Directory of Researchers

MIZUTANI Masaharu
Graduate School of Agricultural Science / Department of Agrobioscience
Associate Professor
Agricultural Chemistry
Last Updated :2024/02/01

Researcher Profile and Settings

Affiliation

  • <Faculty / Graduate School / Others>

    Graduate School of Agricultural Science / Department of Agrobioscience
  • <Related Faculty / Graduate School / Others>

    Faculty of Agriculture / Department of Agrobioscience

Teaching

Research at Kobe

  • 24 Mar. 2021, Toxin in potatoes evolved from a bitter-tasting compound in tomatoes
  • 11 May 2016, Why do tomatoes smell "grassy"? -Researchers identify enzymes that convert the grassy smell into a sweeter scent-

Research Activities

Research Interests

  • dioxygenase
  • cyst nematode
  • strigolactone
  • steroidal glycoalkaloid
  • brassinosteroid
  • cytochrome P450
  • 二次代謝
  • 植物ホルモン

Research Areas

  • Life sciences / Bioorganic chemistry
  • Life sciences / Molecular biology
  • Life sciences / Plants: molecular biology and physiology

Committee Memberships

  • 2020 - Present, 日本植物生理学会, 欧文誌Plant and Cell Physiology、Regular Editor
  • 2018 - Present, 日本植物バイオテクノロジー学会, 代議員
  • 2016 - Present, 植物化学調節学会, 会計幹事
  • 2013 - Present, 日本植物細胞分子生物学会, 学会誌Plant Biotechnology 編集委員
  • 2012 - Present, 日本農芸化学会関西支部, 参与
  • 2017 - 2019, 日本植物生理学会, 欧文誌Plant and Cell Physiology, Advisory Editorial Board
  • 2017, 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, 12th International Symposium on Cytochrome P450 Biodiversity and Biotechnology(Kyoto), Organizing Committee
  • 2012 - 2012, 日本農芸化学会関西支部, 庶務幹事
  • 2011 - 2012, 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)

Awards

  • Sep. 2021 日本バイオテクノロジー学会, Plant Biotechnology paper award, Hatching stimulation activity of steroidal glycoalkaloids toward the potato cyst nematode, Globodera rostochiensis.

    1 Shimizu, K., Kushida, A., Akiyama, R., Lee, H.J., Okamura, Y., Masuda, Y., Sakata, I., Tanino, K., Matsukida, S., Inoue, T., Sugimoto, Y., Mizutani, M.

  • Sep. 2019 日本植物細胞分子生物学会, Plant Biotechnology paper award, Characterization of steroid 5a-reductase involved in a-tomatine biosynthesis in tomatoes.

    2 Akiyama, R., Lee, H.J., Nakayasu, M., Osakabe, K., Osakabe, Y., Umemoto, N., Saito, K., Muranaka, T., Sugimoto, Y., Mizutani, M.

  • Oct. 2006 植物化学調節学会, JSCRP Award for Young Scientists, 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

    Masaharu Mizutani

  • Mar. 2006 日本農芸化学会, B.B.B. paper award, CYP724B2 and CYP90B3 function in the early C-22 hydroxylation steps of brassinosteroid biosynthetic pathway in tomato.

    3 Ohnishi, T., Watanabe, B., Sakata, K., Mizutani, M.

  • Mar. 2001 日本農芸化学会, B.B.B. paper award, Substrate specificity of b-primeverosidase, a key enzyme in aroma formation during oolong tea and black tea manufacturing.

    4 Ma, S.-J., Mizutani, M., Hiratake, J., Hayashi, K., Yagi, K., Watanabe, N., Sakata, K.

Published Papers

  • Naoyuki Umemoto, Shuhei Yasumoto, Muneo Yamazaki, Kenji Asano, Kotaro Akai, Hyoung Jae Lee, Ryota Akiyama, Masaharu Mizutani, Yozo Nagira, Kazuki Saito, Toshiya Muranaka

    Japanese Society for Plant Cell and Molecular Biology, 25 Sep. 2023, Plant Biotechnology, 40 (3), 211 - 218

    Scientific journal

  • Ryota Akiyama, Naoyuki Umemoto, Masaharu Mizutani

    Japanese Society for Plant Cell and Molecular Biology, 25 Sep. 2023, Plant Biotechnology, 40 (3), 185 - 191

    Scientific journal

  • Masato Homma, Takatoshi Wakabayashi, Yoshitaka Moriwaki, Nanami Shiotani, Takumi Shigeta, Kazuki Isobe, Atsushi Okazawa, Daisaku Ohta, Tohru Terada, Kentaro Shimizu, Masaharu Mizutani, Hirosato Takikawa, Yukihiro Sugimoto

    Abstract Strigolactones (SLs) are plant apocarotenoids with diverse functions and structures. The widespread canonical SLs, with distinctive structural variations in their tricyclic lactone known as the ABC-ring, are classified into two types based on the C-ring configurations. The steric C-ring configuration arises during the BC-ring closure downstream of carlactonoic acid (CLA), a biosynthetic intermediate. Most plants stereoselectively produce either type of canonical SLs, e.g., tomato (Solanum lycopersicum) produces orobanchol with α-oriented C-ring. The mechanisms governing SL structural diversification are partly understood, with limited insight into the functional implications. Moreover, the precise molecular mechanism for the stereoselective BC-ring closure reaction remains unknown. Herein, we identified an enzyme called the stereoselective BC-ring-forming factor (SRF) from the dirigent protein (DIR) family, especially the DIR-f subfamily, whose biochemical function was previously unidentified, making it a pivotal enzyme in stereoselective canonical SL biosynthesis with the α-oriented C-ring. We begin by confirming the exact catalytic function of the tomato cytochrome P450 SlCYP722C, which we previously demonstrated to be involved in the orobanchol biosynthesis [Wakabayashi et al.,Sci. Adv.5, eaax9067 (2019)], to convert CLA to 18-oxocarlactonoic acid. Subsequently, we demonstrate that SRF catalyzes the stereoselective BC-ring closure reaction of 18-oxocarlactonoic acid to form orobanchol. Our approach integrates experimental and computational methods, including SRF structure prediction and molecular dynamics simulations, to propose a catalytic mechanism based on the conrotatory 4π-electrocyclic reaction for stereoselective BC-ring formation in orobanchol. The present study provides insight into the molecular basis of how plants produce SLs with specific stereochemistry in a controlled manner.

    Cold Spring Harbor Laboratory, 07 Aug. 2023

  • Takatoshi Wakabayashi, Megumi Nakayama, Yurie Kitano, Masato Homma, Kenji Miura, Hirosato Takikawa, Masaharu Mizutani, Yukihiro Sugimoto

    Societal Impact Statement Parasitic witchweeds (Striga species) pose a serious threat to food security in Africa, attacking cereal grains and legumes. Chemicals released from the host roots that initiate the life cycle of Striga are known as germination stimulants, predominantly strigolactones (SLs). Strigol, the first identified SL, was isolated from the root exudates of cotton (Gossypium hirsutum), a false host of Striga, over 50 years ago. The identification of strigol synthase in cotton establishes the complete biosynthesis pathway of this emblematic SL. This discovery has the potential to advance our understanding of SL‐mediated rhizosphere interactions and enhance cotton's effectiveness as a trap crop.

    Wiley, 02 Aug. 2023, PLANTS, PEOPLE, PLANET

    Scientific journal

  • Miki Hatada, Ryota Akiyama, Moeko Yamagishi, Kimitsune Ishizaki, Masaharu Mizutani

    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), 11 May 2023, Plant And Cell Physiology, 64 (7), 826 - 838

    Scientific journal

  • Kosuke Shimizu, Ryota Akiyama, Yuya Okamura, Chihiro Ogawa, Yuki Masuda, Itaru Sakata, Bunta Watanabe, Yukihiro Sugimoto, Atsuhiko Kushida, Keiji Tanino, Masaharu Mizutani

    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), 17 Mar. 2023, Science Advances, 9 (11)

    Scientific journal

  • Bryophytes as a source of unique natural products and platforms for synthetic biology

    Masaharu Mizutani

    Mar. 2023, Agricultural Biotechnology, 7 (3), 20 - 23, Japanese

    [Invited]

  • Takatoshi Wakabayashi, Daisuke Moriyama, Ayumi Miyamoto, Hironori Okamura, Nanami Shiotani, Nobuhiro Shimizu, Masaharu Mizutani, Hirosato Takikawa, Yukihiro Sugimoto

    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, 14 Dec. 2022, Frontiers in Plant Science, 13

    Scientific journal

  • Evolution of steroid biosynthetic enzymes in plants and their use in metabolic engineering

    水谷正治, 秋山遼太

    Oct. 2022, Regulation of plant growth & development, 57 (2), 84 - 92, Japanese

    [Refereed][Invited]

    Scientific journal

  • Daisuke Moriyama, Takatoshi Wakabayashi, Nanami Shiotani, Shunya Yamamoto, Yui Furusato, Kohki Yabe, Masaharu Mizutani, Hirosato Takikawa, Yukihiro Sugimoto

    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, 13 May 2022, Bioscience, Biotechnology, and Biochemistry

    Scientific journal

  • Ryota Akiyama, Bunta Watanabe, Junpei Kato, Masaru Nakayasu, Hyoung Jae Lee, Naoyuki Umemoto, Toshiya Muranaka, Kazuki Saito, Yukihiro Sugimoto, Masaharu Mizutani

    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.

    13 May 2022, Plant & cell physiology, English, Domestic magazine

    Scientific journal

  • Ryota AKIYAMA, Masaharu MIZUTANI

    Japan Society for Bioscience, Biotechnology, and Agrochemistry, 01 Mar. 2022, KAGAKU TO SEIBUTSU, 60 (3), 107 - 109, Japanese

    [Invited]

    Scientific journal

  • Evolution of potato toxin solanine from a tomato bitter compound

    Ryota Akiyama, Masaharu Mizutani

    Feb. 2022, Chemistry, 77 (2), 12 - 16, Japanese

    [Invited]

  • Gaku Murata, Taketo Uehara, Hyoung Jae Lee, Masaharu Mizutani, Yasuhiro Kadota, Yoshimi Shinmura, Takeo Saito, Kenta Uesugi

    Wiley, 03 Jan. 2022, Journal of Phytopathology

    Scientific journal

  • Takatoshi Wakabayashi, Ryo Yasuhara, Kenji Miura, Hirosato Takikawa, Masaharu Mizutani, Yukihiro Sugimoto

    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, Nov. 2021, Planta, 254 (5), 88 - 88, English, International magazine

    [Refereed]

    Scientific journal

  • Ryota Akiyama, Masaru Nakayasu, Naoyuki Umemoto, Junpei Kato, Midori Kobayashi, Hyoung Jae Lee, Yukihiro Sugimoto, Yoko Iijima, Kazuki Saito, Toshiya Muranaka, Masaharu Mizutani

    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), 01 Oct. 2021, Plant and Cell Physiology, 62 (5), 775 - 783

    [Refereed]

    Scientific journal

  • Isolation of a dioxygenase involved in toxic alkaloid biosynthesis in potato and tomato

    Ryota Akiyama, Masaharu Mizutani

    Sep. 2021, Bioscience and Industry, 79 (5), 384 - 385, Japanese

    [Invited]

  • Masaru Nakayasu, Naoyuki Umemoto, Ryota Akiyama, Kiyoshi Ohyama, Hyoung J. Lee, Haruka Miyachi, Bunta Watanabe, Toshiya Muranaka, Kazuki Saito, Yukihiro Sugimoto, Masaharu Mizutani

    Wiley, 09 Aug. 2021, The Plant Journal, 108 (1), 81 - 92

    [Refereed]

    Scientific journal

  • Potato and tomato are delicious vegetable but contain toxins?

    Ryota Akiyama, Masaru Nakayasu, Masaharu Mizutani

    Aug. 2021, Pharmacia, 57 (8), 726 - 730, Japanese

    [Refereed][Invited]

  • Bunta Watanabe, Katsunori Makino, Masaharu Mizutani, Hikaru Takaya

    Elsevier BV, May 2021, Tetrahedron, 132194 - 132194

    [Refereed]

    Scientific journal

  • Ryosuke Munakata, Alexandre Olry, Tomoya Takemura, Kanade Tatsumi, Takuji Ichino, Cloé Villard, Joji Kageyama, Tetsuya Kurata, Masaru Nakayasu, Florence Jacob, Takao Koeduka, Hirobumi Yamamoto, Eiko Moriyoshi, Tetsuya Matsukawa, Jérémy Grosjean, Célia Krieger, Akifumi Sugiyama, Masaharu Mizutani, Frédéric Bourgaud, Alain Hehn, Kazufumi Yazaki

    Plants produce ∼300 aromatic compounds enzymatically linked to prenyl side chains via C–O bonds. These O-prenylated aromatic compounds have been found in taxonomically distant plant taxa, with some of them being beneficial or detrimental to human health. Although their O-prenyl moieties often play crucial roles in the biological activities of these compounds, no plant gene encoding an aromatic O-prenyltransferase (O-PT) has been isolated to date. This study describes the isolation of an aromatic O-PT gene, CpPT1, belonging to the UbiA superfamily, from grapefruit (Citrus × paradisi, Rutaceae). This gene was shown responsible for the biosynthesis of O-prenylated coumarin derivatives that alter drug pharmacokinetics in the human body. Another coumarin O-PT gene encoding a protein of the same family was identified in Angelica keiskei, an apiaceous medicinal plant containing pharmaceutically active O-prenylated coumarins. Phylogenetic analysis of these O-PTs suggested that aromatic O-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.

    Proceedings of the National Academy of Sciences, 27 Apr. 2021, Proceedings of the National Academy of Sciences, 118 (17), e2022294118 - e2022294118, English, International magazine

    [Refereed]

    Scientific journal

  • Takatoshi Wakabayashi, Shunsuke Ishiwa, Kasumi Shida, Noriko Motonami, Hideyuki Suzuki, Hirosato Takikawa, Masaharu Mizutani, Yukihiro Sugimoto

    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), 02 Apr. 2021, Plant Physiology, 185 (3), 902 - 913, English, International magazine

    [Refereed]

    Scientific journal

  • Ryota Akiyama, Bunta Watanabe, Masaru Nakayasu, Hyoung Jae Lee, Junpei Kato, Naoyuki Umemoto, Toshiya Muranaka, Kazuki Saito, Yukihiro Sugimoto, Masaharu Mizutani

    AbstractPotato (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, Mar. 2021, Nature Communications, 12 (1)

    [Refereed]

    Scientific journal

  • Daichi Watanabe, Ikuo Takahashi, Naiyanate Jaroensanti‐Tanaka, Sho Miyazaki, Kai Jiang, Masaru Nakayasu, Masato Wada, Tadao Asami, Masaharu Mizutani, Kazuma Okada, Masatoshi Nakajima

    Wiley, Feb. 2021, The Plant Journal, 105 (4), 1026 - 1034

    [Refereed]

    Scientific journal

  • Takatoshi WAKABAYASHI, Masaharu MIZUTANI, Yukihiro SUGIMOTO

    Japan Society for Bioscience, Biotechnology, and Agrochemistry, 01 Nov. 2020, KAGAKU TO SEIBUTSU, 58 (11), 628 - 634, Japanese

    [Refereed][Invited]

    Scientific journal

  • Takatoshi Wakabayashi, Hikaru Shinde, Nanami Shiotani, Shunya Yamamoto, Masaharu Mizutani, Hirosato Takikawa, Yukihiro Sugimoto

    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, 09 Oct. 2020, Natural Product Research, 1 - 8, English, International magazine

    [Refereed]

    Scientific journal

  • Kosuke Shimizu, Atsuhiko Kushida, Ryota Akiyama, Hyoung Jae Lee, Yuya Okamura, Yuki Masuda, Itaru Sakata, Keiji Tanino, Seiji Matsukida, Tsutomu Inoue, Yukihiro Sugimoto, Masaharu Mizutani

    Japanese Society for Plant Cell and Molecular Biology, 25 Sep. 2020, Plant Biotechnology, 37 (3), 319 - 325

    [Refereed]

    Scientific journal

  • Kazuma Okada, Masato Wada, Yumiko Takebayashi, Mikiko Kojima, Hitoshi Sakakibara, Masaru Nakayasu, Masaharu Mizutani, Masatoshi Nakajima, Shigeki Moriya, Taku Shimizu, Kazuyuki Abe

    AbstractThe 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), 29 Aug. 2020, Tree Physiology, 40 (9), 1205 - 1216, English, International magazine

    [Refereed]

    Scientific journal

  • Mitsuhiro Aida, Yuka Tsubakimoto, Satoko Shimizu, Hiroyuki Ogisu, Masako Kamiya, Ryosuke Iwamoto, Seiji Takeda, Md Karim, Masaharu Mizutani, Michael Lenhard, Masao Tasaka

    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, 15 Aug. 2020, International Journal of Molecular Sciences, 21 (16), 5864 - 5864, English, International magazine

    [Refereed]

    Scientific journal

  • Shuhei Yasumoto, Satoru Sawai, Hyoung Jae Lee, Masaharu Mizutani, Kazuki Saito, Naoyuki Umemoto, Toshiya Muranaka

    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, 25 Jun. 2020, Plant Biotechnology, 37 (2), 205 - 211, English, Domestic magazine

    [Refereed]

    Scientific journal

  • Structural insights into a key step of brassinosteroid biosynthesis and its inhibition

    Kesuke Fujiyama, Tomoya Hino, Masaharu Mizutani, Shingo Nagano

    Jun. 2020, Regulation of Plant Growth and Development, 56 (1), 35 - 41, Japanese

    [Refereed][Invited]

  • Takatoshi Wakabayashi, Kasumi Shida, Yurie Kitano, Hirosato Takikawa, Masaharu Mizutani, Yukihiro Sugimoto

    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, May 2020, Planta, 251 (5), 97 - 97, English, International magazine

    [Refereed]

    Scientific journal

  • Masaru Nakayasu, Ryota Akiyama, Midori Kobayashi, Hyoung Jae Lee, Takashi Kawasaki, Bunta Watanabe, Shingo Urakawa, Junpei Kato, Yukihiro Sugimoto, Yoko Iijima, Kazuki Saito, Toshiya Muranaka, Naoyuki Umemoto, Masaharu Mizutani

    AbstractTomato 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), 01 Jan. 2020, Plant and Cell Physiology, 61 (1), 21 - 28

    [Refereed]

    Scientific journal

  • Takatoshi Wakabayashi, Misaki Hamana, Ayami Mori, Ryota Akiyama, Kotomi Ueno, Keishi Osakabe, Yuriko Osakabe, Hideyuki Suzuki, Hirosato Takikawa, Masaharu Mizutani, Yukihiro Sugimoto

    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), 18 Dec. 2019, Science Advances, 5 (12), eaax9067 - eaax9067, English, International magazine

    [Refereed]

    Scientific journal

  • Shuhei Yasumoto, Naoyuki Umemoto, Hyoung Jae Lee, Masaru Nakayasu, Satoru Sawai, Tetsushi Sakuma, Takashi Yamamoto, Masaharu Mizutani, Kazuki Saito, Toshiya Muranaka

    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, 25 Sep. 2019, Plant Biotechnology, 36 (3), 167 - 173, English

    [Refereed]

    Scientific journal

  • Yuhta Nomura, Hikaru Seki, Tomonori Suzuki, Kiyoshi Ohyama, Masaharu Mizutani, Tomomi Kaku, Keita Tamura, Eiichiro Ono, Manabu Horikawa, Hiroshi Sudo, Hiroaki Hayashi, Kazuki Saito, Toshiya Muranaka

    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, Sep. 2019, The Plant Journal, 99 (6), 1127 - 1143, English, International magazine

    [Refereed]

    Scientific journal

  • Hijiri Fujioka, Hiroaki Samejima, Masaharu Mizutani, Masanori Okamoto, Yukihiro Sugimoto

    Informa UK Limited, 03 Jul. 2019, Plant Signaling & Behavior, 14 (7), 1605810 - 1605810

    [Refereed]

    Scientific journal

  • Keisuke Fujiyama, Tomoya Hino, Masahiro Kanadani, Bunta Watanabe, Hyoung Jae Lee, Masaharu Mizutani, Shingo Nagano

    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, Jun. 2019, Nature Plants, 5 (6), 589 - 594, English, International magazine

    [Refereed]

    Scientific journal

  • Hyoung Jae Lee, Masaru Nakayasu, Ryota Akiyama, Midori Kobayashi, Haruka Miyachi, Yukihiro Sugimoto, Naoyuki Umemoto, Kazuki Saito, Toshiya Muranaka, Masaharu Mizutani

    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), 01 Jun. 2019, Plant and Cell Physiology, 60 (6), 1304 - 1315, English, Domestic magazine

    [Refereed]

    Scientific journal

  • Hijiri Fujioka, Hiroaki Samejima, Hideyuki Suzuki, Masaharu Mizutani, Masanori Okamoto, Yukihiro Sugimoto

    Striga parasitizes major crops in arid regions, depriving the host crop of nutrients through the transpiration stream and causing vast agricultural damage. Here, we report on the mechanism underlying how Striga maintains high transpiration under drought conditions. We found that Striga did not respond to abscisic acid, the phytohormone responsible for controlling stomatal closure. Protein phosphatase 2C of Striga (ShPP2C1) is not regulated by abscisic acid receptors, and this feature is attributable to specific mutations in its amino acid sequence. Moreover, Arabidopsis transformed with ShPP2C1 showed an abscisic acid-insensitive phenotype, indicating that ShPP2C1 functions as a dominant negative regulator of abscisic acid signal transduction. These findings suggest that ShPP2C1 interrupts abscisic acid signalling in Striga, resulting in high transpiration and subsequent efficient absorption of host nutrients under drought conditions.

    Springer Science and Business Media LLC, Mar. 2019, Nature Plants, 5 (3), 258 - 262, English, International magazine

    [Refereed]

    Scientific journal

  • Ono E, Murata J, Toyonaga H, Nakayasu M, Mizutani M, Yamamoto MP, Umezawa T, Horikawa M

    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.

    Nov. 2018, Plant & cell physiology, 59 (11), 2278 - 2287, English, Domestic magazine

    [Refereed]

    Scientific journal

  • Masaru Nakayasu, Ryota Akiyama, Hyoung Jae Lee, Keishi Osakabe, Yuriko Osakabe, Bunta Watanabe, Yukihiro Sugimoto, Naoyuki Umemoto, Kazuki Saito, Toshiya Muranaka, Masaharu Mizutani

    Elsevier BV, Oct. 2018, Plant Physiology and Biochemistry, 131, 70 - 77

    [Refereed]

    Scientific journal

  • Yamauchi M, Ueno K, Furumoto T, Wakabayashi T, Mizutani M, Takikawa H, Sugimoto Y

    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.

    Aug. 2018, Bioorganic & medicinal chemistry, 26 (14), 4225 - 4233, English, International magazine

    [Refereed]

    Scientific journal

  • Naoyuki UMEMOTO, Masaharu MIZUTANI, Toshiya MURANAKA

    Japan Society for Bioscience, Biotechnology, and Agrochemistry, 20 Jul. 2018, KAGAKU TO SEIBUTSU, 56 (8), 566 - 572, Japanese

    [Refereed][Invited]

    Scientific journal

  • Masaru Nakayasu, Naoki Shioya, Masahito Shikata, Chonprakun Thagun, Ayman Abdelkareem, Yoshihiro Okabe, Tohru Ariizumi, Gen-ichiro Arimura, Masaharu Mizutani, Hiroshi Ezura, Takashi Hashimoto, Tsubasa Shoji

    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, Jun. 2018, The Plant Journal, 94 (6), 975 - 990, English, International magazine

    [Refereed]

    Scientific journal

  • Jun Murata, Eiichiro Ono, Seigo Yoroizuka, Hiromi Toyonaga, Akira Shiraishi, Shoko Mori, Masayuki Tera, Toshiaki Azuma, Atsushi J Nagano, Masaru Nakayasu, Masaharu Mizutani, Tatsuya Wakasugi, Masayuki P Yamamoto, Manabu Horikawa

    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.

    25 May 2018, Nature communications, 9 (1), 2140 - 2140, English, International magazine

    Scientific journal

  • Bunta Watanabe, Hiroaki Kirikae, Takao Koeduka, Yoshinori Takeuchi, Tomoki Asai, Yoshiyuki Naito, Hideya Tokuoka, Shinri Horoiwa, Yoshiaki Nakagawa, Bun-ichi Shimizu, Masaharu Mizutani, Jun Hiratake

    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, 15 May 2018, Bioorganic and Medicinal Chemistry, 26 (9), 2466 - 2474, English

    [Refereed]

    Scientific journal

  • Nakayasu Masaru, Akiyama Ryota, Lee Jae Hyoung, Keishi Osakabe, Yuriko Osakabe, Watanabe Bunta, Sugimoto Yukihiro, Umemoto Naoyuki, Saito Kazuki, Muranaka Toshiya, Mizutani Masaharu

    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.

    Apr. 2018, Plant Physiology and Biochemistry : PPB, Vol.131, 70 - 77, English

    [Refereed]

    Scientific journal

  • Iseki M, Shida K, Kuwabara K, Wakabayashi T, Mizutani M, Takikawa H, Sugimoto Y

    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.

    Apr. 2018, Journal of experimental botany, 69 (9), 2305 - 2318, English, International magazine

    [Refereed]

    Scientific journal

  • Ueno Kotomi, Nakashima Hitomi, Mizutani Masaharu, Takikawa Hirosato, Sugimoto Yukihiro

    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, JOURNAL OF PESTICIDE SCIENCE, 43 (3-4), 198 - 206, English, Domestic magazine

    [Refereed]

    Scientific journal

  • Murata Jun, Ono Eiichiro, Yoroizuka Seigo, Toyonaga Hiromi, Shiraishi Akira, Mori Shoko, Tera Masayuki, Azuma Toshiaki, Nagano Atsushi J, Nakayasu Masaru, Mizutani Masaharu, Wakasugi Tatsuya, Yamamoto Masayuki P, Horikawa Manabu

    (+)-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.

