Faculty Profiles - SAKAMOTO Wataru

Papers - SAKAMOTO Wataru

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The thylakoid membrane remodeling protein VIPP1 forms bundled oligomers in tobacco chloroplasts. International journal

Sarah W Gachie, Alexandre Muhire, Di Li, Akihiro Kawamoto, Noriko Takeda-Kamiya, Yumi Goto, Mayuko Sato, Kiminori Toyooka, Ryo Yoshimura, Tsuneaki Takami, Lingang Zhang, Genji Kurisu, Toru Terachi, Wataru Sakamoto

Plant physiology 2025.4

Language：English Publishing type：Research paper (scientific journal)

The thylakoid membrane (TM) serves as the scaffold for oxygen-evolving photosynthesis, hosting the protein complexes responsible for the light reactions and ATP synthesis. Vesicle Inducing Protein in Plastid 1 (VIPP1), a key protein in TM remodeling, has been recognized as essential for TM homeostasis. In vitro studies of cyanobacterial VIPP1 demonstrated its ability to form large homo-oligomers (> 2 MDa) manifesting as ring-like or filament-like assemblies associated with membranes. Similarly, VIPP1 in Chlamydomonas reinhardtii assembles into rods that encapsulate liposomes or into stacked spiral structures. However, the nature of VIPP1 assemblies in chloroplasts, particularly in Arabidopsis, remains uncharacterized. Here, we expressed Arabidopsis thaliana VIPP1 fused to GFP (AtVIPP1-GFP) in tobacco (Nicotiana tabacum) chloroplasts and performed transmission electron microscopy (TEM). A purified AtVIPP1-GFP fraction was enriched with long filamentous tubule-like structures. Detailed TEM observations of chloroplasts in fixed resin-embedded tissues identified VIPP1 assemblies in situ that appeared to colocalize with GFP fluorescence. Electron tomography demonstrated that the AtVIPP1 oligomers consisted of bundled filaments near membranes, some of which appeared connected to the TM or inner chloroplast envelope at their contact sites. The observed bundles were never detected in wild-type Arabidopsis but were observed in Arabidopsis vipp1 mutants expressing AtVIPP1-GFP. Taken together, we propose that the bundled filaments are the dominant AtVIPP1 oligomers that represent its static state in vivo.

DOI： 10.1093/plphys/kiaf137

The plastidial protein acetyltransferase GNAT1 forms a complex with GNAT2, yet their interaction is dispensable for state transitions

Annika Brünje, Magdalena Füßl, Jürgen Eirich, Jean-Baptiste Boyer, Paulina Heinkow, Ulla Neumann, Minna Konert, Aiste Ivanauskaite, Julian Seidel, Shin-Ichiro Ozawa, Wataru Sakamoto, Thierry Meinnel, Dirk Schwarzer, Paula Mulo, Carmela Giglione, Iris Finkemeier

Molecular & Cellular Proteomics 100850 - 100850 2024.9

Characterization of organelle DNA degradation mediated by DPD1 exonuclease in the rice genome-edited line

Md. Faridul Islam, Hiroshi Yamatani, Tsuneaki Takami, Makoto Kusaba, Wataru Sakamoto

Plant Molecular Biology 114 ( 3 ) 2024.6

Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

Abstract

Mitochondria and plastids, originated as ancestral endosymbiotic bacteria, contain their own DNA sequences. These organelle DNAs (orgDNAs) are, despite the limited genetic information they contain, an indispensable part of the genetic systems but exist as multiple copies, making up a substantial amount of total cellular DNA. Given this abundance, orgDNA is known to undergo tissue-specific degradation in plants. Previous studies have shown that the exonuclease DPD1, conserved among seed plants, degrades orgDNAs during pollen maturation and leaf senescence in Arabidopsis. However, tissue-specific orgDNA degradation was shown to differ among species. To extend our knowledge, we characterized DPD1 in rice in this study. We created a genome-edited (GE) mutant in which OsDPD1 and OsDPD1-like were inactivated. Characterization of this GE plant demonstrated that DPD1 was involved in pollen orgDNA degradation, whereas it had no significant effect on orgDNA degradation during leaf senescence. Comparison of transcriptomes from wild-type and GE plants with different phosphate supply levels indicated that orgDNA had little impact on the phosphate starvation response, but instead had a global impact in plant growth. In fact, the GE plant showed lower fitness with reduced grain filling rate and grain weight in natural light conditions. Taken together, the presented data reinforce the important physiological roles of orgDNA degradation mediated by DPD1.

DOI： 10.1007/s11103-024-01452-x

Other Link： https://link.springer.com/article/10.1007/s11103-024-01452-x/fulltext.html

Dysfunction of Chloroplast Protease Activity Mitigates pgr5 Phenotype in the Green Algae Chlamydomonas reinhardtii. International journal

Shin-Ichiro Ozawa, Guoxian Zhang, Wataru Sakamoto

Plants (Basel, Switzerland) 13 ( 5 ) 2024.2

Language：English Publishing type：Research paper (scientific journal)

Researchers have described protection mechanisms against the photoinhibition of photosystems under strong-light stress. Cyclic Electron Flow (CEF) mitigates electron acceptor-side limitation, and thus contributes to Photosystem I (PSI) protection. Chloroplast protease removes damaged protein to assist with protein turn over, which contributes to the quality control of Photosystem II (PSII). The PGR5 protein is involved in PGR5-dependent CEF. The FTSH protein is a chloroplast protease which effectively degrades the damaged PSII reaction center subunit, D1 protein. To investigate how the PSI photoinhibition phenotype in pgr5 would be affected by adding the ftsh mutation, we generated double-mutant pgr5ftsh via crossing, and its phenotype was characterized in the green algae Chlamydomonas reinhardtii. The cells underwent high-light incubation as well as low-light incubation after high-light incubation. The time course of Fv/Fm values in pgr5ftsh showed the same phenotype with ftsh1-1. The amplitude of light-induced P700 photo-oxidation absorbance change was measured. The amplitude was maintained at a low value in the control and pgr5ftsh during high-light incubation, but was continuously decreased in pgr5. During the low-light incubation after high-light incubation, amplitude was more rapidly recovered in pgr5ftsh than pgr5. We concluded that the PSI photoinhibition by the pgr5 mutation is mitigated by an additional ftsh1-1 mutation, in which plastoquinone pool would be less reduced due to damaged PSII accumulation.

DOI： 10.3390/plants13050606

Characterization of tryptophan oxidation affecting D1 degradation by FtsH in the photosystem II quality control of chloroplasts. International journal

Yusuke Kato, Hiroshi Kuroda, Shin-Ichiro Ozawa, Keisuke Saito, Vivek Dogra, Martin Scholz, Guoxian Zhang, Catherine de Vitry, Hiroshi Ishikita, Chanhong Kim, Michael Hippler, Yuichiro Takahashi, Wataru Sakamoto

eLife 12 2023.11

Language：English Publishing type：Research paper (scientific journal) Publisher：eLife Sciences Publications, Ltd

Light reaction of photosynthesis is one of the most important reactions for sustaining our environment. Photosystem II (PSII) is the initial site of photosynthetic electron transfer by water oxidation. Light in excess, however, causes the simultaneous production of singlet oxygen, a potent reactive oxygen species (ROS), leading to photo-oxidative damage in PSII. To maintain photosynthetic activity, the PSII reaction center protein D1, which is the primary target of unavoidable photo-oxidative damage, is efficiently degraded by FtsH protease. In PSII subunits, photo-oxidative modifications of several amino acids such as Trp have been indeed documented, whereas the linkage between such modifications and D1 degradation remains elusive. Here, we show that an oxidative post-translational modification of Trp residue at the N-terminal tail of D1 is correlated with D1 degradation by FtsH during high-light stress. We revealed that Arabidopsis mutant lacking FtsH2 had increased levels of oxidative Trp residues in D1, among which an N-terminal Trp-14 was distinctively localized in the stromal side. Further characterization of Trp-14 using chloroplast transformation in Chlamydomonas indicated that substitution of D1 Trp-14 to Phe, mimicking Trp oxidation enhanced FtsH-mediated D1 degradation under high light, although the substitution did not affect protein stability and PSII activity. Molecular dynamics simulation of PSII implies that both Trp-14 oxidation and Phe substitution cause fluctuation of D1 N-terminal tail. Furthermore, Trp-14 to Phe modification appeared to have an additive effect in the interaction between FtsH and PSII core in vivo. Together, our results suggest that the Trp oxidation at its N-terminus of D1 may be one of the key oxidations in the PSII repair, leading to processive degradation by FtsH.

In photosynthetic organisms, maintenance of photosynthetic light reaction is manifested by so called Photosystem II (PSII) repair system, where the reaction center protein D1 is targeted to photo-oxidative damage and rapidly degraded by the processive protease FtsH. While this system is well known to cope with photoinhibition, the actual oxidation within the D1 polypeptide and its association to degradation remained elusive. Here, we characterized oxidative modification of tryptophan (Trp) residues in the PSII core, and hypothesize that the oxidation of N-terminal Trp is one of the key oxidations in the PSII repair, likely enhancing D1’s accessibility to FtsH.

DOI： 10.7554/eLife.88822

Plastid inheritance revisited: emerging role of organelle DNA degradation in angiosperms.

Wataru Sakamoto, Tsuneaki Takami

Plant & cell physiology 2023.9

Language：English Publishing type：Research paper (scientific journal)

Plastids are essential organelles in angiosperms and show non-Mendelian inheritance due to their evolution as endosymbionts. In approximately 80% of angiosperms, plastids are thought to be inherited from the maternal parent, whereas other species transmit plastids biparentally. Maternal inheritance can be generally explained by the stochastic segregation of maternal plastids after fertilization because the zygote is overwhelmed by the maternal cytoplasm. In contrast, biparental inheritance shows transmission of organelles from both parents. In some species, maternal inheritance is not absolute and paternal leakage occurs at a very low frequency (~10-5). A key process controlling the inheritance mode lies in the behavior of plastids during male gametophyte (pollen) development, with accumulating evidence indicating that the plastids themselves or their DNAs are eliminated during pollen maturation or at fertilization. Cytological observations in numerous angiosperm species have revealed several critical steps that mutually influence the degree of plastid transmission quantitatively among different species. This review revisits plastid inheritance and focuses on the mechanistic viewpoint. Particularly, we focus on a recent finding demonstrating that both low temperature and plastid DNA degradation mediated by the organelle exonuclease DPD1 influence the degree of paternal leakage significantly in tobacco. Given these findings, we also highlight the emerging role of DPD1 in organelle DNA degradation.

