2021/08/17 更新

写真a

オケガワ ユキ
桶川 友季
Okegawa Yuki
所属
資源植物科学研究所 助教
職名
助教
外部リンク

学位

  • 博士(農学) ( 九州大学 )

研究キーワード

  • 光合成

研究分野

  • ライフサイエンス / 植物分子、生理科学

経歴

  • 岡山大学   資源植物科学研究所   助教

    2021年4月 - 現在

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  • 京都産業大学   研究員

    2020年4月 - 2021年3月

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  • 京都産業大学 総合生命科学部   Faculty of Life Sciences   研究助教

    2015年4月 - 2020年3月

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  • 京都産業大学 総合生命科学部   Faculty of Life Sciences   プロジェクト助教

    2010年4月 - 2015年3月

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所属学協会

 

論文

  • The evolutionary conserved iron-sulfur protein TCR controls P700 oxidation in photosystem I. 査読 国際誌

    Mai Duy Luu Trinh, Daichi Miyazaki, Sumire Ono, Jiro Nomata, Masaru Kono, Hiroyuki Mino, Tatsuya Niwa, Yuki Okegawa, Ken Motohashi, Hideki Taguchi, Toru Hisabori, Shinji Masuda

    iScience   24 ( 2 )   102059 - 102059   2021年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    In natural habitats, plants have developed sophisticated regulatory mechanisms to optimize the photosynthetic electron transfer rate at the maximum efficiency and cope with the changing environments. Maintaining proper P700 oxidation at photosystem I (PSI) is the common denominator for most regulatory processes of photosynthetic electron transfers. However, the molecular complexes and cofactors involved in these processes and their function(s) have not been fully clarified. Here, we identified a redox-active chloroplast protein, the triplet-cysteine repeat protein (TCR). TCR shared similar expression profiles with known photosynthetic regulators and contained two triplet-cysteine motifs (CxxxCxxxC). Biochemical analysis indicated that TCR localizes in chloroplasts and has a [3Fe-4S] cluster. Loss of TCR limited the electron sink downstream of PSI during dark-to-light transition. Arabidopsis pgr5-tcr double mutant reduced growth significantly and showed unusual oxidation and reduction of plastoquinone pool. These results indicated that TCR is involved in electron flow(s) downstream of PSI, contributing to P700 oxidation.

    DOI: 10.1016/j.isci.2021.102059

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  • As Clear As Night And Day: Redox-Dependent Metabolic Switching In Chloroplasts.

    Yuki Okegawa

    Plant & cell physiology   2021年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1093/pcp/pcab015

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  • Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins. 査読 国際誌

    Takatoshi Sekiguchi, Keisuke Yoshida, Yuki Okegawa, Ken Motohashi, Ken-Ichi Wakabayashi, Toru Hisabori

    Biochimica et biophysica acta. Bioenergetics   1861 ( 11 )   148261 - 148261   2020年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The activity of the molecular motor enzyme, chloroplast ATP synthase, is regulated in a redox-dependent manner. The γ subunit, CF1-γ, is the central shaft of this enzyme complex and possesses the redox-active cysteine pair, which is reduced by thioredoxin (Trx). In light conditions, Trx transfers the reducing equivalent obtained from the photosynthetic electron transfer system to the CF1-γ. Previous studies showed that the light-dependent reduction of CF1-γ is more rapid than those of other Trx target proteins in the stroma. Although there are multiple Trx isoforms in chloroplasts, it is not well understood as to which chloroplast Trx isoform primarily contributes to the reduction of CF1-γ, especially under physiological conditions. We therefore performed direct assessment of the CF1-γ reduction capacity of each of the Trx isoforms. The kinetic analysis of the reduction process showed no significant difference in the reduction efficiency between two major chloroplast Trxs, namely Trx-f and Trx-m. Based on the thorough analyses of the CF1-γ redox dynamics in Arabidopsis thaliana Trx mutant plants, we found that lack of Trx-f or Trx-m had no significant impact on the in vivo light-dependent reduction of CF1-γ. The results showed that CF1-γ can accept the reducing power from both Trx-f and Trx-m in chloroplasts.

    DOI: 10.1016/j.bbabio.2020.148261

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  • Molecular and Biochemical Differences in Leaf Explants and the Implication for Regeneration Ability in Rorippa aquatica (Brassicaceae). 査読 国際誌

    Rumi Amano, Risa Momoi, Emi Omata, Taiga Nakahara, Kaori Kaminoyama, Mikiko Kojima, Yumiko Takebayashi, Shuka Ikematsu, Yuki Okegawa, Tomoaki Sakamoto, Hiroyuki Kasahara, Hitoshi Sakakibara, Ken Motohashi, Seisuke Kimura