    18 Dec. 2017, NATURE COMMUNICATIONS, 8 (1), 2155 - 2155, English, International magazine

    [Refereed]

    Scientific journal

  • John L Bowman, Takayuki Kohchi, Katsuyuki T Yamato, Jerry Jenkins, Shengqiang Shu, Kimitsune Ishizaki, Shohei Yamaoka, Ryuichi Nishihama, Yasukazu Nakamura, Frédéric Berger, Catherine Adam, Shiori Sugamata Aki, Felix Althoff, Takashi Araki, Mario A Arteaga-Vazquez, Sureshkumar Balasubrmanian, Kerrie Barry, Diane Bauer, Christian R Boehm, Liam Briginshaw, Juan Caballero-Perez, Bruno Catarino, Feng Chen, Shota Chiyoda, Mansi Chovatia, Kevin M Davies, Mihails Delmans, Taku Demura, Tom Dierschke, Liam Dolan, Ana E Dorantes-Acosta, D Magnus Eklund, Stevie N Florent, Eduardo Flores-Sandoval, Asao Fujiyama, Hideya Fukuzawa, Bence Galik, Daniel Grimanelli, Jane Grimwood, Ueli Grossniklaus, Takahiro Hamada, Jim Haseloff, Alexander J Hetherington, Asuka Higo, Yuki Hirakawa, Hope N Hundley, Yoko Ikeda, Keisuke Inoue, Shin-Ichiro Inoue, Sakiko Ishida, Qidong Jia, Mitsuru Kakita, Takehiko Kanazawa, Yosuke Kawai, Tomokazu Kawashima, Megan Kennedy, Keita Kinose, Toshinori Kinoshita, Yuji Kohara, Eri Koide, Kenji Komatsu, Sarah Kopischke, Minoru Kubo, Junko Kyozuka, Ulf Lagercrantz, Shih-Shun Lin, Erika Lindquist, Anna M Lipzen, Chia-Wei Lu, Efraín De Luna, Robert A Martienssen, Naoki Minamino, Masaharu Mizutani, Miya Mizutani, Nobuyoshi Mochizuki, Isabel Monte, Rebecca Mosher, Hideki Nagasaki, Hirofumi Nakagami, Satoshi Naramoto, Kazuhiko Nishitani, Misato Ohtani, Takashi Okamoto, Masaki Okumura, Jeremy Phillips, Bernardo Pollak, Anke Reinders, Moritz Rövekamp, Ryosuke Sano, Shinichiro Sawa, Marc W Schmid, Makoto Shirakawa, Roberto Solano, Alexander Spunde, Noriyuki Suetsugu, Sumio Sugano, Akifumi Sugiyama, Rui Sun, Yutaka Suzuki, Mizuki Takenaka, Daisuke Takezawa, Hirokazu Tomogane, Masayuki Tsuzuki, Takashi Ueda, Masaaki Umeda, John M Ward, Yuichiro Watanabe, Kazufumi Yazaki, Ryusuke Yokoyama, Yoshihiro Yoshitake, Izumi Yotsui, Sabine Zachgo, Jeremy Schmutz

    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.

    05 Oct. 2017, Cell, 171 (2), 287 - 304, English, International magazine

    [Refereed]

    Scientific journal

  • Masaru Nakayasu, Naoyuki Umemoto, Kiyoshi Ohyama, Yoshinori Fujimoto, Hyoung Jae Lee, Bunta Watanabe, Toshiya Muranaka, Kazuki Saito, Yukihiro Sugimoto, Masaharu Mizutani

    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), Sep. 2017, Plant Physiology, 175 (1), 120 - 133, English, International magazine

    [Refereed]

    Scientific journal

  • Ayman Abdelkareem, Chonprakun Thagun, Masaru Nakayasu, Masaharu Mizutani, Takashi Hashimoto, Tsubasa Shoji

    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, Jul. 2017, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 489 (2), 206 - 210, English

    [Refereed]

    Scientific journal

  • Ryota Akiyama, Masaru Nakayasu, Naoyuki Umemoto, Toshiya Muranaka, Masaharu Mizutani

    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.

    The Japanese Society for Chemical Regulation of Plants, Jun. 2017, Regulation of Plant Growth and Development, 52 (1), 92 - 98, Japanese

    [Refereed][Invited]

  • Essential role of the PSI-LHCII supercomplex in photosystem acclimation to light and/or heat conditions by state transitions.

    Marutani, Y, Yamauchi, Y, Higashiyama, M, Miyoshi, A, Akimoto, S, Inoue, K, Ikeda, K, Mizutani, M, Sugimoto, Y

    2017, Photosynthesis Research, 131, 41 - 50, English

    [Refereed]

    Scientific journal

  • Hyoung Jae Lee, Bunta Watanabe, Masaru Nakayasu, Michio Onjo, Yukihiro Sugimoto, Masaharu Mizutani

    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, Bioscience, Biotechnology and Biochemistry, 81 (12), 2253 - 2260, English

    [Refereed]

    Scientific journal

  • Akane Kamiyama, Mado Nakajima, Liyou Han, Kei Wada, Masaharu Mizutani, Yukiko Tabuchi, Akiko Kojima-Yuasa, Isao Matsui-Yuasa, Hideyuki Suzuki, Keiichi Fukuyama, Bunta Watanabe, Jun Hiratake

    Elsevier BV, Nov. 2016, Bioorganic & Medicinal Chemistry, 24 (21), 5340 - 5352

    [Refereed]

    Scientific journal

  • Yosuke KAWAI, Eiichiro ONO, Masaharu MIZUTANI

    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.

    Japan Society for Bioscience, Biotechnology, and Agrochemistry, Sep. 2016, KAGAKU TO SEIBUTSU, 54 (9), 640 - 649, Japanese

    [Refereed][Invited]

    Scientific journal

  • Naoyuki Umemoto, Masaru Nakayasu, Kiyoshi Ohyama, Mari Yotsu-Yamashita, Masaharu Mizutani, Hikaru Seki, Kazuki Saito, Toshiya Muranaka

    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, Aug. 2016, PLANT PHYSIOLOGY, 171 (4), 2458 - 2467, English

    [Refereed]

    Scientific journal

  • Mikiko Kunishima, Yasuo Yamauchi, Masaharu Mizutani, Masaki Kuse, Hirosato Takikawa, Yukihiro Sugimoto

    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, Jul. 2016, JOURNAL OF BIOLOGICAL CHEMISTRY, 291 (27), 14023 - 14033, English

    [Refereed]

    Scientific journal

  • Mikiko Kunishima, Yasuo Yamauchi, Masaharu Mizutani, Masaki Kuse, Hirosato Takikawa, Yukihiro Sugimoto

    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, Jul. 2016, JOURNAL OF BIOLOGICAL CHEMISTRY, 291 (27), 14023 - 14033, English

    [Refereed]

    Scientific journal

  • Masaru Nakayasu, Takashi Kawasaki, Hyoung Jae Lee, Yukihiro Sugimoto, Michio Onjo, Toshiya Muranaka, Masaharu Mizutani

    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.

    JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY, Dec. 2015, PLANT BIOTECHNOLOGY, 32 (4), 299 - U141, English

    [Refereed]

    Scientific journal

  • Kotomi Ueno, Shunsuke Ishiwa, Hitomi Nakashima, Masaharu Mizutani, Hirosato Takikawa, Yukihiro Sugimoto

    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, Sep. 2015, BIOORGANIC & MEDICINAL CHEMISTRY, 23 (18), 6100 - 6110, English

    [Refereed]

    Scientific journal

  • Kotomi Ueno, Shunsuke Ishiwa, Hitomi Nakashima, Masaharu Mizutani, Hirosato Takikawa, Yukihiro Sugimoto

    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, Sep. 2015, BIOORGANIC & MEDICINAL CHEMISTRY, 23 (18), 6100 - 6110, English

    [Refereed]

    Scientific journal

  • Shoji Ohgami, Eiichiro Ono, Manabu Horikawa, Jun Murata, Koujirou Totsuka, Hiromi Toyonaga, Yukie Ohba, Hideo Dohra, Tatsuo Asai, Kenji Matsui, Masaharu Mizutani, Naoharu Watanabe, Toshiyuki Ohnishi

    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, Jun. 2015, PLANT PHYSIOLOGY, 168 (2), 464 - 477, English

    [Refereed]

    Scientific journal

  • Yasuo Yamauchi, Mikiko Kunishima, Masaharu Mizutani, Yukihiro Sugimoto

    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, Jan. 2015, SCIENTIFIC REPORTS, 5, 8030, English

    [Refereed]

    Scientific journal

  • Yasuo Yamauchi, Mikiko Kunishima, Masaharu Mizutani, Yukihiro Sugimoto

    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, Jan. 2015, SCIENTIFIC REPORTS, 5, 8030, English

    [Refereed]

    Scientific journal

  • Yoko Marutani, Yasuo Yamauchi, Akihito Miyoshi, Kanako Inoue, Ken-ichi Ikeda, Masaharu Mizutani, Yukihiro Sugimoto

    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, Dec. 2014, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 15 (12), 23042 - 23058, English

    [Refereed]

    Scientific journal

  • Kotomi Ueno, Toshio Furumoto, Shuhei Umeda, Masaharu Mizutani, Hirosato Takikawa, Rossitza Batchvarova, Yukihiro Sugimoto

    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, Dec. 2014, PHYTOCHEMISTRY, 108, 122 - 128, English

    Scientific journal

  • Akio Noguchi, Manabu Horikawa, Jun Murata, Masayuki Tera, Yosuke Kawai, Masaji Ishiguro, Toshiaki Umezawa, Masaharu Mizutani, Eiichiro Ono

    (+)-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, Dec. 2014, Plant Biotechnology, 31 (5), 493 - 503, English

    [Refereed]

    Scientific journal

  • Atsushi Okazawa, Tatsuya Kusunose, Eiichiro Ono, Hyun Jung Kim, Honoo Satake, Bun-ichi Shimizu, Masaharu Mizutani, Hikaru Seki, Toshiya Muranaka

    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, Dec. 2014, PLANT BIOTECHNOLOGY, 31 (5), 561 - 566, English

    [Refereed]

    Scientific journal

  • Yosuke Kawai, Eiichiro Ono, Masaharu Mizutani

    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, Dec. 2014, PLANT BIOTECHNOLOGY, 31 (5), 579 - 584, English

    [Refereed]

    Scientific journal

  • S. Kondo, S. Sugaya, M. Kittikorn, Y. Todoroki, M. Mizutani, N. Hirai

    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.

    20 Jul. 2014, Acta Horticulturae, 1042, 151 - 157

    [Refereed]

    Scientific journal

  • Hiromichi Saino, Tetsuya Shimizu, Jun Hiratake, Toru Nakatsu, Hiroaki Kato, Kanzo Sakata, Masaharu Mizutani

    Elsevier BV, Jun. 2014, Journal of Biological Chemistry, 289 (24), 16826 - 16834

    Scientific journal

  • Yosuke Kawai, Eiichiro Ono, Masaharu Mizutani

    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, Apr. 2014, PLANT JOURNAL, 78 (2), 328 - 343, English

    [Refereed]

    Scientific journal

  • Saki Nomura, Hitomi Nakashima, Masaharu Mizutani, Hirosato Takikawa, Yukihiro Sugimoto

    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.

    Jun. 2013, Plant Cell Reports, 32 (6), 829 - 838, English

    Scientific journal

  • Noriko Motonami, Kotomi Ueno, Hitomi Nakashima, Saki Nomura, Masaharu Mizutani, Hirosato Takikawa, Yukihiro Sugimoto

    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, Phytochemistry, 93, 41 - 48, English

    Scientific journal

  • Tomoaki Sakamoto, Toshiyuki Ohnishi, Shozo Fujioka, Bunta Watanabe, Masaharu Mizutani

    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, Sep. 2012, PLANT PHYSIOLOGY AND BIOCHEMISTRY, 58, 220 - 226, English

    [Refereed]

    Scientific journal

  • Toshiyuki Ohnishi, Blanka Godza, Bunta Watanabe, Shozo Fujioka, Lidia Hategan, Kouhei Ide, Kiyomi Shibata, Takao Yokota, Miklos Szekeres, Masaharu Mizutani

    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, Sep. 2012, JOURNAL OF BIOLOGICAL CHEMISTRY, 287 (37), 31551 - 31560, English

    [Refereed]

    Scientific journal

  • Marutani Yoko, Yamauchi Yasuo, Kimura Yukihiro, Mizutani Masaharu, Sugimoto Yukihiro

    Aug. 2012, PLANTA, 236 (2), 753 - 761

    [Refereed]

  • Masaharu Mizutani

    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, Jun. 2012, BIOLOGICAL & PHARMACEUTICAL BULLETIN, 35 (6), 824 - 832, English

    [Refereed]

    Scientific journal

  • Yasushi Todoroki, Kumi Naiki, Taku Muramatsu, Toshiyuki Ohnishi, Kotomi Ueno, Masaharu Mizutani, Nobuhiro Hirai

    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, May 2012, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 22 (9), 3240 - 3243, English

    [Refereed]

    Scientific journal

  • Mariko Okazaki, Monrudee Kittikorn, Kotomi Ueno, Masaharu Mizutani, Nobuhiro Hirai, Satoru Kondo, Toshiyuki Ohnishi, Yasushi Todoroki

    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, May 2012, BIOORGANIC & MEDICINAL CHEMISTRY, 20 (10), 3162 - 3172, English

    [Refereed]

    Scientific journal

  • Guilhem Vialart, Alain Hehn, Alexandre Olry, Kyoko Ito, Celia Krieger, Romain Larbat, Cedric Paris, Bun-Ichi Shimizu, Yukihiro Sugimoto, Masaharu Mizutani, Frederic Bourgaud

    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.

    May 2012, Plant Journal, 70 (3), 460 - 470, English

    [Refereed]

    Scientific journal

  • Bornali Gohain, Sangeeta Borchetia, Priyadarshini Bhorali, Niraj Agarwal, L. P. Bhuyan, A. Rahman, K. Sakata, M. Mizutani, B. Shimizu, G. Gurusubramaniam, R. Ravindranath, M. C. Kalita, M. Hazarika, Sudripta Das

    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, Apr. 2012, PLANT MOLECULAR BIOLOGY, 78 (6), 577 - 597, English

    [Refereed]

    Scientific journal

  • Yasuo Yamauchi, Ayaka Hasegawa, Masaharu Mizutani, Yukihiro Sugimoto

    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, Apr. 2012, FEBS LETTERS, 586 (8), 1208 - 1213, English

    [Refereed]

    Scientific journal

  • Seitaro Matsumoto, Masaharu Mizutani, Kanzo Sakata, Bun-Ichi Shimizu

    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, Feb. 2012, PHYTOCHEMISTRY, 74, 49 - 57, English

    [Refereed]

    Scientific journal

  • Satoru Kondo, Sumiko Sugaya, Shun Sugawa, Maki Ninomiya, Monrudee Kittikorn, Katsuya Okawa, Hitoshi Ohara, Kotomi Ueno, Yasushi Todoroki, Masaharu Mizutani, Nobuhiro Hirai

    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, Feb. 2012, JOURNAL OF PLANT PHYSIOLOGY, 169 (3), 234 - 241, English

    [Refereed]

    Scientific journal

  • Ery O. Fukushima, Hikaru Seki, Kiyoshi Ohyama, Eiichiro Ono, Naoyuki Umemoto, Masaharu Mizutani, Kazuki Saito, Toshiya Muranaka

    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, Dec. 2011, PLANT AND CELL PHYSIOLOGY, 52 (12), 2050 - 2061, English

    [Refereed]

    Scientific journal

  • Hikaru Seki, Satoru Sawai, Kiyoshi Ohyama, Masaharu Mizutani, Toshiyuki Ohnishi, Hiroshi Sudo, Ery Odette Fukushima, Tomoyoshi Akashi, Toshio Aoki, Kazuki Saito, Toshiya Muranaka

    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, Nov. 2011, PLANT CELL, 23 (11), 4112 - 4123, English

    [Refereed]

    Scientific journal

  • Kotomi Ueno, Saki Nomura, Satoru Muranaka, Masaharu Mizutani, Hirosato Takikawa, Yukihiro Sugimoto

    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, Oct. 2011, JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 59 (19), 10485 - 10490, English

    Scientific journal

  • Ueno Kotomi, Nomura Saki, Fujiwara Mami, Muranaka Satoru, Mizutani Masaharu, Sasaki Mitsuru, Takikawa Hirosato, Sugimoto Yukihiro

    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).

    Symposium on the chemistry of natural products, 02 Sep. 2011, Symposium on the Chemistry of Natural Products, symposium papers, (53), 313 - 318, Japanese

  • Kotomi Ueno, Mami Fujiwara, Saki Nomura, Masaharu Mizutani, Mitsuru Sasaki, Hirosato Takikawa, Yukihiro Sugimoto

    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, Sep. 2011, JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 59 (17), 9226 - 9231, English

    [Refereed]

    Scientific journal

  • Yasuo Yamauchi, Yukihiro Kimura, Seiji Akimoto, Yoko Marutani, Masaharu Mizutani, Yukihiro Sugimoto

    AbstractPlants 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, 01 Aug. 2011, Nature Precedings

    Scientific journal

  • Tomoaki Sakamoto, Ayami Kawabe, Asako Tokida-Segawa, Bun-ichi Shimizu, Suguru Takatsuto, Yukihisa Shimada, Shozo Fujioka, Masaharu Mizutani

    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, Jul. 2011, PLANT JOURNAL, 67 (1), 1 - 12, English

    [Refereed]

    Scientific journal

  • Yasushi Todoroki, Kenta Narita, Taku Muramatsu, Hajime Shimomura, Toshiyuki Ohnishi, Masaharu Mizutani, Kotomi Ueno, Nobuhiro Hirai

    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, Mar. 2011, BIOORGANIC & MEDICINAL CHEMISTRY, 19 (5), 1743 - 1750, English

    [Refereed]

    Scientific journal

  • Yasuo Yamauchi, Ayaka Hasegawa, Ai Taninaka, Masaharu Mizutani, Yukihiro Sugimoto

    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, Mar. 2011, JOURNAL OF BIOLOGICAL CHEMISTRY, 286 (9), 6999 - 7009, English

    [Refereed]

    Scientific journal

  • Mariko Okazaki, Hataitip Nimitkeatkai, Taku Muramatsu, Hikaru Aoyama, Kotomi Ueno, Masaharu Mizutani, Nobuhiro Hirai, Satoru Kondo, Toshiyuki Ohnishi, Yasushi Todoroki

    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, Jan. 2011, BIOORGANIC & MEDICINAL CHEMISTRY, 19 (1), 406 - 413, English

    [Refereed]

    Scientific journal

  • Yasushi Todoroki, Kumi Naiki, Hikaru Aoyama, Minaho Shirakura, Kotomi Ueno, Masaharu Mizutani, Nobuhiro Hirai

    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, Sep. 2010, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 20 (18), 5506 - 5509, English

    [Refereed]

    Scientific journal

  • Tadashi Fujiwara, Sylvie Maisonneuve, Masayuki Isshiki, Masaharu Mizutani, Letian Chen, Hann Ling Wong, Tsutomu Kawasaki, Ko Shimamoto

    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, Apr. 2010, JOURNAL OF BIOLOGICAL CHEMISTRY, 285 (15), 11308 - 11313, English

    [Refereed]

    Scientific journal

  • Toshiyuki Ohnishi, Takao Yokota, Masaharu Mizutani

    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, Dec. 2009, PHYTOCHEMISTRY, 70 (17-18), 1918 - 1929, English

    [Refereed]

    Scientific journal

  • Yasushi Todoroki, Hikaru Aoyama, Saori Hiramatsu, Minaho Shirakuru, Hataitip Nimitkeatkai, Satoru Kondo, Kotomi Ueno, Masaharu Mizutani, Nobuhiro Hirai

    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, Oct. 2009, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 19 (19), 5782 - 5786, English

    [Refereed]

    Scientific journal

  • Yasushi Todoroki, Kyotaro Kobayashi, Minaho Shirakura, Hikaru Aoyama, Kokichi Takatori, Hataitip Nimitkeatkai, Mei-Hong Jin, Saori Hiramatsu, Kotomi Ueno, Satoru Kondo, Masaharu Mizutani, Nobuhiro Hirai

    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, Sep. 2009, BIOORGANIC & MEDICINAL CHEMISTRY, 17 (18), 6620 - 6630, English

    [Refereed]

    Scientific journal

  • Mikiko Kojima, Tomoe Kamada-Nobusada, Hirokazu Komatsu, Kentaro Takei, Takeshi Kuroha, Masaharu Mizutani, Motoyuki Ashikari, Miyako Ueguchi-Tanaka, Makoto Matsuoka, Koji Suzuki, Hitoshi Sakakibara

    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, Jul. 2009, PLANT AND CELL PHYSIOLOGY, 50 (7), 1201 - 1214, English

    [Refereed]

    Scientific journal

  • Hiromi Daiyasu, Hiromichi Saino, Hiroo Tomoto, Masaharu Mizutani, Kanzo Sakata, Hiroyuki Toh

    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, Oct. 2008, JOURNAL OF BIOCHEMISTRY, 144 (4), 467 - 475, English

    [Refereed]

    Scientific journal

  • Kosuke Kai, Masaharu Mizutani, Naohiro Kawamura, Ryotaro Yamamoto, Michiko Tamai, Hikaru Yamaguchi, Kanzo Sakata, Bun-ichi Shimizu

    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, Sep. 2008, PLANT JOURNAL, 55 (6), 989 - 999, English

    [Refereed]

    Scientific journal

  • Hikaru Seki, Kiyoshi Ohyama, Satoru Sawai, Masaharu Mizutani, Toshiyuki Ohnishi, Hiroshi Sudo, Tomoyoshi Akashi, Toshio Aoki, Kazuki Saito, Toshiya Muranaka

    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, Sep. 2008, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 105 (37), 14204 - 14209, English

    [Refereed]

    Scientific journal

  • Biosynthetic Origin of the 1-Oxygen of Umbelliferone in the Root Tissue of Sweet Potato

    Bun-ichi Shimizu, Kosuke Kai, Michiko Tamai, Hikaru Yamaguchi, Masaharu Mizutani, Kanzo Sakata

    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, Sep. 2008, ZEITSCHRIFT FUR NATURFORSCHUNG SECTION C-A JOURNAL OF BIOSCIENCES, 63 (9-10), 687 - 690, English

    [Refereed]

    Scientific journal

  • Tomoyuki Nakatsubo, Yu Kitamura, Norikazu Sakakibara, Masaharu Mizutani, Takefumi Hattori, Nozomu Sakurai, Daisuke Shibata, Shiro Suzuki, Toshiaki Umezawa

    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, Aug. 2008, JOURNAL OF WOOD SCIENCE, 54 (4), 312 - 317, English

    [Refereed]

    Scientific journal

  • Tomoyuki Nakatsubo, Masaharu Mizutani, Shiro Suzuki, Takefumi Hattori, Toshiaki Umezawa

    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, Jun. 2008, JOURNAL OF BIOLOGICAL CHEMISTRY, 283 (23), 15550 - 15557, English

    [Refereed]

    Scientific journal

  • Yasushi Todoroki, Kyotaro Kobayashi, Hidetaka Yoneyama, Saori Hiramatsu, Mei-Hong Jin, Bunta Watanabe, Masaharu Mizutani, Nobuhiro Hirai

    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, Mar. 2008, BIOORGANIC & MEDICINAL CHEMISTRY, 16 (6), 3141 - 3152, English

    [Refereed]

    Scientific journal

  • Hirornichl Saino, Masaharu Mizutani, Jun Hiratake, Kanzo Sakata

    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, Feb. 2008, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 72 (2), 376 - 383, English

    [Refereed]

    Scientific journal

  • Katsuhiko Sekimata, Toshiyuki Ohnishi, Masaharu Mizutani, Yasushi Todoroki, Sun-Young Han, Jun Uzawa, Shozo Fujioka, Koichi Yoneyama, Yasutomo Takeuchi, Suguru Takatsuto, Kanzo Sakata, Shigeo Yoshida, Tadao Asami

    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, Jan. 2008, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 72 (1), 7 - 12, English

    [Refereed]

    Scientific journal

  • Hajime Shimomura, Hideo Etoh, Masaharu Mizutani, Nobuhiro Hirai, Yasushi Todoroki

    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, Sep. 2007, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 17 (17), 4977 - 4981, English

    [Refereed]

    Scientific journal

  • Kotomi Ueno, Hidetaka Yoneyama, Masaharu Mizutani, Nobuhiro Hirai, Yasushi Todoroki

    \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, Sep. 2007, BIOORGANIC & MEDICINAL CHEMISTRY, 15 (18), 6311 - 6322, English

    [Refereed]

    Scientific journal

  • Young Ock Ahn, Hiromichi Saino, Masaharu Mizutani, Bun-ichi Shimizu, Kanzo Sakata

    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, Jul. 2007, PLANT AND CELL PHYSIOLOGY, 48 (7), 938 - 947, English

    [Refereed]

    Scientific journal

  • Jeong-Yong Cho, Masaharu Mizutani, Bun-ichi Shimizu, Tomomi Kinoshita, Miharu Ogura, Kazuhiko Tokoro, Mu-Lien Lin, Kanzo Sakata

    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, Jun. 2007, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 71 (6), 1476 - 1486, English

    [Refereed]

    Scientific journal

  • Asada Takayuki, Sonoda Tetsuya, Honma Tamaki, Furujyo Atsushi, Shibata Masaru, Mizutani Masaharu, Hirai Nobuhiro, Tomizawa Ken'ichi, Sakaino Makoto

    THE JAPANESE FORESTRY SOCIETY, 2007, The Japanese Forest Society Congress, 118, 119 - 119, Japanese

  • Ide Kohei, Ohnishi Toshiyuki, Yokota Takao, Sakata Kanzo, Mizutani Masaharu

    Brassinosteroids (BRs) are thought to be biosynthesized from campesterol via campestanol in plants. Recently, we have characterized that Arabidopsis CYP90C1 and CYP90D1 are redundant C-23 hydroxylases. Analyses of their substrate specificities revealed a novel shortcut in BR biosynthesis, which allows direct conversion of early 22-hydroxylated intermediates to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol via C-23 hydroxylation. Here, we report biochemical characterization of this novel shortcut, focusing on 5α-reductase (DET2) and C-3 oxidase. These recombinant enzymes were heterogenously expressed, and their activities were measured in vitro. DET2 catalyzed 5α-reduction of not only (24R)-ergost-4-en-3-one (4-en-3-one) but also 22-hydroxy-4-en-3-one and 22,23-dihydroxy-4-en-3-one. The substrate specificity of C-3 oxidase showed that (22S)-22-hydroxycampesterol is a better substrate than (22R,23R)-22,23-hydroxycampesterol, whereas campesterol is not metabolized. Thus, the shortcut is likely a main route of BR biosynthetic pathway.

