研究者詳細 - 桶川　友季

Functional division of f-type and m-type thioredoxins to regulate the Calvin cycle and cyclic electron transport around photosystem I. 査読

Yuki Okegawa, Wataru Sakamoto, Ken Motohashi

Journal of plant research 135 ( 4 ) 543 - 553 2022年3月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Redox regulation of chloroplast proteins is necessary to adjust photosynthetic performance with changes in light. The thioredoxin (Trx) system plays a central role in this process. Chloroplast-localized classical Trx is a small redox-active protein that regulates many target proteins by reducing their disulfide bonds in a light-dependent manner. Arabidopsis thaliana mutants lacking f-type Trx (trx f1f2) or m-type Trx (trx m124-2) have been reported to show delayed reduction of Calvin cycle enzymes. As a result, the trx m124-2 mutant exhibits growth defects. Here, we characterized a quintuple mutant lacking both Trx f and Trx m to investigate the functional complementarity of Trx f and Trx m. The trx f1f2 m124-2 quintuple mutant was newly obtained by crossing, and is analyzed here for the first time. The growth defects of the trx m124-2 mutant were not enhanced by the lack of Trx f. In contrast, deficiencies of both Trxs additively suppressed the reduction of Calvin cycle enzymes, resulting in a further delay in the initiation of photosynthesis. Trx f appeared to be necessary for the rapid activation of the Calvin cycle during the early induction of photosynthesis. To perform effective photosynthesis, plants seem to use both Trxs in a coordinated manner to activate carbon fixation reactions. In contrast, the PROTON GRADIENT REGULATION 5 (PGR5)-dependent cyclic electron transport around photosystem I was regulated by Trx m, but not by Trx f. Lack of Trx f did not affect the activity and regulation of the PGR5-dependent pathway. Trx f may have a higher specificity for target proteins, whereas Trx m has a variety of target proteins to regulate overall photosynthesis and other metabolic reactions in the chloroplasts.

DOI： 10.1007/s10265-022-01388-7

Maintaining the Chloroplast Redox Balance through the PGR5-Dependent Pathway and the Trx System Is Required for Light-Dependent Activation of Photosynthetic Reactions. 査読

Yuki Okegawa, Natsuki Tsuda, Wataru Sakamoto, Ken Motohashi

Plant & cell physiology 63 ( 1 ) 92 - 103 2022年1月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Light-dependent activation of chloroplast enzymes is required for the rapid induction of photosynthesis after a shift from dark to light. The thioredoxin (Trx) system plays a central role in this process. In chloroplasts, the Trx system consists of two pathways: the ferredoxin (Fd)/Trx pathway and the NADPH-Trx reductase C (NTRC) pathway. In Arabidopsis (Arabidopsis thaliana) mutants defective in either pathway, photoreduction of thiol enzymes was impaired, resulting in decreased carbon fixation. The close relationship between the Fd/Trx pathway and PROTON GRADIENT REGULATION 5 (PGR5)-dependent photosystem I cyclic electron transport (PSI CET) in the induction of photosynthesis was recently elucidated. However, how the PGR5-dependent pathway is involved in the NTRC pathway is unclear, although NTRC has been suggested to physically interact with PGR5. In this study, we analyzed Arabidopsis mutants lacking either the PGR5 or the NADH dehydrogenase-like (NDH)-dependent PSI CET pathway in the ntrc mutant background. The ntrc pgr5 double mutant suppressed both the growth defects and the high non-photochemical quenching (NPQ) phenotype of the ntrc mutant when grown under long-day conditions. By contrast, inactivation of NDH activity with the chlororespiratory reduction 2-2 (crr2-2) mutant failed to suppress either phenotype. We discovered that the phenotypic rescue of ntrc by pgr5 is caused by the partial restoration of Trx-dependent reduction of thiol enzymes during the induction of photosynthesis. These results suggest that electron partitioning to the PGR5-dependent pathway and the Trx system needs to be properly regulated for activation of the Calvin-Benson-Bassham cycle enzymes during the induction of photosynthesis.

DOI： 10.1093/pcp/pcab148

As Clear as Night and Day: Redox-Dependent Metabolic Switching in Chloroplasts. 査読

Yuki Okegawa

Plant & cell physiology 62 ( 3 ) 389 - 391 2021年7月

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

Evaluation of CBSX Proteins as Regulators of the Chloroplast Thioredoxin System. 査読国際誌

Ryota Murai, Yuki Okegawa, Nozomi Sato, Ken Motohashi

Frontiers in plant science 12 530376 - 530376 2021年2月

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

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 ) 1372 2020年10月

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 32 ( 12 ) 3866 - 3883 2020年10月

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 184 ( 3 ) 1291 - 1302 2020年9月

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

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

Scopus

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

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

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

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

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

Scopus

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

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

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

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

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

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

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

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

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