DOI： 10.1093/pcp/pcad104

x- and y-type thioredoxins maintain redox homeostasis on photosystem I acceptor side under fluctuating light. International journal

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

Plant physiology 2023.8

Language：English Publishing type：Research paper (scientific journal)

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

Greetings from the PCP Editor-in-Chief.

Wataru Sakamoto

Plant & cell physiology 64 ( 7 ) 701 - 703 2023.7

Maternal plastid inheritance: two abating factors identified. International journal

Wataru Sakamoto, Tsuneaki Takami

Trends in genetics : TIG 39 ( 5 ) 342 - 343 2023.5

Language：English Publishing type：Research paper (scientific journal)

Organelle DNAs (orgDNAs) in mitochondria and plastids are generally inherited from the maternal parent; however, it is unclear how their inheritance mode is controlled, particularly in the plastids of seed plants. Chung et al. identify two factors that affect maternal inheritance in tobacco plastids: cold temperature and DNA amount in pollen.

DOI： 10.1016/j.tig.2023.03.002

Identification of quantitative trait loci associated with sorghum susceptibility to Asian stem borer damage

Cyprian Osinde, Wataru Sakamoto, Hiromi Kajiya-Kanegae, Islam S. Sobhy, Arthur K. Tugume, Anthony M. Nsubuga, Ivan Galis

Journal of Plant Interactions 18 ( 1 ) 2023

Publishing type：Research paper (scientific journal)

Sorghum (Sorghum bicolor (L.) Moench) is an important crop originated in Africa that shows susceptibility to herbivores. In this study, we identified two sorghum genotypes with highly contrasting levels of stem damage caused by the caterpillars of Asian stem borer (Ostrinia furnacalis Guenée). Recombinant inbred lines (RILs) from genetic cross between resistant (BTx623) and susceptible (NOG) sorghum were used to perform a quantitative trait locus (QTL) analysis in the field. Two major QTLs responsible for higher NOG infestation by stem borer in three independent field seasons were detected on chromosomes 7 and 9, interestingly in positions that overlapped with two major QTLs for plant height. As plant height and stem borer damage were highly correlated, we propose that sorghum height-associated morphological or physiological traits could be important for stem borer establishment and/or damage in sorghum.

DOI： 10.1080/17429145.2022.2153182

FZL, a dynamin-like protein localized to curved grana edges, is required for efficient photosynthetic electron transfer in Arabidopsis. International journal

Yu Ogawa, Megumi Iwano, Toshiharu Shikanai, Wataru Sakamoto

Frontiers in plant science 14 1279699 - 1279699 2023

Language：English Publishing type：Research paper (scientific journal)

Photosynthetic electron transfer and its regulation processes take place on thylakoid membranes, and the thylakoid of vascular plants exhibits particularly intricate structure consisting of stacked grana and flat stroma lamellae. It is known that several membrane remodeling proteins contribute to maintain the thylakoid structure, and one putative example is FUZZY ONION LIKE (FZL). In this study, we re-evaluated the controversial function of FZL in thylakoid membrane remodeling and in photosynthesis. We investigated the sub-membrane localization of FZL and found that it is enriched on curved grana edges of thylakoid membranes, consistent with the previously proposed model that FZL mediates fusion of grana and stroma lamellae at the interfaces. The mature fzl thylakoid morphology characterized with the staggered and less connected grana seems to agree with this model as well. In the photosynthetic analysis, the fzl knockout mutants in Arabidopsis displayed reduced electron flow, likely resulting in higher oxidative levels of Photosystem I (PSI) and smaller proton motive force (pmf). However, nonphotochemical quenching (NPQ) of chlorophyll fluorescence was excessively enhanced considering the pmf levels in fzl, and we found that introducing kea3-1 mutation, lowering pH in thylakoid lumen, synergistically reinforced the photosynthetic disorder in the fzl mutant background. We also showed that state transitions normally occurred in fzl, and that they were not involved in the photosynthetic disorders in fzl. We discuss the possible mechanisms by which the altered thylakoid morphology in fzl leads to the photosynthetic modifications.

DOI： 10.3389/fpls.2023.1279699

DOMINANT AWN INHIBITOR Encodes the ALOG Protein Originating from Gene Duplication and Inhibits AWN Elongation by Suppressing Cell Proliferation and Elongation in Sorghum.

Hideki Takanashi, Hiromi Kajiya-Kanegae, Asuka Nishimura, Junko Yamada, Motoyuki Ishimori, Masaaki Kobayashi, Kentaro Yano, Hiroyoshi Iwata, Nobuhiro Tsutsumi, Wataru Sakamoto

Plant & cell physiology 63 ( 7 ) 901 - 918 2022.7

Language：English Publishing type：Research paper (scientific journal)

The awn, a needle-like structure extending from the tip of the lemma in grass species, plays a role in environmental adaptation and fitness. In some crops, awns appear to have been eliminated during domestication. Although numerous genes involved in awn development have been identified, several dominant genes that eliminate awns are also known to exist. For example, in sorghum (Sorghum bicolor), the dominant awn-inhibiting gene has been known since 1921; however, its molecular features remain uncharacterized. In this study, we conducted quantitative trait locus analysis and a genome-wide association study of awn-related traits in sorghum and identified DOMINANT AWN INHIBITOR (DAI), which encodes the ALOG family protein on chromosome 3. DAI appeared to be present in most awnless sorghum cultivars, likely because of its effectiveness. Detailed analysis of the ALOG protein family in cereals revealed that DAI originated from a duplication of its twin paralog (DAIori) on chromosome 10. Observations of immature awns in near-isogenic lines revealed that DAI inhibits awn elongation by suppressing both cell proliferation and elongation. We also found that only DAI gained a novel function to inhibit awn elongation through an awn-specific expression pattern distinct from that of DAIori. Interestingly, heterologous expression of DAI with its own promoter in rice inhibited awn elongation in the awned cultivar Kasalath. We found that DAI originated from gene duplication, providing an interesting example of gain-of-function that occurs only in sorghum but shares its functionality with rice and sorghum.

DOI： 10.1093/pcp/pcac057

Sorghum Ionomics Reveals the Functional SbHMA3a Allele that Limits Excess Cadmium Accumulation in Grains.

Fiona Wacera Wahinya, Kiyoshi Yamazaki, Zihuan Jing, Tsuneaki Takami, Takehiro Kamiya, Hiromi Kajiya-Kanegae, Hideki Takanashi, Hiroyoshi Iwata, Nobuhiro Tsutsumi, Toru Fujiwara, Wataru Sakamoto

Plant & cell physiology 63 ( 5 ) 713 - 728 2022.5

Language：English Publishing type：Research paper (scientific journal)

Understanding uptake and redistribution of essential minerals or sequestering of toxic elements is important for optimized crop production. Although the mechanisms controlling mineral transport have been elucidated in rice and other species, little is understood in sorghum-an important C4 cereal crop. Here, we assessed the genetic factors that govern grain ionome profiles in sorghum using recombinant inbred lines (RILs) derived from a cross between BTx623 and NOG (Takakibi). Pairwise correlation and clustering analysis of 22 elements, measured in sorghum grains harvested under greenhouse conditions, indicated that the parental lines, as well as the RILs, show different ionomes. In particular, BTx623 accumulated significantly higher levels of cadmium (Cd) than NOG, because of differential root-to-shoot translocation factors between the two lines. Quantitative trait locus (QTL) analysis revealed a prominent QTL for grain Cd concentration on chromosome 2. Detailed analysis identified SbHMA3a, encoding a P1B-type ATPase heavy metal transporter, as responsible for low Cd accumulation in grains; the NOG allele encoded a functional HMA3 transporter (SbHMA3a-NOG) whose Cd-transporting activity was confirmed by heterologous expression in yeast. BTx623 possessed a truncated, loss-of-function SbHMA3a allele. The functionality of SbHMA3a in NOG was confirmed by Cd concentrations of F2 grains derived from the reciprocal cross, in which the NOG allele behaved in a dominant manner. We concluded that SbHMA3a-NOG is a Cd transporter that sequesters excess Cd in root tissues, as shown in other HMA3s. Our findings will facilitate the isolation of breeding cultivars with low Cd in grains or in exploiting high-Cd cultivars for phytoremediation.

DOI： 10.1093/pcp/pcac035

Genetic analysis of chlorophyll synthesis and degradation regulated by BALANCE of CHLOROPHYLL METABOLISM. International journal

Hiroshi Yamatani, Takeshi Ito, Kenji Nishimura, Tetsuya Yamada, Wataru Sakamoto, Makoto Kusaba

Plant physiology 189 ( 1 ) 419 - 432 2022.5

Language：English Publishing type：Research paper (scientific journal)

Chlorophyll (Chl) serves a number of essential functions, capturing and converting light energy as a component of photosystem supercomplexes. Chl degradation during leaf senescence is also required for adequate degeneration of chloroplasts and salvaging of nutrients from senescent leaves. In this study, we performed genetic analysis to determine the functions of BALANCE of CHLOROPHYLL METABOLISM1 (BCM1) and BCM2, which control Chl levels by regulating synthesis and degradation, and STAY-GREEN (SGR)1 (also known as NON-YELLOWING1 [NYE1]) and SGR2, which encode Mg-dechelatase and catalyze Chl a degradation in Arabidopsis (Arabidopsis thaliana). Analysis of bcm1 bcm2 revealed that both BCM1 and BCM2 are involved in the regulation of Chl levels in presenescent leaves and Chl degradation in senescing leaves. Analysis of bcm1 bcm2 nye1 nye2 suggested that BCMs repress Chl-degrading activity in both presenescent and senescing leaves by regulating SGR activity. Furthermore, transactivation analysis and chromatin immunoprecipitation (ChIP) assay revealed that GOLDEN2-LIKE1 (GLK1), a central transcription factor regulating the expression of genes encoding photosystem-related proteins, such as light-harvesting Chl a/b-binding proteins (LHCPs), directly regulates the transcription of BCM1. LHCPs are stabilized by Chl binding, suggesting that GLKs control the amount of LHCP through transcriptional and post-translational regulation via BCM-mediated Chl-level regulation. Meanwhile, we generated a mutant of the BCM ortholog in lettuce (Lactuca sativa) by genome editing and found that it showed an early yellowing phenotype, but only a slight reduction in Chl in presenescent leaves. Thus, this study revealed a conserved but slightly diversified regulation of Chl and LHCP levels via the GLK-BCM pathway in eudicots.