    The Japanese Society of Plant Physiologists, 2007, Plant and Cell Physiology Supplement, 2007 (0), 409 - 409

  • Ohnishi Toshiyuki, Bancos Simona, Watanabe Bunta, Fujioka Shozo, Yokota Takao, Sakata Kanzo, Szekeres Miklos, Mizutani Masaharu

    Brassinosteroids (BRs) are plant steroid hormones that are essential for normal plant growth and development. Cytochrome P450 monooxygenases (P450s) play crucial roles in BR biosynthesis from campesterol to brassinolide. Recent molecular genetic studies for BR-deficient mutants of Arabidopsis, rice, tomato and garden pea have identified several P450 genes (CYP85A, 90A, 90B, 90C, 90D, and 724B) so far. Recently, we have characterized Arabidopsis CYP90C1 and CYP90D1 functionally expressed in insect cells and found that CYP90C1 and CYP90D1 are redundant C-23 hydroxylases. Here, we report biochemical characterization of a P450, C-3 oxidase. The P450 was expressed in insect cells using a baculovirus expression system. The P450 activity was measured in an in vitro assay and the reaction products were analyzed by GC-MS. These results indicated that the P450 is BR C-3 oxidase. To our knowledge, this is the first report of the biochemical characterization of the C-3 oxidation by the P450.

    The Japanese Society of Plant Physiologists, 2007, Plant and Cell Physiology Supplement, 2007 (0), 733 - 733

  • T. Morikawa, M. Mizutani, D. Ohta

    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, Dec. 2006, BIOCHEMICAL SOCIETY TRANSACTIONS, 34, 1202 - 1205, English

    [Refereed]

    Scientific journal

  • Toshiyuki Ohnishi, Anna-Maria Szatmari, Bunta Watanabe, Satomi Fujita, Simona Bancos, Csaba Koncz, Marcel Lafos, Kyomi Shibata, Takao Yokota, Kanzo Sakata, Miklos Szekeres, Masaharu Mizutani

    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, Nov. 2006, PLANT CELL, 18 (11), 3275 - 3288, English

    [Refereed]

    Scientific journal

  • Toshiyuki Ohnishi, Takahito Nomura, Bunta Watanabe, Daisaku Ohta, Takao Yokota, Hisashi Miyagawa, Kanzo Sakata, Masaharu Mizutani

    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, Sep. 2006, PHYTOCHEMISTRY, 67 (17), 1895 - 1906, English

    [Refereed]

    Scientific journal

  • Toshiyuki Ohnishi, Bunta Watanabe, Kanzo Sakata, Masaharu Mizutani

    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, Sep. 2006, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 70 (9), 2071 - 2080, English

    [Refereed]

    Scientific journal

  • CHO Jeong Yong, Mizutani Masaharu, Shimizu Bun-ichi, Kinoshita Tomomi, Ogura Miharu, Tokoro Kazuhiko, Lin Mu-Lien, Sakata Kanzo

    Symposium on the chemistry of natural products, 23 Jul. 2006, International Symposium on the Chemistry of Natural Products, 2006, "P - 357", English

  • Shigeki Saito, Masanori Okamoto, Shoko Shinoda, Tetsuo Kushiro, Tomokazu Koshiba, Yuji Kamiya, Nobuhiro Hirai, Yasushi Todoroki, Kanzo Sakata, Eiji Nambara, Masaharu Mizutani

    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, Jul. 2006, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 70 (7), 1731 - 1739, English

    [Refereed]

    Scientific journal

  • Yoshiharu Araki, Arisa Miyawaki, Tomoyuki Miyashita, Masaharu Mizutani, Nobuhiro Hirai, Yasushi Todoroki

    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, Jun. 2006, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 16 (12), 3302 - 3305, English

    [Refereed]

    Scientific journal

  • Masaharu Mizutani, Yasushi Todoroki

    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.

    Jun. 2006, Phytochemistry Reviews, 5 (2-3), 385 - 404, English

    [Refereed]

    Scientific journal

  • T Morikawa, M Mizutani, N Aoki, B Watanabe, H Saga, S Saito, A Oikawa, H Suzuki, N Sakurai, D Shibata, A Wadano, K Sakata, D Ohta

    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, Apr. 2006, PLANT CELL, 18 (4), 1008 - 1022, English

    [Refereed]

    Scientific journal

  • S Fujita, T Ohnishi, B Watanabe, T Yokota, S Takatsuto, S Fujioka, S Yoshida, K Sakata, M Mizutani

    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, Mar. 2006, PLANT JOURNAL, 45 (5), 765 - 774, English

    [Refereed]

    Scientific journal

  • K Kai, B Shimizu, M Mizutani, K Watanabe, K Sakata

    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, Feb. 2006, PHYTOCHEMISTRY, 67 (4), 379 - 386, English

    [Refereed]

    Scientific journal

  • Identification and enzymatic characterization of the plant sterol C-22 desaturase

    N Aoki, T Morikawa, B Watanabe, S Saito, D Ohta, K Sakata, M Mizutani

    OXFORD UNIV PRESS, 2006, PLANT AND CELL PHYSIOLOGY, 47, S195 - S195, English

    [Refereed]

  • Cytochrome P450CYP710A encodes the sterol C-22 desaturase in plants

    T Morikawa, M Mizutani, N Aoki, B Watanabe, H Saga, S Saito, A Oikawa, H Suzuki, N Sakurai, D Shibata, A Wadano, K Sakata, D Ohta

    OXFORD UNIV PRESS, 2006, PLANT AND CELL PHYSIOLOGY, 47, S139 - S139, English

    [Refereed]

  • T Sakamoto, Y Morinaka, T Ohnishi, H Sunohara, S Fujioka, M Ueguchi-Tanaka, M Mizutani, K Sakata, S Takatsuto, S Yoshida, H Tanaka, H Kitano, M Matsuoka

    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, Jan. 2006, NATURE BIOTECHNOLOGY, 24 (1), 105 - 109, English

    [Refereed]

    Scientific journal

  • K Ueno, H Yoneyama, S Saito, M Mizutani, K Sakata, N Hirai, Y Todoroki

    (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, Dec. 2005, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 15 (23), 5226 - 5229, English

    [Refereed]

    Scientific journal

  • N Kitahata, S Saito, Y Miyazawa, T Umezawa, Y Shimada, YK Min, M Mizutani, N Hirai, K Shinozaki, S Yoshida, T Asami

    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, Jul. 2005, BIOORGANIC & MEDICINAL CHEMISTRY, 13 (14), 4491 - 4498, English

    [Refereed]

    Scientific journal

  • K Ueno, Y Araki, N Hirai, S Saito, M Mizutani, K Sakata, Y Todoroki

    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, May 2005, BIOORGANIC & MEDICINAL CHEMISTRY, 13 (10), 3359 - 3370, English

    [Refereed]

    Scientific journal

  • Ohnishi Toshiyuki, Watanabe bunta, Nakamura Masanobu, Nagatani Akira, Yokota Takao, Sakata Kanzo, Mizutani Masaharu

    Brassinosteroids (BRs) are steroidal plant hormones that regulate growth and development. The Arabidopsis mutant chibi2 was obtained by activation T-DNA tagging and showed a dwarf phenotype due to BR-deficiency. The chibi2 mutant has been found to overexpress a cytochrome P450 gene (CYP72C1), suggesting that the P450 catalyzes the inactivation of endogenous BRs. To investigate the biochemical properties of CHIBI2, the recombinant CHIBI2 protein was expressed using baculovirus expression system in insect cells (Sf9). CHIBI2 activity was measured in an assay mixture reconstituted with the recombinant CHIBI2, NADPH-P450 reductase, and NADPH generating system. We have identified several metabolites of BRs catalyzed by CHIBI2. Analysis of the structures of the metabolites is in progress.

    The Japanese Society of Plant Physiologists, 2005, Plant and Cell Physiology Supplement, 2005 (0), 764 - 764

  • Ohnishi Toshiyuki, Watanabe Bunta, Sakata Kanzo, Mizutani Masaharu

    Brassinosteroids (BRs) are plant steroid hormones that influence of important developmental processes. In BR biosynthesis pathway, several cytochrome P450 monooxygenases (P450) catalyze essential oxidative reactions, C-22 and C-23 hydroxylation of the BR side-chain and C-6 oxidation of 6-deoxointermediates. These P450 genes have been identified from Arabidopsis, tomato and rice. Phylogenitic analysis of plant P450s revealed that these P450s are closely related and clustered. In this cluster, there are several P450 genes whose functions are still unknown. Among them, we isolated a tomato cDNA encoding a novel P450 homologous to CYP90s. To investigate the biochemical properties of the novel P450, the recombinant P450 protein was expressed using a baculovirus expression system in insect cells. The enzyme activity was measured in an assay mixture reconstituted with the recombinant P450 protein, NADPH-P450 reductase and NADPH generating system. We have identified that the novel P450 catalyzes C-22 hydroxylation of BRs.

    The Japanese Society of Plant Physiologists, 2005, Plant and Cell Physiology Supplement, 2005 (0), 767 - 767

  • Mizutani Masaharu, Ahn Young-Ock, Saino Hiromichi, Sakata Kanzo

    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.

    Symposium on the chemistry of natural products, 01 Oct. 2004, Symposium on the Chemistry of Natural Products, symposium papers, (46), 665 - 670, Japanese

  • Redundancy or flexibility: Molecular diversity of the electron transfer components for P450 monooxygenases in higher plants

    D Ohta, M Mizutani

    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, May 2004, FRONTIERS IN BIOSCIENCE-LANDMARK, 9, 1587 - 1597, English

    [Refereed]

    Scientific journal

  • YO Ahn, M Mizutani, H Saino, K Sakata

    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, May 2004, JOURNAL OF BIOLOGICAL CHEMISTRY, 279 (22), 23405 - 23414, English

    [Refereed]

    Scientific journal

  • S Saito, N Hirai, C Matsumoto, H Ohigashi, D Ohta, K Sakata, M Mizutani

    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, Apr. 2004, PLANT PHYSIOLOGY, 134 (4), 1439 - 1449, English

    [Refereed]

    Scientific journal

  • Characterization of cytochromes P450 involved in ABA catabolism

    S Saito, C Matsumoto, N Hirai, H Ohigashi, D Ohta, M Mizutani, K Sakata

    OXFORD UNIV PRESS, 2004, PLANT AND CELL PHYSIOLOGY, 45, S76 - S76, English

    [Refereed]

  • Sonoda Tetsuya, Mikame Mariko, Honma Tamaki, Hirai Nobuhiro, Mizutani Masaharu, Tomizawa Kenichi, Asada Takayuki

    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.

    The Janapese Society for Chemical Regulation of Plants, 2004, The Janapese Society for Chemical Regulation of Plants, Abstract, 39, 44 - 44, Japanese

  • Improvement of Flavour Quality of CTC Black Tea By Glycosidases in Tea Leaves

    Sakata K, Mizutani Masaharu, Ma S-J, Guo W

    2004, Internat. J. Tea Sci, 3, 167 - 173, English

    [Refereed]

    Scientific journal

  • 馬 勝?, 水谷 正治, 平竹 潤, 林 謙太郎, 八木 健介, 渡辺 修治, 坂田 完三

    Japan Society for Bioscience, Biotechnology, and Agrochemistry, 01 Jul. 2003, 日本農芸化学会誌, 77 (7), 666 - 667, Japanese

  • K Takubo, T Morikawa, Y Nonaka, M Mizutani, S Takenaka, K Takabe, MA Takahashi, D Ohta

    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, Jul. 2003, PLANT MOLECULAR BIOLOGY, 52 (4), 817 - 830, English

    [Refereed]

    Scientific journal

  • K Inoue, J Hiratake, M Mizutani, M Takada, M Yamamoto, K Sakata

    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, Jul. 2003, CARBOHYDRATE RESEARCH, 338 (14), 1477 - 1490, English

    [Refereed]

    Scientific journal

  • T Asami, M Mizutani, Y Shimada, H Goda, N Kitahata, K Sekimata, SY Han, S Fujioka, S Takatsuto, K Sakata, S Yoshida

    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, Jan. 2003, BIOCHEMICAL JOURNAL, 369 (1), 71 - 76, English

    [Refereed]

    Scientific journal

  • M Mizutani, H Nakanishi, J Ema, SJ Ma, E Noguchi, M Inohara-Ochiai, M Fukuchi-Mizutani, M Nakao, K Sakata

    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, Dec. 2002, PLANT PHYSIOLOGY, 130 (4), 2164 - 2176, English

    [Refereed]

    Scientific journal

  • S Suzuki, M Mizutani, K Suzuki, M Yamada, M Kojima, H Hatanaka, S Koizumi

    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, Jun. 2002, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 294 (5), 1087 - 1092, English

    [Refereed]

    Scientific journal

  • Identification and characterization of a mitochondrial electron transfer chain in plants comprising of an adrenocortical ferredoxin homologue and its oxidoreductase

    K Takubo, Y Nonaka, M Mizutani, S Takenaka, S Tsuyama, D Ohta

    OXFORD UNIV PRESS, 2002, PLANT AND CELL PHYSIOLOGY, 43, S96 - S96, English

    [Refereed]

  • Functional analysis of tomato CYP72B involved in brassinosteroid catabolism

    O Toshiyuki, M Mizutani, D Ohta, K Sakata

    OXFORD UNIV PRESS, 2002, PLANT AND CELL PHYSIOLOGY, 43, S184 - S184, English

    [Refereed]

  • Asami Tadao, Mizutani Masaharu, Fujioka Shozo, Shimada Yukihisa, Takatsuto Suguru, Sakata Kanzo, Yoshida Shigeo

    The Japanese Society for Chemical Regulation of Plants, 2002, Regulation of Plant Growth & Development, 37 (2), 227 - 228, Japanese

  • Association of SH2-B to phosphorylated tyrosine residues in the activation loop of TRKB

    Kenji Suzuki, Masaharu Mizutani, Yoshiaki Hitomi, Takako Kizaki, Hideki Ohno, Hitoshi Ishida, Shukoh Haga, Shinichi Koizumi

    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, Research Communications in Molecular Pathology and Pharmacology, 111 (1-4), 27 - 39, English

    [Refereed]

    Scientific journal

  • SJ Ma, M Mizutani, J Hiratake, K Hayashi, K Yagi, N Watanabe, K Sakata

    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, Dec. 2001, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 65 (12), 2719 - 2729, English

    [Refereed]

    Scientific journal

  • T Asami, M Mizutani, S Fujioka, H Goda, YK Min, Y Shimada, T Nakano, S Takatsuto, T Matsuyama, N Nagata, K Sakata, S Yoshida

    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, Jul. 2001, JOURNAL OF BIOLOGICAL CHEMISTRY, 276 (28), 25687 - 25691, English

    [Refereed]

    Scientific journal

  • Analysis of tyrosine phosphorylation-dependent protein-protein interactions in TrkB-mediated intracellular signaling using modified yeast two-hybrid system

    M Yamada, K Suzuki, M Mizutani, A Asada, T Matozaki, T Ikeuchi, S Koizumi, H Hatanaka

    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, Jul. 2001, JOURNAL OF BIOCHEMISTRY, 130 (1), 157 - 165, English

    [Refereed]

    Scientific journal

  • Molecular cloning and characterization of ATP-phosphoribosyl transferase from Arabidopsis, a key enzyme in the histidine biosynthetic pathway

    D Ohta, K Fujimori, M Mizutani, Y Nakayama, R Kunpaisal-Hashimoto, S Munzer, A Kozaki

    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, Mar. 2000, PLANT PHYSIOLOGY, 122 (3), 907 - 914, English

    [Refereed]

    Scientific journal

  • M Fukuchi-Mizutani, M Mizutani, Y Tanaka, T Kusumi, D Ohta

    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, Jan. 1999, PLANT PHYSIOLOGY, 119 (1), 353 - 361, English

    [Refereed]

    Scientific journal

  • M Mizutani, E Ward, D Ohta

    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, May 1998, PLANT MOLECULAR BIOLOGY, 37 (1), 39 - 52, English

    [Refereed]

    Scientific journal

  • M Mizutani, D Ohta

    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, Jan. 1998, PLANT PHYSIOLOGY, 116 (1), 357 - 367, English

    [Refereed]

    Scientific journal

  • M Mizutani, D Ohta, R Sato

    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, Mar. 1997, PLANT PHYSIOLOGY, 113 (3), 755 - 763, English

    [Refereed]

    Scientific journal

  • PURIFICATION AND CHARACTERIZATION OF A CYTOCHROME-P450 (TRANS-CINNAMIC ACID 4-HYDROXYLASE) FROM ETIOLATED MUNG BEAN SEEDLINGS

    M MIZUTANI, D OHTA, R SATO

    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, Apr. 1993, PLANT AND CELL PHYSIOLOGY, 34 (3), 481 - 488, English

    [Refereed]

    Scientific journal

  • M MIZUTANI, E WARD, J DIMAIO, D OHTA, J RYALS, R SATO

    ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, Feb. 1993, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 190 (3), 875 - 880, English

    [Refereed]

    Scientific journal

  • A Sensitive and Microscale Method for Boron Determination of Plant Materials

    Matoh T, Matsunaga W, Mizutani Masaharu, Ohno K

    1993, Jpn. J. Soil Sci. Plant Nutr., 64, 71 - 73, English

    [Refereed]

    Scientific journal

  • 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

    T MATOH, K ISHIGAKI, M MIZUTANI, W MATSUNAGA, K TAKABE

    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, Dec. 1992, PLANT AND CELL PHYSIOLOGY, 33 (8), 1135 - 1141, English

    [Refereed]

    Scientific journal

MISC

  • 柑橘の薬物動態撹乱成分の生合成を担う芳香族基質O-プレニル化酵素遺伝子の同定

    棟方涼介, 棟方涼介, 棟方涼介, OLRY Alexandre, 竹村知陽, 巽奏, 市野琢爾, VILLARD Cloe, 影山丈士, 倉田哲也, 中安大, 森吉英子, 松川哲也, 松川哲也, GROSJEAN Jeremy, KRIGER Celia, 杉山暁史, 水谷正治, HEHN Alain, 矢崎一史

    2022, イソプレノイド研究会例会講演要旨集, 32nd (CD-ROM)

  • 2-oxoglutarate dependent dioxygenases involved in biosynthesis of steroidal glycoalkaloids

    秋山遼太, 中安大, 中安大, 渡辺文太, 加藤純平, LEE Hyoung Jae, 飯島陽子, 梅基直行, 村中俊哉, 齊藤和季, 齊藤和季, 杉本幸裕, 水谷正治

    2022, 日本植物生理学会年会(Web), 63rd

  • The apple gene responsible for columnar tree shape reduces biologically-active gibberellin

    中嶋正敏, 渡辺大智, 高橋郁夫, JAROENSANTI-TAKANA Naiyanate, 宮崎翔, JIANG Kai, 中安大, 和田雅人, 浅見忠男, 水谷正治, 岡田和馬

    2021, 日本農薬学会大会講演要旨集, 46th (CD-ROM)

  • The apple gene responsible for columnar tree shape reduces biologically active gibberellin

    中嶋正敏, 渡辺大智, 高橋郁夫, JAROENSANTI-TAKANA Naiyanate, 宮崎翔, JIANG Kai, 中安大, 和田雅人, 浅見忠男, 水谷正治, 岡田和馬

    2021, 日本農芸化学会大会講演要旨集(Web), 2021

  • 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

    岡田和馬, 和田雅人, 竹林裕美子, 小嶋美紀子, 榊原均, 中安大, 水谷正治, 中嶋正敏, 森谷茂樹, 清水拓, 阿部和幸, 渡辺大智, 高橋郁夫, JAROENSANTI-TAKANA Naiyanate, 宮崎翔, 姜凱, 浅見忠男

    2020, 農研機構果樹茶業研究部門成果情報(Web), 2020

  • Elucidation of biosynthetic pathway of canonical strigolactone

    若林孝俊, 若林孝俊, 濱名実咲, 森采美, 北野友里恵, 支田香澄, 秋山遼太, 刑部敬史, 刑部祐里子, 水谷正治, 杉本幸裕, 杉本幸裕

    2020, 日本農芸化学会大会講演要旨集(Web), 2020

  • Masaharu Mizutani

    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.

    01 Dec. 2019, Plant biotechnology (Tokyo, Japan), 36 (4), 253 - 263, English, Domestic magazine

  • 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

    野田蒼空, 秋山遼太, 李榮宰, 中安大, 刑部敬史, 刑部祐里子, 梅基直行, 斉藤和季, 村中俊哉, 杉本幸裕, 水谷正治

    2019, 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におけるソラニダン骨格形成機構の解析

    秋山遼太, 中安大, 李けい宰, 加藤純平, 梅基直行, 渡辺文太, 村中俊哉, 斉藤和季, 斉藤和季, 杉本幸裕, 水谷正治

    2019, 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

    清水宏祐, 増田裕貴, 秋山遼太, 坂田至, 串田篤彦, 谷野圭持, 刑部敬史, 刑部祐里子, 杉本幸裕, 水谷正治

    2019, 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

  • Metabolic Engineering of Glycoalkaloid-Free Potatoes Accumulating Useful Steroidal Saponins by Genome Editing

    Mizutani, M, Akiyama, R, Nakayasu, M, Lee, H.-J, Sugimoto, Y, Yasumoto, S, Sawai, S, Seki, H, Asano, K, Osakabe, K, Osakabe, Y, Watanabe, B, Umemoto, N, Saito, K, Muranaka, T

    May 2018, 10th World Potato Congress, Cusco, Peru, English

    Summary international conference

  • 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

    秋山遼太, 加藤純平, 李栄宰, 渡辺文太, 中安大, 小林緑, 飯島陽子, 梅基直行, 村中俊哉, 杉本幸裕, 水谷正治

    16 Mar. 2018, 日本農芸化学会2018年度大会,名古屋, 2018, Japanese

    Summary national conference

  • ゴマリグナン新規酸化酵素の同定

    村田純, 小埜栄一郎, 鎧塚清吾, 豊永宏美, 白石慧, 森祥子, 寺正行, 東鋭明, 永野惇, 永野惇, 中安大, 水谷正治, 若杉達也, 山本将之, 堀川学

    05 Mar. 2018, 日本農芸化学会大会講演要旨集(Web), 2018, ROMBUNNO.2A25p10 (WEB ONLY), Japanese

  • 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

    中安大, LEE Hyoung Jae, 刑部敬史, 刑部祐里子, 杉本幸裕, 村中俊哉, 水谷正治

    05 Mar. 2018, 日本農芸化学会大会講演要旨集(Web), 2018, ROMBUNNO.2A27p07 (WEB ONLY), Japanese

  • 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

    秋山遼太, 李ヒョン宰, 中安大, 刑部敬史, 刑部祐里子, 梅基直行, 村中俊哉, 杉本幸裕, 水谷正治

    2018, 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

    水谷正治, 秋山遼太, 加藤純平, 中安大, 李栄宰, 渡辺文太, 浅野賢治, 梅基直行, 斉藤和季, 村中俊哉

    2018, 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

  • 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

    藤本大輝, 秋山遼太, 中安大, 安本周平, 澤井学, 李榮宰, 李榮宰, 刑部祐里子, 刑部敬史, 梅基直行, 斉藤和季, 水谷正治, 村中俊哉

    2018, 日本農芸化学会関西支部講演会講演要旨集, 506th

  • 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

    野田蒼空, 秋山遼太, 李榮宰, 中安大, 刑部敬史, 刑部祐里子, 梅基直行, 村中俊哉, 杉本幸裕, 水谷正治

    2018, 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

  • ゲノム編集によるジャガイモCYP88B1遺伝子の破壊

    安本周平, LEE Hyoung Jae, 秋山遼太, 澤井学, 島津知華, 關光, 梅基直行, 水谷正治, 斉藤和季, 村中俊哉

    2018, 日本農芸化学会大会講演要旨集(Web), 2018

  • 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

    小埜栄一郎, 村田純, 鎧塚清吾, 豊永宏美, 白石慧, 森祥子, 寺正之, 東鋭明, 永野惇, 中安大, 水谷正治, 若杉達也, 山本将之, 堀川学

    2018, 日本植物生理学会年会(Web), 59th, ROMBUNNO.3aJ06, Japanese

  • MIZUTANI MASAHARU

    公益社団法人 日本農芸化学会, 2018, 化学と生物, 56 (8), 566 - 71, Japanese

    [Refereed]

    Introduction scientific journal

  • 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

    横田美晴, 村松佳祐, 野村崇人, 横田孝雄, 渡辺文太, 水谷正治, 大西利幸

    28 Oct. 2017, 植物化学調節学会第52回大会,鹿児島, 52 (Supplement), Japanese

    Summary national conference

  • 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

    加藤純平, 李栄宰, 渡辺文太, 中安大, 小林緑, 飯島陽子, 村中俊哉, 杉本幸裕, 水谷正治

    31 Aug. 2017, 第35回日本植物細胞分子生物学会大会,さいたま, 35th, Japanese

    Summary national conference

  • ジャガイモα-ソラニン生合成遺伝子St16DOXのゲノム編集

    秋山遼太, 中安大, 李栄宰, 刑部敬史, 刑部祐里子, 渡辺文太, 梅基直行, 村中俊哉, 斉藤和季, 杉本幸裕, 水谷正治

    29 Aug. 2017, 第35回日本植物細胞分子生物学会大会,さいたま, 35th, Japanese

    Summary national conference

  • 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

    秋山遼太, 中安大, LEE Hyong Jae, 刑部敬史, 刑部祐里子, 梅基直行, 村中俊哉, 斉藤和季, 杉本幸裕, 水谷正治

    05 Mar. 2017, 日本農芸化学会大会講演要旨集(Web), 2017, ROMBUNNO.2J31p06 (WEB ONLY), Japanese

  • 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

    中安大, LEE Hyoung Jae, 杉本幸裕, 鈴木秀幸, 遠城道雄, 村中俊哉, 水谷正治

    2017, 植物の生長調節, 52 (Supplement)

  • 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

    庄司翼, ABDELKAREEM Ayman, CHONPRAKUN Thagun, 中安大, 水谷正治, 橋本隆

    2017, 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

    中安大, LEE Hyoung Jae, 杉本幸裕, 鈴木秀幸, 遠城道雄, 村中俊哉, 水谷正治

    2017, 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

    LEE Hyoung Jae, 小林緑, 中安大, 杉本幸裕, 水谷正治

    2017, 日本農芸化学会大会講演要旨集(Web), 2017

  • 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

    中安大, 李けい宰, 杉本幸裕, 鈴木秀幸, 遠城道雄, 村中俊哉, 水谷正治

    2017, 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

  • TALENによるジャガイモCYP88B1遺伝子の破壊

    安本周平, 澤井学, 島津知華, 關光, 梅基直行, 水谷正治, 斉藤和季, 村中俊哉

    2017, 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

    梅基直行, 安本周平, 澤井学, 關光, 李けい宰, 水谷正治, 浅野賢治, 斉藤和季, 村中俊哉

    2017, 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

    清水宏祐, 中安大, 李伶, 梅野太輔, 杉本幸裕, 水谷正治

    2017, 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

  • Oxidation of (+)-sesamin by CYP92B14 in sesame

    村田純, 小埜栄一郎, 鎧塚清吾, 豊永宏美, 白石慧, 森祥子, 寺正行, 東鋭明, 永野惇, 永野惇, 中安大, 水谷正治, 若杉達也, 山本将之, 堀川学

    2017, 天然有機化合物討論会講演要旨集(Web), 59th

  • MIZUTANI MASAHARU

    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.