    Plants (Basel, Switzerland)   9 ( 10 )   2020年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Plants have a high regeneration capacity and some plant species can regenerate clone plants, called plantlets, from detached vegetative organs. We previously outlined the molecular mechanisms underlying plantlet regeneration from Rorippa aquatica (Brassicaceae) leaf explants. However, the fundamental difference between the plant species that can and cannot regenerate plantlets from vegetative organs remains unclear. Here, we hypothesized that the viability of leaf explants is a key factor affecting the regeneration capacity of R. aquatica. To test this hypothesis, the viability of R. aquatica and Arabidopsis thaliana leaf explants were compared, with respect to the maintenance of photosynthetic activity, senescence, and immune response. Time-course analyses of photosynthetic activity revealed that R. aquatica leaf explants can survive longer than those of A. thaliana. Endogenous abscisic acid (ABA) and jasmonic acid (JA) were found at low levels in leaf explant of R. aquatica. Time-course transcriptome analysis of R. aquatica and A. thaliana leaf explants suggested that senescence was suppressed at the transcriptional level in R. aquatica. Application of exogenous ABA reduced the efficiency of plantlet regeneration. Overall, our results propose that in nature, plant species that can regenerate in nature can survive for a long time.

    DOI: 10.3390/plants9101372

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  • M-type Thioredoxins Regulate the PGR5/PGRL1-dependent Pathway by Forming a Disulfide-linked Complex with PGRL1. 査読 国際誌

    Yuki Okegawa, Ken Motohashi

    The Plant cell   2020年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    In addition to linear electron transport, photosystem I cyclic electron transport (PSI-CET) contributes to photosynthesis and photoprotection. In Arabidopsis thaliana, PSI-CET consists of two partially redundant pathways, one of which is the PROTON GRADIENT REGULATION 5 (PGR5)/PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE 1 (PGRL1)-dependent pathway. Although the physiological significance of PSI-CET is widely recognized, the regulatory mechanism behind these pathways remains largely unknown. Here, we report on the regulation of the PGR5/PGRL1-dependent pathway by the m-type thioredoxins (Trx m). Genetic and phenotypic characterization of multiple mutants indicated the physiological interaction between Trx m and the PGR5/PGRL1-dependent pathway in vivo. Using purified Trx proteins and ruptured chloroplasts, in vitro, we showed that the reduced form of Trx m specifically decreased the PGR5/PGRL1-dependent plastoquinone reduction. In planta, Trx m4 directly interacted with PGRL1 via disulfide complex formation. Analysis of the transgenic plants expressing PGRL1 cysteine variants demonstrated that cysteine 123 of PGRL1 is required for Trx m4-PGRL1 complex formation. Furthermore, the Trx m4-PGRL1 complex was transiently dissociated during the induction of photosynthesis. We propose that Trx m directly regulates the PGR5/PGRL1-dependent pathway by complex formation with PGRL1.

    DOI: 10.1105/tpc.20.00304

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  • Cyclic electron transport around photosystem I contributes to photosynthetic induction with Thioredoxin f. 査読 国際誌

    Yuki Okegawa, Leonardo Basso, Toshiharu Shikanai, Ken Motohashi

    Plant physiology   2020年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    In response to light, plants efficiently induce photosynthesis. Light activation of thiol enzymes by the thioredoxin (Trx) systems and cyclic electron transport by the PROTON GRADIENT REGULATION 5 (PGR5)-dependent pathway contribute substantially to regulation of photosynthesis. Arabidopsis thaliana mutants lacking f-type Trxs (trx f1f2) show delayed activation of carbon assimilation due to impaired photoreduction of Calvin-Benson cycle enzymes. To further study regulatory mechanisms that contribute to efficiency during the induction of photosynthesis, we analyzed the contributions of PSI donor- and acceptor-side regulation in the trx f1f2 mutant background. The cytochrome b6f complex is involved in PSI donor-side regulation, whereas PGR5-dependent PSI cyclic electron transport is required for both donor and acceptor functions. Introduction of the pgr1 mutation, which is conditionally defective in cytochrome b6f complex activity, into the trx f1f2 mutant background did not further affect the induction of photosynthesis, but the combined deficiency of Trx f and PGR5 severely impaired photosynthesis and suppressed plant growth under long-day conditions. In the pgr5 trx f1f2 mutant, the acceptor-side of PSI was almost completely reduced, and quantum yields of PSII and PSI hardly increased during the induction of photosynthesis. We also compared the photoreduction of thiol enzymes between the trx f1f2 and pgr5 trxf1f2 mutants. The pgr5 mutation did not result in further impaired photoreduction of Calvin-Benson cycle enzymes or ATP synthase in the trx f1f2 mutant background. These results indicated that acceptor-side limitations in the pgr5 trx f1f2 mutant suppress photosynthesis initiation, suggesting that PGR5 is required for efficient photosynthesis induction.