DOI： 10.1093/plphys/kiac059

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

Yuki Okegawa, Wataru Sakamoto, Ken Motohashi

Journal of plant research 135 ( 4 ) 543 - 553 2022.3

Language：English Publishing type：Research paper (scientific journal)

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

Distinctive in vitro ATP Hydrolysis Activity of AtVIPP1, a Chloroplastic ESCRT-III Superfamily Protein in Arabidopsis. International journal

Norikazu Ohnishi, Manabu Sugimoto, Hideki Kondo, Ken-Ichi Shioya, Lingang Zhang, Wataru Sakamoto

Frontiers in plant science 13 949578 - 949578 2022

Language：English Publishing type：Research paper (scientific journal)

Vesicle-inducing protein in plastid 1 (VIPP1), characteristic to oxygenic photosynthetic organisms, is a membrane-remodeling factor that forms homo-oligomers and functions in thylakoid membrane formation and maintenance. The cyanobacterial VIPP1 structure revealed a monomeric folding pattern similar to that of endosomal sorting complex required for transport (ESCRT) III. Characteristic to VIPP1, however, is its own GTP and ATP hydrolytic activity without canonical domains. In this study, we found that histidine-tagged Arabidopsis VIPP1 (AtVIPP1) hydrolyzed GTP and ATP to produce GDP and ADP in vitro, respectively. Unexpectedly, the observed GTPase and ATPase activities were biochemically distinguishable, because the ATPase was optimized for alkaline conditions and dependent on Ca2+ as well as Mg2+, with a higher affinity for ATP than GTP. We found that a version of AtVIPP1 protein with a mutation in its nucleotide-binding site, as deduced from the cyanobacterial structure, retained its hydrolytic activity, suggesting that Arabidopsis and cyanobacterial VIPP1s have different properties. Negative staining particle analysis showed that AtVIPP1 formed particle or rod structures that differed from those of cyanobacteria and Chlamydomonas. These results suggested that the nucleotide hydrolytic activity and oligomer formation of VIPP1 are common in photosynthetic organisms, whereas their properties differ among species.

DOI： 10.3389/fpls.2022.949578

NB-LRR-encoding genes conferring susceptibility to organophosphate pesticides in sorghum International journal

Zihuan Jing, Fiona Wacera W., Tsuneaki Takami, Hideki Takanashi, Fumi Fukada, Yoji Kawano, Hiromi Kajiya-Kanegae, Hiroyoshi Iwata, Nobuhiro Tsutsumi, Wataru Sakamoto

Scientific Reports 11 ( 1 ) 19828 - 19828 2021.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

<title>Abstract</title>Organophosphate is the commonly used pesticide to control pest outbreak, such as those by aphids in many crops. Despite its wide use, however, necrotic lesion and/or cell death following the application of organophosphate pesticides has been reported to occur in several species. To understand this phenomenon, called organophosphate pesticide sensitivity (OPS) in sorghum, we conducted QTL analysis in a recombinant inbred line derived from the Japanese cultivar NOG, which exhibits OPS. Mapping OPS in this population identified a prominent QTL on chromosome 5, which corresponded to <italic>Organophosphate-Sensitive Reaction</italic> (<italic>OSR</italic>) reported previously in other mapping populations. The <italic>OSR</italic> locus included a cluster of three genes potentially encoding nucleotide-binding leucine-rich repeat (NB-LRR, NLR) proteins, among which <italic>NLR-C</italic> was considered to be responsible for OPS in a dominant fashion. <italic>NLR-C</italic> was functional in NOG, whereas the other resistant parent, BTx623, had a null mutation caused by the deletion of promoter sequences. Our finding of <italic>OSR</italic> as a dominant trait is important not only in understanding the diversified role of NB-LRR proteins in cereals but also in securing sorghum breeding free from OPS.

DOI： 10.1038/s41598-021-98908-7

Other Link： https://www.nature.com/articles/s41598-021-98908-7

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

Yuki Okegawa, Natsuki Tsuda, Wataru Sakamoto, Ken Motohashi

Plant & cell physiology 63 ( 1 ) 92 - 103 2021.10

Language：English Publishing type：Research paper (scientific journal)

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

Mutations in a Golden2-Like Gene Cause Reduced Seed Weight in Barley albino lemma 1 Mutants

Shin Taketa, Momoko Hattori, Tsuneaki Takami, Eiko Himi, Wataru Sakamoto

Plant and Cell Physiology 62 ( 3 ) 447 - 457 2021.7

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press ({OUP})

The albino lemma 1 (alm1) mutants of barley (Hordeum vulgare L.) exhibit obvious chlorophyll-deficient hulls. Hulls are seed-enclosing tissues on the spike, consisting of the lemma and palea. The alm1 phenotype is also expressed in the pericarp, culm nodes and basal leaf sheaths, but leaf blades and awns are normal green. A single recessive nuclear gene controls tissue-specific alm1 phenotypic expression. Positional cloning revealed that the ALM1 gene encodes a Golden 2-like (GLK) transcription factor, HvGLK2, belonging to the GARP subfamily of Myb transcription factors. This finding was validated by genetic evidence indicating that all 10 alm1 mutants studied had a lesion in functionally important regions of HvGLK2, including the three alpha-helix domains, an AREAEAA motif and the GCT box. Transmission electron microscopy revealed that, in lemmas of the alm1.g mutant, the chloroplasts lacked thylakoid membranes, instead of stacked thylakoid grana in wild-type chloroplasts. Compared with wild type, alm1.g plants showed similar levels of leaf photosynthesis but reduced spike photosynthesis by 34%. The alm1.g mutant and the alm1.a mutant showed a reduction in 100-grain weight by 15.8% and 23.1%, respectively. As in other plants, barley has HvGLK2 and a paralog, HvGLK1. In flag leaves and awns, HvGLK2 and HvGLK1 are expressed at moderate levels, but in hulls, HvGLK1 expression was barely detectable compared with HvGLK2. Barley alm1/Hvglk2 mutants exhibit more severe phenotypes than glk2 mutants of other plant species reported to date. The severe alm1 phenotypic expression in multiple tissues indicates that HvGLK2 plays some roles that are nonredundant with HvGLK1.

DOI： 10.1093/pcp/pcab001

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity. International journal

Tilak Kumar Gupta, Sven Klumpe, Karin Gries, Steffen Heinz, Wojciech Wietrzynski, Norikazu Ohnishi, Justus Niemeyer, Benjamin Spaniol, Miroslava Schaffer, Anna Rast, Matthias Ostermeier, Mike Strauss, Jürgen M Plitzko, Wolfgang Baumeister, Till Rudack, Wataru Sakamoto, Jörg Nickelsen, Jan M Schuller, Michael Schroda, Benjamin D Engel

Cell 184 ( 14 ) 3643 - 3659 2021.7

Language：English Publishing type：Research paper (scientific journal)

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.

DOI： 10.1016/j.cell.2021.05.011

With Greetings and Hope for a Recoverable 2021: From the PCP Editor-In-Chief

Wataru Sakamoto

Plant and Cell Physiology 62 ( 2 ) 219 - 221 2021.5

Editorial Feature: Meet the PCP Editor-In-Chief—Wataru Sakamoto

Wataru Sakamoto

Plant and Cell Physiology 62 ( 2 ) 222 - 223 2021.5

Genetic dissection of QTLs associated with spikelet-related traits and grain size in sorghum. International journal

Hideki Takanashi, Mitsutoshi Shichijo, Lisa Sakamoto, Hiromi Kajiya-Kanegae, Hiroyoshi Iwata, Wataru Sakamoto, Nobuhiro Tsutsumi

Scientific reports 11 ( 1 ) 9398 - 9398 2021.4

Language：English Publishing type：Research paper (scientific journal)

Although spikelet-related traits such as size of anther, spikelet, style, and stigma are associated with sexual reproduction in grasses, no QTLs have been reported in sorghum. Additionally, there are only a few reports on sorghum QTLs related to grain size, such as grain length, width, and thickness. In this study, we performed QTL analyses of nine spikelet-related traits (length of sessile spikelet, pedicellate spikelet, pedicel, anther, style, and stigma; width of sessile spikelet and stigma; and stigma pigmentation) and six grain-related traits (length, width, thickness, length/width ratio, length/thickness ratio, and width/thickness ratio) using sorghum recombinant inbred lines. We identified 36 and 7 QTLs for spikelet-related traits and grain-related traits, respectively, and found that most sorghum spikelet organ length- and width-related traits were partially controlled by the dwarf genes Dw1 and Dw3. Conversely, we found that these Dw genes were not strongly involved in the regulation of grain size. The QTLs identified in this study aid in understanding the genetic basis of spikelet- and grain-related traits in sorghum.

DOI： 10.1038/s41598-021-88917-x

Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane. International journal

Keiji Nishioka, Yusuke Kato, Shin-Ichiro Ozawa, Yuichiro Takahashi, Wataru Sakamoto

Photosynthesis research 147 ( 1 ) 107 - 124 2021.1

Language：English Publishing type：Research paper (scientific journal)

Protein phosphorylation is a fundamental post-translational modification in all organisms. In photoautotrophic organisms, protein phosphorylation is essential for the fine-tuning of photosynthesis. The reversible phosphorylation of the photosystem II (PSII) core and the light-harvesting complex of PSII (LHCII) contribute to the regulation of photosynthetic activities. Besides the phosphorylation of these major proteins, recent phosphoproteomic analyses have revealed that several proteins are phosphorylated in the thylakoid membrane. In this study, we utilized the Phos-tag technology for a comprehensive assessment of protein phosphorylation in the thylakoid membrane of Arabidopsis. Phos-tag SDS-PAGE enables the mobility shift of phosphorylated proteins compared with their non-phosphorylated isoform, thus differentiating phosphorylated proteins from their non-phosphorylated isoforms. We extrapolated this technique to two-dimensional (2D) SDS-PAGE for detecting protein phosphorylation in the thylakoid membrane. Thylakoid proteins were separated in the first dimension by conventional SDS-PAGE and in the second dimension by Phos-tag SDS-PAGE. In addition to the isolation of major phosphorylated photosynthesis-related proteins, 2D Phos-tag SDS-PAGE enabled the detection of several minor phosphorylated proteins in the thylakoid membrane. The analysis of the thylakoid kinase mutants demonstrated that light-dependent protein phosphorylation was mainly restricted to the phosphorylation of the PSII core and LHCII proteins. Furthermore, we assessed the phosphorylation states of the structural domains of the thylakoid membrane, grana core, grana margin, and stroma lamella. Overall, these results demonstrated that Phos-tag SDS-PAGE is a useful biochemical tool for studying in vivo protein phosphorylation in the thylakoid membrane protein.