    The Japanese Society for Chemical Regulation of Plants, 2017, 植物の化学調節, 52 (2), 92 - 98, Japanese

    [Refereed][Invited]

    Introduction scientific journal

  • 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

    秋山遼太, 中安大, 李榮宰, 刑部敬史, 刑部祐里子, 梅基直行, 村中俊哉, 斉藤和季, 杉本幸裕, 水谷正治

    01 Dec. 2016, 日本農芸化学会関西支部講演会講演要旨集, 497th, 10, Japanese

  • 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

    加藤純平, 李栄宰, 渡辺文太, 中安大, 小林緑, 飯島陽子, 村中俊哉, 杉本幸裕, 水谷正治

    02 Sep. 2016, 第34回日本植物細胞分子生物学会大会,上田, 34th, Japanese

    Summary national conference

  • 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

    水谷正治, 秋山遼太, 中安大, 李栄宰, 刑部敬史, 刑部祐里子, 梅基直行, 斉藤和希, 杉本幸裕, 村中俊哉

    01 Sep. 2016, 日本植物学会大会研究発表記録, 80th, 178, Japanese

  • 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

    秋山遼太, 中安大, 李えい宰, 刑部敬史, 刑部祐里子, 梅基直行, 斉藤和季, 村中俊哉, 杉本幸裕, 水谷正治

    20 Aug. 2016, 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, Japanese

  • 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

    中安大, 梅基直行, 大山清, 宮地陽香, 渡辺文太, 村中俊哉, 斉藤和季, 杉本幸裕, 水谷正治

    29 Mar. 2016, 日本農芸化学会2016年度大会,札幌, 2016, Japanese

    Summary national conference

  • 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

    中安大, 梅基直行, 大山清, 宮地陽香, 渡辺文太, 村中俊哉, 斉藤和季, 杉本幸裕, 水谷正治

    19 Mar. 2016, 第57回日本植物生理学会年会,岩手, Japanese

    Summary national conference

  • 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

    秋山遼太, 中安大, LEE Hyoung‐Jae, 刑部敬史, 刑部祐里子, 梅基直行, 村中俊哉, 斉藤和希, 斉藤和希, 杉本幸裕, 水谷正治

    05 Mar. 2016, 日本農芸化学会大会講演要旨集(Web), 2016, 2H043 (WEB ONLY), Japanese

  • 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

    井上直人, 李榮宰, 中安大, 梅基直行, 斉藤和季, 村中俊哉, 鈴木秀幸, 杉本幸裕, 水谷正治

    2016, 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

    宮田朋子, 李えい宰, 中安大, 梅基直行, 斉藤和季, 澤井学, 大山清, 大山清, 村中俊哉, 杉本幸裕, 水谷正治

    2016, 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

    中安大, 李えい宰, 杉本幸裕, 鈴木秀幸, 遠城道雄, 村中俊哉, 水谷正治

    2016, 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

  • 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

    中安大, 李ヒョンゼ, 杉本幸裕, 鈴木秀幸, 遠城道雄, 村中俊哉, 水谷正治

    2016, 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ステロイド3位酸化異性化酵素の解析

    LEE Hyoung Jae, 小林緑, 中安大, 杉本幸裕, 水谷正治

    2016, 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A環酸化還元酵素の機能解析

    李ヒョンゼ, 小林緑, 中安大, 杉本幸裕, 水谷正治

    2016, 日本植物学会大会研究発表記録, 80th

  • 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

    宮地陽香, LEE Hyoung Jae, 山村理恵, 川崎崇, 中安大, 遠城道雄, 杉本幸裕, 水谷正治

    2016, 日本農芸化学会大会講演要旨集(Web), 2016

  • 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

    東山真理, 青木仁美, 山内靖雄, 中安大, 水谷正治, 杉本幸裕

    2016, 日本農芸化学会関西支部講演会講演要旨集, 497th

  • 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

    梅基直行, 安本周平, 澤井学, 關光, 李えい宰, 水谷正治, 佐久間哲史, 山本卓, 斉藤和季, 村中俊哉

    2016, 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

  • P099 Germination and stomatal responses of Striga hermonthica to abscisic acid

    Fujioka Hijiri, Inoue Tomoe, Samejima Hiroaki, Ueno Kotomi, Mizutani Masaharu, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2015, 植物化学調節学会研究発表記録集, 50, 117 - 117, Japanese

  • P089 Identification of 3-hydroxysteroid dehydrogenase involved in α-tomatine biosynthesis from tomato

    Lee Hyoung Jae, Kobayashi Midori, Nakayasu Masaru, Sugimoto Yukihiro, Mizutani Masaharu

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2015, 植物化学調節学会研究発表記録集, 50, 107 - 107, Japanese

  • P037 RNA-seq analysis to investigate orobanchol biosynthesis genes in cowpea

    Mori Ayami, Ueno Kotomi, Suzuki Hideyuki, Mizutani Masaharu, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2015, 植物化学調節学会研究発表記録集, 50, 55 - 55, Japanese

  • P036 Analysis of strigolactone biosynthetsis in various plants

    Iseki Moe, Mizutani Masaharu, Takikawa Hirosato, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2015, 植物化学調節学会研究発表記録集, 50, 54 - 54, Japanese

  • P035 RNA-seq screening of sorgomol synthase involved in the conversion of 5-deoxystrigol to sorgomol in sorghum

    Ishiwa Shunsuke, Ueno Kotomi, Suzuki Hideyuki, Mizutani Masaharu, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2015, 植物化学調節学会研究発表記録集, 50, 53 - 53, Japanese

  • P034 Bioorganic chemical studies on strigolactone biosynthesis

    Ueno Kotomi, Nakashima Hitomi, Mizutani Masaharu, Takikawa Hirosato, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2015, 植物化学調節学会研究発表記録集, 50, 52 - 52, Japanese

  • P079 Identification and characterization of a novel enzyme, hexenal isomerase in response to wounding in plants

    Kunishima Mikiko, Yamauchi Yasuo, Mizutani Masaharu, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2015, 植物化学調節学会研究発表記録集, 50, 97 - 97, Japanese

  • Kawai Yosuke, Ono Eiichiro, MIZUTANI MASAHARU

    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, Sep. 2015, 化学と生物, 54 (9), 640 - 649, Japanese

    [Refereed][Invited]

    Introduction scientific journal

  • 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

    中安大, 梅基直行, 大山清, 宮地陽香, 渡辺文太, 村中俊哉, 斉藤和季, 杉本幸裕, 水谷正治

    11 Aug. 2015, 第33回日本植物細胞分子生物学会大会,東京, 33rd, Japanese

    Summary national conference

  • 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

    中安大, 梅基直行, 大山清, 渡辺文太, 村中俊哉, 斉藤和季, 杉本幸裕, 水谷正治

    27 Mar. 2015, 日本農芸化学会2014年度大会,岡山, 2015, Japanese

    Summary national conference

  • 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

    中安大, 梅基直行, 大山清, 大山清, 渡辺文太, 村中俊哉, 斉藤和季, 杉本幸裕, 水谷正治

    2015, 日本植物生理学会年会要旨集, 56th

  • 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

    梅基直行, 大山清, 中安大, 水谷正治, 村中俊哉, 斉藤和季, 斉藤和季

    2015, 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

  • α-トマチン生合成に関わる3位酸化異性化酵素の解析

    李ヒョンゼ, 小林緑, 中安大, 杉本幸裕, 水谷正治

    2015, 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

  • 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

    LEE Hyoung Jae, 小林緑, 中安大, 杉本幸裕, 水谷正治

    2015, 植物の生長調節, 50 (Supplement)

  • 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

    佐原瑞穂, 渡辺文太, 横田孝雄, 渡辺修治, 水谷正治, 大西利幸

    18 Oct. 2014, 植物化学調節学会第49回大会,京都, Japanese

    Summary national conference

  • 67. Heliolactone, a non-sesquiterpene lactone germination stimulant for root parasitic weeds from sunflower

    Ueno Kotomi, Furumoto Toshio, Umeda Shuhei, Mizutani Masaharu, Takikawa Hirosato, Batchvarova Rossitza, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2014, 植物化学調節学会研究発表記録集, 49, 85 - 85, Japanese

  • 69. Regioselective and stereospecific hydroxylation of GR24 by sorghum

    Ishiwa Shunsuke, Ueno Kotomi, Nakashima Hitomi, Mizutani Masaharu, Takikawa Hirosato, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2014, 植物化学調節学会研究発表記録集, 49, 87 - 87, Japanese

  • 47. Functional characterization of monoterpene alcohol biosynthases in Cammelia sinensis

    Inoue Tomohiro, Sakai Naoya, Totsuka Koujirou, Mizutani Masaharu, Watanabe Naoharu, Ohnishi Toshiyuki

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2014, 植物化学調節学会研究発表記録集, 49, 65 - 65, Japanese

  • 50. Functional analysis of ALD1 involved in biosynthsesis of pipecoric acid, a signal mediator of plant defense

    Matsuda Tatsuya, Miyake Ryoma, Kawabata Jun, Sugimoto Yukihiro, Mizutani Masaharu

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2014, 植物化学調節学会研究発表記録集, 49, 68 - 68, Japanese

  • 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

    中安大, 梅基直行, 大山清, 渡辺文太, 村中俊哉, 斉藤和季, 杉本幸裕, 水谷正治

    20 Sep. 2014, 2014年度日本農芸化学会関西支部大会, 486th, Japanese

    Summary national conference

  • 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

    中安大, 梅基直行, 大山清, 渡辺文太, 村中俊哉, 斉藤和季, 杉本幸裕, 水谷正治

    29 Mar. 2014, 日本農芸化学会2014年度大会,東京, 2014, Japanese

    Summary national conference

  • 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

    中安大, 梅基直行, 大山清, 渡辺文太, 村中俊哉, 斉藤和季, 杉本幸裕, 水谷正治

    18 Mar. 2014, 第55回日本植物生理学会,富山, 55th, Japanese

    Summary national conference

  • 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

    李栄宰, 渡辺文太, 中安大, 山村理恵, 岡田実佳, 杉本幸裕, 水谷正治

    Mar. 2014, 日本農芸化学会2014年度大会,東京, 2014, Japanese

    Summary national conference

  • トマト果実におけるα-トマチン代謝酵素の探索

    小林緑, 中安大, 飯島陽子, 梅基直行, 杉本幸裕, 水谷正治

    2014, 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

    中安大, 梅基直行, 大山清, 大山清, 渡辺文太, 村中俊哉, 村中俊哉, 斉藤和季, 斉藤和季, 杉本幸裕, 水谷正治

    2014, 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

    大山清, 大山清, 梅基直行, 中安大, 李けい宰, 水谷正治, 斉藤和季, 斉藤和季, 村中俊哉, 村中俊哉

    2014, 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

  • 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

    東久保諒, GUIRIMAND Gregory, 若林孝俊, 關光, 村中俊哉, 水谷正治, 杉本幸裕, 杉本幸裕, 岡澤敦司, 岡澤敦司

    2014, 日本農薬学会大会講演要旨集, 39th

  • 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

    東久保諒, GUIRIMAND Gregory, 若林孝俊, 水谷正治, 杉本幸裕, 杉本幸裕, 關光, 村中俊哉, 岡澤敦司, 岡澤敦司

    2014, 日本農芸化学会大会講演要旨集(Web), 2014

  • 56. Searching for D14-like gene in S. gesnerioides and their hydrolysis activity toward strigoractone

    Inoue Tomoyuki, Fujioka Hijiri, Mizutani Masaharu, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 04 Oct. 2013, 植物化学調節学会研究発表記録集, 48, 71 - 71, Japanese

  • 50. The bioconversion of 5-deoxystrigol to mono-hydroxylated strigolactones in plants

    Ueno Kotomi, Motonami Noriko, Nakashima Hitomi, Mizutani Masaharu, Takikawa Hirosato, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 04 Oct. 2013, 植物化学調節学会研究発表記録集, 48, 65 - 65, Japanese

  • 29. Functional characterization of Arabidopsis CYP72C1 involved in brassinosteroid catabolism

    Sahara Mizuho, Watanabe Bunta, Yokota Takao, Watanabe Naoharu, Mizutani Masaharu, Ohnishi Toshiyuki

    The Japanese Society for Chemical Regulation of Plants, 04 Oct. 2013, 植物化学調節学会研究発表記録集, 48, 44 - 44, Japanese

  • 14. Ligand recognition mechanism of CYP714 family involved in gibberellin biosynthesis and metabolism

    Shinma Yuko, Kubijiri Yuki, Ohnishi Toshiyuki, Magome Hiroshi, Yamaguchi Shinjiro, Mizutani Masaharu, Todoroki Yasushi

    The Japanese Society for Chemical Regulation of Plants, 04 Oct. 2013, 植物化学調節学会研究発表記録集, 48, 29 - 29, Japanese

  • 18. Synthesis of fluorinated substrate analogs as inhibitors of phaseic acid 4'-reductase

    Usami Kenta, Fujii Yusuke, Mizutani Masaharu, Kondo Satoru, Todoroki Yasushi, Hirai Nobuhiro

    The Japanese Society for Chemical Regulation of Plants, 04 Oct. 2013, 植物化学調節学会研究発表記録集, 48, 33 - 33, Japanese

  • 45. Characterization of germination stimulants for root parasitic weed seeds exuded by sunflower

    Umeda Shuhei, Ueno Kotomi, Mizutani Masaharu, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 04 Oct. 2013, 植物化学調節学会研究発表記録集, 48, 60 - 60, Japanese

  • 69. Investigation of P450 genes involved in biosynthesis of steroidal saponins from Dioscorea esculenta(Togedokoro)

    Lee Hyoung Jae, Yamamura Rie, Kawasaki Takashi, Nakayasu Masaru, Onjyo Michio, Sugimoto Yukihiro, Mizutani Masaharu

    The Japanese Society for Chemical Regulation of Plants, 04 Oct. 2013, 植物化学調節学会研究発表記録集, 48, 84 - 84, Japanese

  • 49. In vivo production of strigolactone biosynthetic intermediates in a β-carotene producing strain of E. coli

    Kuwabara Kazuma, Ueno Kotomi, Misawa Norihiko, Mizutani Masaharu, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 04 Oct. 2013, 植物化学調節学会研究発表記録集, 48, 64 - 64, Japanese

  • 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

    山村理恵, 李栄宰, 川崎崇, 中安大, 渡辺文太, 遠城道雄, 杉本幸裕, 水谷正治

    Oct. 2013, 植物化学調節学会第48回大会,新潟, 48 (Supplement), Japanese

    Summary national conference

  • 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

    關 光, 大山 清, 水谷 正治, 須藤 浩, 斉藤 和季, 村中 俊哉

    (一社)日本生薬学会, Aug. 2013, 日本生薬学会年会講演要旨集, 60回, 141 - 141, Japanese

  • Identification and Quantification of Steroidal Saponins in Dioscorea esculenta

    Lee, H. J, Watanabe, B, Nakayasu, M, Yamamura, R, Okada, M, Sugimoto, Y, Mizutani, M

    Jun. 2013, TERPNET 2013, Crete, Greece, 31st, English

    Summary international conference

  • 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

    LEE Hyoung Jae, 山村理恵, 川崎崇, 中安大, 遠城道雄, 杉本幸裕, 水谷正治

    2013, 植物の生長調節, 48 (Supplement)

  • 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

    中安大, 川崎崇, 山村理恵, LEE Hyoung Jae, 遠城道雄, 杉本幸裕, 水谷正治

    2013, 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

    中安大, LEE Hyoung-Jae, 杉本幸裕, 水谷正治

    2013, 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

    水谷正治, 中安大, LEE Hyoung Jae, 山村理恵, 川崎崇, 遠城道雄, 杉本幸裕

    2013, 日本農芸化学会大会講演要旨集(Web), 2013

  • 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

    岡田実佳, 中安大, 遠城道雄, 杉本幸裕, 水谷正治

    2013, 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

  • 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

    中安大, 梅基直行, 大山清, 大山清, 渡辺文太, 村中俊哉, 村中俊哉, 斉藤和季, 斉藤和季, 杉本幸裕, 水谷正治

    2013, 植物の生長調節, 48 (Supplement)

  • トマト果実におけるα-トマチン代謝酵素遺伝子の探索

    小林緑, 飯島陽子, 中安大, 杉本幸裕, 水谷正治

    2013, 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

    山村理恵, 川崎崇, LEE Hyoung Jae, 中安大, 岡田実佳, 遠城道雄, 杉本幸裕, 水谷正治

    2013, 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

    東久保諒, GUIRIMAND Gregory, 若林孝俊, 水谷正治, 杉本幸裕, 關光, 村中俊哉, 岡澤敦司, 岡澤敦司

    2013, 日本農芸化学会大会講演要旨集(Web), 2013

  • 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

    楠瀬達也, 畑直樹, 畑直樹, 小埜栄一郎, 佐竹炎, 清水文一, 水谷正治, 關光, 村中俊哉, 岡澤敦司, 岡澤敦司

    2013, 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

    梅基直行, 佐々木勝徳, 大山清, 大山清, 山下まり, 水谷正治, 關光, 斉藤和季, 斉藤和季, 村中俊哉

    2013, 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

  • 3P-221 Identification of a glycosyltransferase involved in the biosynthesis of glycyrrhizin

    seki Hikaru, Ohyama Kiyoshi, Mizutani Masaharu, Sudo Hiroshi, Saito Kazuki, Muranaka Toshiya

    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 243 - 243, Japanese

  • Nakayasu Masaru, Umemoto Naoyuki, Ohyama Kiyoshi, Watanabe Bunta, Muranaka Toshiya, Saito Kazuki, Sugimoto Yukihiro, Mizutani Masaharu

    一般社団法人 植物化学調節学会, 2013, Regulation of Plant Growth & Development, 48 (0), 86 - 86, Japanese

  • Daisaku Ohta, Masaharu Mizutani

    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, 01 Jan. 2013, Isoprenoid Synthesis in Plants and Microorganisms: New Concepts and Experimental Approaches, 381 - 391, English

    [Refereed]

    Introduction commerce magazine

  • 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

    中安大, 梅基直行, 大山清, 渡辺文太, 杉本幸裕, 水谷正治

    27 Oct. 2012, 植物化学調節学会第47回大会,鶴岡, 47 (Supplement), Japanese

    Summary national conference

  • 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

    大西利幸, Godza, B, 渡辺文太, 藤岡昭三, Hategan, L, 柴田恭美, 横田孝雄, Szekeres, M, 水谷正治

    27 Oct. 2012, 植物化学調節学会第47回大会,鶴岡, Japanese

    Summary national conference

  • 75. Substrate specifi city of phaseic acid 4'-reductase

    Usami Kenta, Fujii Yusuke, Mizutani Masaharu, Kondo Satoru, Todoroki Yasushi, Hirai Nobuhiro

    The Japanese Society for Chemical Regulation of Plants, 05 Oct. 2012, 植物化学調節学会研究発表記録集, 47, 92 - 92, Japanese

  • 51. Purifi cation of strigolactones produced by sunfl ower

    Umeda Shuhei, Ueno Kotomi, Mizutani Masaharu, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 05 Oct. 2012, 植物化学調節学会研究発表記録集, 47, 68 - 68, Japanese

  • 47. Essential structure of agonistic strigolactone analogs

    Inoue Tomoyuki, Nakashima Hitomi, Sasaki Mitsuru, Takikawa Hirosato, Mizutani Masaharu, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 05 Oct. 2012, 植物化学調節学会研究発表記録集, 47, 64 - 64, Japanese

  • 62. Bioconversion of 5-deoxystrigol to sorgomol in sorghum

    Motonami Noriko, Nakashima Hitomi, Ueno Kotomi, Mizutani Masaharu, Takikawa Hirosato, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 05 Oct. 2012, 植物化学調節学会研究発表記録集, 47, 79 - 79, Japanese

  • 49. Design and synthesis of germination inhibitors for Striga gesnerioides

    Nakashima Hitomi, Nomura Saki, Mizutani Masaharu, Takikawa Hirosato, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 05 Oct. 2012, 植物化学調節学会研究発表記録集, 47, 66 - 66, Japanese

  • 48. Structural requirements of strigolactones for germination induction and inhibition of Striga gesnerioides seeds

    Nomura Saki, Nakashima Hitomi, Ueno Kotomi, Muranaka Satoru, Mizutani Masaharu, Takikawa Hirosato, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 05 Oct. 2012, 植物化学調節学会研究発表記録集, 47, 65 - 65, Japanese

  • 63. Oxidative metabolism of 5-deoxystrigol in plants

    Ueno Kotomi, Nakashima Hitomi, Nomura Saki, Mizutani Masaharu, Takikawa Hirosato, Sugimoto Yukihiro

    The Japanese Society for Chemical Regulation of Plants, 05 Oct. 2012, 植物化学調節学会研究発表記録集, 47, 80 - 80, Japanese

  • 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

    中安大, 川崎崇, LEE Hyoung Jae, 山村理恵, 遠城道雄, 杉本幸裕, 水谷正治

    2012, 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

    中安大, 川崎崇, 山村理恵, 遠城道雄, 杉本幸裕, 水谷正治

    2012, 日本農芸化学会大会講演要旨集(Web), 2012

  • CYP716A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

    福島エリオデット, 福島エリオデット, 福島エリオデット, 關光, 關光, 關光, 大山清, 大山清, 小埜栄一郎, 梅本直行, 水谷正治, 斉藤和季, 斉藤和季, 村中俊哉, 村中俊哉, 村中俊哉

    2012, 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。

    小埜栄一郎, 福島エリオデッド, 福島エリオデッド, 福島エリオデット, 梅基直行, 關光, 關光, 關光, 村中俊哉, 村中俊哉, 村中俊哉, 水谷正治

    2012, 日本植物生理学会年会要旨集, 53rd

  • グリコアルカロイドを非常に低下させたジャガイモ

    梅基直行, 佐々木勝徳, 大山清, 大山清, 山下まり, 水谷正治, 關光, 斉藤和季, 斉藤和季, 村中俊哉

    2012, 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

  • 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

    鈴木 史朗, 田中 功二, 河合 真吾, 鈴木 秀幸, サフェンドリ コマラ ラガムスタリ, 小埜 栄一郎, 水谷 正治, 清水 文一, Takefumi Hattori, 横田 信三, 中村 正治, 星 比呂志, 柴田 大輔, 梅澤 俊明

    2012, 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, Japanese

    Others

  • 53. Analysis of GR24 conversion in plant

    Hiragakiuchi Masaki, Motonami Noriko, Ueno Kotomi, Nakashima Hitomi, Takikawa Hirosato, Mizutani Masaharu, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 03 Oct. 2011, 植物化学調節学会研究発表記録集, (46), 69 - 69, Japanese

  • 54. Synthesis and identification of hydroxylated GR24

    Nakashima Hitomi, Hiragakiuchi Masaki, Motonami Noriko, Ueno Kotomi, Mizutani Masaharu, Takikawa Hirosato, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 03 Oct. 2011, 植物化学調節学会研究発表記録集, (46), 70 - 70, Japanese

  • 5. An optically active abscinazole-E2B, a specific inhibitor of ABA 8'-hydroxylase CYP707A

    Okazaki Mariko, Nimitkeatkai Hataitip, Kondo Satoru, Mizutani Masaharu, Hirai Nobuhiro, Ohnishi Toshiyuki, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 03 Oct. 2011, 植物化学調節学会研究発表記録集, (46), 21 - 21, Japanese

  • 3. A selective inhibitor of CYP701A involved in gibberellin biosynthesis

    Naiki Kumi, Ohnishi Toshiyuki, Mizutani Masaharu, Ueno Kotomi, Hirai Nobuhiro, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 03 Oct. 2011, 植物化学調節学会研究発表記録集, (46), 19 - 19, Japanese

  • 56. Essential structure of strigolactone as branching inhibitor

    Inoue Tomoyuki, Sasaki Mitsuru, Takikawa Hirosato, Mizutani Masaharu, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 03 Oct. 2011, 植物化学調節学会研究発表記録集, (46), 72 - 72, Japanese

  • 57. Structural requirements of strigolactones for germination induction of Striga gesnerioides seeds

    Ueno Kotomi, Nomura Saki, Fujiwara Mami, Mizutani Masaharu, Sasaki Mitsuru, Takikawa Hirosato, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 03 Oct. 2011, 植物化学調節学会研究発表記録集, (46), 73 - 73, Japanese

  • 55. Oxidation of 5-deoxystrigol in sorghum

    Motonami Noriko, Nakashima Hitomi, Ueno Kotomi, Mizutani Masaharu, Takikawa Hirosato, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 03 Oct. 2011, 植物化学調節学会研究発表記録集, (46), 71 - 71, Japanese

  • 58. Germination stimulants for Striga gesnerioides from cowpea (Vigna unguiculata)

    Nomura Saki, Ueno Kotomi, Muranaka Satoru, Mizutani Masaharu, Takikawa Hirosato, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 03 Oct. 2011, 植物化学調節学会研究発表記録集, (46), 74 - 74, Japanese

  • 4. An inhibitor of the 13-hydroxylases in gibberellin biosynthesis

    Sakai Anna, Shinma Yuko, Ohnishi Toshiyuki, Mizutani Masaharu, Hirai Nobuhiro, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 03 Oct. 2011, 植物化学調節学会研究発表記録集, (46), 20 - 20, Japanese

  • 98. The screening of genes for steroidal saponin biosynthesis from Dioscorea spp.