    DOI: 10.1104/pp.20.00741

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  • Comparative transcriptomics with self-organizing map reveals cryptic photosynthetic differences between two accessions of North American Lake cress 査読

    Hokuto Nakayama, Tomoaki Sakamoto, Yuki Okegawa, Kaori Kaminoyama, Manabu Fujie, Yasunori Ichihashi, Tetsuya Kurata, Ken Motohashi, Ihsan Al-Shehbaz, Neelima Sinha, Seisuke Kimura

    Scientific Reports   8 ( 1 )   3302   2018年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Publishing Group  

    Because natural variation in wild species is likely the result of local adaptation, it provides a valuable resource for understanding plant-environmental interactions. Rorippa aquatica (Brassicaceae) is a semi-aquatic North American plant with morphological differences between several accessions, but little information available on any physiological differences. Here, we surveyed the transcriptomes of two R. aquatica accessions and identified cryptic physiological differences between them. We first reconstructed a Rorippa phylogeny to confirm relationships between the accessions. We performed large-scale RNA-seq and de novo assembly
    the resulting 87,754 unigenes were then annotated via comparisons to different databases. Between-accession physiological variation was identified with transcriptomes from both accessions. Transcriptome data were analyzed with principal component analysis and self-organizing map. Results of analyses suggested that photosynthetic capability differs between the accessions. Indeed, physiological experiments revealed between-accession variation in electron transport rate and the redox state of the plastoquinone pool. These results indicated that one accession may have adapted to differences in temperature or length of the growing season.

    DOI: 10.1038/s41598-018-21646-w

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  • チオレドキシンシステムによる光合成の調節機構 ~変動する光環境で植物はどのように効率良く光合成を行っているのか?~ 招待 査読

    桶川 友季, 本橋 健

    化学と生物 (日本農芸化学会)   56 ( 7 )   452 - 453   2018年6月

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    記述言語:日本語  

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  • Expression of spinach ferredoxin-thioredoxin reductase using tandem T7 promoters and application of the purified protein for in vitro light-dependent thioredoxin-reduction system. 査読

    Okegawa Y, Motohashi K

    Protein expression and purification   121   46 - 51   2016年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.pep.2016.01.005

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  • Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies 査読

    Yuki Okegawa, Masanori Koshino, Teruya Okushima, Ken Motohashi

    PROTEIN EXPRESSION AND PURIFICATION   118   77 - 82   2016年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams). (C) 2015 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.pep.2015.10.008

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  • A simple and ultra-low cost homemade seamless ligation cloning extract (SLiCE) as an alternative to a commercially available seamless DNA cloning kit 査読

    Yuki Okegawa, Ken Motohashi

    Biochemistry and Biophysics Reports   4   148 - 151   2015年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier B.V.  

    The seamless ligation cloning extract (SLiCE) method is a novel seamless DNA cloning tool that utilizes homologous recombination activities in Escherichia coli cell lysates to assemble DNA fragments into a vector. Several laboratory E. coli strains can be used as a source for the SLiCE extract
    therefore, the SLiCE-method is highly cost-effective. The SLiCE has sufficient cloning ability to support conventional DNA cloning, and can simultaneously incorporate two unpurified DNA fragments into vector. Recently, many seamless DNA cloning kits have become commercially available
    these are generally very convenient, but expensive. In this study, we evaluated the cloning efficiencies between a simple and highly cost-effective SLiCE-method and a commercial kit under various molar ratios of insert DNA fragments to vector DNA. This assessment identified that the SLiCE from a laboratory E. coli strain yielded 30-85% of the colony formation rate of a commercially available seamless DNA cloning kit. The cloning efficiencies of both methods were highly effective, exhibiting over 80% success rate under all conditions examined. These results suggest that SLiCE from a laboratory E. coli strain can efficiently function as an effective alternative to commercially available seamless DNA cloning kits.

    DOI: 10.1016/j.bbrep.2015.09.005

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  • Chloroplastic thioredoxin m functions as a major regulator of Calvin cycle enzymes during photosynthesis in vivo. 査読

    Okegawa Y, Motohashi K

    The Plant journal : for cell and molecular biology   84 ( 5 )   900 - 913   2015年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1111/tpj.13049

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  • Evaluation of seamless ligation cloning extract preparation methods from an Escherichia coli laboratory strain 査読

    Yuki Okegawa, Ken Motohashi

    ANALYTICAL BIOCHEMISTRY   486   51 - 53   2015年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Seamless ligation cloning extract (SLiCE) is a simple and efficient method for DNA cloning without the use of restriction enzymes. Instead, SLiCE uses homologous recombination activities from Escherichia coil cell lysates. To date, SLiCE preparation has been performed using an expensive commercially available lytic reagent. To expand the utility of the SLiCE method, we evaluated different methods for SLiCE preparation that avoid using this reagent. Consequently, cell extracts prepared with buffers containing Triton X-100, which is a common and low-cost nonionic detergent, exhibited sufficient cloning activity for seamless gene incorporation into a vector. (C) 2015 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.ab.2015.06.031

