アブシジン酸代謝不活性化酵素CYP707Aの特異的阻害剤アブシナゾールF
口頭発表（一般）
- 農芸化学会2009年度大会, 2009年03月, 日本語, 国内会議アブシジン酸代謝不活性化酵素CYP707Aの特異的阻害剤アブシナゾールE
口頭発表（一般）
- 農芸化学会2009年度大会, 2009年03月, 日本語, 国内会議ABA代謝物質ファゼイン酸の還元酵素の性質
口頭発表（一般）
所属学協会
植物バイオテクノロジー学会
2000年03月 - 現在植物化学調節学会
1998年 - 現在American Society of Plant Biologists
日本生化学会
日本農芸化学会
日本植物生理学会
共同研究・競争的資金等の研究課題
- 学術研究助成基金助成金／基盤研究(C), 2018年04月 - 2021年03月, 研究代表者
競争的資金
- 国立研究開発法人理化学研究所, SIP戦略的イノベーション創造プログラム(次世代農林水産業創造技術), 2017年戦略的オミクス育種技術体系の構築
競争的資金
- 国立大学法人筑波大学, SIP戦略的イノベーション創造プログラム(次世代農林水産業創造技術), 2017年, 研究代表者ゲノム編集技術等を用いた農水産物の画期的育種改良
競争的資金
- 農林水産業・食品産業科学技術研究推進事業, 2016年, 研究代表者高度機能分化した植物組織培養による有用サポニン生産技術開発
競争的資金
- 戦略的イノベーション創造プログラム（SIP）, 2016年, 研究代表者ゲノム編集技術等を用いた農水産物の画期的育種改良
競争的資金
- 科学研究費補助金／新学術領域研究, 2011年, 研究代表者
競争的資金
- 学術研究助成基金助成金／基盤研究(C), 2011年, 研究代表者
競争的資金
- 2010年, 研究代表者植物におけるクマリン類およびフラノクマリン類生合成の解明
競争的資金
- 植物ホルモン生合成に関与するチトクロムP450に関する研究
競争的資金
- 植物の香気生成酵素β-プリメベロシダーゼに関する研究
競争的資金