    Kawasaki Takashi, Yamamura Rie, Nakayasu Masaru, Onjyou Michio, Sugimoto Yukihiro, Mizutani Masaharu

    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.

    The Japanese Society for Chemical Regulation of Plants, 03 Oct. 2011, 植物化学調節学会研究発表記録集, (46), 114 - 114, Japanese

  • Masaharu Mizutani, Fumihiko Sato

    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, Mar. 2011, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 507 (1), 194 - 203, English

    [Refereed]

    Book review

  • 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

    川崎崇, 山村理恵, 中安大, 遠城道雄, 杉本幸裕, 水谷正治

    2011, 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

    中安大, 川崎崇, 浦川晋吾, 杉本幸裕, 水谷正治

    2011, 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に関与する遺伝子の探索

    川崎崇, 山村理恵, 中安大, 遠城道雄, 杉本幸裕, 水谷正治

    2011, 植物の生長調節, 46 (Supplement)

  • トマト果実におけるα-トマチンの代謝酵素の探索

    浦川晋吾, 飯島陽子, 青木考, 川崎崇, 中安大, 杉本幸裕, 水谷正治

    2011, 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

  • 80. Purification and molecular analysis of α,β-unsaturated carbonyl-reducing enzymes from cucumber leaves

    Hasegawa Ayaka, Yamauchi Yasuo, Mizutani Masaharu, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2010, 植物化学調節学会研究発表記録集, (45), 97 - 97, Japanese

  • 42. Synthesis, biological activity, and action of amino acid conjugates of abscisic acid

    Narita Kenta, Muramatsu Taku, Ohnishi Toshiyuki, Mizutani Masaharu, Ueno Kotomi, Hirai Nobuhiro, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2010, 植物化学調節学会研究発表記録集, (45), 59 - 59, Japanese

  • 83. Investigation of cinnamates ortho-hydroxylase, a key enzyme of coumarins biosynthesis in plants

    Ito Kyoko, Mizutani Masaharu, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2010, 植物化学調節学会研究発表記録集, (45), 100 - 100, Japanese

  • 34. Purification of seed germination inhibitors from mesquite leaves against root parasitic plants

    Yamamoto Chika, Ueno Kotomi, Babiker Adbel Gabar, Mizutani Masaharu, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2010, 植物化学調節学会研究発表記録集, (45), 51 - 51, Japanese

  • 32. Investigation of the regulation mechanism of the de-differentiation and re-differentiation in the devepolmental process of Orobanche minor

    Ohta Sayaka, Mizutani Masaharu, Sakakibara Hitoshi, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2010, 植物化学調節学会研究発表記録集, (45), 49 - 49, Japanese

  • 44. Generation of novel chemical regulators of plant growth and development using a chemical inducer of dimerization (CID) system

    Aoyama Hikaru, Okazaki Mariko, Ohnishi Toshiyuki, Mizutani Masaharu, Ueno Kotomi, Hirai Nobuhiro, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2010, 植物化学調節学会研究発表記録集, (45), 61 - 61, Japanese

  • 45. In vitro screening of selective inhibitors for P450 involved in gibberellins biosynthesis

    Muramatsu Taku, Ohnishi Toshiyuki, Mizutani Masaharu, Ueno Kotomi, Hirai Nobuhiro, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2010, 植物化学調節学会研究発表記録集, (45), 62 - 62, Japanese

  • 43. Abscinazole-E2B, a practical inhibitor of ABA 8'-hydroxylase CYP707A

    Okazaki Mariko, Muramatsu Taku, Aoyama Hikaru, Ueno Kotomi, Mizutani Masaharu, Hirai Nobuhiro, Nimitkeatkai Hataitip, Kondo Satoru, Ohnishi Toshiyuki, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 2010, 植物化学調節学会研究発表記録集, (45), 60 - 60, Japanese

  • Masaharu Mizutani, Daisaku Ohta

    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, ANNUAL REVIEW OF PLANT BIOLOGY, VOL 61, 61, 291 - 315, English

    [Refereed]

    Introduction scientific journal

  • 38. Screening of bioactive secondary metabolites produced by mesquite

    Yamamoto Chika, Mizutani Masaharu, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2009, 植物化学調節学会研究発表記録集, (44), 52 - 52, Japanese

  • 41. Exploration of the biosynthetic enzymes of coumarins in citrus and legume plants

    Ito Kyoko, Mizutani Masaharu, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2009, 植物化学調節学会研究発表記録集, (44), 55 - 55, Japanese

  • 42. Isolation and characterization of cytochrome P450 cDNAs involved in isoquinoline alkaloid biosynthesis

    Katsurada Takuto, Sugimoto Gen, Mizutani Masaharu, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2009, 植物化学調節学会研究発表記録集, (44), 56 - 56, Japanese

  • 57. Morphological investigation of the developmental process of Orobanche minor

    Ohta Sayaka, Mizutani Masaharu, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2009, 植物化学調節学会研究発表記録集, (44), 71 - 71, Japanese

  • 47. Functional analysis of putative brassinosteroid-biosynthetic P450s from Physcomitrella patents and Selaginella moellendorffii

    Nishigaki Naoto, Urakawa Shingo, Mizutani Masaharu, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2009, 植物化学調節学会研究発表記録集, (44), 61 - 61, Japanese

  • 71. Abscinazole E as a specific inhibitor of abscisic acid 8'-hydroxylase, CYP707A

    Okazaki Mariko, Aoyama Hikaru, Shirakura Minaho, Mizutani Masaharu, Hirai Nobuhiro, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2009, 植物化学調節学会研究発表記録集, (44), 85 - 85, Japanese

  • 73. Purification of phaseic acid 4'-reductase

    Fujii Yusuke, Kondo Satoru, Mizutani Masaharu, Todoroki Yasushi, Hirai Nobuhiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2009, 植物化学調節学会研究発表記録集, (44), 87 - 87, Japanese

  • 60. Effects of modifications at the B-ring of strigolactones on the bioactivity toward seed germination of root parasitic plants

    Fukutomi Tatsuya, Tanaka Aya, Takikawa Hirosato, Sasaki Mitsuru, Muranaka Satoru, Mizutani Masaharu, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2009, 植物化学調節学会研究発表記録集, (44), 74 - 74, Japanese

  • 58. Effects of rhizobial symbiosis on Orobanche aegyptiaca parasitism to Lotus japonicus

    Doi Tomoko, Mizutani Masaharu, Sugimoto Yukihiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2009, 植物化学調節学会研究発表記録集, (44), 72 - 72, Japanese

  • 72. Highly selective plant P450 inhibitors with a disubstituted azole ring

    Naiki Kumi, Aoyama Hikaru, Shirakura Minaho, Mizutani Masaharu, Hirai Nobuhiro, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2009, 植物化学調節学会研究発表記録集, (44), 86 - 86, Japanese

  • 9B25 Structural basis of AHI1 (abscisic acid 8'-hydroxylase inhibitor) that provides drought tolerance to plants.

    Ueno Kotomi, Mizutani Masaharu, Hirai Nobuhiro, Todoroki Yasushi

    Pesticide Science Society of Japan, 27 Feb. 2009, 講演要旨集, (34), 126 - 126, Japanese

  • アブシシン酸8’水酸化酵素制御とリンゴの乾燥耐性

    須川瞬, 大川克哉, 小原均, 上野琴巳, 水谷正治, 轟泰司, 平井伸博, 近藤悟

    2009, 園芸学研究 別冊, 8 (1)

  • 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

    内藤喜之, 竹内良徳, 内藤喜之, 清水文一, 平竹 潤, 水谷正治

    30 Oct. 2008, 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, Japanese

  • 54. Development of highly selective plant P450 inhibitors by restricting conformation and modifying azole ring

    Shirakura Minaho, Kobayashi Kyotaro, Aoyama Hikaru, Hiramatsu Saori, Mzutani Masaharu, Hirai Nobuhiro, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2008, 植物化学調節学会研究発表記録集, (43), 68 - 68, Japanese

  • 55. Development of azole-containing specific P450 inhibitors using click reaction

    Hiramatsu Saori, Shirakura Minaho, Mizutani Masaharu, Hirai Nobuhiro, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2008, 植物化学調節学会研究発表記録集, (43), 69 - 69, Japanese

  • 52. Biological activities of ABA-amino acid conjugate

    Muramatsu Taku, Mizutani Masaharu, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2008, 植物化学調節学会研究発表記録集, (43), 66 - 66, Japanese

  • 53. Characterization of phaseic acid 4'-reductase

    Fujii Yusuke, Kondo Satoshi, Mizutani Masaharu, Todoroki Yasushi, Hirai Nobuhiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2008, 植物化学調節学会研究発表記録集, (43), 67 - 67, Japanese

  • 40. Cloning and functional analysis of ortho-hydroxylases involved in coumarin biosynthesis in sweet potato

    Matsumoto Seitaro, Yamamoto Ryotaro, Mizutani Masaharu, Shimizu Bun-ichi

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2008, 植物化学調節学会研究発表記録集, (43), 54 - 54, Japanese

  • 39. Biochemical characterization of rice CYP734As involved in brassinosteroid catabolism

    Kawabe Ayami, Shimizu Bun-ichi, Shimada Yukihisa, Fujioka Shozo, Sakamoto Tomoaki, Mizutani Masaharu

    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.

    The Japanese Society for Chemical Regulation of Plants, 06 Oct. 2008, 植物化学調節学会研究発表記録集, (43), 53 - 53, Japanese

  • SHIMIZU Bun-ichi, KAI Kosuke, MIZUTANI Masaharu

    Last, Japan Society for Bioscience, Biotechnology, and Agrochemistry, Aug. 2008, 化学と生物, 46 (8), 518 - 520, Japanese

  • 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

    平竹 潤, 竹内良徳, 内藤喜之, 水谷正治, 清水文一

    28 Mar. 2008, 日本農芸化学会 2008 年度(平成 20 年度)大会 ,名古屋(名城大学), Japanese

  • 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

    内藤喜之, 齊野廣通, 水谷正治, 清水文一, 平竹 潤

    28 Mar. 2008, 日本農芸化学会 2008 年度(平成 20 年度)大会 ,名古屋(名城大学), Japanese

  • 竹内良徳, 内藤喜之, 水谷正治, 清水文一, 平竹 潤

    20 Mar. 2008, 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, Japanese

  • 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

    澤井学, 澤井学, 關光, 關光, 大山清, 須藤浩, 須藤浩, 水谷正治, 大西利幸, 明石智義, 青木俊夫, 村中俊哉, 村中俊哉, 斉藤和季, 斉藤和季

    2008, 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

    關光, 關光, 大山清, 須藤浩, 須藤浩, 水谷正治, 大西利幸, 高上馬希重, 櫻井望, 鈴木秀幸, 柴田大輔, 豊田敦, 十時泰, 榊佳之, 林宏明, 澤井学, 澤井学, 柴田敏郎, 飯田修, 明石智義, 青木俊夫, 斉藤和季, 斉藤和季, 村中俊哉, 村中俊哉

    2008, 日本生薬学会年会講演要旨集, 55th

  • 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)

    關光, 關光, 大山清, 澤井学, 澤井学, 明石智義, 水谷正治, 大西利幸, 須藤浩, 須藤浩, 青木俊夫, 斉藤和季, 斉藤和季, 村中俊哉, 村中俊哉

    2008, 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

  • 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

    須川瞬, 大川克哉, 小原均, 上野琴巳, 水谷正治, 轟泰司, 平井伸博, 近藤悟

    2008, 園芸学研究 別冊, 7 (1)

  • 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

    中坪朋文, 水谷正治, 鈴木史朗, 服部武文, 梅澤俊明

    2008, 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.

  • Arabidopsis thalianaのリグナン生合成酵素遺伝子の機能解析

    中坪朋文, 水谷正治, 鈴木史朗, 服部武文, 梅澤俊明

    2008, 第58回日本木材学会大会講演要旨集,p. 70,2008.

  • Arabidopsis thaliana pinoresinol reductases control enantiomeric compositions of lariciresinol

    Nakatsubo, T, Mizutani, M, Suzuki, S, Hattori, T, Umezawa, T

    2008, Proceedings of Phytochemical Society of North America 2008 Annual Meeting, p. 47

    [Refereed]

  • Chemical tools to control auxin homeostasis : IAA-amino acid synthetase (GH3) inhibitors and their in vivo activities

    TAKEUCHI Yoshinori, TAI Liz, MIZUTANI Masaharu, SHIMIZU Bun-ichi, HIRATAKE Jun

    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.

    The Japanese Society for Chemical Regulation of Plants, 29 Oct. 2007, 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, Japanese

  • The ligand structure required for binding to abscisic acid 8'-hydroxylase

    UENO Kotomi, MIZUTANI Masaharu, HIRAI Nobuhiro, TODOROKI Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 05 Oct. 2007, 植物の生長調節 = Regulation of plant growth & development, 42, 57 - 57, Japanese

  • Uniconazoleの立体配座とABA 8'-hydroxylase阻害活性の関係

    小林享太郎, 米山英高, 平松佐織, 金美紅, 渡辺文太, 水谷正治, 平井伸博, 轟泰司

    Oct. 2007, 植物化学調節学会第42回大会,静岡, Japanese

    Summary national conference

  • 吉田彩子, 甲斐建次, 水谷正治, 平竹 潤, 宮川 恒, 福田裕穂

    28 Mar. 2007, 第48回日本植物生理学会年会 ,愛媛大学, Japanese

  • 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

    水谷正治, Liz Tai, 平竹 潤, 清水文一, 坂田完三

    28 Mar. 2007, 第48回日本植物生理学会年会 ,愛媛大学, Japanese

  • 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

    平竹 潤, Tai, Liz, 水谷正治, 清水文一, 坂田完三

    25 Mar. 2007, 日本農芸化学会 2007 年度大会 ,東京農業大学, Japanese

  • SAINO HIROMICHI, MIZUTANI SHOJI, HIRATAKE JUN, SHIMIZU TETSUYA, KATO HIROAKI, SAKATA KANZO

    Disaccharide glycoside-specific glycosidases (diglycosidases) specifically hydrolyze the &beta;-glycosidic bond between disaccharide and aglycons. Diglycosidases belong to glycosidase family 1, in which various monoglucosidases (&beta;-glucosidases) are also classified. Disaccharide glycosides are found in diverse plants, suggesting the wide distribution of diglycosidases in the plant kingdom. Diglycosidases show over 50% amino acid identities with &beta;-glucosidases, indicating that diglycosidases have evolved from &beta;-glucosidases. To elucidate structural basis of alteration in their glycone specificities, we carried out X-ray crystallographic analysis of &beta;-primeverosidase (PD, a diglycosidase from <I>Camellia sinensis</I>). The 3-D structure of PD complexed with a substrate analog inhibitor was determined at 1.8 &Aring; resolution. The conformation of most catalytic residues was conserved between PD and &beta;-glucosidases. Interestingly, Ser473 and Gln477 in PD were found to form the hydrogen bonds with the second xylose of primeverose. Thus, diglycosidases evolved from monooglucosidases as the consequence of slight structural change in the substrate binding pocket.

    日本植物生理学会, 15 Mar. 2007, 日本植物生理学会年会要旨集, 48th (0), 298 - 814, Japanese

  • 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

    SAINO HIROMICHI, MIZUTANI MASAHARU, HIRATAKE JUN, SHIMIZU TETSUYA, KATO HIROAKI, SAKATA KANZO

    05 Mar. 2007, 日本農芸化学会大会講演要旨集, 2007, 205, Japanese

  • 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

    關光, 關光, 大山清, 水谷正治, 大西利幸, 須藤浩, 登谷友紀子, 斉藤和季, 斉藤和季, 村中俊哉, 村中俊哉

    2007, 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

  • Structural basis for evolution of diglycosidases from monoglucosidases in plants

    Hiromichi Saino, Masaharu Mizutani, Jun Hiratake, Tetzuya Shimizu, Hiroaki Kato, Kanzo Sakata

    OXFORD UNIV PRESS, 2007, PLANT AND CELL PHYSIOLOGY, 48, S220 - S220, English

    Summary international conference

  • Identification of ortho-hydroxylase of cinnamates involved in coumarin biosynthesis in Arabidopsis

    Kosuke Kai, Bun-ichi Shimizu, Masaharu Mizutani, Ryotaro Yamamoto, Naohiro Kawamura, Kanzo Sakata

    OXFORD UNIV PRESS, 2007, PLANT AND CELL PHYSIOLOGY, 48, S64 - S64, English

    Summary international conference

  • シトクロムP450の分子進化と植物の化学的多様性の獲得

    MIZUTANI MASAHARU

    共立出版, 2007, 蛋白質・核酸・酵素, 52 (12), 1454 - 1464, Japanese

    Introduction scientific journal

  • MIZUTANI MASAHARU

    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.

    The Japanese Society for Chemical Regulation of Plants, 2007, 植物の生長調節, 42 (1), 260 - 268, Japanese

    Introduction scientific journal

  • Biochemical studies on cytochrome P450 monooxygenases in biosynthesis and catabolism of abscisic acid and plant steroids

    MIZUTANI Masaharu

    The Japanese Society for Chemical Regulation of Plants, 04 Oct. 2006, 植物の生長調節 = Regulation of plant growth & development, 41, 15 - 15, Japanese

  • 7'-Hydroxy-ABA does not function as an inhibitor of ABA 8'-hydroxylase

    SHIMOMURA Hajime, MIZUTANI Masaharu, HIRAI Nobuhiro, ETOH Hideo, TODOROKI Yasushi

    The Japanese Society for Chemical Regulation of Plants, 04 Oct. 2006, 植物の生長調節 = Regulation of plant growth & development, 41, 30 - 30, Japanese

  • Structure-activity studies of uniconazole analogs

    YONEYAMA Hidetaka, WATANABE Bunta, MIZUTANI Masaharu, HIRAI Nobuhiro, TODOROKI Yasushi

    The Japanese Society for Chemical Regulation of Plants, 04 Oct. 2006, 植物の生長調節 = Regulation of plant growth & development, 41, 29 - 29, Japanese

  • Biochemical characterization of brassinosteroid C-3 oxidase

    OHNISHI Toshiyuki, BANCOS Simona, WATANABE Bunta, FUJIOKA Shozo, YOKOTA Takao, SAKATA Kanzo, SZEKERES Miklos, MIZUTANI Masaharu

    The Japanese Society for Chemical Regulation of Plants, 04 Oct. 2006, 植物の生長調節 = Regulation of plant growth & development, 41, 39 - 39, Japanese

  • 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

    戸本浩央, 齊野広道, 清水文一, 水谷正治, 平竹 潤, 坂田完三

    01 Oct. 2006, 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, Japanese

  • Inhibitors of IAA-Amino Acid Conjugate Synthetases and Hydrolases as Chemical Probes to Study IAA Homeostasis

    Tai, L. H, Hiratake J, Mizutani M, Shimizu, B, Sakata K

    08 Aug. 2006, 11th IUPAC International Congress of Pesticide Chemistry ,Kobe, Japan, English

  • Arabidopsis thaliana CAldOMTの機能について

    北村悠, 中坪朋文, 榊原紀和, 水谷正治, 柴田大輔, 服部武文, 梅澤俊明

    Aug. 2006, 日本木材学会

  • CYP724Bs and CYP90Bs are Redundant C-22 Hydroxylases in the Early C-22 Hydroxylation Routes of Brassinosteroid Biosynthesis

    Ohnishi, T, Watanabe, B, Sakata, K, Mizutani, M

    Jul. 2006, 8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology ,Swansea, Wales, English

    Summary international conference

  • Functional Characterization of Arabidopsis CYP90C1 and CYP90D1 Encode Brassinosteroids C-23 Hydroxylazes

    Ohnishi, T, Bancos, S, Watanabe, B, Yokota, T, Sakata, K, Szekeres, M, Mizutani, M

    Jul. 2006, 8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology, Swansea, Wales, English

    Summary international conference

  • FUNCTIONS OF ARABIDOPSIS CAldOMT

    Nakatsubo,T, Kitamura, Y, Sakakibara, N, Mizutani, M, Shibata, D, Hattori, T, Umezawa, T

    Jul. 2006, ICOB-5 & ISCNP-25 IUPAC International Conference on Biodiversity and Natural Products

  • 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

    榊 優子, 平竹 潤, 水谷正治, 清水文一, 坂田完三

    20 Mar. 2006, 第 47 回日本植物生理学会年会 ,筑波大学, Japanese

  • 植物由来ジグリコシダーゼのX線結晶構造解析

    SAINO HIROMICHI, MIZUTANI MASAHARU, HIRATAKE JUN, SHIMIZU TETSUYA, KATO HIROAKI, SAKATA KANZO

    05 Mar. 2006, 日本農芸化学会大会講演要旨集, 2006, 306, Japanese

  • Diglycosidase deeply concerned with the tea aroma formation and its application for food industries

    MIZUTANI Masaharu, SAKATA Kanzo

    バイオインダストリー協会, 01 Mar. 2006, Bioscience & industry, 64 (3), 145 - 150, Japanese

  • Ohnishi Toshiyuki, Bancos Simona, Watanabe Bunta, Yokota Takao, Sakata Kanzo, Szekeres Miklos, Mizutani Masaharu

    Cytochrome P450 monooxygenases (P450s) play crucial roles in Brassinosteroid (BR) biosynthesis from campesterol to brassinolide. Recent molecular genetic studies for BR-deficient mutants of Arabidopsis, rice, tomato and garden pea have identified several P450 genes (CYP85A, 90A, 90B, 90C, 90D, and 724B) so far. Among the BR-biosynthetic P450 genes, CYP85As and CYP90B1 have been characterized by functional expression in yeast cells and E. coli, respectively. Here, we report biochemical characterization of a P450, C-23 hydroxylase. The P450 was expressed in insect cells. The P450 activity was measured in an in vitro assay reconstituted with NADPH:cytochrome P450 reductase. The reaction products were analyzed by GC-MS. The P450 catalyzed the C-23 hydroxylation of 6-deoxocathasterone and cathasterone to 6-deoxoteasterone and teasterone, respectively. We have also determined the substrate specificity of the C-23 hydroxylase. To our knowledge, this is the first report of the biochemical characterization of the C-23 hydroxylation by the P450.

    The Japanese Society of Plant Physiologists, Mar. 2006, Plant and Cell Physiology Supplement, 2006 (0), 485 - 485, Japanese

    Summary national conference

  • 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

    青木望, 森川智美, 渡辺文太, 齋藤茂樹, 太田大策, 坂田完三, 水谷正治

    Mar. 2006, 第47回日本植物生理学会,つくば, Japanese

    Summary national conference

  • 森川智美, 水谷正治, 青木望, 渡辺文太, 嵯峨寛久, 斎藤茂樹, 及川彰, 鈴木秀幸, 櫻井望, 柴田大輔, 和田野晃, 坂田完三, 太田大策

    Mar. 2006, 第47回日本植物生理学会,つくば, Japanese

    Summary national conference

  • 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

    嵯峨寛久, 森川智美, 水谷正治, 青木望, 渡辺文太, 斎藤茂樹, 及川彰, 鈴木秀幸, 櫻井望, 柴田大輔, 和田野晃, 坂田完三, 太田大策

    Mar. 2006, 日本農芸化学会2006年度大会,京都, Japanese

    Summary national conference

  • Methyltransferase related to biosynthesis of coumarins

    K Kai, B Shimizu, N Kawamura, H Yamaguchi, M Mizutani, K Sakata

    OXFORD UNIV PRESS, 2006, PLANT AND CELL PHYSIOLOGY, 47, S199 - S199, English

    Summary international conference

  • Chemical tools for probing into IAA homeostasis-synthesis of IAA-amino acid synthetase inhibitors

    Y Sakaki, J Hiratake, M Mizutani, B Shimizu, K Sakata

    OXFORD UNIV PRESS, 2006, PLANT AND CELL PHYSIOLOGY, 47, S116 - S116, English

    Summary international conference

  • Glucosylation of scopoletin in Arabidopsis roots

    K Kai, B Shimizu, H Yamaguchi, M Mizutani, K Sakata

    OXFORD UNIV PRESS, 2006, PLANT AND CELL PHYSIOLOGY, 47, S198 - S198, English

    Summary international conference

  • 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

    甲斐 光輔, 清水 文一, 山口 晃, 水谷 正治, 坂田 完三

    20 Dec. 2005, 根の研究 = Root research, 14 (4), 183 - 183, Japanese

  • 56. Non-azole inhibitors of ABA 8'-hydroxylase

    Yoneyama Hidetaka, Araki Yoshiharu, Shimomura Hajime, Ueno Kotomi, Saito Sigeki, Mizutani Masaharu, Sakata Kanzo, Hirai Nobuhiro, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 13 Oct. 2005, 植物化学調節学会研究発表記録集, (40), 75 - 75, Japanese

  • 27. Analysis of coumarin biosynthesis pathway in Arabidopsis thaliana

    Kai Kosuke, Shimizu Bunichi, Yamaguchi Hikaru, Mizutani Masaharu, Sakata Kanzo

    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.

    The Japanese Society for Chemical Regulation of Plants, 13 Oct. 2005, 植物化学調節学会研究発表記録集, (40), 46 - 46, Japanese

  • 55. The functional groups of ABA required for chiral recognition by ABA 8'-hydroxylase

    Ueno Kotomi, Saito Shigeki, Mizutani Masaharu, Sakata Kanzo, Hirai Nobuhiro, Todoroki Yasushi

    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.

    The Japanese Society for Chemical Regulation of Plants, 13 Oct. 2005, 植物化学調節学会研究発表記録集, (40), 74 - 74, Japanese

  • ブラシノステロイドC-23位水酸化酵素の酵素学的解析

    大西利幸, Simona, B, 渡辺文太, 横田孝雄, 坂田完三, Szekeres, M, 水谷正治

    Oct. 2005, 植物調節化学会第40回大会,東京, Japanese

    Summary national conference

  • Biosynthesis and Catabolism of Brassinosteroids by Cytochrome P450 in Plants

    Mizutani, M, Ohnishi,T, Watanabe, B, Sakata, K

    Sep. 2005, The Second Yamada Symposium on Key Natural Molecules in Biological Systems, Hyogo, Japan, English

    Summary international conference

  • The substrate specificity of ABA 8'-hydroxylase

    UENO Kotomi, ARAKI Yoshiharu, TODOROKI Yasushi, SAITO Shigeki, MIZUTANI Masaharu, SAKATA Kanzo, HIRAI Nobuhiro

    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.