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  • Method for enhancement of plant redox-related protein expression and its application for in vitro reduction of chloroplastic thioredoxins 査読

    Ken Motohashi, Yuki Okegawa

    PROTEIN EXPRESSION AND PURIFICATION   101   152 - 156   2014年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Plant redox-related proteins were overexpressed using a genetic codon substitution downstream of the translation initiation codon. This method significantly improved recombinant protein expression levels of Arabidopsis chloroplastic thioredoxins and cytosolic nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (E.C. 1.8.1.9) in Escherichia coli. Using these proteins, the in vitro chloroplastic thioredoxins-reduction system was reconstituted in an NADPH-dependent manner. This system could convert the five classes of chloroplastic Arabidopsis thioredoxins and two chloroplastic Spinach thioredoxins to their reduced forms, independent of dithiothreitol and the photosynthetic electron transport system. (C) 2014 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.pep.2014.07.001

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  • A Single Amino Acid Alteration in PGR5 Confers Resistance to Antimycin A in Cyclic Electron Transport around PSI 査読

    Kazuhiko Sugimoto, Yuki Okegawa, Akihiko Tohri, Terri A. Long, Sarah F. Covert, Toru Hisabori, Toshiharu Shikanai

    PLANT AND CELL PHYSIOLOGY   54 ( 9 )   1525 - 1534   2013年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    In Arabidopsis thaliana, the main route of cyclic electron transport around PSI is sensitive to antimycin A, but the site of inhibition has not been clarified. We discovered that ferredoxin-dependent plastoquinone reduction in ruptured chloroplasts was less sensitive to antimycin A in Arabidopsis that overaccumulated PGR5 (PROTON GRADIENT REGULATION 5) originating from Pinus taeda (PtPGR5) than that in the wild type. Consistent with this in vitro observation, infiltration of antimycin A reduced PSII yields and the non-photochemical quenching (NPQ) of Chl fluorescence in wild-type leaves but not in leaves accumulating PtPGR5. There are eight amino acid differences between PGR5 of Arabidopsis (AtPGR5) and PtPGR5 in their mature forms. To determine the site conferring antimycin A resistance, a series of AtPGR5 and PtPGR5 variants was introduced into the Arabidopsis pgr5 mutant. We determined that the presence of lysine rather than valine at the third amino acid position was necessary and sufficient for resistance to antimycin A. High levels of resistance to antimycin A required overaccumulation of PtPGR5 in ruptured chloroplasts, suggesting that PtPGR5 is partly resistant to antimycin A. In contrast, PSII yield was almost fully resistant to antimycin A in leaves accumulating endogenous levels of PtPGR5 or AtPGR5 V3K that had lysine instead of valine at the third position. NPQ was also dramatically recovered in leaves of these lines. These results imply that partial recovery of PSI cyclic electron transport is sufficient for maintaining redox homeostasis in photosynthesis. Our discovery suggests that antimycin A inhibits the function of PGR5 or proteins localized close to PGR5.

    DOI: 10.1093/pcp/pct098

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  • Antimycin A-like molecules inhibit cyclic electron transport around photosystem I in ruptured chloroplasts 査読

    Yoshichika Taira, Yuki Okegawa, Kazuhiko Sugimoto, Masato Abe, Hideto Miyoshi, Toshiharu Shikanai

    FEBS OPEN BIO   3   406 - 410   2013年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE LONDON  

    Antimycin A(3) (AA) is used as an inhibitor of cyclic electron transport around photosystem I. However, the high concentrations of AA that are needed for inhibition have secondary effects, even in chloroplasts. Here, we screened for chemicals that inhibited ferredoxin-dependent plastoquinone reduction in ruptured chloroplasts at lower concentrations than those required for AA. We identified two AA-like compounds: AAL1 and AAL2. AAL1 likely shares an inhibitory site with AA, most probably in the PGR5-PGRL1 protein complex, and enhances O-2 evolution in photosystem II, most likely via an uncoupler-like effect. AAL1 and AAL2 are unlikely to penetrate intact leaves. In ruptured chloroplasts, AALs are superior to AA as inhibitors of cyclic electron transport. (C) 2013 The Authors. Published by Elsevier BM, on behalf of Federation of European Biochemical Societies. All rights reserved.