DOI： 10.1007/s11120-020-00803-1

Phototropin- and photosynthesis-dependent mitochondrial positioning in Arabidopsis thaliana mesophyll cells. International journal

Md Sayeedul Islam, Toan Van Nguyen, Wataru Sakamoto, Shingo Takagi

Journal of integrative plant biology 62 ( 9 ) 1352 - 1371 2020.9

Language：English Publishing type：Research paper (scientific journal)

Mitochondria are frequently observed in the vicinity of chloroplasts in photosynthesizing cells, and this association is considered necessary for their metabolic interactions. We previously reported that, in leaf palisade cells of Arabidopsis thaliana, mitochondria exhibit blue-light-dependent redistribution together with chloroplasts, which conduct accumulation and avoidance responses under the control of blue-light receptor phototropins. In this study, precise motility analyses by fluorescent microscopy revealed that the individual mitochondria in palisade cells, labeled with green fluorescent protein, exhibit typical stop-and-go movement. When exposed to blue light, the velocity of moving mitochondria increased in 30 min, whereas after 4 h, the frequency of stoppage of mitochondrial movement markedly increased. Using different mutant plants, we concluded that the presence of both phototropin1 and phototropin2 is necessary for the early acceleration of mitochondrial movement. On the contrary, the late enhancement of stoppage of mitochondrial movement occurs only in the presence of phototropin2 and only when intact photosynthesis takes place. A plasma-membrane ghost assay suggested that the stopped mitochondria are firmly adhered to chloroplasts. These results indicate that the physical interaction between mitochondria and chloroplasts is cooperatively mediated by phototropin2- and photosynthesis-dependent signals. The present study might add novel regulatory mechanism for light-dependent plant organelle interactions.

DOI： 10.1111/jipb.12910

RAD-seq-Based High-Density Linkage Map Construction and QTL Mapping of Biomass-Related Traits in Sorghum using the Japanese Landrace Takakibi NOG.

Hiromi Kajiya-Kanegae, Hideki Takanashi, Masaru Fujimoto, Motoyuki Ishimori, Norikazu Ohnishi, Fiona Wacera W, Everlyne A Omollo, Masaaki Kobayashi, Kentaro Yano, Michiharu Nakano, Toshiaki Kozuka, Makoto Kusaba, Hiroyoshi Iwata, Nobuhiro Tsutsumi, Wataru Sakamoto

Plant & cell physiology 61 ( 7 ) 1262 - 1272 2020.7

Language：English Publishing type：Research paper (scientific journal)

Sorghum [Sorghum bicolor (L.) Moench] grown locally by Japanese farmers is generically termed Takakibi, although its genetic diversity compared with geographically distant varieties or even within Takakibi lines remains unclear. To explore the genomic diversity and genetic traits controlling biomass and other physiological traits in Takakibi, we focused on a landrace, NOG, in this study. Admixture analysis of 460 sorghum accessions revealed that NOG belonged to the subgroup that represented Asian sorghums, and it was only distantly related to American/African accessions including BTx623. In an attempt to dissect major traits related to biomass, we generated a recombinant inbred line (RIL) from a cross between BTx623 and NOG, and we constructed a high-density linkage map based on 3,710 single-nucleotide polymorphisms obtained by restriction-site-associated DNA sequencing of 213 RIL individuals. Consequently, 13 fine quantitative trait loci (QTLs) were detected on chromosomes 2, 3, 6, 7, 8 and 9, which included five QTLs for days to heading, three for plant height (PH) and total shoot fresh weight and two for Brix. Furthermore, we identified two dominant loci for PH as being identical to the previously reported dw1 and dw3. Together, these results corroborate the diversified genome of Japanese Takakibi, while the RIL population and high-density linkage map generated in this study will be useful for dissecting other important traits in sorghum.

DOI： 10.1093/pcp/pcaa056

A 2020 Vision of the Next Four Years—From the PCP’s New Editor-in-Chief

Wataru Sakamoto

Plant and Cell Physiology 61 ( 4 ) 671 - 672 2020.4

Overexpression of BUNDLE SHEATH DEFECTIVE 2 improves the efficiency of photosynthesis and growth in Arabidopsis. International journal

Florian A Busch, Jun Tominaga, Masato Muroya, Norihiko Shirakami, Shunichi Takahashi, Wataru Yamori, Takuya Kitaoka, Sara E Milward, Kohji Nishimura, Erika Matsunami, Yosuke Toda, Chikako Higuchi, Atsuko Muranaka, Tsuneaki Takami, Shunsuke Watanabe, Toshinori Kinoshita, Wataru Sakamoto, Atsushi Sakamoto, Hiroshi Shimada

The Plant journal : for cell and molecular biology 102 ( 1 ) 129 - 137 2020.4

Language：English Publishing type：Research paper (scientific journal)

Bundle Sheath Defective 2, BSD2, is a stroma-targeted protein initially identified as a factor required for the biogenesis of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in maize. Plants and algae universally have a homologous gene for BSD2 and its deficiency causes a RuBisCO-less phenotype. As RuBisCO can be the rate-limiting step in CO2 assimilation, the overexpression of BSD2 might improve photosynthesis and productivity through the accumulation of RuBisCO. To examine this hypothesis, we produced BSD2 overexpression lines in Arabidopsis. Compared with wild type, the BSD2 overexpression lines BSD2ox-2 and BSD2ox-3 expressed 4.8-fold and 8.8-fold higher BSD2 mRNA, respectively, whereas the empty-vector (EV) harbouring plants had a comparable expression level. The overexpression lines showed a significantly higher CO2 assimilation rate per available CO2 and productivity than EV plants. The maximum carboxylation rate per total catalytic site was accelerated in the overexpression lines, while the number of total catalytic sites and RuBisCO content were unaffected. We then isolated recombinant BSD2 (rBSD2) from E. coli and found that rBSD2 reduces disulfide bonds using reductants present in vivo, for example glutathione, and that rBSD2 has the ability to reactivate RuBisCO that has been inactivated by oxidants. Furthermore, 15% of RuBisCO freshly isolated from leaves of EV was oxidatively inactivated, as compared with 0% in BSD2-overexpression lines, suggesting that the overexpression of BSD2 maintains RuBisCO to be in the reduced active form in vivo. Our results demonstrated that the overexpression of BSD2 improves photosynthetic efficiency in Arabidopsis and we conclude that it is involved in mediating RuBisCO activation.

DOI： 10.1111/tpj.14617

Recent advances and perspectives in plant mitochondrial biology for plant breeding

Kubo Tomohiko, Arimura Shin-ichi, Handa Hirokazu, Koizuka Nobuya, Arakawa Takumi, Kitazaki Kazuyoshi, Kazama Tomohiko, Takenaka Mizuki, Sakamoto Wataru, Ishihara Naotada, Nakamura Takahiro, Niikura Satoshi

Breeding Research 22 ( 1 ) 87 - 94 2020

High temperature causes breakdown of S haplotype-dependent stigmatic self-incompatibility in self-incompatible Arabidopsis thaliana. International journal

Masaya Yamamoto, Kenji Nishimura, Hiroyasu Kitashiba, Wataru Sakamoto, Takeshi Nishio

Journal of experimental botany 70 ( 20 ) 5745 - 5751 2019.10

Language：English Publishing type：Research paper (scientific journal)

Commercial seeds of Brassicaceae vegetable crops are mostly F1 hybrids, the production of which depends on self-incompatibility during pollination. Self-incompatibility is known to be weakened by exposure to elevated temperatures, which may compromise future breeding and seed production. In the Brassicaceae, self-incompatibility is controlled by two genes, SRK and SCR, which function as female and male determinants of recognition specificity, respectively. However, the molecular mechanisms underlying the breakdown of self-incompatibility under high temperature are poorly understood. In this study, we examined the self-incompatibility phenotypes of self-incompatible Arabidopsis thaliana SRK-SCR transformants under normal (23 °C) and elevated (29 °C) temperatures. Exposure to elevated temperature caused defects in the stigmatic, but not the pollen, self-incompatibility response. In addition, differences in the response to elevated temperature were observed among different S haplotypes. Subcellular localization revealed that high temperature disrupted the targeting of SRK to the plasma membrane. SRK localization in plants transformed with different S haplotypes corresponded to their self-incompatibility phenotypes, further indicating that defects in SRK localization were responsible for the breakdown in the self-incompatibility response at high temperature. Our results provide new insights into the causes of instability in self-incompatibility phenotypes.

DOI： 10.1093/jxb/erz343

Photosynthetic Responses to High Temperature and Strong Light Suggest Potential Post-flowering Drought Tolerance of Sorghum Japanese Landrace Takakibi.

Norikazu Ohnishi, Fiona Wacera W, Wataru Sakamoto

Plant & cell physiology 60 ( 9 ) 2086 - 2099 2019.9

Language：English Publishing type：Research paper (scientific journal)

Sorghum [Sorghum bicolor (L.) Moench] is a C4 crop known to be adaptable to harsh environments such as those under high temperature and water deficit. In this study, we focused on a Japanese sorghum landrace Takakibi (NOG) and employed chlorophyll fluorescence measurements to assess its response to environmental stress. Comparison of photosynthetic rate evaluated using two parameters (effective quantum yield and electron transfer rate) indicated that NOG showed less activity than BTx623 in the pre-flowering stage, which was consistent with the higher susceptibility of NOG seedlings to drought than BTx623. The observed differences in photosynthetic activity between the two cultivars were detectable without drought conditions on days with high temperature and strong light. Interestingly, the photosynthetic activity of NOG leaves in stress conditions increased soon after heading, and the trend was similar to that in BTx642, a well-characterized post-flowering drought-tolerant cultivar. In contrast, BTx623 showed a gradual decline in photosynthetic rate. Thus, we inferred that Japanese Takakibi has the potential to show pre-flowering drought susceptibility and post-flowering drought tolerance, through which it adapts to local climates with high temperature and strong light at harvest.