    The Japanese Society for Chemical Regulation of Plants, 28 May 2005, 植物の生長調節 = Regulation of plant growth & development, 39, 43 - 43, Japanese

  • Screening of ABA catabolic inhibitor

    KITAHATA Nobutaka, SAITO Shigeki, MIZUTANI Masaharu, MIN Yong-Ki, MIYAZAWA Yutaka, Hirai Nobuhiro, SHIMADA Yukihisa, SHINOZAKI Kazuo, HIRAI Nobuhiro, YOSHIDA Shigeo, ASAMI Tadao

    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.

    The Japanese Society for Chemical Regulation of Plants, 28 May 2005, 植物の生長調節 = Regulation of plant growth & development, 39, 52 - 52, Japanese

  • 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

    吉田宏一, 清水文一, 鶴喰寿孝, 森 茂治, 高田正保, 水谷正治, 平竹 潤, 坂田完三

    30 Mar. 2005, 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, Japanese

  • 榊 優子, 平竹 潤, 水谷正治, 清水文一, 坂田完三

    26 Mar. 2005, 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, Japanese

  • 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

    渡辺文太, 斎藤茂樹, 坂田完三, 水谷正治

    Mar. 2005, 日本農芸化学会2005年度大会,札幌, Japanese

    Summary national conference

  • Structural requirements for substrate recognition of ABA 8 '-hydroxylase

    K Ueno, Y Araki, Y Todoroki, S Saito, M Mizutani, K Sakata, N Hirai

    OXFORD UNIV PRESS, 2005, PLANT AND CELL PHYSIOLOGY, 46, S50 - S50, English

    Summary international conference

  • Uniconazole-P is a potent inhibitor for ABA catabolism

    S Saito, M Okamoto, S Shinoda, T Kushiro, Y Kamiya, E Nambara, N Hirai, K Sakata, M Mizutani

    OXFORD UNIV PRESS, 2005, PLANT AND CELL PHYSIOLOGY, 46, S215 - S215, English

    Summary international conference

  • Analysis of coumarin biosynthesis pathway in Arabidopsis thaliana

    K Kai, B Shimizu, M Mizutani, K Sakata

    OXFORD UNIV PRESS, 2005, PLANT AND CELL PHYSIOLOGY, 46, S240 - S240, English

    Summary international conference

  • Chemical knockout for probing into IAA homeostasis - Synthesis of the inhibitors of IAA-amino acid amidohydrolases

    Y Kubota, J Hiratake, M Mizutani, B Shimizu, K Sakata

    OXFORD UNIV PRESS, 2005, PLANT AND CELL PHYSIOLOGY, 46, S207 - S207, English

    Summary international conference

  • Chemical knockout for probing into IAA homeostasis - Synthesis of IAA-amino acid synthetase inhibitor

    Y Sakaki, J Hiratake, M Mizutani, B Shimizu, K Sakata

    OXFORD UNIV PRESS, 2005, PLANT AND CELL PHYSIOLOGY, 46, S208 - S208, English

    Summary international conference

  • 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?!—

    MIZUTANI MASAHARU, 斎藤茂樹

    日本農芸化学会, 2005, 化学と生物, 43 (10), 628 - 630, Japanese

    Introduction scientific journal

  • MIZUTANI MASAHARU

    The Japanese Society for Chemical Regulation of Plants, 2005, 植物の生長調節, 40 (1), 67 - 82, Japanese

    Introduction scientific journal

  • 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

    水谷正治, 藤田聡美, 大西利幸, 渡辺文太, 横田孝雄, 高津戸秀, 藤岡昭三, 吉田茂男, 坂田完三

    Oct. 2004, 植物化学調節学会第39回大会,秋田, Japanese

    Summary national conference

  • Arabidopsis CYP90B1 Catalyzes the C-22 Hydroxylation of C27-, C28-, and C29-Brassinosteroids

    Fujita, S, Ohnishi, T, Watanabe, B, Yokota, T, Fujioka, S, Takatsuto, S, Yoshida, S, Sakata, K, Mizutani, M

    Sep. 2004, 18th International Conference on Plant Growth Substances, Canberra, Australia, English

    Summary international conference

  • 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?

    MIZUTANI MASAHARU, 安栄玉, 坂田完三

    日本農芸化学会, 2004, 化学と生物, 42(12) (12), 774 - 776, Japanese

    Introduction scientific journal

  • Characterization of Cytochromes P450 involved in ABA Catabolism

    Saito Shigeki, Matsumoto Chiaki, Hirai Nobuhiro, Ohigashi Hajime, Ohta Daisaku, Mizutani Masaharu, Sakata Kanzo

    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.

    The Japanese Society for Chemical Regulation of Plants, 10 Oct. 2003, 植物化学調節学会研究発表記録集, (38), 43 - 43, Japanese

  • β-Primeverosidase and its contribution to formation of floral tea aromas during oolong and black tea manufacturing

    MIZUTANI M.

    FFIジャーナル編集委員会, 2003, Foods & food ingredients journal of Japan, 208 (12), 991 - 1003, Japanese

  • K Sakata, M Mizutani, SJ Ma, J Hiratake

    ACADEMIC PRESS INC, 2003, RECOGNITION OF CARBOHYDRATES IN BIOLOGICAL SYSTEMS, PART B: SPECIFIC APPLICATIONS, 363, 444 - 459, English

    [Refereed]

    Book review

  • TRIADIMEFON, A FUNGICIDAL TRIAZOLE-TYPE P450 INHIBITOR, INDUCES BRASSINOSTEROID DEFICIENCY-LIKE PHENOTYPES IN PLANTS AND BINDS TO DWF4 PROTEIN IN THE BRASSINOSTEROID BIOSYNTHESIS PATHWAY

    Asami Tadao, Mizutani Masaharu, Fujioka Shozo, Shimada Yukihisa, Takatsuto Suguru, Sakata Kanzo, Yoshida Shigeo

    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.

    The Japanese Society for Chemical Regulation of Plants, 31 Oct. 2002, 植物化学調節学会研究発表記録集, (37), 197 - 198, Japanese

  • Design, Synthesis and Applications of Selective β-Glycosidase Inhibitors, β-Glycosylamidines (MOLECULAR BIOFUNCTION - Chemistry of Molecular Biocatalysts)

    SAKATA Kanzo, HIRATAKE Jun, MIZUTANI Masaharu, SHIMIZU Bun-ichi, SINGH Inder Pal

    Institute for Chemical Research, Kyoto University, Mar. 2002, ICR annual report, 8, 44 - 45, English

  • CHARACTERIZATION OF CYTOCHROMES P450 HIGHLY EXPRESSED IN TOMATO OVARY

    Saito Shigeki, Mizutan Masaharu, Ohta Daisaku, Hiratake Jun, Sataka Kanzo

    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.

    The Japanese Society for Chemical Regulation of Plants, 09 Oct. 2001, 植物化学調節学会研究発表記録集, (36), 39 - 40, Japanese

  • TARGET SITES OF BRASSINOSTEROID BIOSYNTHESIS INHIBITOR IN PLANTS

    Asami Tadao, Mizutani Masaharu, Fujioka Shozo, Nagata Noriko, Nakano Takeshi, Goda Hideki, Shimada Yukihisa, Takatsuto Suguru, Sakata Kanzo, Yoshida Shigeo

    The Japanese Society for Chemical Regulation of Plants, 09 Oct. 2001, 植物化学調節学会研究発表記録集, (36), 19 - 20, Japanese

  • Functional analysis of tomato CYP72B involved in brassinosteroid metabolism

    Ohnishi Toshiyuki, Mizutani1 Masaharu, Ohta Daisaku, Sakata Kanzo

    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.

    The Japanese Society for Chemical Regulation of Plants, 09 Oct. 2001, 植物化学調節学会研究発表記録集, (36), 17 - 18, Japanese

  • 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

    馬 勝ジン, 平竹 潤, 水谷正治, 清水文一, 渡辺修治, 坂田完三

    03 Oct. 2001, 第43回天然有機化合物討論会 ,大阪, Japanese

  • 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

    齋藤茂樹, 水谷正治, 太田大策, 平竹 潤, 坂田完三

    25 Mar. 2001, 日本農芸化学会 2001 年度大会 ,京都, Japanese

  • Target site of brassinosteroid biosynthesis inhibitor in plants

    ASAMI Tadao, MIZUTANI Masaharu, FUJIOKA Shozo, NGATA Noriko, NAKANO Takeshi, GODA Hideki, SHIMADA Yukihisa, TAKATSUTO Suguru, SAKATA Kanzo, YOSHIDA Shigeo

    Pesticide Science Society of Japan, 22 Feb. 2001, 講演要旨集, 26, 107 - 107, Japanese

  • Characterization of cytochrome P450 cDNAs involved in brassinosteroid biosynthesis and metabolism from tomato

    MIZUTANI Masaharu, OHTA Daisaku, SAKATA Kanzo

    The Japanese Society for Chemical Regulation of Plants, 25 Dec. 2000, Chemical Regulation of Plants, 35 (2), 229 - 229, Japanese

  • Improvement of a yeast three-hybrid system to isolate an anxin receptor

    SATO Tadashi, MIZUTANI Masaharu, HIRATAKE Jun, SAKATA Kanzo

    The Japanese Society for Chemical Regulation of Plants, 25 Dec. 2000, Chemical Regulation of Plants, 35 (2), 236 - 237, Japanese

  • Improvement of a yeast three-hybrid system to isolate an auxin receptor

    Sato Tadashi, Mizutani Masaharu, Hiratake Jun, Sakata Kanzo

    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.

    The Japanese Society for Chemical Regulation of Plants, 02 Nov. 2000, 植物化学調節学会研究発表記録集, (35), 109 - 110, Japanese

  • Characterization of cytochrome P450 cDNAs involved in brassinosteroid biosynthesis and metabolism from tomato

    Mizutani Masaharu, Ohta Daisaku, Sakata Kanzo

    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.

    The Japanese Society for Chemical Regulation of Plants, 02 Nov. 2000, 植物化学調節学会研究発表記録集, (35), 63 - 64, Japanese

  • INTRACELLULAR LOCALIZATION STUDY OF TWO NADPH-CYT P450 REDUCTASE PROTEINS IN ARABIDOPSIS :

    Murai Jun, Yamasita Naoki, Prieto Rafael, Mizutani Masaharu, HASHIMOTO Takashi, Ohta Daisaku

    Japanese Society of Plant Physiologists, 2000, Plant and cell physiology, 41, s167, English

  • CHARACTERIZATION OF THE DWF4 PROTEIN :

    Mizutani Masaharu, Asami Tadao, Fujioka Shozo, Yoshida Shigeo, Sakata Kanzo

    Japanese Society of Plant Physiologists, 2000, Plant and cell physiology, 41, s201, English

  • Structure-activity relationships and target site (s) of brassinosteroid biosynthesis inhibitors.

    SEKIMATA Katsuhiko, ASAMI Tadao, MIZUTANI Masaharu, NAGATA Noriko, YAMAGISHI Kazutoshi, MIYAGAI Mashu, MATSUYAMA Tomoki, NAKANO Takeshi, SHIMADA Yukihisa, NOGUCHI Takahiro, FUJINOKA Shozo, YONEYAMA Koichi, TAKEUCHI Yasutomo, SAKATA Kanzo, YOSHIDA Shigeo

    The Japanese Society for Chemical Regulation of Plants, 25 Dec. 1999, Chemical Regulation of Plants, 34 (2), 349 - 349, Japanese

  • Three-hybrid system to isolate a cDNA encoding an auxin receptor.

    SATO Tadashi, MIZUTANI Masaharu, SAKATA Kanzo

    The Japanese Society for Chemical Regulation of Plants, 25 Dec. 1999, Chemical Regulation of Plants, 34 (2), 336 - 336, Japanese

  • 15 Three-hybrid system to isolate a cDNA encoding an auxin receptor

    Sato Tadashi, Mizutani Masaharu, Sakata kanzo

    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.

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 1999, 植物化学調節学会研究発表記録集, (34), 34 - 35, Japanese

  • 53 Structure-activity relationships and target site(s) of brassinosteroid biosynthesis inhibitors

    Sekimata Katsuhiko, Asami Tadao, Mizutani Masaharu, Nagata Noriko, Yamagishi Kazutoshi, Miyagai Masyu, Matsuyama Tomoki, Nakano Takeshi, Shimada Yukihisa, Noguchi Takahiro, Fujioka Shozo, Yoneyama Koichi, Takeuchi Yasutomo, Sakata Kanzo, Yoshida Shigeo

    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.

    The Japanese Society for Chemical Regulation of Plants, 01 Oct. 1999, 植物化学調節学会研究発表記録集, (34), 107 - 108, Japanese

  • 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

    佐藤 文彦, 田中 勝, 永吉 正典, 水谷 正治, 太田 大策, 水谷 正子, 田中 良和, 久住 高章

    05 Mar. 1999, 日本農芸化学会誌, 73, 474 - 474, Japanese

  • Cloning and expression of a β-primeverosidase concerned with the alcoholic aroma formation from the tea plant : Camellia sinensis var. sinensis cv. Yabukita

    NAKANISHI Hidemitsu, MIZUTANI Masaharu, SAKATA Kanzo

    Mar. 1999, Plant and cell physiology, 40, s125 - s125, English

  • 太田 大策, MIZUTANI MASAHARU

    学会出版センタ-, 1999, 化学と生物, 37 (2), 128 - 134, Japanese

    [Refereed]

    Introduction scientific journal

  • CHARACTERIZATION OF TWO ISOFORMS OF NADPH-P450 REDUCTASE FROM Arabidopsis thaliana

    MIZUTANI Masaharu, OHTA Daisaku

    Mar. 1997, Plant and cell physiology, 38, s67, English

  • ISOLATION AND CHARACTERIZATION OF NOVELP450S IN ARABIDOPSIS

    MIZUTANI Masaharu, OHTA Daisaku, WARD Eric, SATO Ryo

    Mar. 1995, Plant and cell physiology, 36, S105, English

Books etc

  • 基礎から学ぶ植物代謝生化学

    MIZUTANI MASAHARU

    Joint editor, 羊土社, Jan. 2019, Japanese, 植物天然物化学・植物生化学の教科書, ISBN: 9784758120906

    Textbook

  • 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

    大西利幸, MIZUTANI MASAHARU

    Others, 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, Japanese

    Scholarly book

  • 第2章 植物栄養学を理解するために: 植物ホルモン

    MIZUTANI MASAHARU

    Others, 文永堂植物栄養学(間藤徹、馬建鋒、藤原徹編)、, 2010, Japanese

    Textbook

  • 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)

    MIZUTANI MASAHARU, 坂田完三

    Others, 廣川書店廣川タンパク質化学 第4巻 酵素 4.3P.232-240, 2004, Japanese

    Scholarly book

Presentations

  • ソルガムにおける5-deoxystrigolからsorgomolへの変換反応

    上野琴巳, 本並宜子, 中嶌瞳, 水谷正治, 滝川浩郷, 杉本幸裕

    日本農芸化学会大会2014年度東京大会, Mar. 2014, Japanese, Domestic conference

    [Invited]

    Invited oral presentation

  • Steroidal Glycoalkaloid biosynthesis in Solanaceae

    MIZUTANI MASAHARU, 中安 大, 梅基直行, 大山 清, 渡辺文太, 村中俊哉, 斉藤和季, 杉本幸裕

    10th JSOL International Symposium on Solanaceae Genomics, Nov. 2013, English, International conference

    [Invited]

    Invited oral presentation

  • Diversity and evolution of steroidal saponin biosynthesis in plants

    Masaharu Mizutani

    奈良先端未来開拓コロキウム, Nov. 2013, English, International conference

    [Invited]

    Invited oral presentation

  • 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

    上野琴巳, 本並宜子, 中嶌瞳, 水谷正治, 滝川浩郷, 杉本幸裕

    第48回大会植物化学調節学会, Oct. 2013, Japanese, International conference

    Oral presentation

  • 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

    桑原一真, 上野琴巳, 三沢典彦, 水谷正治, 杉本幸裕

    第48回大会植物化学調節学会, Oct. 2013, Japanese, Domestic conference

    Oral presentation

  • 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

    李滎宰, 山村理恵, 川崎崇, 中安大, 道雄遠城, 杉本幸裕, MIZUTANI MASAHARU

    第48回植物化学調節学会, Oct. 2013, Japanese, Domestic conference

    Poster presentation

  • 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

    山村理恵, Lee Hyoung Jae, 川崎崇, 中安大, 渡辺文太, 遠城道雄, 杉本幸裕, 水谷正治

    植物化学調節学会第48回大会, Oct. 2013, Japanese, Domestic conference

    Oral presentation

  • 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

    中安大, 梅基直行, 大山清, 渡辺文太, 村中俊哉, 斎藤和季, 杉本幸裕, 水谷正治

    植物化学調節学会第48回大会, Oct. 2013, Japanese, Domestic conference

    Oral presentation

  • Diversity and evolution of steroidal saponin biosynthesis in plants

    Masaharu Mizutani

    INRA_Colmerセミナー, Oct. 2013, English, International conference

    Invited oral presentation

  • Diversity and Evolution of Steroidal Saponin Biosynthesis in Plants

    Masaharu Mizutani

    ボルドー大学セミナー, Oct. 2013, English, International conference

    Invited oral presentation

  • 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

    關 光, 大山 清, MIZUTANI MASAHARU, 斉藤和季, 村中俊哉

    日本生薬学会第60回年会, Sep. 2013, Japanese, Domestic conference

    Oral presentation

  • 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

    關 光, 大山 清, MIZUTANI MASAHARU, 斉藤和季, 村中俊哉

    第65回日本生物工学会大会, Sep. 2013, Japanese, Domestic conference

    Oral presentation

  • 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

    關 光, 大山 清, MIZUTANI MASAHARU, 斉藤和季, 村中俊哉

    第31回植物細胞分子生物学会, Sep. 2013, Japanese, Domestic conference

    Oral presentation

  • 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

    中安大, 川崎崇, 山村理恵, Lee Hyoung Jae, 遠城道雄, 杉本幸裕, 水谷正治

    第31回植物細胞分子生物学会, Sep. 2013, Japanese, Domestic conference

    Oral presentation

  • 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

    山村理恵, 川崎崇, Lee Hyoung Jae, 中安大, 岡田実佳, 遠城道雄, 杉本幸裕, 水谷正治

    第31回植物細胞分子生物学会, Sep. 2013, Japanese, Domestic conference

    Oral presentation

  • トマト果実におけるα-トマチン代謝酵素遺伝子の探索

    小林緑, 飯島陽子, 中安大, 杉本幸裕, 水谷正治

    第31回植物細胞分子生物学会, Sep. 2013, Japanese, Domestic conference

    Poster presentation

  • 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

    中安大, Lee Hyoung Jae, 杉本幸裕, 水谷正治

    第31回植物細胞分子生物学会, Sep. 2013, Japanese, Domestic conference

    Poster presentation

  • グリコアルカロイド生合成遺伝子群の同定について

    梅基直行, 佐々木勝徳, 大山 清, 山下まり, MIZUTANI MASAHARU, 關 光, 斉藤和季, 村中俊哉

    第31回植物細胞分子生物学会, Sep. 2013, Japanese, Domestic conference

    Oral presentation

  • 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

    川口大地, 山内靖雄, MIZUTANI MASAHARU, 杉本幸裕

    第31回植物細胞分子生物学会, Sep. 2013, Japanese, Domestic conference

    Poster presentation

  • The bioconversion of 5-deoxystrigol to monohydroxylated strigolactones by plants

    Ueno K, Motonami N, Nakashima H, Nomura S, Mizutani M, Takikawa H, Sugimoto Y

    World Congress of Parasitic Plants, Jul. 2013, English, International conference

    Oral presentation

  • Structural requirements of strigolactones for germination induction and inhibition of Striga gesnerioides seeds

    Sugimoto Y, Nomura S, Nakashima H, Mizutani M, Takikawa H

    12th World Congress of Parasitic Plants, Jul. 2013, English, International conference

    Oral presentation

  • 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

    MIZUTANI MASAHARU, 小埜栄一郎, 河合洋介

    蛋白質科学会, Jun. 2013, Japanese, Domestic conference

    Poster presentation

  • Transgenic potatoes remarkably decreasing content of glycoalkaloids

    Naoyuki Umemoto, Katsunori Sasaki, Kiyoshi Ohyama, Mari Yotsu-Yamashita, Masaharu Mizutani, Hikaru Seki, Kazuki Saito, Toshiya Muranaka

    TERPNET2013, Jun. 2013, English, International conference

    [Invited]

    Invited oral presentation

  • Identification and quantification of steroidal saponins in Dioscorea esculenta

    李 滎宰, 渡辺 文太, 中安 大, 山村 理恵, 岡田 実佳, 杉本 幸裕, MIZUTANI MASAHARU

    TERPNET2013, Jun. 2013, English, International conference

    Poster presentation

  • Evolution and diversity of the 2-oxoglutarate-dependent dioxygenase superfamily in plants

    MIZUTANI MASAHARU, 小埜栄一郎, 河合洋介

    TERPNET2013, Jun. 2013, English, International conference

    Poster presentation

  • Characterization of furostanol glycoside 26-O-β-glucosidase involved in hydrolysis of protodioscin from Dioscorea esculenta,

    Nakayasu M, Kawasaki T, Lee H-J, Yamamura R, Onjyo M, Sugimoto Y, Mizutani M

    TERPNET2013, Jun. 2013, English, International conference

    Oral presentation

  • Characterization of furostanol glycoside 26-O-β-glucosidase involved in hydrolysis of protodioscin from Dioscorea esculenta

    Nakayasu M, Kawasaki T, Lee H-J, Yamamura R, Onjyo M, Sugimoto Y, Mizutani M

    TERPNET2013, Jun. 2013, English, International conference

    Oral presentation

  • Characterization of De90B, cholesterol 22-hydroxylase involved in steroidal saponin biosynthesis in the tubers of Dioscorea esculenta

    Yamamura R, Kawasaki T, Lee H-J, Nakayasu M, Okada M, Onjyo M, Sugimoto Y, Mizutani M

    TERPNET2013, Jun. 2013, English, International conference

    Oral presentation

  • 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

    MIZUTANI MASAHARU, 中安大, 李榮宰, 山村理恵, 岡田実佳, 川崎崇, 遠城道雄, 杉本幸裕

    岩手生物工学研究所セミナー, Apr. 2013, Japanese, Domestic conference

    Invited oral presentation

  • 植物二次代謝の多様性とシトクロムP450の分子進化

    MIZUTANI MASAHARU

    次世代創薬プロジェクトワークショップ, Feb. 2013, Japanese, Domestic conference

    [Invited]

    Invited oral presentation

  • Remodeling of photosystems to induce thermal dissipation and state transition accompanied by structural changes of thylakoid membranes in heat-stressed wheat

    Yoko Marutani, Yasuo Yamauchi, Akimoto S, Kanako Inoue, Ken-ichi Ikeda, Masaharu Mizutani, Yukihiro Sugimoto

    The 6th Asia and Oceania Conference on Photobiology, 2013, English, International conference

    Poster presentation

  • Cytochromes P450 in Steroid Glycoalkaloids Biosynthesis in Solanum Plants

    MIZUTANI MASAHARU

    P450発見50周年記念シンポジウム, Dec. 2012, Japanese, Domestic conference

    Poster presentation

  • 植物二次代謝(適応代謝)の多様性とP450の分子進化

    MIZUTANI MASAHARU

    第28回ユーグレナ研究会, Nov. 2012, Japanese, Domestic conference

    [Invited]

    Invited oral presentation

  • Oxidative Bioconversion of a Synthetic Strigolactone Analog GR24 in the Root Cultures of Menispermum dauricum

    Masaharu Mizutani, M. Hiragakiuchi, H. Nakashima, N. Motonami, K. Ueno, H. Takikawa, Y. Sugimoto

    10th International Congress on Plant Molecular Biology, Oct. 2012, English, International conference

    Poster presentation

  • Oxidation of 5-deoxystrigol in sorghum; Sorghum bicolor (L.) Moech

    N. Motonami, H. Nakashima, K. Ueno, Masaharu Mizutani, H. Takikawa, Y. Sugimoto

    10th International Congress on Plant Molecular Biology, Oct. 2012, English, International conference

    Poster presentation

  • D1 protein is degraded in heat-stressed plants in the dark; presumably due to electron inflow of the stromal reducing power into the plastoquinone

    Y. Marutani, Y. Yamauchi, Y. Kimura, Masaharu Mizutani, Y. Sugimoto

    10th International Congress on Plant Molecular Biology, Oct. 2012, English, International conference

    Poster presentation

  • 植物ステロイドサポニン生合成の解明

    MIZUTANI MASAHARU

    第1回植物二次代謝フロンティア, Sep. 2012, Japanese, Domestic conference

    [Invited]

    Invited oral presentation

  • 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

    中安大, 川崎崇, Lee Hyoung Jae, 山村理恵, 遠城道雄, 杉本幸裕, MIZUTANI MASAHARU

    第30回日本植物細胞分子学会, Aug. 2012, Japanese, Domestic conference

    Oral presentation

  • 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

    宅間悠紀, 巳浪真輝, 中嶌瞳, 山内靖雄, MIZUTANI MASAHARU, 滝川浩郷, 杉本幸裕

    第30回日本植物細胞分子学会, Aug. 2012, Japanese, Domestic conference

    Oral presentation

  • 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

    Masaru Nakayasu, Takashi Kawasaki, Hyoung-Jae Lee, Rie Yamamura, Michio Onjyo, Yukihiro Sugimoto, Masaharu Mizutani

    11th International Symposium on Cytochrome P450, Jun. 2012, English, International conference