    DOI: 10.1016/j.fob.2013.09.007

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  • PGR5-Dependent Cyclic Electron Transport Around PSI Contributes to the Redox Homeostasis in Chloroplasts Rather Than CO2 Fixation and Biomass Production in Rice 査読

    Yuri Nishikawa, Hiroshi Yamamoto, Yuki Okegawa, Shinya Wada, Nozomi Sato, Yoshichika Taira, Kazuhiko Sugimoto, Amane Makino, Toshiharu Shikanai

    PLANT AND CELL PHYSIOLOGY   53 ( 12 )   2117 - 2126   2012年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    The PGR5 (PROTON GRADIENT REGULATION 5) gene that is required for PSI cyclic electron transport in Arabidopsis was knocked down in rice (Oryza sativa). In three PGR5 knockdown (KD) lines, the PGR5 protein level was reduced to 5-8% of that in the wild type, resulting in a 50% reduction in PGRL1 (PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE 1) protein levels. In ruptured chloroplasts, ferredoxin-dependent plastoquinone reduction activity was partially impaired; the phenotype was mimicked by addition of antimycin A to wild-type chloroplasts. As occurred in the Arabidopsis pgr5 mutant, non-photochemical quenching of Chl fluorescence (NPQ) induction was impaired in the leaves, but the electron transport rate (ETR) was only mildly affected at high light intensity. The P700(+) level was reduced even at low light intensity, suggesting that the PGR5 function was severely disturbed as in the Arabidopsis pgr5 mutant and that the other alternative routes of electrons could not compensate the stromal redox balance. The amplitude of the light-dark electrochromic shift (ECS) signal (ECSt), which reflects the total size of the proton motive force in steady-state photosynthesis, was reduced by 13-25% at approximately the growth light intensity. The CO2 fixation rate was only slightly reduced in the PGR5 KD lines. Despite the drastic reduction in NPQ and P700(+) levels, total biomass was only slightly reduced in PGR5 KD lines grown at 370 mu mol photons m(-2) s(-1). These results suggest that CO2 fixation and growth rate are very robust in the face of alterations in the fundamental reactions of photosynthesis under constant light conditions in rice.

    DOI: 10.1093/pcp/pcs153

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  • Physiological links among alternative electron transport pathways that reduce and oxidize plastoquinone in Arabidopsis 査読

    Yuki Okegawa, Yoshichika Kobayashi, Toshiharu Shikanai

    PLANT JOURNAL   63 ( 3 )   458 - 468   2010年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    P>In addition to linear electron transport from water to NADP+, alternative electron transport pathways are believed to regulate photosynthesis. In the two routes of photosystem I (PSI) cyclic electron transport, electrons are recycled from the stromal reducing pool to plastoquinone (PQ), generating additional delta pH (proton gradient across thylakoid membranes). Plastid terminal oxidase (PTOX) accepts electrons from PQ and transfers them to oxygen to produce water. Although both electron transport pathways share the PQ pool, it is unclear whether they interact in vivo. To investigate the physiological link between PSI cyclic electron transport-dependent PQ reduction and PTOX-dependent PQ oxidation, we characterized mutants defective in both functions. Impairment of PSI cyclic electron transport suppressed leaf variegation in the Arabidopsis immutans (im) mutant, which is defective in PTOX. The im variegation was more effectively suppressed in the pgr5 mutant, which is defective in the main pathway of PSI cyclic electron transport, than in the crr2-2 mutant, which is defective in the minor pathway. In contrast to this chloroplast development phenotype, the im defect alleviated the growth phenotype of the crr2-2 pgr5 double mutant. This was accompanied by partial suppression of stromal over-reduction and restricted linear electron transport. We discuss the function of the alternative electron transport pathways in both chloroplast development and photosynthesis in mature leaves.

    DOI: 10.1111/j.1365-313X.2010.04252.x

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  • Arabidopsis thaliana PGR7 Encodes a Conserved Chloroplast Protein That Is Necessary for Efficient Photosynthetic Electron Transport 査読

    Hou-Sung Jung, Yuki Okegawa, Patrick M. Shih, Elizabeth Kellogg, Salah E. Abdel-Ghany, Marinus Pilon, Kimmen Sjoelander, Toshiharu Shikanai, Krishna K. Niyogi

    PLOS ONE   5 ( 7 )   e11688   2010年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    A significant fraction of a plant's nuclear genome encodes chloroplast-targeted proteins, many of which are devoted to the assembly and function of the photosynthetic apparatus. Using digital video imaging of chlorophyll fluorescence, we isolated proton gradient regulation 7 (pgr7) as an Arabidopsis thaliana mutant with low nonphotochemical quenching of chlorophyll fluorescence (NPQ). In pgr7, the xanthophyll cycle and the PSBS gene product, previously identified NPQ factors, were still functional, but the efficiency of photosynthetic electron transport was lower than in the wild type. The pgr7 mutant was also smaller in size and had lower chlorophyll content than the wild type in optimal growth conditions. Positional cloning located the pgr7 mutation in the At3g21200 (PGR7) gene, which was predicted to encode a chloroplast protein of unknown function. Chloroplast targeting of PGR7 was confirmed by transient expression of a GFP fusion protein and by stable expression and subcellular localization of an epitope-tagged version of PGR7. Bioinformatic analyses revealed that the PGR7 protein has two domains that are conserved in plants, algae, and bacteria, and the N-terminal domain is predicted to bind a cofactor such as FMN. Thus, we identified PGR7 as a novel, conserved nuclear gene that is necessary for efficient photosynthetic electron transport in chloroplasts of Arabidopsis.