DOI： 10.1093/pcp/pcz107

Impaired PSII Proteostasis Promotes Retrograde Signaling via Salicylic Acid. International journal

Jianli Duan, Keun Pyo Lee, Vivek Dogra, Siyuan Zhang, Kaiwei Liu, Carlos Caceres-Moreno, Shanshan Lv, Weiman Xing, Yusuke Kato, Wataru Sakamoto, Renyi Liu, Alberto P Macho, Chanhong Kim

Plant physiology 180 ( 4 ) 2182 - 2197 2019.8

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society of Plant Biologists ({ASPB})

Photodamage of the PSII reaction center (RC) is an inevitable process in an oxygen-rich environment. The damaged PSII RC proteins (Dam-PSII) undergo degradation via the thylakoid membrane-bound FtsH metalloprotease, followed by posttranslational assembly of PSII. While the effect of Dam-PSII on gene regulation is described for cyanobacteria, its role in land plants is largely unknown. In this study, we reveal an intriguing retrograde signaling pathway by using the Arabidopsis (Arabidopsis thaliana) yellow variegated2-9 mutant, which expresses a mutated FtsH2 (FtsH2G267D) metalloprotease, specifically impairing its substrate-unfolding activity. This lesion leads to the perturbation of PSII protein homeostasis (proteostasis) and the accumulation of Dam-PSII. Subsequently, this results in an up-regulation of salicylic acid (SA)-responsive genes, which is abrogated by inactivation of either an SA transporter in the chloroplast envelope membrane or extraplastidic SA signaling components as well as by removal of SA. These results suggest that the stress hormone SA, which is mainly synthesized via the chloroplast isochorismate pathway in response to the impaired PSII proteostasis, mediates the retrograde signaling. These findings reinforce the emerging view of chloroplast function toward plant stress responses and suggest SA as a potential plastid factor mediating retrograde signaling.

DOI： 10.1104/pp.19.00483

Targeted proteome analysis of microalgae under high-light conditions by optimized protein extraction of photosynthetic organisms.

Masakazu Toyoshima, Masumi Sakata, Kazuki Ohnishi, Yuma Tokumaru, Yusuke Kato, Ryutaro Tokutsu, Wataru Sakamoto, Jun Minagawa, Fumio Matsuda, Hiroshi Shimizu

Journal of bioscience and bioengineering 127 ( 3 ) 394 - 402 2019.3

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier {BV}

Cell disruption and protein solubilization protocols for the relative quantification of individual subunits in photosystems were developed for photosynthetic organisms including cyanobacterium Synechocystis sp. PCC 6803, green-algae Chlamydomonas reinhardtii, and seed plant Arabidopsis thaliana. The optimal methods for the disruption of Chlamydomonas, Synechocystis, and Arabidopsis cells were sonication, microbeads (Φ approximately 0.1 mm), and large beads (Φ = 5.0 mm), respectively. Extraction of the total proteins exceeded 90% using each optimal cell disruption method. Solubilization efficiency of membrane proteins was improved by the phase transfer surfactant (PTS) method. Ninety seven and 114 proteins from Chlamydomonas and Synechocystis, respectively, including membrane proteins such as photosystem proteins, ATP synthase, and NADH dehydrogenase, were successfully analyzed by nano-liquid chromatography tandem mass spectrometry. These results also indicated the improved efficiency of solubilization and trypsin digestion using PTS buffer. The results of the relative quantitative evaluation of photosystem subunits in Chlamydomonas and Synechocystis grown under high-light conditions were consistent with those of previous studies. Thus, the optimal cell disruption and PTS methods allow for comprehensive relative quantitative proteome analysis of photosynthetic organisms. Additionally, NdhD1 and NdhF1, which are NDH-1 subunit homologs, were increased under high-light conditions, suggesting that the NDH-1L complex, including NdhD1 and NdhF1, is increased under high-light conditions. The relative quantitative proteome analysis of individual subunits indicates the diverse functions of NDH-1 protein.

DOI： 10.1016/j.jbiosc.2018.09.001

Phosphorylation of the Chloroplastic Metalloprotease FtsH in Arabidopsis Characterized by Phos-Tag SDS-PAGE. International journal

Yusuke Kato, Wataru Sakamoto

Frontiers in plant science 10 1080 - 1080 2019

Language：English Publishing type：Research paper (scientific journal)

FtsH is an essential ATP-dependent metalloprotease for protein quality control in the thylakoid membrane of Arabidopsis thaliana chloroplasts. It is required for chloroplast development during leaf growth, and particularly for the specific degradation of photo-damaged D1 protein in the photosystem II (PSII) complex to maintain photosynthesis activity. In the thylakoid membrane, the reversible phosphorylation of proteins is known to control the activity and remodeling of photosynthetic complexes, and previous studies implicate that FtsH is also phosphorylated. We therefore assessed the phosphorylation status of FtsH and its possible role in the regulatory mechanism in this study. The phosphorylation level of FtsHs that compose the FtsH heterohexameric complex was investigated by phosphate-affinity gel electrophoresis using a Phos-Tag molecule. Phos-tag SDS-PAGE of thylakoid proteins and subsequent immunoblot analysis showed that both type A (FtsH1/5) and type B (FtsH2/8) subunits were separable into phosphorylated and non-phosphorylated forms. Neither different light conditions nor the lack of two major thylakoid kinases, STN7 and STN8, resulted in any clear difference in FtsH phosphorylation, suggesting that this process is independent of the light-dependent regulation of photosynthesis-related proteins. Site-directed mutagenesis of putatively phosphorylated Ser or Thr residues into Ala demonstrated that Ser-212 may play a role in FtsH stability in the thylakoid membranes. Different phosphorylation status of FtsH oligomers analyzed by two-dimensional clear-native/Phos-tag SDS-PAGE implied that phosphorylation partially affects FtsH complex formation or its stability.

DOI： 10.3389/fpls.2019.01080

Organelle DNA degradation contributes to the efficient use of phosphate in seed plants. International journal

Tsuneaki Takami, Norikazu Ohnishi, Yuko Kurita, Shoko Iwamura, Miwa Ohnishi, Makoto Kusaba, Tetsuro Mimura, Wataru Sakamoto

Nature plants 4 ( 12 ) 1044 - 1055 2018.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media {LLC}

Mitochondria and chloroplasts (plastids) both harbour extranuclear DNA that originates from the ancestral endosymbiotic bacteria. These organelle DNAs (orgDNAs) encode limited genetic information but are highly abundant, with multiple copies in vegetative tissues, such as mature leaves. Abundant orgDNA constitutes a substantial pool of organic phosphate along with RNA in chloroplasts, which could potentially contribute to phosphate recycling when it is degraded and relocated. However, whether orgDNA is degraded nucleolytically in leaves remains unclear. In this study, we revealed the prevailing mechanism in which organelle exonuclease DPD1 degrades abundant orgDNA during leaf senescence. The DPD1 degradation system is conserved in seed plants and, more remarkably, we found that it was correlated with the efficient use of phosphate when plants were exposed to nutrient-deficient conditions. The loss of DPD1 compromised both the relocation of phosphorus to upper tissues and the response to phosphate starvation, resulting in reduced plant fitness. Our findings highlighted that DNA is also an internal phosphate-rich reservoir retained in organelles since their endosymbiotic origin.

DOI： 10.1038/s41477-018-0291-x

The Photosystem II Repair Cycle Requires FtsH Turnover through the EngA GTPase International journal

Yusuke Kato, Kiwamu Hyodo, Wataru Sakamoto

Plant Physiology 178 ( 2 ) 596 - 611 2018.10

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society of Plant Biologists ({ASPB})

Specific degradation of photodamaged D1, the photosystem II (PSII) reaction center protein, is a crucial step in the PSII repair cycle to maintain photosynthesis activity. Processive proteolysis by the FtsH protease is fundamental to cooperative D1 degradation. Here, we attempted to purify the FtsH complex to elucidate its regulation mechanisms and substrate recognition in Arabidopsis (Arabidopsis thaliana). Unlike previously reported prokaryotic and mitochondrial FtsHs, the Arabidopsis chloroplastic FtsH does not appear to form a megacomplex with prohibition-like proteins but instead accumulates as smaller complexes. The copurified fraction was enriched with a partial PSII intermediate presumably undergoing repair, although its precise properties were not fully clarified. In addition, we copurified a bacteria-type GTPase localized in chloroplasts, EngA, and confirmed its interaction with FtsH by subsequent pull-down and bimolecular fluorescence complementation assays. While the engA mutation is embryo lethal, the transgenic lines overexpressing EngA (EngA-OX) showed leaf variegation reminiscent of the variegated mutant lacking FtsH2. EngA-OX was revealed to accumulate more cleaved D1 fragments and reactive oxygen species than the wild type, indicative of compromised PSII repair. Based on these results and the fact that FtsH becomes more stable in EngA-OX, we propose that EngA negatively regulates FtsH stability. We demonstrate that proper FtsH turnover is crucial for PSII repair in the chloroplasts of Arabidopsis. Consistent with the increased turnover of FtsH under high-light conditions in Chlamydomonas reinhardtii, our findings underline the rapid turnover of not only D1 but also FtsH proteases in the PSII repair cycle.

DOI： 10.1104/pp.18.00652

VIPP1 Involved in Chloroplast Membrane Integrity Has GTPase Activity in Vitro International journal

Norikazu Ohnishi, Lingang Zhang, Wataru Sakamoto

Plant Physiology 177 ( 1 ) 328 - 338 2018.5

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society of Plant Biologists ({ASPB})

VESICLE-INDUCING PROTEIN IN PLASTID1 (VIPP1) is conserved among oxygenic photosynthetic organisms and appears to have diverged from the bacterial PspA protein. VIPP1 localizes to the chloroplast envelope and thylakoid membrane, where it forms homooligomers of high molecular mass. Although multiple roles of VIPP1 have been inferred, including thylakoid membrane formation, envelope maintenance, membrane fusion, and regulation of photosynthetic activity, its precise role in chloroplast membrane quality control remains unknown. VIPP1 forms an oligomer through its amino-terminal domain and triggers membrane fusion in an Mg2+-dependent manner. We previously demonstrated that Arabidopsis (Arabidopsis thaliana) VIPP1 also exhibits dynamic complex disassembly in response to osmotic and heat stresses in vivo. These results suggest that VIPP1 mediates membrane fusion/remodeling in chloroplasts. Considering that protein machines that regulate intracellular membrane fusion/remodeling events often require a capacity for GTP binding and/or hydrolysis, we questioned whether VIPP1 has similar properties. We conducted an in vitro assay using a purified VIPP1-His fusion protein expressed in Escherichia coli cells. VIPP1-His showed GTP hydrolysis activity that was inhibited competitively by an unhydrolyzable GTP analog, GTPγS, and that depends on GTP binding. It is particularly interesting that the ancestral PspA from E. coli also possesses GTP hydrolysis activity. Although VIPP1 does not contain a canonical G domain, the amino-terminal α-helix was found to be important for both GTP binding and GTP hydrolysis as well as for oligomer formation. Collectively, our results reveal that the properties of VIPP1/PspA are similar to those of GTPases.