    Poster presentation

  • 根寄生雑草Striga gesnerioides種子発芽誘導物質alectrolの単離と構造決定

    野村早紀, 上野琴巳, 村中聡, MIZUTANI MASAHARU, 滝川浩郷, 杉本幸裕

    日本農芸化学会大会2012年度大会, Mar. 2012, Japanese, Domestic conference

    Oral presentation

  • 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

    中嶌瞳, 平垣内雅規, 本並宜子, 上野琴巳, MIZUTANI MASAHARU, 滝川浩郷, 杉本幸裕

    日本農芸化学会大会2012年度大会, Mar. 2012, Japanese, Domestic conference

    Oral presentation

  • 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

    丸谷曜子, 山内靖雄, 秋本誠志, MIZUTANI MASAHARU, 杉本幸裕

    第52回日本植物生理学会, Mar. 2012, Japanese, Domestic conference

    Oral presentation

  • 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

    中安大, 川崎崇, 山村理恵, 遠城道雄, 杉本幸裕, MIZUTANI MASAHARU

    日本農芸化学会大会2012年度大会, Mar. 2012, Japanese, Domestic conference

    Oral presentation

  • ソルガムにおける5-deoxystrigolの酸化的代謝の解析

    本並宜子, 中嶌瞳, 上野琴巳, MIZUTANI MASAHARU, 滝川浩郷, 杉本幸裕

    日本農芸化学会大会2012年度大会, Mar. 2012, Japanese, Domestic conference

    Oral presentation

  • LC-MS/MSによる新奇ストリゴラクトン探索法の確立

    上野 琴巳, MIZUTANI MASAHARU, TAKIKAWA HIROSATO, SUGIMOTO YUKIHIRO

    日本農芸化学会2012年大会, Mar. 2012, Japanese, Domestic conference

    Oral presentation

  • 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

    Marutani Yoko, Yamauchi Yasuo, Akimoto Seiji, Kimura Yukihiro, Mizutani Masaharu, Sugimoto Yukihiro

    第53回日本植物生理学会年会, 2012, Japanese, Domestic conference

    Oral presentation

  • 植物の酸素添加酵素の多彩な機能と代謝工学への展開

    MIZUTANI MASAHARU

    植物化学シンポジウム, Nov. 2011, Japanese, Domestic conference

    [Invited]

    Invited oral presentation

  • 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

    上野 琴巳, 野村 早紀, 藤原 真美, MIZUTANI MASAHARU, SASAKI MITSURU, TAKIKAWA HIROSATO, SUGIMOTO YUKIHIRO

    植物化学調節学会第46回大会, Nov. 2011, Japanese, Domestic conference

    Oral presentation

  • 根寄生雑草Striga gesnerioides種子発芽刺激物質の立体化学

    上野 琴巳, 野村 早紀, 藤原 真美, 村中 聡, 水谷 正治, 佐々木 満, 滝川 浩郷, 杉本 幸裕

    天然有機化合物討論会, Sep. 2011, Japanese, Domestic conference

    Oral presentation

  • 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

    川崎崇, 山村理恵, 中安大, 遠城道雄, 杉本幸裕, MIZUTANI MASAHARU

    第29回植物細胞分子生物学会, Sep. 2011, Japanese, Domestic conference

    Oral presentation

  • トマト果実におけるα-トマチンの代謝酵素の探索

    浦川晋吾, 飯島陽子, 青木考, 川崎崇, 中安大, 杉本幸裕, MIZUTANI MASAHARU

    第29回植物細胞分子生物学会, Sep. 2011, Japanese, Domestic conference

    Oral presentation

  • トマトにおけるα-トマチン生合成酵素の探索

    中安大, 川崎崇, 浦川晋吾, 杉本幸裕, MIZUTANI MASAHARU

    第29回植物細胞分子生物学会, Sep. 2011, Japanese, Domestic conference

    Oral presentation

  • 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

    MIZUTANI MASAHARU, 伊藤杏子, 清水文一, 杉本幸裕

    第29回植物細胞分子生物学会, Sep. 2011, Japanese, Domestic conference

    Oral presentation

  • Structural requirements of strigolactones for induction of germination in root parasitic plants

    Ueno K, Fujiwara M, Nomura S, Mizutani Masaharu, Sasaki M, Takikawa H, Sugimoto Y

    11th World Congress on Parasitic Plants, Sep. 2011, English, International conference

    Poster presentation

  • 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

    長谷川綾香, 山内靖雄, MIZUTANI MASAHARU, 杉本幸裕

    日本農芸化学会大会2011年度大会, Mar. 2011, Japanese, Domestic conference

    Oral presentation

  • 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

    Marutani Yoko, Yamauchi Yasuo, Akimoto Seiji, Kimura Yukihiro, Mizutani Masaharu, Sugimoto Yukihiro

    日本農芸化学会関西・中部支部合同大会, 2011, Japanese, Domestic conference

    Oral presentation

  • 根寄生雑草Striga gesnerioides種子発芽刺激物質の立体化学

    上野 琴巳, 野村 早紀, 藤原 真美, 村中 聡, MIZUTANI MASAHARU, SASAKI MITSURU, TAKIKAWA HIROSATO, SUGIMOTO YUKIHIRO

    第53回天然有機化合物討論会, 2011, Japanese, 大阪, Domestic conference

    Poster presentation

  • Transcriptome analysis of the rhizomes of Dioscorea spp. to investigate steroidal saponin biosynthesis

    Mizutani Masaharu, Nakayasu M, Kawasaki T, Sugimoto Y

    Terpnet2011, 2011, English, International conference

    Poster presentation

  • 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

    伊藤杏子, MIZUTANI MASAHARU, 杉本幸裕

    植物化学調節学会第45回大会, Oct. 2010, Japanese, Domestic conference

    Poster presentation

  • 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

    青山光, 岡崎真理子, 大西利幸, MIZUTANI MASAHARU, 上野琴巳, 平井伸博, 轟泰司

    植物化学調節学会第45回大会, Oct. 2010, Japanese, Domestic conference

    Poster presentation

  • 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

    村松卓, 大西利幸, MIZUTANI MASAHARU, 上野琴巳, 平井伸博, 轟泰司

    植物化学調節学会第45回大会, Oct. 2010, Japanese, Domestic conference

    Poster presentation

  • 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

    長谷川綾香, 山内靖雄, MIZUTANI MASAHARU, 杉本幸裕

    植物化学調節学会第45回大会, Oct. 2010, Japanese, Domestic conference

    Poster presentation

  • Exploring P450s involved in ABA catabolism from Bryophyte and Pteridophyte

    Mizutani Masaharu, Urakawa S, Nishigaki N, Sugimoto Y

    P450 Biodiversity and Biotechnology; 10th International Symposium, Oct. 2010, English, International conference

    Poster presentation

  • ABAアミノ酸複合体の合成と生物活性

    成田健太, 村松卓, 大西利幸, MIZUTANI MASAHARU, 上野琴巳, 平井伸博, 轟泰司

    植物化学調節学会第45回大会, Oct. 2010, Japanese, Domestic conference

    Poster presentation

  • 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

    岡崎真理子, 村松卓, 青山光, 上野琴巳, MIZUTANI MASAHARU, 平井伸博, Hataitip Nimikeatkai, 近藤悟, 大西利幸, 轟泰司

    植物化学調節学会第45回大会, Oct. 2010, Japanese, Domestic conference

    Poster presentation

  • Qualitative and quantitative analysis of strigolactones using LC-MS/MS

    UenoK, Mizutani Masaharu, Sugimoto Y

    SATREPS-JSPS AA Science Platform Program Joint Seminar on Striga spp.; the food security scourge in Africa, Jul. 2010, English, International conference

    Oral presentation

  • 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

    太田 早矢香, MIZUTANI MASAHARU, 榊原 均, 杉本 幸裕

    日本農芸化学会大会2010年度大会, Mar. 2010, Japanese, Domestic conference

    Oral presentation

  • コケおよびシダ植物におけるABA不活性化P450の探索

    MIZUTANI MASAHARU, 浦川晋吾, 西垣直人, 杉本幸裕

    第51回日本植物生理学会年会, Mar. 2010, Japanese, Domestic conference

    Oral presentation

  • キュウリ緑葉由来NADPH 依存性Alkenal/one oxidoreductase の精製と遺伝子解析

    長谷川綾香, 山内靖雄, MIZUTANI MASAHARU, 杉本幸裕

    日本農芸化学会大会2010年度大会, Mar. 2010, Japanese, Domestic conference

    Oral presentation

  • ABAアミノ酸複合体の生理作用発現機構

    成田健太, 村松卓, MIZUTANI MASAHARU, 上野琴巳, 轟泰司

    日本農芸化学会大会2010年度大会, Mar. 2010, Japanese, Domestic conference

    Oral presentation

  • 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

    岡崎真理子, 青山光, 白倉美奈穂, 上野琴巳, MIZUTANI MASAHARU, 平井伸博, 轟泰司

    日本農芸化学会大会2010年度大会, Mar. 2010, Japanese, Domestic conference

    Oral presentation

  • 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

    内記久美, 青山光, 白倉美奈穂, 上野琴巳, MIZUTANI MASAHARU, 平井伸博, 轟泰司

    日本農芸化学会大会2010年度大会, Mar. 2010, Japanese, Domestic conference

    Oral presentation

  • キュウリ緑葉由来NADPH 依存性Alkenal/one oxidoreductase の精製と遺伝子解析

    長谷川綾香, 山内靖雄, MIZUTANI MASAHARU, 杉本幸裕

    日本農芸化学会関西支部例会, Dec. 2009, Japanese, Domestic conference

    Oral presentation

  • 根粒共生がオロバンキの寄生に及ぼす影響

    土井智子, MIZUTANI MASAHARU, 杉本幸裕

    植物化学調節学会第44回大会, Oct. 2009, Japanese, Domestic conference

    Poster presentation

  • 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

    太田早矢香, MIZUTANI MASAHARU, 杉本幸裕

    植物化学調節学会第44回大会, Oct. 2009, Japanese, Domestic conference

    Poster presentation

  • 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

    福冨達也, MIZUTANI MASAHARU, 杉本幸裕

    植物化学調節学会第44回大会, Oct. 2009, Japanese, Domestic conference

    Poster presentation

  • 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

    伊藤杏子, MIZUTANI MASAHARU, 杉本幸裕

    植物化学調節学会第44回大会, Oct. 2009, Japanese, Domestic conference

    Poster presentation

  • メスキートが生産する有用二次代謝産物の検索

    山本知佳, MIZUTANI MASAHARU, 杉本幸裕

    植物化学調節学会第44回大会, Oct. 2009, Japanese, Domestic conference

    Poster presentation

  • 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

    杉本元, MIZUTANI MASAHARU, 杉本幸裕

    農芸化学会合同支部会, Oct. 2009, Japanese, Domestic conference

    Oral presentation

  • 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

    西垣直人, 浦川晋吾, MIZUTANI MASAHARU, 杉本幸裕

    植物化学調節学会第44回大会, Oct. 2009, Japanese, Domestic conference

    Poster presentation

  • 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

    桂田拓人, 杉本元, MIZUTANI MASAHARU, 杉本幸裕

    植物化学調節学会第44回大会, Oct. 2009, Japanese, Domestic conference

    Poster presentation

  • 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

    岡崎真理子, 青山光, 白倉美奈穂, MIZUTANI MASAHARU, 平井伸博, 轟泰司

    植物化学調節学会第44回大会, Oct. 2009, Japanese, Domestic conference

    Poster presentation

  • 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

    内記久美, 青山光, 白倉美奈穂, MIZUTANI MASAHARU, 平井伸博, 轟泰司

    植物化学調節学会第44回大会, Oct. 2009, Japanese, Domestic conference

    Poster presentation

  • ABA代謝物質ファゼイン酸の還元酵素の精製

    藤井佑典, 近藤悟, MIZUTANI MASAHARU, 平井伸博

    植物化学調節学会第44回大会, Oct. 2009, Japanese, Domestic conference

    Poster presentation

  • Unravelling coumarin biosynthesis in plants and in vivo synthesis in E. coli

    Mizutani, M, Itoh, K, Sugimoto, Y

    25th Naito Conference, Sep. 2009, English, International conference

    Oral presentation

  • Rice CYP734As are multifunctional P450s in brassinosteroid catabolism.

    Mizutani Masaharu, Kawabe A, Shimizu B, Shimada Y, Fujioka S, Sakamoto T

    Terpnet 2009, Jun. 2009, English, International conference

    [Invited]

    Invited oral presentation

  • 東方美人茶における香気成分hortrienol前駆体の単離と精製

    谷田彩, 木下朋美, MIZUTANI MASAHARU, 渡辺修治

    農芸化学会2009年度大会, Mar. 2009, Japanese, Domestic conference

    Oral presentation

  • 新規ブラシノステロイド不活性化経路の解明

    MIZUTANI MASAHARU, 川邉綾美, 清水文一, 嶋田幸久, 藤岡昭三, 坂本知昭

    第50回植物生理学会, Mar. 2009, Japanese, Domestic conference

    Oral presentation

  • 植物免疫信号伝達系におけるセロトニンの機能解析

    藤原幹, Sylvie, M, 一色正之, MIZUTANI MASAHARU, 川崎努, 島本功

    第50回植物生理学会, Mar. 2009, Japanese, Domestic conference

    Oral presentation

  • 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

    清水文一, 田口悟朗, 山本亮太郎, 甲斐光輔, MIZUTANI MASAHARU

    農芸化学会2009年度大会, Mar. 2009, Japanese, Domestic conference

    Oral presentation

  • 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

    清水文一, 田口悟朗, 山本亮太郎, 甲斐光輔, MIZUTANI MASAHARU

    第50回植物生理学会, Mar. 2009, Japanese, Domestic conference

    Oral presentation

  • 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

    青山光, 小林亨太郎, 白倉美奈保, 平松佐織, MIZUTANI MASAHARU, 平井伸博, 轟泰司

    農芸化学会2009年度大会, Mar. 2009, Japanese, Domestic conference

    Oral presentation

  • 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

    轟泰司, 平松佐織, 白倉美奈保, MIZUTANI MASAHARU, 平井伸博

    農芸化学会2009年度大会, Mar. 2009, Japanese, Domestic conference

    Oral presentation

  • ABA代謝物質ファゼイン酸の還元酵素の性質

    藤井佑典, 近藤悟, MIZUTANI MASAHARU, 轟泰司, 平井伸博

    農芸化学会2009年度大会, Mar. 2009, Japanese, Domestic conference

    Oral presentation

Association Memberships

  • Japanese Society for Plant Biotechnology

    Mar. 2000 - Present
  • Tha Japanese Society for Chemical Regulation of Plants

    1998 - Present
  • 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

Research Projects

  • ジャガイモシストセンチュウに対する新奇孵化促進物質の同定と生合成の解明

    水谷 正治, 串田 篤彦, 坂田 至

    日本学術振興会, 科学研究費助成事業, 基盤研究(B), 神戸大学, 01 Apr. 2021 - 31 Mar. 2024

  • Elucidation of the BC-ring formation mechanisms in the biosynthesis of strigolactones

    SUGIMOTO YUKIHIRO

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Kobe University, 01 Apr. 2019 - 31 Mar. 2022

    Strigolactones (SLs) are carotenoid-derived phytohormones and rhizosphere signaling molecules for arbuscular mycorrhizal fungi and root parasitic weeds. Cytochrome P450 CYP722C was identified as a key enzyme that catalyzes the reaction of BC-ring closure and the conversion of carlactonoic acid (CLA) to 5-deoxystrigol in cotton and orobanochol in cowpea and tomato. By knocking out the gene in tomato plants, orobanchol was undetectable in the root exudates but CLA, which was not observed in root exudates of wild-type (WT) plants, was detected. Root exudates of the knockout plants reduced the induction of germination of seeds of root parasitic weeds compared to WT. The architecture of the knockout and WT plants was comparable. These findings suggest the possibility of generating crops with greater resistance to infection by noxious root parasitic weeds.

  • 水谷 正治

    学術研究助成基金助成金/基盤研究(C), Apr. 2018 - Mar. 2021, Principal investigator

    Competitive research funding

  • Identification of new biosynthetic and metabolic genes on strigolactones

    Umehara Mikihisa, MIZUTANI Masaharu

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Toyo University, 01 Apr. 2014 - 31 Mar. 2017

    Strigolactones (SLs) are derived from beta-carotene. Beta-carotene isomerase, carotenoid cleavage dioxygenase (CCD) 7, CCD8, and cytochrome P450 are involved in SL biosynthesis. Plant roots can highly produce SLs in response to phosphorus and/or nitrogen deficiencies. In addition to the nutrient deficiencies, this study demonstrated that sulfur deficiency stimulated SL production in plant roots. The increased SL level is due to marked expression of D27 that encodes b-carotene isomerase.

  • The statistical verification of the minimum experimental datasets for the isolation of plant secondary metabolites related transcription factors using RNA-Seq analysis technique.

    Suzuki Hideyuki, Mannen Kazuto

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Kazusa DNA Research Institute, 01 Apr. 2014 - 31 Mar. 2017

    We established standalone ConfeitoGUIplus software for detecting local communities from a correlation network involving size sensitivity. The developed software accepts any kind of a multivariate dataset and a correlation matrix. Using the public Arabidopsis DNA array resource data (more than 9 thousands), we have shown an application of our developed software to isolate gene co-expression network module in Arabidopsis flavonol biosynthesis. We performed RNA-Seq analysis in Tanacetum cinerariifolium for gene discovery to isolate biosynthetic emzyme involved in natural pyrethrins biosynthesis. We have investigated the effects of the blend of the volatile organic compounds (VOCs) on gene expressions of seven biosynthetic enzymes.

  • Elucidation of biosynthetic pathway from carlactone to strigolactones

    SUGIMOTO Yukihiro, MIZUTANI Masaharu

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Kobe University, 01 Apr. 2013 - 31 Mar. 2017

    Plausible biosynthetic precursors of strigolactones (SLs) were applied to SL-producing plants and conversion of the precursors to SLs was analyzed. The conversion of carlactone (CL) to carlactonoic acid (CLA) was a common reaction among the plants. Sorghum converted CL and CLA to 5-deoxystrigol (5-DS) and sorgomol, and 5-DS to sorgomol. Moonseed converted CL and 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. Coincidently, moonseed converted exogenously applied CL to an unknown metabolite. Molecular mass of the metabolite was larger than that of CL by 16 and its major fragment ion smaller than that of CL by 2, suggesting that it is most probably a hydroxylated product of CL. Similarly, 4-deoxyorobanchol is not a precursor of orobanchol in cowpea. These results demonstrate that biosynthetic pathway of hydroxyl SLs does not necessarily pass through their respective deoxy SL as a precursor.

  • 戦略的オミクス育種技術体系の構築

    山崎 将紀

    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

    Competitive research funding

  • ゲノム編集技術等を用いた農水産物の画期的育種改良

    水谷 正治

    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, Principal investigator

    Competitive research funding

  • 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

    水谷 正治

    農林水産業・食品産業科学技術研究推進事業, 2016, Principal investigator

    Competitive research funding

  • ゲノム編集技術等を用いた農水産物の画期的育種改良

    水谷 正治

    戦略的イノベーション創造プログラム(SIP), 2016, Principal investigator

    Competitive research funding

  • 水谷 正治

    科学研究費補助金/新学術領域研究, 2011, Principal investigator

    Competitive research funding

  • 水谷 正治

    学術研究助成基金助成金/基盤研究(C), 2011, Principal investigator

    Competitive research funding

  • 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

    水谷 正治

    2010, Principal investigator

    Competitive research funding

  • Development of chemicals that control the glutathione metabolism and oxidative stress and their use for chemical biology

    HIRATAKE Jun, WATANABE Bunta, MIZUTANI Masaharu, SHIMIZU Bunichi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Kyoto University, 2007 - 2009

    Thisstudy concerns the development and applications of chemical tools that regulate the redox status of cells by designing and synthesizing specific inhibitors of γ-glutamylcysteine synthetase (GCS), therate-determiningenzymeinglutathione synthesis, and γ-glutamyl transpeptidase (GGT), the initial andprimarily important enzyme in the glutathione metabolism.The inhibitors are to serve as chemical probes to investigate the relationships between cell redox potential and various diseases and as leads to agrochemicals and pharmaceuticals, as well as to antiaging cosmetics.

  • Development of drugs protecting plants from environmental stress in semi-arid land

    HIRAI Nobuhiro, MIZUTANI Masaharu, TODOROKI Yasushi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Kyoto University, 2006 - 2008

    半乾燥地帯における植物の環境ストレス保護剤の開発を目的として、環境ストレスへの適応反応を誘導する植物ホルモン・アブシシン酸の代謝不活性化酵素に関する研究を行った。その結果、アブシシン酸水酸化酵素の選択的阻害剤の開発に成功するとともに、アブシシン酸の最終不活性化酵素であるファゼイン酸還元酵素活性の諸性質を明らかにした。これにより、アブシシン酸の不活性化制御による植物保護の道を拓いた。

  • Construction of plant oxygenase libraries and their functional characterization

    MIZUTANI Masaharu

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Kyoto University, 2006 - 2007

    The aim of this study was to establish the protein libraries of plant oxygenases consisiting of cytochrome P450monooxygenases,2-oxoglutarate dependent dioxygenases, and flavin monooxygenases and to elucidate their biochemical functions. (1) Plant Monooxygenase Database: The gene sequences as well as the deduced amino acid sequences of three monooxygenases (P450/FMO/2OGD) were extracted from the genome database of Arabidopsis. The phylogenetic analyses of the protein sequences were performed. In addition, the data of the gene expression in various treatments were extracted from MPSS and TAIR database. (2) Isolation of full ORFs and expression of the recombinant proteins: The full-length cDNAs of P450/FMO/2OGD have been collected from RIKEN-BRC, and the full-ORFs of the other clones were amplified and isolated by RT-PCR approach. After all, about 70% of total oxygenase ORFs have been isolated. The ORFs of FMOs and2OGDs were introduced into the expression vector pETs, and the recombinant proteins with N-terminal His-tag were expressed in E. coli and were purified with affinity chromatography. (3) Construction of the co-expression system of plant P450s and P450-reductase; P450s require the electron-transfer from P450-reductase (CPR) for the P450 activities, but E. coli does not have the reductase. The co-expression system of P450s and CPR was constructed with the co-transformation of the two plasmids (pCWori-P450 and pACYC-CPR) into the bacterial cells. The co-expression system enabled us to detect P450 activities in in vitro and in vivo assays in E. coli. (4) Analysis of At3g13610: We previously found that Arabidopsis roots accumulate large amounts of scopolin, a β-glucoside of coumarin derivtive scopoletin. At3g13610, which is one of the 2OGD genes and exclusively expressed in roots, was found to catalyze ortho-hydroxylation of feruloyl-CoA thioester to form scopoletin. Thus, At3g13610 is Feruloyl-CoA 6'-hydroxylase in Arabidopsis, and this is an ortho-hydrozylase, which is a key enzyme in coumarins biosynthesis in hiaher plants.

  • Elucidation of the aroma formation of Darjeeling tea "Second flash" and development of new manufacturing process of black tea

    MIZUTANI Masaharu, SHIMIZU Bun-ichi, KINOSHITA Tomomi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Kyoto University, 2006 - 2007

    We made the joint羊esearch contract of two遥ears collaboration with Indian Tea Research Association (TRA) and Tocklai Tea Experimental Station (TIES). Accordingly, we performed the researches as follows: 1) Investigation of the manufacturing of Darjeeling tea “Second flash"in India. At the end of May 2006, three researchers from Japan visited TTES at Tocklai, Assam and also Darjeeling in India. We, together with two researchers from TTES, visited several tea gardens in Darjeeling to see the tea manufacturing and the state of the insect擁nfestation. We also collected the tea samples at two gardens. We observed that two kinds of insects, jassid (green leaffly: Empoasca flavescens Fabr.) and thrips (Taeniothrips setiventris Bagnall), are mainly involved in the insect infestation in Darjeeling. In June 2007, Japanese researchers visited TTES. The in様aboratry breeding of the insects was performed at TTES, and the tea leaves artificially infested by the insects were prepared to analyze the effects of the infestation on the characteristic aroma formation of the tea. 2) Elucidation of the characteristic aroma formation of Darjeeling tea “Second flash"produced with the insect擁nfested leaves. The samples collected at the Darjeeling tea gardens in 2006 were analyzed by GC-MS, and it was suggested that insect infestation is involved in the aroma formation such as monoterpene alcohols and hotrienol. It was also evident from the experiments in 2007that insect infestation is mainly concerned with the characteristic aroma formation. The other experiments conducted at a Japanese tea garden demonstrated that the infestation by tea green様eafhopper (a very similar insect to jassid) is involved in the formation of 2,6-dimethylocta-3,7-diene-2,6-diol. 3) Attempts to develop a new manufacturing process of CTC black tea. We suggested to the TTES researchers that they should apply the concept in manufacturing Taiwan oolong tea to Indian black tea manufacturing to improve the quality of the flavor of CTC black tea. According to our suggestion, they tried the tea manufacturing with a pilot plant at TTES, and found that the additional processes of some mechanical damages on tea leaves during the withering may improve the quality of the tea flavor.

  • Applied metabolomics based on the Fourie transform ion cyclotron mass spectrometry

    OHTA Daisaku, MIZUTANI Masaharu, KANAYA Shigehiko

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Osaka Prefecture University, 2006 - 2007

    With the aid of the extreme resolving power of Fourier-transform ion-cyclotron-resonance mass spectrometry (FT-ICR/MS), we have developed a metabolomics platform for high-throughput metabolic profiling and metabolite candidate identification integrating a data-processing system, the Dr.DMASS program (http://kanaya.naist.jp/DrDMASS/), and a metabolite-species database, KNApSAcK (http://kanaya.aist-nara.ac.jp/KNApSAcK/). The entire scheme was applied to study key metabolic events and enzymes involved therefore. First, this FT-ICR/MS-based metabolic profiling scheme was used to clarity plant metabolic disorders and specific metabolite accumulation patterns caused by herbicidal enzyme inhibitors. Also, the scheme was applied to differentiate metabolic activities through detailed comparison of metabolite accumulation patterns reflecting light/dark conditions in Arabidopsis T87 cell culture. Furthermore, several plant cytochromes P450 functions have been demonstrated using the FT-ICR/MS based metabolomics studies.