    DOI: 10.1371/journal.pone.0011688

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  • Conserved role of PROTON GRADIENT REGULATION 5 in the regulation of PSI cyclic electron transport 査読

    Terri A. Long, Yuki Okegawa, Toshiharu Shikanai, Gregory W. Schmidt, Sarah F. Covert

    PLANTA   228 ( 6 )   907 - 918   2008年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    There are at least two photosynthetic cyclic electron transport (CET) pathways in most C(3) plants: the NAD(P)H dehydrogenase (NDH)-dependent pathway and a pathway dependent upon putative ferredoxin:plastoquinone oxidoreductase (FQR) activity. While the NDH complex has been identified, and shown to play a role in photosynthesis, especially under stress conditions, less is known about the machinery of FQR-dependent CET. Recent studies indicate that FQR-dependent CET is dependent upon PGR5, a small protein of unknown function. In a previous study we found that overexpression of PGR5 causes alterations in growth and development associated with decreased chloroplast development and a transient increase in nonphotochemical quenching (NPQ) after the shift from dark to light. In the current study we examine the spatiotemporal expression pattern of PGR5, and the effects of overexpression of PGR5 in Arabidopsis under a host of light and stress conditions. To investigate the conserved function of PGR5, we cloned PGR5 from a species which apparently lacks NDH, loblolly pine, and overexpressed it in Arabidopsis. Although greening of cotyledons was severely delayed in overexpressing lines under low light, mature plants survived exposure to high light and drought stress better than wild-type. In addition, PSI was more resistant to high light in the PGR5 overexpressors than in wild-type plants, while PSII was more sensitive to this stress. These complex responses corresponded to alterations in linear and cyclic electron transfer, suggesting that over-accumulation of PGR5 induces pleiotropic effects, probably via elevated CET. We conclude that PGR5 has a developmentally-regulated, conserved role in mediating CET.

    DOI: 10.1007/s00425-008-0789-y

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  • Characterization of factors affecting the activity of photosystem I cyclic electron transport in chloroplasts 査読

    Yuki Okegawa, Yugo Kagawa, Yoshichika Kobayashi, Toshiharu Shikanai

    PLANT AND CELL PHYSIOLOGY   49 ( 5 )   825 - 834   2008年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    PSI cyclic electron transport is essential for photosynthesis and photoprotection. In higher plants, the antimycin A-sensitive pathway is the main route of electrons in PSI cyclic electron transport. Although a small thylakoid protein, PGR5 (PROTON GRADIENT REGULATION 5), is essential for this pathway, its function is still unclear, and there are numerous debates on the rate of electron transport in vivo and its regulation. To assess how PGR5-dependent PSI cyclic electron transport is regulated in vivo, we characterized its activity in ruptured chloroplasts isolated from Arabidopsis thaliana. The activity of ferredoxin (Fd)-dependent plastoquinone (PQ) reduction in the dark is impaired in the pgr5 mutant. Alkalinization of the reaction medium enhanced the activity of Fd-dependent PQ reduction in the wild type. Even weak actinic light (AL) illumination also markedly activated PGR5-dependent PSI cyclic electron transport in ruptured chloroplasts. Even in the presence of linear electron transport [11 mu mol O(2) (mg Chl)(-1) h(-1)], PGR5-dependent PSI electron transport was detected as a difference in Chl fluorescence levels in ruptured chloroplasts. In the wild type, PGR5-dependent PSI cyclic electron transport competed with NADP(+) photoreduction. These results suggest that the rate of PGR5-dependent PSI cyclic electron transport is high enough to balance the production ratio of ATP and NADPH during steady-state photosynthesis, consistently with the pgr5 mutant phenotype. Our results also suggest that the activity of PGR5-dependent PSI cyclic electron transport is regulated by the redox state of the NADPH pool.

    DOI: 10.1093/pcp/pcn055

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  • A balanced PGR5 level is required for chloroplast development and optimum operation of cyclic electron transport around photosystem I 査読

    Yuki Okegawa, Terri A. Long, Megumi Iwano, Seiji Takayama, Yoshichika Kobayashi, Sarah F. Covert, Toshiharu Shikanai