DOI： 10.1104/pp.18.00145

Cellular Dynamics: Cellular Systems in the Time Domain. International journal

Dan Szymanski, Diane Bassham, Teun Munnik, Wataru Sakamoto

Plant physiology 176 ( 1 ) 12 - 15 2018.1

Chloroplast DNA Dynamics: Copy Number, Quality Control and Degradation

Sakamoto, Wataru, Takami, Tsuneaki

Plant and Cell Physiology 59 ( 6 ) 1120 - 1127 2018

Language：English Publishing type：Research paper (scientific journal)

Endosymbiotically originated chloroplast DNA (cpDNA) encodes part of the genetic information needed to fulfill chloroplast function, including fundamental processes such as photosynthesis. In the last two decades, advances in genome analysis led to the identification of a considerable number of cpDNA sequences from various species. While these data provided the consensus features of cpDNA organization and chloroplast evolution in plants, how cpDNA is maintained through development and is inherited remains to be fully understood. In particular, the fact that cpDNA exists as multiple copies despite its limited genetic capacity raises the important question of how copy number is maintained or whether cpDNA is subjected to quantitative fluctuation or even developmental degradation. For example, cpDNA is abundant in leaves, where it forms punctate structures called nucleoids, which seemingly alter their morphologies and numbers depending on the developmental status of the chloroplast. In this review, we summarize our current understanding of 'cpDNA dynamics', focusing on the changes in DNA abundance. A special focus is given to the cpDNA degradation mechanism, which appears to be mediated by Defective in Pollen organelle DNA degradation 1 (DPD1), a recently discovered organelle exonuclease. The physiological significance of cpDNA degradation in flowering plants is also discussed.

DOI： 10.1093/pcp/pcy084

Taiwan-Japan Plant Biology 2017 Spotlight Issue: From Light Signals/Signaling to Photosynthesis and Chloroplast Development

Nishimura, Kenji, Matsushita, Tomonao, Shikanai, Toshiharu, Sakamoto, Wataru

Plant and Cell Physiology 59 ( 6 ) 1099 - 1103 2018

Impairment of Lhca4, a subunit of LHCI, causes high accumulation of chlorophyll and the stay-green phenotype in rice International journal

Yamatani, Hiroshi, Kohzuma, Kaori, Nakano, Michiharu, Takami, Tsuneaki, Kato, Yusuke, Hayashi, Yoriko, Monden, Yuki, Okumoto, Yutaka, Abe, Tomoko, Kumamaru, Toshihiro, Tanaka, Ayumi, Sakamoto, Wataru, Kusabal, Makoto

Journal of Experimental Botany 69 ( 5 ) 1027 - 1035 2018

Language：English Publishing type：Research paper (scientific journal)

Chlorophyll is an essential molecule for acquiring light energy during photosynthesis. Mutations that result in chlorophyll retention during leaf senescence are called 'stay-green' mutants. One of the several types of stay-green mutants, Type E, accumulates high levels of chlorophyll in the pre-senescent leaves, resulting in delayed yellowing. We isolated delayed yellowing1-1 (dye1-1), a rice mutant whose yellowing is delayed in the field. dye1-1 accumulated more chlorophyll than the wild-type in the pre-senescent and senescent leaves, but did not retain leaf functionality in the 'senescent green leaves', suggesting that dye1-1 is a Type E stay-green mutant. Positional cloning revealed that DYE1 encodes Lhca4, a subunit of the light-harvesting complex I (LHCI). In dye1-1, amino acid substitution occurs at the location of a highly conserved amino acid residue involved in pigment binding; indeed, a severely impaired structure of the PSI-LHCI super-complex in dye1-1 was observed in a blue native PAGE analysis. Nevertheless, the biomass and carbon assimilation rate of dye1-1 were comparable to those in the wild-type. Interestingly, Lhcb1, a trimeric LHCII protein, was highly accumulated in dye1-1, in the chlorophyll-protein complexes. The high accumulation of LHCII in the LHCI mutant dye1 suggests a novel functional interaction between LHCI and LHCII.

DOI： 10.1093/jxb/erx468

Overexpression of the protein disulfide isomerase AtCYO1 in chloroplasts slows dark-induced senescence in Arabidopsis International journal

Tominaga, Jun, Nakahara, Yasutoshi, Horikawa, Daisuke, Tanaka, Ayumi, Kondo, Maki, Kamei, Yasuhiro, Takami, Tsuneaki, Sakamoto, Wataru, Unno, Kazutoshi, Sakamoto, Atsushi, Shimada, Hiroshi

Bmc Plant Biology 18 ( 1 ) 80 - 80 2018

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Chlorophyll breakdown is the most obvious sign of leaf senescence. The chlorophyll catabolism pathway and the associated proteins/genes have been identified in considerable detail by genetic approaches combined with stay-green phenotyping. Arabidopsis CYO1 (AtCYO1), a protein disulfide reductase/isomerase localized in the thylakoid membrane, is hypothesized to assemble the photosystem by interacting with cysteine residues of the subunits. RESULTS: In this study, we report that ectopic overexpression of AtCYO1 in leaves induces a stay-green phenotype during darkness, where oxidative conditions favor catabolism. In AtCYO1ox leaves, Fv/Fm and both chlorophyll a and chlorophyll b content remained high during dark-induced senescence. The thylakoid ultrastructure was preserved for a longer time in AtCYO1ox leaves than in wild type leaves. AtCYO1ox leaves maintained thylakoid chlorophyll-binding proteins associated with both PSII (D1, D2, CP43, CP47, LHCB2, and Cyt f) and PSI (PSA-A/B), as well as stromal proteins (Rubisco and ferredoxin-NADP+ reductase). AtCYO1ox did not affect senescence-inducible gene expression for chlorophyll catabolism or accumulation of chlorophyll catabolites. CONCLUSIONS: Our results suggest that ectopic overexpression of AtCYO1 had a negative impact on the initiation of chlorophyll degradation and proteolysis within chloroplasts. Our findings cast new light on the redox regulation of protein disulfide bonds for the maintenance of functional chloroplasts.

DOI： 10.1186/s12870-018-1294-5

Selective Elimination of Membrane-Damaged Chloroplasts via Microautophagy International journal

Nakamura, Sakuya, Hidema, Jun, Sakamoto, Wataru, Ishida, Hiroyuki, Izumi, Masanori

Plant Physiology 177 ( 3 ) 1007 - 1026 2018

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society of Plant Biologists ({ASPB})

Plant chloroplasts constantly accumulate damage caused by visible wavelengths of light during photosynthesis. Our previous study revealed that entire photodamaged chloroplasts are subjected to vacuolar digestion through an autophagy process termed chlorophagy; however, how this process is induced and executed remained poorly understood. In this study, we monitored intracellular induction of chlorophagy in Arabidopsis (Arabidopsis thaliana) leaves and found that mesophyll cells damaged by high visible light displayed abnormal chloroplasts with a swollen shape and 2.5 times the volume of normal chloroplasts. In wild-type plants, the activation of chlorophagy decreased the number of swollen chloroplasts. In the autophagy-deficient autophagy mutants, the swollen chloroplasts persisted, and dysfunctional chloroplasts that had lost chlorophyll fluorescence accumulated in the cytoplasm. Chloroplast swelling and subsequent induction of chlorophagy were suppressed by the application of exogenous mannitol to increase the osmotic pressure outside chloroplasts or by overexpression of VESICLE INDUCING PROTEIN IN PLASTID1, which maintains chloroplast envelope integrity. Microscopic observations of autophagy-related membranes showed that swollen chloroplasts were partly surrounded by autophagosomal structures and were engulfed directly by the tonoplast, as in microautophagy. Our results indicate that an elevation in osmotic potential inside the chloroplast due to high visible light-derived envelope damage results in chloroplast swelling and serves as an induction factor for chlorophagy, and this process mobilizes entire chloroplasts via tonoplast-mediated sequestering to avoid the cytosolic accumulation of dysfunctional chloroplasts.

DOI： 10.1104/pp.18.00444

FtsH Protease in the Thylakoid Membrane: Physiological Functions and the Regulation of Protease Activity. International journal

Yusuke Kato, Wataru Sakamoto

Frontiers in plant science 9 855 - 855 2018

Language：English Publishing type：Research paper (scientific journal)

Protein homeostasis in the thylakoid membranes is dependent on protein quality control mechanisms, which are necessary to remove photodamaged and misfolded proteins. An ATP-dependent zinc metalloprotease, FtsH, is the major thylakoid membrane protease. FtsH proteases in the thylakoid membranes of Arabidopsis thaliana form a hetero-hexameric complex consisting of four FtsH subunits, which are divided into two types: type A (FtsH1 and FtsH5) and type B (FtsH2 and FtsH8). An increasing number of studies have identified the critical roles of FtsH in the biogenesis of thylakoid membranes and quality control in the photosystem II repair cycle. Furthermore, the involvement of FtsH proteolysis in a singlet oxygen- and EXECUTER1-dependent retrograde signaling mechanism has been suggested recently. FtsH is also involved in the degradation and assembly of several protein complexes in the photosynthetic electron-transport pathways. In this minireview, we provide an update on the functions of FtsH in thylakoid biogenesis and describe our current understanding of the D1 degradation processes in the photosystem II repair cycle. We also discuss the regulation mechanisms of FtsH protease activity, which suggest the flexible oligomerization capability of FtsH in the chloroplasts of seed plants.

DOI： 10.3389/fpls.2018.00855

Structure and Molecular Characterization of Diadenosine Polyphosphate Hydrolase in Brachypodium distachyon Reviewed

Motohiro Tanaka, Igor Iamshchikov, Yusuke Kato, Rushan Sabirov, Oleg Gusev, Wataru Sakamoto, Manabu Sugimoto

Journal of Plant Biochemistry & Physiology 06 ( 03 ) 2018

The Non-Mendelian Green Cotyledon Gene in Soybean Encodes a Small Subunit of Photosystem II International journal

Kohzuma, Kaori, Sato, Yutaka, Ito, Hisashi, Okuzaki, Ayako, Watanabe, Mai, Kobayashi, Hideki, Nakano, Michiharu, Yamatani, Hiroshi, Masuda, Yu, Nagashima, Yumi, Fukuoka, Hiroyuki, Yamada, Tetsuya, Kanazawa, Akira, Kitamura, Keisuke, Tabei, Yutaka, Ikeuchi, Masahiko, Sakamoto, Wataru, Tanaka, Ayumi, Kusaba, Makoto

Plant Physiology 173 ( 4 ) 2138 - 2147 2017

Language：English Publishing type：Research paper (scientific journal)

Chlorophyll degradation plays important roles in leaf senescence including regulation of degradation of chlorophyll-binding proteins. Although most genes encoding enzymes of the chlorophyll degradation pathway have been identified, the regulation of their activity has not been fully understood. Green cotyledon mutants in legume are stay-green mutants, in which chlorophyll degradation is impaired during leaf senescence and seed maturation. Among them, the soybean (Glycine max) green cotyledon gene cytG is unique because it is maternally inherited. To isolate cytG, we extensively sequenced the soybean chloroplast genome, and detected a 5-bp insertion causing a frame-shift in psbM, which encodes one of the small subunits of photosystem II. Mutant tobacco plants (Nicotiana tabacum) with a disrupted psbM generated using a chloroplast transformation technique had green senescent leaves, confirming that cytG encodes PsbM. The phenotype of cytG was very similar to that of mutant of chlorophyll b reductase catalyzing the first step of chlorophyll b degradation. In fact, chlorophyll b-degrading activity in dark-grown cytG and psbM-knockout seedlings was significantly lower than that of wild-type plants. Our results suggest that PsbM is a unique protein linking photosynthesis in presenescent leaves with chlorophyll degradation during leaf senescence and seed maturation. Additionally, we discuss the origin of cytG, which may have been selected during domestication of soybean.