  • Molecular evolution of plant β-glucosidases deduced from clarification of substrate specificities and catalytic mechanisms of β-diglycosidases (glycosidases specific to disacchalide glycosides)

    SAKATA Kanzo, HIRATAKE Jun, MIZUTANI Masaharu, SHIMIZU Bunichi, KATO Hiroaki

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Kyoto University, 2004 - 2006

    1) Clarification of substrate specificities and catalytic mechanisms of diglycosidases 1-a) X-Ray crystallographic analysis of β-primeverosidase from a tea plant We have succeeded in synteshis of a potent glycosidase inhibitor, β-primeverosyl amidine, for a representative diglycosidase, β-primeverosidase. Single crystalls of β-primeverosidase binding the inhibitor was obtained and subjected to X-ray crystallographic analysis to obtain the structure of the protein with resolution of 1.8 A based on the structure of a family 1 β-glucosidase from maize. The structure allowed us to identify the amino acid residures that interact with disaccharide moiety of the substrates and to show the detailed structure of the opening part of catalytic site. Based on these observations and the substrate specificities of the enzyme we could have understood the substrate recognition mechanism of the enzyme in detail. 1-b) Clarification of the substrate recognition mechanism of a diglycosidase by the use of 6'-substituted β-D-glucopyranosides Pseudo-disaccharide glycosides with various kinds of S-substituents (alcohols of C_<2_4> and D-xylopyranosyl) at C-6'of p-nitrophenyl (pNP) β-D-glucopyranosides were synthesized and subjected to hydrolysis by a diglycosidase, vicianin hydrolase. This diglycosidase shows high reactivity against pNP β-primeveroside, but little activity against 6'-S-substituted pNP β-primeveroside as well as pNPβ-D-glucopyranoside. This enzyme shows high activity against pseudo-diglycosides with a straight chain C3-alcohol substituent at C-6'. These observations suggest that the hydroxy group of the C_3-alcohol substituent interacts with the amino acid residues concerned with the recognition of the disaccharide moeiety of natural substrates. 2) Distribution of diglycosidases in plant kingdom and analysis of molecular evolution of diglycosidases By the use of pNP P-primeveroside as a substrate diglycosidases were screened among plant crude enzymes randomly selected. Out of 18 kinds of plant species β-primeverosidase-like activity was found in the crude enzyme of 7 kinds of plants, indicating considerably wide distribution of diglycosidases among plant kingdom. Phylogenetic tree analysis of diglycosidases, β-primeverosidase, furcatin hydrolase and vicianin hydrolase in family 1 of glycosyl hydrolases were carried out based on their full amino acid sequences, indicating that these diglycosidases cluster with each other.

  • Bioorganic studies on plant glycosidases by using p-glycosylamidines as research tools

    HIRATAKE Jun, MIZUTANI Masaharu, SHIMIZU Bun'ichi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Kyoto University, 2004 - 2006

    The function and the physiological roles of plant β-glycosidases have been studied by using p-glycosylamidines as molecular probes. p-Glycosylamidines, newly developed p-glycosidase inhibitors that selectively inhibitβ-glycosidases according to their glycon substrate specificities, have been successfully used as ligand for affinity chromatography of glycosidases. High fidelities of β-glycosylamidines as specific binders toward theβ-glycosidases with the corresponding glycon substrate specificities enabled one-step affinity purification of specific β-glycosidases from natural samples according to their glycon substrate specificities as a sole marker. This method has been successfully used for the isolation of diglycoside-specific p-glycosidases from microbes and a p-N-acetylhexosaminidase from insect cells. The latter glycosidase is a key biosynthetic enzyme in processing insect-cell-specific N-glycans. With this research tool in hands, we have purified and characterized diglycoside-specific β-glycosidases (diglycosidases) from plants. The diglycosidases are thought to play a key role in self-defense system in plants by emitting defense chemicals such as linalool, 2-phenylethanol and mandelonitrile by cleaving their precursor diglycosides. β-Primeverosidase (PD), a typical diglycosidase found in tea leaves, has been purified to homogeneity by a newly developed pH-controlled affinity chromatography with p-primeverosylamidline as an affinity ligand. The enzymatic properties of PD and the binding mechanism of the β-primeverosylamidine inhibitor have been studied extensively. Furthermore, a gene encoding PD was cloned from tea leaves and was analyzed to find that the diglycosidases were a member of family 1 glycoside hydrolase with significant sequence similarities to the β-glucosidases in this family. This result suggests that plant diglycosidases have been evolved from family 1 β-glucosidases for cleavage of specific diglycosides and serve as a key enzyme in plant self-defense system. The recombinant PD was expressed and highly purified by the affinity chromatography for X-ray structural analysis. The high resolution X-ray crystal structure (1.8 Å) of PD in complex with the β-primeverosylamidine inhibitor revealed the amino acid residues recognizing the β-xylosyl moiety of β-primeveroside and the unique binding mode of β-primeverosylamidine ligand in the active site. These results cogently suggested the structural basis for their unique substrate specificities and the evolutional link between plant diglycosidases and family 1 p-glucosidases.

  • インドール酢酸(IAA)複合体の生合成・分解を化学的にノッアウトする

    平竹 潤, 水谷 正治

    日本学術振興会, 科学研究費助成事業, 萌芽研究, 京都大学, 2005 - 2005

    本研究は、オーキシン(インドール酢酸、IAA)のホメオスタシスに重要な役割を果たしていると思われる、インドール酢酸-アミノ酸複合体(IAA-aa)の合成酵素および加水分解酵素の特異的阻害剤を設計・合成し、遺伝子ノックアウトの代わりに、阻害剤によって化学的に酵素活性をノックアウトするような化合物を開発することを目的としたものである。オーキシン早期誘導遺伝子のGH3ファミリーの一群が、IAA-aa合成酵素ではないかとの予測をふまえ、その推定反応機構にもとづき、IAAのアデニル化中間体のアナログとして、IAAのスルファモイルアデノシン誘導体(IAA-SA)を合成した。さらに、天然および人工オーキシンであるフェニル酢酸、ナフタレン-1-酢酸、2,4-DそれぞれのSA誘導体(PA-SA,NAA-SA,2,4-D-SA)を合成した。得られた化合物は、大腸菌で発現させたGH3-5およびGH3-6に対する阻害活性を測定した。その結果、いずれの化合物も、GH3-5およびGH3-6を両方とも阻害する強い活性を示し、特に、PA-SAはIC_<50>値にして3μM以下の強い阻害活性を示した。これら一連のSA誘導体を培地に溶かし、シロイヌナズナの幼植物体を培養したところ、2,4-D-SAが1μMの低濃度で、根の著しい形態変化を引き起こすことがわかった。これは、IAA過剰時に見られる異常発根(adventitious rooting)に酷似しており、化合物が植物体に取り込まれ、GH-3を阻害することによってオーキシンのホメオスタシスに影響を与えている可能性が強く示唆された。 一方、IAA-aa加水分解酵素についての知見はきわめて乏しいが、IAA-aa添加によってIAA過剰症が出ない変異体の解析から、IAR3,ILR1,ILL2などの遺伝子が見いだされ、それが金属プロテアーゼの一種であるとの知見をもとに、アミノ酸部分の構造の異なるIAAホスフィン酸誘導体5種類を合成した。ホスフィン酸誘導体は、濃度依存的にILR1を阻害することが判明し、合成した化合物が、少なくとも、IAA-aa加水分解酵素の障害剤として機能することが確かめられた。 以上のように、酵素の推定反応機構を唯一の手がかりに、反応機構依存的に設計したオーキシンのSA誘導体および、IAAホスフィン酸誘導体が、予想通り、IAA-aa合成酵素および加水分解酵素の阻害剤として機能し、特に、SA誘導体はin vivoでも活性を示す、化学ノックアウト剤として有望な化合物であることがわかった。

  • 植物テルペノイド生合成酵素のオペロン様遺伝子クラスターの解明

    水谷 正治

    日本学術振興会, 科学研究費助成事業, 若手研究(B), 京都大学, 2003 - 2004

    本研究では、シロイヌナズナゲノム上クラスターを形成するテルペン環化酵素とP450遺伝子が植物テルペノイド生合成経路上でも密接に関連していることを実験的に証明することを目指す。今年度は、(1)各遺伝子発現様式のデータベース解析、(2)バキュロウイルス昆虫細胞系によるP450の組み換え酵素の作成、(3)モデル酵素を利用したアッセイ系の確立、を行った。またシロイヌナズナ以外の植物でも同様の事象を検証するため(4)イネゲノム上のテルペン環化酵素とP450遺伝子クラスターの解析を行った。 (1)理研、マックスプランクおよびABRCから提供されているマイクロアレイデータベースを利用して発現様式を解析した。遺伝子クラスターを形成する各遺伝子は各ホルモンおよびストレス処理下でも協調して発現していた。根で発現しているクローンの多くはジャスモン酸によっても協調的に発現が誘導されていた。これらの事実からクラスターを形成するP450とテルペン合成酵素遺伝子は同じ転写制御を受けている可能性が高いと思われる。(2)CYP706A3,CYP705A1,CYP716A1,CYP708A2,CYP71A16,CYP708A3の各P450 cDNAをpFastBac1発現ベクターに挿入し昆虫細胞Sf9に導入して各組み換えウイルスを作成した。SDS-PAGEにより組み換え酵素の発現を確認した後、C0差スペクトルを測定することにより、活性型P450の発現を確認した。今回、CYP706A3、CYP716A1、CYP71A16、CYP708A3の活性型P450を調製することができた。(3)アブシジン酸の代謝するCYP707A、またステロールを代謝するCYP90BおよびCYP710Aを用いて、in vitroアッセイ系のモデル実験を行った。昆虫細胞ミクロソームに基質を加えることによりP450活性を検出できることが確認できた。これは昆虫細胞由来のP450還元酵素が植物P450に電子伝達できることを示している。(4)イネゲノムを検索した結果、ジテルペン型ファイトアレキシンの生成に関わるジテルペン環化酵素遺伝子とP450(CYP71Zs,CYP76Ms,CYP99As)がイネゲノム上でクラスターを形成していることが分かった。これらのP450はファイトカサンおよびモミラクトン生成に関わる酸化反応を触媒している可能性が高いと思われる。

  • Clarification of Molecular Basis of the Characteristic Aroma Formation in the Formosa Oolong Ted Produced from Tea leaves Infested by the Tea Green Leafhopper

    SAKATA Kanzo, MIZUTANI Masaharu, SHIMIZU Bun-ichi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), KYOTO UNIVERSITY, 2003 - 2004

    Oriental Beauty is a flavor-rich oolong tea produced from tea leaves infested by the tea green leafhopper (Jacobiasca formosana) in Taiwan. We have studied to clarify the molecular basis of the characteristic aroma formation of the tea by various approaches such as natural product chemistry, biochemistry, and molecular biology. Oolong tea samples were prepared from tea leaves infeted/noninfested by the insects. Samples were obtained at each step of the manufacturing process and subjected to evaluation tests by professional tea tasters and to GC-MS analysis. The tea produced from tea leaves infested by the insects was found to be superior in the quality and quantity of aroma to that from tea leaves without or with much less the insect attack. Hotrienol and its related compound, 2,6-dimethyl-3.7-octadiene-2,6-diol, were confirmed to be responsible for the insect attack. Genes induced in response to the insect attack and the tea manufacturing processes were identified by the differential screening based on the Megasort analysis. Among them our interests were focused into the genes encoding the stress-responded ones and the amounts of the stress-responded compounds such as raffinose and abscisic acid in tea samples obtained at each step of the tea processing were quantitatively analyzed by HPLC. Both compounds were found to increase dramatically at the solar withering step in good correlation with the expression of the genes responsible for their biosynthesis at the step. These results have revealed that the tea leaves of Oriental Beauty are greatly affected by the stresses of the insect attack and the tea manufacturing processes such as solar withering and turning-over, and these stresses are important factors to increase the production of the aroma compounds characteristic to this characteristic oolong tea.

  • 植物の生理現象にかかわるグルコシダーゼの網羅的検索 ―特異的グルコシダーゼ阻害剤をツールとしたvivoアッセイ―

    平竹 潤, 清水 文一, 水谷 正治

    日本学術振興会, 科学研究費助成事業, 萌芽研究, 京都大学, 2003 - 2003

    本研究は、グルコース、ガラクトースおよびキシロースから合成した一連のβ-グリコシルアミジン誘導体を、直接、植物(シロイヌナズナ)に与え、その結果観察される、植物の生育および形態上の変化を手がかりに、植物の生理現象にどのような種類のグリコシダーゼが関与しているか、その手がかりを得ることを目的としたものである。そこで、ベンジル基を共通のアグリコン部とし、グルコース、ガラクトースおよびキシロースをグリコン部とする3種類のβ-グリコシルアミジン誘導体(Glc-amidine,Gal-amidineおよびXyl-amidine)を合成し、それぞれの化合物を固形寒天培地(GM培地)に適量加えて、シロイヌナズナ(野生株)を播種あるいは幼植物体を培養し、植物の生育状況と形態を経時的に観察した。その結果、Xyl-amidineを加えた場合のみ、葉の色が黄色くなり、葉の厚みが薄くて垂れ下がった異常な形態を示すことが観察された。また、これらの変化には濃度依存性が見られ、アッセイに用いた最小濃度である0.1μMのXyl-amidine存在下でも、顕著な変化が見られた。一方、グリコン部の構造の異なるGlc-およびGal-amidineでは、このような異常な形態は観察されず、また、構造的に類似したβ-キシロシルアミド(Xyl-amide)でも、この変化は見られなかった。Xyl-amideにはβ-キシロシダーゼ阻害活性がほとんどないことから、シロイヌナズナに見られた形態上の変化はXyl-amidineに特徴的なもので、これら形態上の変化には、何らかの形でβ-キシロシダーゼの阻害が関与していることが示唆された。 一方、植物から抽出した粗酵素液には、高いβ-グルコシダーゼ活性およびβ-ガラクトシダーゼ活性とともに、低いレベルではあるがβ-キシロシダーゼ活性が観測された。In vitroでの阻害実験により、それぞれの酵素活性は、対応するアミジン誘導体によって強く阻害され、特に、粗抽出液中のβ-キシロシダーゼ活性は、Xyl-amidineによって完全に阻害されることが判明した。この結果は、植物の形態異常を引き起こす原因が、β-キシロシダーゼの阻害にあるというin vivoでの結果を裏付けるものである。

  • Substrate-specific Inhibitors of Glycosidases as Tools for Bioorganic Chemical Studies on Glycosidases

    HIRATAKE Jun, TAKADA Masayasu, MIZUTANI Masaharu

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), KYOTO UNIVERSITY, 2001 - 2003

    This study aims to develop novel glycosidase inhibitors and to use these inhibitors as versatile research tools in glycosidases studies from a bioorganic point of view. For this purpose, we have designed and synthesized f -glycosylamidine derivatives as highly potent and glycon-selective inhibitors of β-glycosidases. β-Glycosylamidines are a substrate analog in which a positively charged amidino group is incorporated into a sugar surrogate in such a way as to maintain the whole structure of the glycon, including the stereochemistry at C-1. The β-glycosylamidines are readily synthesized from various sugars n two steps without using any protective groups. The β-glycosylamidines with each glycon moiety serve as highly potent inhibitors (K_i<0.1μM) toward the β-glycosidases with the corresponding glycon substrate specificity, while the β-glycosidases with different glycon substrate specificity are not inhibited significantly. Furthermore, varying structures can be introduced into the "aglycon" moiety of the inhibitor to increase or tune the inhibition potency and selectivity towards various β-glycosidases. Therefore the β-glycosyamidines are versatile β-glycosidase inhibitors in which both the glycon and aglyon moieties can be adjusted according to the enzyme to be inhibited. This property can be successfully used as a ligand for affinity chromatography of glycosidases.. Thus, tea leaf β-glucosidases were purified to homogeneity in one step by the affinity adsorbent with β-glucosylamidine as ligand, while a β-galactosidase from mold was also purified in one step by an affinity chromatography with β-galactosylarnidine as ligand. Other β-glycosidases such as β-primeverosidase, a novel diglycoside-specific glycosidase, was also affinity-purified successfully by using the corresponding glycosylamidine as ligand. The purified tea leaf β-glucosidases were subjected to amino acid sequence analysis to clone the genes. Hence the β-glycosylamidines are versatile research tools in glycosidase studies spanning from the isolation, the characterization and the gene cloning.

  • Establishment of A New Group of Glycosyl Hydrolase Family 1 Specific to Disaccharide Glycosides in Plant Kingdom

    SAKATA Kanzo, HIRATAKE Jun, MIZUTANI Masaharu, SHIMIZU Bunichi, KATO Hiroaki, SAKATA Kanzo

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Kyoto University, 2001 - 2003

    The following research results have been obtained by this research project. (A)Clarification of catalytic mechanisms of the diglycosidases and their stereostructures 1)Substrate specificities of diglycosidases such as tea leaf β-primeverosidase (PRD), furcatin hydrolase (FH) in Viburnum furcatum, Vicianin hydrolase (VH) in Vicia angustifolia var. segetalis have been studied with various kinds of synthetic and natural substrates to show that these diglycosdiases are very specific to disaccharide glycosides with β-1,6 linkage. 2)The full-length cDNA of PRD and VH were overexpressed in insect cells and that of. FH in E.coli. The recombinant PRD was obtained at ca. 30 mg/liter of the medium. This allowed us to try crystallization of the protein for X-ray crystallographic analysis. 3)We have developed a new type of glycosidase inhibitors, glycosylamidines, with potent and selective activities. An affinity adsorbent with a ligand of β-primeverosyl has been prepared and was found to be quite effective for purification of PRD as well as the other diglycosidases. (B)Establishment of a new family of diglycosidases in plant kingdom and clarification of their physiological roles The anti-PRD antibody prepared with the recombinant PRD was found to be highly selective and sensitive. Application of this- antibody allowed us to know the expression of the PRD gene at higher level in the younger tea leaves and the localization of the PRD in cell membrane or intercellular space. Distribution of diglycosidases was surveyed by using. β-primeverosidase activity, detection of disaccharide formation with TLC and immunoblotting analysis with the anti-PRD antibody among several kinds of plants most of which are known to contain some disaccharide glycosides. The results strongly suggest wide range of distribution of diglycosidases in plant kingdom.

  • 植物ホルモン活性化/不活性化機構の分子基盤-ブラシノステロイド生合成・代謝酵素による制御-

    水谷 正治

    日本学術振興会, 科学研究費助成事業, 特定領域研究, 京都大学, 2001 - 2002

    今年度は、ブラシノステロイド不活性化に関わるP450を酵素化学的に解析することを中心として実験を進めた。これまでにトマトからブラシノステロイド生合成に関わるP450(CYP90A, B, C)と不活性化に関わる新規CYP72B2および72B3の全長cDNAを単離した。トマトをブラシノステロイド処理すると生合成遺伝子の転写量は減少したのに対し、CYP72B2および72B3の転写は増加した。また72B2cDNAをタバコで過剰発現させたところ矮性表現型を示した。内生ブラシノステロイド含量をGC-MSにより定量し野生株と比較したところ、組換え体においてカスタステロンが顕著に減少していたことから、過剰発現している72B2がカスタステロンを不活性化したために矮化していることが示唆された。昆虫細胞ムバキュロウイルス発現系により作製した72B2酵素を用いて生合成中間体に対する基質結合アッセイを行った結果、72B2はカスタステロンに対して強い親和性(6μM)を示したが、一方、活性型ブラシノライドや生合成前駆体6-デオキソカスタステロンはやや弱い親和性(11μM)しか示さなかった。そこで、72B2酵素を発現している昆虫細胞のミクロソーム膜を用いてNADPH-P450還元酵素共存のもとで代謝実験を行った。カスタステロンを基質として酵素アッセイを行った結果、GC-MSにより水酸化生成物を検出する事ができた。MS解裂パターンからカスタステロンの側鎖に水酸基が一つ導入されていることが示された。現在、生成物を分取してNMRによる構造解析を進めている。

  • 植物ホルモン活性化/不活性化機構の分子基盤-ブラシノステロイド生合成/代謝酵素による制御-

    水谷 正治

    日本学術振興会, 科学研究費助成事業, 特定領域研究(A), 京都大学, 2000 - 2000

    ブラシノステロイド(BR)生合成・代謝経路には多数のチトクロムP450酵素が関与している。本研究では、BR活性化/不活性化に関与するP450酵素を単離同定し、その発現制御機構を解析することによりBR分子の作用部位における経時的消長を遺伝子、酵素レベルで解析しBRの生理機能の分子基盤を解明することを目的とする。 A.cDNAクローニング:相同性検索によりBR生合成・代謝に関与する可能性の高いと推定された候補P450遺伝子をシロイヌナズナおよびトマトからクローニングした。シロイヌナズナから14クローン、トマトから8クローンの全長cDNAを単離した。 B.BR応答性の検討:シロイヌナズナおよびトマトを用いて、BR処理、BR生合成阻害剤処理による各遺伝子の転写変動を検討した。BR添加によりCYP90、CYP708ファミリーに属する各遺伝子は発現が抑制され、逆に阻害剤により発現が増加したことから、これらの遺伝子はBR生合成に関与していることが示唆された。またトマトCYP72B2はBR処理により発現が増加しBR代謝に関わっている可能性が示唆された。 C.組み替え酵素の発現:候補P450の組み替え酵素を発現させるための予備検討を大腸菌を用いて行った結果、一部の分子種は大量に発現するものの発現しない分子種も多く、またN末領域のアミノ酸配列を改変する必要があるため発現ベクターの構築にも多大な労力を要することが予想された。そこで、より安定して多量のP450を発現することが可能で、かつ全長cDNAをそのまま組み込むことができるバキュロウイルスー昆虫細胞発現系を用いて上記候補P450の発現を行うこととした。pFastBacl発現ベクターに各P450の全長cDNAを組み込み昆虫細胞を用いて発現させた結果、全てのP450について発現タンパク質由来のバンドをSDS-PAGE上で確認することができた。そこで培地にヘムの前駆物質を加えて昆虫細胞を培養しウイルス感染を行ったところ、ほとんどのP450について活性型酵素を検出した。

  • 酵母Three-Hybridシステムによる植物ホルモン受容体のクローニング

    水谷 正治

    日本学術振興会, 科学研究費助成事業, 奨励研究(A), 京都大学, 1999 - 2000

    酵母Three-hybrid法は、低分子リガンド(bait)とタンパク質の相互作用を酵母細胞内で検出し、リガンド受容体候補遺伝子をcDNAライブラリーから直接クローニングする方法である。本研究では、baitとして用いるオーキシンリガンドの合成とその生理活性の検討を行った。またスクリーニングの感度を向上させる目的でThree-hybrid法の改良を試みた。 1)オーキシンリガンドの合成と生理活性;まずデキサメタソン(Dex)との架橋を目的とし、NAAの構造活性相関を化合物のオーキシン活性をシロイヌナズナの根の伸長阻害試験により評価した。NAAのアセトキシメチルエステル誘導体は生体内で速やかに加水分解されるためNAAと同等の活性を持つこと、一方NAAのメチルエステルはIC_<50>にしてNAAの約1/6に低下することから、活性にはカルボキシル基が重要であることが判った。そこでNAAを基本骨格として側鎖メチレン基から炭素鎖を伸ばしてDexと架橋可能な官能基を導入した化合物を合成した。そのメチルエステル体は最終濃度10^<-4>Mで顕著に根の伸長を阻害し、10^<-5>Mでは根の重力応答に異常を与えたことから、低濃度ではオーキシンとしてよりもむしろオーキシン輸送の阻害剤として作用している可能性が示唆された。一方、その加水分解物は10^<-5>Mで顕著に根の伸長を阻害し強いオーキシン活性を示した。今後、この化合物を元にオーキシンリガンド創製を目指す。 2)酵母Three-hybrid法の改良;スクリーニング感度の向上を目的とし、グルココルチコイド受容体とDexとの相互作用よりもさらに強固なNi-NTAとHis-tag相互作用をThree-hybrid法に応用した新しいスクリーニング法の構築を試みた。Dexとニトリロ三酢酸(NTA)を架橋した複合体(Dex-NTA)およびその細胞膜透過性を向上させたアセトキシメチルエステル誘導体を合成した。His-tagを付加したGAL4-BDおよびGAL4-ADとグルココルチコイド受容体との融合タンパク質を発現させた酵母をリガンド添加培地上で培養したが、リガンドに依存したレポーター遺伝子の転写活性化は検出されなかった。現在リガンドの構造、スペーサーの長さも含めてレポーター遺伝子の転写活性化が起こる条件を引き続き検討している。

  • Development of New Technology for Plant Aroma Emission with New Type of Plant

    SAKATA Kanzo, YAMAMOTO Sigeru, KOIKEDA Satoshi, MIZUTANI Masaharu

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B)., KYOTO UNIVERSITY, 1999 - 2000

    Floral tea aroma of oolong tea and black tea has been clarified to be generated form aroma precursors, most of which are present as β-primeverosides in tea leaves, by the action of β-primeverosidase during their manufacturing, so called fermentation processes. Recently these aroma formation mechanisms have been found to be concemed with the attractive aroma formation in flower, fruits, wine, etc. Based on these new findings, new type of aroma emission technology was attempted to be established by screening a new disaccharide specific glycosidase among microbial metabolites, its mass production and its application as follows : 1) Clarification of substrate specificities of the tea β-primeverosidase, cloning of its cDNA and a trial for its large scale overexpression in E.coli. Several kinds of disaccharide glycosides with 2-phenylethanol as an aglycon were obtained by chemical synthesis or from nature. They were subjected to hydrolysis with the β-primeverosidase to know its substrate specificities. The β-primeverosidase showed very high substrate specificity towards β-primeveroside. All the synthetic unntural disaccharide glycosides were not hydrolyzed at all. Only other natural disaccharide (β 1-6) glycosides were hydrolyzed some extent (0.07-3.0%). The amino acid alignment of the β-primeverosidase obtained by its gene cloning suggested some N-glycosyl modifications and fairly high homology (60%) with that of the β-glucosidase which hydrolyzes cyanogenic β-D-glucopyranosides. The recombinant enzyme easily agglutinated to loose its activity, suggested that the N-glycosyl modifications are necessary for the enzyme stability. 2) Screening of microbials with the β-primeveroside hydrolyzing activity Soil-born microbial strains were screened using eugenyl β-primeveroside as a sole carbon sauce to give 4 active strains with the activity. The most active strain was identified as Aspergillus fumigatus Pr-20. The glycosidase purified from the culture medium of the strain showed a single band at 47 kDa in SDS-PAGE analysis and was found to hydrolyze other β (1→6)-disaccharide glycosides as well as 6-acylated β-D-glucopyranoslde. 3) Gene cloning of the β-primeverosidase-like diglycosidase from A.fumigatus The amine acid alignment of the diglycosidase revealed by the gene cloning showed only 14% identity with that of the tea leave β-primeverosidase. The overexpression of the gene was attempted in a yeast and a fungus to give a successful result with a fungus. 4) A few trials to enhancing aroma from endogenous plant aroma precursors by treatment with the diglycosidase The diglycosidase was applied to crude tea leave extracts and grape juice and liberated aroma was analyzed by GC.Many kinds of aroma compounds were found to be much enhanced by this treatment, suggesting that this new enzyme will be applicable to enhance aroma in black tea and wine production and their related industries.

  • Studies on Gtochromes P450 involoed in plant hovmne biosynthsis

    Competitive research funding

  • Studies on β-primeverosidase mvolved in aroma formation in plants

    Competitive research funding