    PLANT AND CELL PHYSIOLOGY   48 ( 10 )   1462 - 1471   2007年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    PSI cyclic electron transport contributes markedly to photosynthesis and photoprotection in flowering plants. Although the thylakoid protein PGR5 (Proton Gradient Regulation 5) has been shown to be essential for the main route of PSI cyclic electron transport, its exact function remains unclear. In transgenic Arabidopsis plants overaccumulating PGR5 in the thylakoid membrane, chloroplast development was delayed, especially in the cotyledons. Although photosynthetic electron transport was not affected during steady-state photosynthesis, a high level of non-photochemical quenching (NPQ) was transiently induced after a shift of light conditions. This phenotype was explained by elevated activity of PSI cyclic electron transport, which was monitored in an in vitro system using ruptured chloroplasts, and also in leaves. The effect of overaccumulation of PGR5 was specific to the antimycin A-sensitive pathway of PSI cyclic electron transport but not to the NAD(P)H dehydrogenase (NDH) pathway. We propose that a balanced PGR5 level is required for efficient regulation of the rate of antimycin A-sensitive PSI cyclic electron transport, although the rate of PSI cyclic electron transport is probably also regulated by other factors during steady-state photosynthesis.

    DOI: 10.1093/pcp/pcm116

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  • The pgr1 mutation in the Rieske subunit of the cytochrome b(6)f complex does not affect PGR5-dependent cyclic electron transport around photosystem I 査読

    Y Okegawa, M Tsuyama, Y Kobayashi, T Shikanai

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 31 )   28332 - 28336   2005年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Although photosystem I ( PSI) cyclic electron transport is essential for plants, our knowledge of the route taken by electrons is very limited. To assess whether ferredoxin (Fd) donates electrons directly to plastoquinone (PQ) or via a Q-cycle in the cytochrome (cyt) b(6)f complex in PSI cyclic electron transport, we characterized the activity of PSI cyclic electron transport in an Arabidopsis mutant, pgr1 (proton gradient regulation). In pgr1, Q-cycle activity was hypersensitive to acidification of the thylakoid lumen because of an amino acid alteration in the Rieske subunit of the cyt b6f complex, resulting in a conditional defect in Q-cycle activity. In vitro assays using ruptured chloroplasts did not show any difference in the activity of PGR5-dependent PQ reduction by Fd, which functions in PSI cyclic electron transport in vivo. In contrast to the pgr5 defect, the pgr1 defect did not show any synergistic effect on the quantum yield of photosystem II in crr2-2, a mutant in which NDH (NAD(P) H dehydrogenase) activity was impaired. Furthermore, the simultaneous determination of the quantum yields of both photosystems indicated that the ratio of linear and PSI cyclic electron transport was not significantly affected in pgr1. All the results indicated that the pgr1 mutation did not affect PGR5-dependent PQ reduction by Fd. The phenotypic differences between pgr1 and pgr5 indicate that maintenance of the proper balance of linear and PSI cyclic electron transport is essential for preventing over-reduction of the stroma.

    DOI: 10.1074/jbc.M505703200

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MISC

  • シロイヌナズナ[CO2]変動応答性遺伝子発現に対する光化学系I サイクリック電子伝達(CEF-PSI)の関与

    山本 宏, 桶川 友季, 深澤 美津江, 林 誠, 鹿内 利治

    第50回日本植物生理学会年会   2009年3月

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    記述言語:日本語  

    DOI: 10.14841/jspp.2009.0.0613.0

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共同研究・競争的資金等の研究

  • psbA欠失変異体の相補を利用したマーカーフリーな葉緑体の遺伝子組換え植物の作出

    研究課題/領域番号:20H02961  2020年04月 - 2024年03月

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    寺地 徹, 山岸 博, 木村 成介, 桶川 友季

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    配分額:17550000円 ( 直接経費:13500000円 、 間接経費:4050000円 )

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  • m型チオレドキシンによる光化学系Iサイクリック電子伝達の制御機構解明

    研究課題/領域番号:19H04733  2019年04月 - 2021年03月

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    桶川 友季

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    配分額:5330000円 ( 直接経費:4100000円 、 間接経費:1230000円 )

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  • チオレドキシンによる光化学系Iサイクリック電子伝達の制御機構解明

    研究課題/領域番号:17H05730  2017年04月 - 2019年03月

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    桶川 友季

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    配分額:6240000円 ( 直接経費:4800000円 、 間接経費:1440000円 )