DOI： 10.1104/pp.16.01589

The Rubisco Chaperone BSD2 May Regulate Chloroplast Coverage in Maize Bundle Sheath Cells International journal

Salesse, Coralie, Sharwood, Robert, Sakamoto, Wataru, Sterna, David

Plant Physiology 175 ( 4 ) 1624 - 1633 2017

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society of Plant Biologists ({ASPB})

In maize (Zea mays), Bundle Sheath Defective2 (BSD2) plays an essential role in Rubisco biogenesis and is required for correct bundle sheath (BS) cell differentiation. Yet, BSD2 RNA and protein levels are similar in mesophyll (M) and BS chloroplasts, although Rubisco accumulates only in BS chloroplasts. This raises the possibility of additional BSD2 roles in cell development. To test this hypothesis, transgenic lines were created that overexpress and underexpress BSD2 in both BS and M cells, driven by the cell type-specific Rubisco Small Subunit (RBCS) or Phosphoenolpyruvate Carboxylase (PEPC) promoters or the ubiquitin promoter. Genetic crosses showed that each of the transgenes could complement Rubisco deficiency and seedling lethality conferred by the bsd2 mutation. This was unexpected, as RBCS-BSD2 lines lacked BSD2 in M chloroplasts and PEPC-BSD2 lines expressed half the wild-type BSD2 level in BS chloroplasts. We conclude that BSD2 does not play a vital role in M cells and that BS BSD2 is in excess of requirements for Rubisco accumulation. BSD2 levels did affect chloroplast coverage in BS cells. In PEPC-BSD2 lines, chloroplast coverage decreased 30% to 50%, whereas lines with increased BSD2 content exhibited a 25% increase. This suggests that BSD2 has an ancillary role in BS cells related to chloroplast size. Gas exchange showed decreased photosynthetic rates in PEPC-BSD2 lines despite restored Rubisco function, correlating with reduced chloroplast coverage and pointing to CO2 diffusion changes. Conversely, increased chloroplast coverage did not result in increased Rubisco abundance or photosynthetic rates. This suggests another limitation beyond chloroplast volume, most likely Rubisco biogenesis and/or turnover rates.

DOI： 10.1104/pp.17.01346

Essentials of Proteolytic Machineries in Chloroplasts International journal

Nishimura, Kenji, Kato, Yusuke, Sakamoto, Wataru

Molecular Plant 10 ( 1 ) 4 - 19 2017

Language：English Publishing type：Research paper (scientific journal)

Plastids are unique organelles that can alter their structure and function in response to environmental and developmental stimuli. Chloroplasts are one type of plastid and are the sites for various metabolic processes, including photosynthesis. For optimal photosynthetic activity, the chloroplast proteome must be properly shaped and maintained through regulated proteolysis and protein quality control mechanisms. Enzymatic functions and activities are conferred by protein maturation processes involving consecutive proteolytic reactions. Protein abundances are optimized by the balanced protein synthesis and degradation, which is depending on the metabolic status. Malfunctioning proteins are promptly degraded. Twenty chloroplast proteolytic machineries have been characterized to date. Specifically, processing peptidases and energy-driven processive proteases are the major players in chloroplast proteome biogenesis, remodeling, and maintenance. Recently identified putative proteases are potential regulators of photosynthetic functions. Here we provide an updated, comprehensive overview of chloroplast protein degradation machineries and discuss their importance for photosynthesis. Wherever possible, we also provide structural insights into chloroplast proteases that implement regulated proteolysis of substrate proteins/peptides.

DOI： 10.1016/j.molp.2016.08.005

Chloroplast Proteases: Updates on Proteolysis within and across Suborganellar Compartments International journal

Kenji Nishimura, Yusuke Kato, Wataru Sakamoto

Plant Physiology 171 ( 4 ) 2280 - 2293 2016.8

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society of Plant Biologists ({ASPB})

Chloroplasts originated from the endosymbiosis of ancestral cyanobacteria and maintain transcription and translation machineries for around 100 proteins. Most endosymbiont genes, however, have been transferred to the host nucleus, and the majority of the chloroplast proteome is composed of nucleus-encoded proteins that are biosynthesized in the cytosol and then imported into chloroplasts. How chloroplasts and the nucleus communicate to control the plastid proteome remains an important question. Protein-degrading machineries play key roles in chloroplast proteome biogenesis, remodeling, and maintenance. Research in the past few decades has revealed more than 20 chloroplast proteases, which are localized to specific suborganellar locations. In particular, two energy-dependent processive proteases of bacterial origin, Clp and FtsH, are central to protein homeostasis. Processing endopeptidases such as stromal processing peptidase and thylakoidal processing peptidase are involved in the maturation of precursor proteins imported into chloroplasts by cleaving off the amino-terminal transit peptides. Presequence peptidases and organellar oligopeptidase subsequently degrade the cleaved targeting peptides. Recent findings have indicated that not only intraplastidic but also extraplastidic processive protein-degrading systems participate in the regulation and quality control of protein translocation across the envelopes. In this review, we summarize current knowledge of the major chloroplast proteases in terms of type, suborganellar localization, and diversification. We present details of these degradation processes as case studies according to suborganellar compartment (envelope, stroma, and thylakoids). Key questions and future directions in this field are discussed.

DOI： 10.1104/pp.16.00330

Rice CYO1, an ortholog of Arabidopsis thaliana cotyledon chloroplastbiogenesis factor AtCYO1, is expressed in leaves and involved in photosynthetic performance International journal

Tominaga, Jun, Mizutani, Haruka, Horikawa, Daisuke, Nakahara, Yasutoshi, Takami, Tsuneaki, Sakamoto, Wataru, Sakamoto, Atsushi, Shimada, Hiroshi

Journal of Plant Physiology 207 78 - 83 2016

Language：English Publishing type：Research paper (scientific journal)

In the dicotyledonous plant Arabidopsis thaliana, the cotyledon chloroplast biogenesis factor AtCYO1 is crucial for the biogenesis of cotyledon chloroplasts. Arabidopsis mutants lacking AtCYO1 have pale cotyledons but develop normal mature leaves. In the monocotyledonous plant Oryza sativa, the gene OsCYO1 has high sequence identity to AtCYO1, but its function is unknown. We examined the role of OsCYO1 in O. sativa. We first confirmed that transformation with OsCYO1 could recover the phenotype of the Arabidopsis cyo1 mutant. Similar to AtCYO1, recombinant OsCYO1 has protein disulfide reductase (PDR) activity, which increased as a function of dieosin glutathione disulfide concentration with an apparent Km of 3.2μM and Kcat of 0.53min-1. The PDR activity was reduced when NADPH or NADH was used as an electron donor; however, PDR activity was observed with OsCYO1 and glutathione, suggesting that glutathione may serve as a reducing agent for OsCYO1 in vivo. In O. sativa, the OsCYO1 transcript level was higher in leaves compared with the coleoptile, which is the first leaf-like organ that forms during rice embryogenesis. Many OsCYO1 mutant lines defective in RNA interference had green leaves, however, three mutant lines had not only albino coleoptile but also albino leaves. Those having green leaves reduced photosynthetic performance in leaves. Our results demonstrate that OsCYO1 is enzymatically equivalent to AtCYO1 but that the physiological role of OsCYO1 in monocotyledonous plants may differ from that of AtCYO1 in dicotyledonous plants.

DOI： 10.1016/j.jplph.2016.10.005

Protection of Chloroplast Membranes by VIPP1 Rescue sAberrant Seedling Development in Arabidopsis nyc1 Mutant International journal

Zhang, Lingang, Kusaba, Makoto, Tanaka, Ayumi, Sakamoto, Wataru

Frontiers in Plant Science 7 533 - 533 2016

Language：English Publishing type：Research paper (scientific journal)

Chlorophylls (Chl) in photosynthetic apparatuses, along with other macromolecules in chloroplasts, are known to undergo degradation during leaf senescence. Several enzymes involved in Chl degradation, by which detoxification of Chl is safely implemented, have been identified. Chl degradation also occurs during embryogenesis and seedling development. Some genes encoding Chl degradation enzymes such as Chl b reductase (CBR) function during these developmental stages. Arabidopsis mutants lacking CBR (NYC1 and NOL) have been reported to exhibit reduced seed storability, compromised germination, and cotyledon development. In this study, we examined aberrant cotyledon development and found that NYC1 is solely responsible for this phenotype. We inferred that oxidative damage of chloroplast membranes caused the aberrant cotyledon. To test the inference, we attempted to trans-complement nyc1 mutant with overexpressing VIPP1 protein that is unrelated to Chl degradation but which supports chloroplast membrane integrity. VIPP1 expression actually complemented the aberrant cotyledon of nyc1, whereas stay-green phenotype during leaf senescence remained. The swollen chloroplasts observed in unfixed cotyledons of nyc1, which are characteristics of chloroplasts receiving envelope membrane damage, were recovered by overexpressing VIPP1. These results suggest that chloroplast membranes are a target for oxidative damage caused by the impairment in Chl degradation. Trans-complementation of nyc1 with VIPP1 also suggests that VIPP1 is useful for protecting chloroplasts against oxidative stress.

