研究者詳細 - 桶川　友季

論文 - 桶川　友季

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PCP Research Highlights: Regulatory Role of Three Important Post-Translational Modifications in Chloroplast Proteins. 査読

Yuki Okegawa

Plant & cell physiology 64 ( 10 ) 1119 - 1123 2023年9月

x- and y-type thioredoxins maintain redox homeostasis on photosystem I acceptor side under fluctuating light. 査読国際誌

Yuki Okegawa, Nozomi Sato, Rino Nakakura, Ryota Murai, Wataru Sakamoto, Ken Motohashi

Plant physiology 193 ( 4 ) 2498 - 2512 2023年8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）

Plants cope with sudden increases in light intensity through various photoprotective mechanisms. Redox regulation by thioredoxin (Trx) systems also contributes to this process. Whereas the functions of f- and m-type Trxs in response to such fluctuating light conditions have been extensively investigated, those of x- and y-type Trxs are largely unknown. Here, we analyzed the trx x single, trx y1 trx y2 double, and trx x trx y1 trx y2 triple mutants in Arabidopsis (Arabidopsis thaliana). A detailed analysis of photosynthesis revealed changes in photosystem I (PSI) parameters under low light in trx x and trx x trx y1 trx y2. The electron acceptor side of PSI was more reduced in these mutants than in the wild type. This mutant phenotype was more pronounced under fluctuating light conditions. During both low- and high-light phases, the PSI acceptor side was largely limited in trx x and trx x trx y1 trx y2. After fluctuating light treatment, we observed more severe PSI photoinhibition in trx x and trx x trx y1 trx y2 than in the wild type. Furthermore, when grown under fluctuating light conditions, trx x and trx x trx y1 trx y2 plants showed impaired growth and decreased level of PSI subunits. These results suggest that Trx x and Trx y prevent redox imbalance on the PSI acceptor side, which is required to protect PSI from photoinhibition, especially under fluctuating light. We also propose that Trx x and Trx y contribute to maintaining the redox balance even under constant low-light conditions to prepare for sudden increases in light intensity.

DOI： 10.1093/plphys/kiad466

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In vitro analysis of the PGR5-dependent cyclic electron transport 査読

Yuki Okegawa, Yugo Kagawa, Yoshichika Kobayashi, Toshiharu Shikanai

PLANT AND CELL PHYSIOLOGY 48 S128 - S128 2007年

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

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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