    光化学系I(PSI)サイクリック電子伝達は、リニア電子伝達と同様に光合成と光防御機構に不可欠であることが証明されているが、その制御機構についてはあまり明らかになっていない。プロトン駆動力の形成に寄与するPSI サイクリック電子伝達の制御機構を明らかにすることはプロトン駆動力制御のネットワークの全貌解明に必要である。
    本年度は前年度に引き続き、チオレドキシン(Trx)がPSI サイクリック電子伝達を制御することを明らかにすることと、その制御においてTrx が標的とするタンパク質を同定することを目的に研究を進めた。
    前年度の研究から葉緑体ストロマに5グループあるTrxのうちm型Trx(Trx m3を除く)が特異的にPSIサイクリック電子伝達活性を抑制することを明らかにすることができた。そこで本年度はm型Trx が2 つあるPSI サイクリック電子伝達経路のどちらを抑制するかを調べるために、PSI サイクリック電子伝達の2つの変異株、crr2、pgr5 を用いて解析をおこなった結果、m型Trx がPGR5/PGRL1 依存の経路を抑制することを明らかにした。
    また、m 型Trx がどのタンパク質を標的とし、PSI サイクリック電子伝達を抑制しているかを明らかにすることを試みた。植物個体から抽出したタンパク質を非還元条件で電気泳動し、m型Trx 特異的な抗体を用いて検出した結果、m型Trx が何らかのタンパク質と相互作用している結果が得られた。分子量からPGRL1 がその候補の1 つとして考えられたため、PGRL1 特異的な抗体を用いて同様の実験をおこなったところ、同じ分子量の位置にバンドが確認できた。またそれぞれの欠損変異株でそのバンドが検出できないという結果から、PGRL1 がm 型Trx の標的タンパク質である可能性が示唆する結果を得ることができた。

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  • 葉緑体チラコイド内腔におけるレドックス制御機構の解明

    研究課題/領域番号:16K18573  2016年04月 - 2019年03月

    日本学術振興会  科学研究費助成事業 若手研究(B)  若手研究(B)

    桶川 友季, 本橋 健

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    配分額:4290000円 ( 直接経費:3300000円 、 間接経費:990000円 )

    高等植物の葉緑体では、ストロマだけでなくチラコイド膜を隔てた内腔にもレドックス制御システムが存在することがわかってきた。本研究の目的はこれまでにストロマからチラコイド内腔への還元力伝達に関与することが示唆されているHCF164とCcdAタンパク質を含めた還元力伝達システム全体の分子機構を明らかにすることである。シロイヌナズナの変異株を用いた表現型回復実験から、チラコイド内腔でのレドックス制御の重要性が明らかになった。またチラコイド内腔への還元力伝達に関与するチオレドキシンのアイソフォームを同定するためにチオレドキシンの欠損変異株および過剰発現株を作出し、評価をおこなった。

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  • 葉緑体ストロマからチラコイド内腔への還元力伝達機構の解明

    研究課題/領域番号:23770049  2011年 - 2012年

    日本学術振興会  科学研究費助成事業 若手研究(B)  若手研究(B)

    桶川 友季

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    配分額:4550000円 ( 直接経費:3500000円 、 間接経費:1050000円 )

    植物の葉緑体内の酸化還元状態は昼と夜で大きく異なる。そのため、光合成関連酵素を初めとし、様々なタンパク質がその活性をレドックスによって制御される。しかしそれらのタンパク質がどのように制御されているかの詳細はわかっていない。本研究では、葉緑体のストロマにおいてタンパク質の制御に関与するチオレドキシンに注目した。突然変異株の解析からチオレドキシンが光合成の制御に大きく寄与していることが明らかになった。

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  • PGR5依存の光化学系Iサイクリック電子伝達経路の解明

    研究課題/領域番号:07J08015  2007年 - 2009年

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    桶川 友季

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    配分額:2700000円 ( 直接経費:2700000円 )

    光化学系Iサイクリック電子伝達はリニア電子伝達と同様、光合成と葉緑体の光阻害回避の両方に必須である。これまでにモデル植物であるシロイヌナズナを使って変異株の解析が行われてきた。この重要性から考えて、サイクリック電子伝達の機能はイネでも当然保存されていると考えられる。本年度、私はイネにおけるサイクリック電子伝達の生報機能を解明することを目的に実験を進めた。サイクリック電子伝達に必須なタンパク質をノックダウンさせた形質転換植物ではサイクリック電子伝達活性が減少していた。それに伴い、光合成電子伝達速度も減少していた。これはシロイヌナズナの表現型と一致する。以上のことからイネにおいてもサイクリック電子伝達は光合成と光阻害の回避に必須であるということが証明された。
    またもう一つの研究として葉緑体分化と光合成に対するサイクリック電子伝達と葉緑体のプラスチドターミナルオキシダーゼであるPTOXの相互作用について遺伝学的に証明することを目的とし実験を行った。サイクリック電子伝達とPTOXの生理学的な関係を調べるために、両方の機能を欠損する突然変異株の解析を行ったところ、PSIサイクリック電子伝達の減少はPTOXを欠損するシロイヌナズナの突然変異株、immutansにおける葉の斑入りを抑制した。対照的にPTOXの欠損はcrr2pgr5における成長阻害の表現型を緩和した。crr2 immutans spgr5ではストロマの過還元と電子伝達の阻害が部分的に軽減されていた。本研究において様々な変異株のバックグラウンドを使うことによってオルタナティブな電子伝達の機能が初めて明らかとなったと言える。以上の結果から研究最終年度の目的を達成する結果が得られたと言える。本研究の研究成果については11月の関西光合成研究会および、3月の日本植物生理学会において口頭発表を行った。

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