DOI： 10.3389/fpls.2016.00533

VIPP1 Has a Disordered C-Terminal Tail Necessary for Protecting Photosynthetic Membranes against Stress International journal

Zhang, Lingang, Kondo, Hideki, Kamikubo, Hironari, Kataoka, Mikio, Sakamoto, Wataru

Plant Physiology 171 ( 3 ) 1983 - 1995 2016

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society of Plant Biologists ({ASPB})

Integrity of biomembranes is vital to living organisms. In bacteria, PspA is considered to act as repairing damaged membrane by forming large supercomplexes in Arabidopsis (Arabidopsis thaliana). Vulnerable to oxidative stress, photosynthetic organisms also contain a PspA ortholog called VIPP1, which has an additional C-terminal tail (Vc). In this study, Vc was shown to coincide with an intrinsically disordered region, and the role of VIPP1 in membrane protection against stress was investigated. We visualized VIPP1 by fusing it to GFP (VIPP1-GFP that fully complemented lethal vipp1 mutations), and investigated its behavior in vivo with live imaging. The intrinsically disordered nature of Vc enabled VIPP1 to form what appeared to be functional particles along envelopes, whereas the deletion of Vc caused excessive association of the VIPP1 particles, preventing their active movement for membrane protection. Expression of VIPP1 lacking Vc complemented vipp1 mutation, but exhibited sensitivity to heat shock stress. Conversely, transgenic plants over-expressing VIPP1 showed enhanced tolerance against heat shock, suggesting that Vc negatively regulates VIPP1 particle association and acts in maintaining membrane integrity. Our data thus indicate that VIPP1 is involved in the maintenance of photosynthetic membranes. During evolution, chloroplasts have acquired enhanced tolerance against membrane stress by incorporating a disordered C-terminal tail into VIPP1.

DOI： 10.1104/pp.16.00532

Amyloplast Membrane Protein SUBSTANDARD STARCH GRAIN6 Controls Starch Grain Size in Rice Endosperm International journal

Matsushima, Ryo, Maekawa, Masahiko, Kusano, Miyako, Tomita, Katsura, Kondo, Hideki, Nishimura, Hideki, Crofts, Naoko, Fujita, Naoko, Sakamoto, Wataru

Plant Physiology 170 ( 3 ) 1445 - 59 2016

Language：English Publishing type：Research paper (scientific journal)

Starch is a biologically and commercially important polymer of glucose. Starch is organized into starch grains (SGs) inside amyloplasts. The SG size differs depending on the plant species and is one of the most important factors for industrial applications of starch. There is limited information on genetic factors regulating SG sizes. In this study, we report the rice (Oryza sativa) mutant substandard starch grain6 (ssg6), which develops enlarged SGs in endosperm. Enlarged SGs are observed starting at 3 d after flowering. During endosperm development, a number of smaller SGs appear and coexist with enlarged SGs in the same cells. The ssg6 mutation also affects SG morphologies in pollen. The SSG6 gene was identified by map-based cloning and microarray analysis. SSG6 encodes a protein homologous to aminotransferase. SSG6 differs from other rice homologs in that it has a transmembrane domain. SSG6-green fluorescent protein is localized in the amyloplast membrane surrounding SGs in rice endosperm, pollen, and pericarp. The results of this study suggest that SSG6 is a novel protein that controls SG size. SSG6 will be a useful molecular tool for future starch breeding and applications.

DOI： 10.1104/pp.15.01811

C2-P-07Functions of phototropins and photosynthesis in the light induced mitochondria chloroplasts co-localization inArabidopsis thaliana

Islam Md. Sayeedul, Nguyen Van Toan, Kato Yusuke, Sakamoto Wataru, Takagi Shingo

Microscopy 64 ( suppl 1 ) i126.1 - i126 2015.11

Physical interaction between peroxisomes and chloroplasts elucidated by in situ laser analysis (vol 1, 15035, 2015) Reviewed

Kazusato Oikawa, Shigeru Matsunaga, Shoji Mano, Maki Kondo, Kenji Yamada, Makoto Hayashi, Takatoshi Kagawa, Akeo Kadota, Wataru Sakamoto, Shoichi Higashi, Masakatsu Watanabe, Toshiaki Mitsui, Akinori Shigemasa, Takanori Iino, Yoichiroh Hosokawa, Mikio Nishimura

NATURE PLANTS 1 ( 4 ) 2015.4

D1 fragmentation in photosystem II repair caused by photo-damage of a two-step model International journal

Kato, Yusuke, Ozawa, Shin-ichiro, Takahashi, Yuichiro, Sakamoto, Wataru

Photosynthesis Research 126 ( 2-3 ) 409 - 16 2015

Language：English Publishing type：Research paper (scientific journal)

Light energy drives photosynthesis, but it simultaneously inactivates photosynthetic mechanisms. A major target site of photo-damage is photosystem II (PSII). It further targets one reaction center protein, D1, which is maintained efficiently by the PSII repair cycle. Two proteases, FtsH and Deg, are known to contribute to this process, respectively, by efficient degradation of photo-damaged D1 protein processively and endoproteolytically. This study tested whether the D1 cleavage accomplished by these proteases is affected by different monochromic lights such as blue and red light-emitting-diode light sources, remaining mindful that the use of these lights distinguishes the current models for photoinhibition: the excess-energy model and the two-step model. It is noteworthy that in the two-step model, primary damage results from the absorption of light energy in the Mn-cluster, which can be enhanced by a blue rather than a red light source. Results showed that blue and red lights affect D1 degradation differently. One prominent finding was that D1 fragmentation that is specifically generated by luminal Deg proteases was enhanced by blue light but not by red light in the mutant lacking FtsH2. Although circumstantial, this evidence supports a two-step model of PSII photo-damage. We infer that enhanced D1 fragmentation by luminal Deg proteases is a response to primary damage at the Mn-cluster.

DOI： 10.1007/s11120-015-0144-7

Possible function of VIPP1 in maintaining chloroplast membranes International journal

Zhang, Lingang, Sakamoto, Wataru

Biochimica Et Biophysica Acta-Bioenergetics 1847 ( 9 ) 831 - 7 2015

Language：English Publishing type：Research paper (scientific journal)

A protein designated as VIPP1 is found widely in organisms performing oxygenic photosynthesis, but its precise role in chloroplasts has remained somewhat mysterious. Based on its structural similarity, it presumably has evolved from bacterial Phage shock protein A (PspA) with a C-terminal extension of approximately 40 amino acids. Both VIPP1 and PspA are membrane-associated despite the lack of transmembrane helices. They form an extremely large homo-complex that consists of an oligomeric ring unit. Although PspA is known to respond to membrane stress and although it acts in maintaining proton motive force through membrane repair, the multiple function of VIPP1, such as vesicle budding from inner envelope to deliver lipids to thylakoids, maintenance of photosynthetic complexes in thylakoid membranes, biogenesis of Photosystem I, and protective role of inner envelope against osmotic stress, has been proposed. Whatever its precise function in chloroplasts, it is an important protein because depletion of VIPP1 in mutants severely affects photoautotrophic growth. Recent reports of the relevant literature describe that VIPP1 becomes highly mobile when chloroplasts receive hypotonic stress, and that VIPP1 is tightly bound to lipids, which implies a crucial role of VIPP1 in membrane repair through lipid transfer. This review presents a summary of our current knowledge related to VIPP1, particularly addressing the dynamic behavior of complexes against stress and its property of lipid binding. Those data altogether suggest that VIPP1 acts a priori in chloroplast membrane maintenance through its activity to transfer lipids rather than in thylakoid formation through vesicles. This article is part of a Special Issue titled: Chloroplast Biogenesis.

DOI： 10.1016/j.bbabio.2015.02.013

Geometrical Formation of Compound Starch Grains in Rice Implements Voronoi Diagram

Matsushima, Ryo, Maekawa, Masahiko, Sakamoto, Wataru

Plant and Cell Physiology 56 ( 11 ) 2150 - 7 2015

Language：English Publishing type：Research paper (scientific journal)

Starch forms transparent grains, called starch grains (SGs), in amyloplasts. One of the major morphological SG forms in Poaceae, called a compound SG, is formed by assemblies of small starch granules in an amyloplast. Starch granules assemble as a well-ordered structure; however, the mechanism that regulates this organization has not been identified. In this study, we examined how starch granules grow and converge into the final SG morphology. First, we found that the number of starch granules in an amyloplast is almost constant from the early developmental stage until endosperm maturity. Next, we quantitatively evaluated the geometrical similarities between starch granules and a Voronoi diagram, which is a mathematical tessellation of space based on the distance to a specific set of points in the space. The in silico growth simulation showed that the geometrical patterns of compound SGs resembling a Voronoi diagram is determined by physical interactions among the free-growing starch granules and the amyloplast envelope membrane. The geometrical similarity between compound SGs and a Voronoi diagram is likely a result of maximum loading and storage of starch in the amyloplast. The simulation described in this study provides a greater understanding of how compound SGs are formed and also has the potential to explain morphological variations of SGs.

DOI： 10.1093/pcp/pcv123

Physical interaction between peroxisomes and chloroplasts elucidated by in situ laser analysis International journal

Oikawa, Kazusato, Matsunaga, Shigeru, Mano, Shoji, Kondo, Maki, Yamada, Kenji, Hayashi, Makoto, Kagawa, Takatoshi, Kadota, Akeo, Sakamoto, Wataru, Higashi, Shoichi, Watanabe, Masakatsu, Mitsui, Toshiaki, Shigemasa, Akinori, Iino, Takanori, Hosokawa, Yoichiroh, Nishimura, Mikio

Nature Plants 1 ( 4 ) 15035 - 15035 2015

Language：English Publishing type：Research paper (scientific journal)

Life on earth relies upon photosynthesis, which consumes carbon dioxide and generates oxygen and carbohydrates. Photosynthesis is sustained by a dynamic environment within the plant cell involving numerous organelles with cytoplasmic streaming. Physiological studies of chloroplasts, mitochondria and peroxisomes show that these organelles actively communicate during photorespiration, a process by which by-products produced by photosynthesis are salvaged. Nevertheless, the mechanisms enabling efficient exchange of metabolites have not been clearly defined. We found that peroxisomes along chloroplasts changed shape from spherical to elliptical and their interaction area increased during photorespiration. We applied a recent femtosecond laser technology to analyse adhesion between the organelles inside palisade mesophyll cells of Arabidopsis leaves and succeeded in estimating their physical interactions under different environmental conditions. This is the first application of this estimation method within living cells. Our findings suggest that photosynthetic-dependent interactions play a critical role in ensuring efficient metabolite flow during photorespiration.

DOI： 10.1038/NPLANTS.2015.35

A Mutation in GIANT CHLOROPLAST Encoding a PARC6 Homolog Affects Spikelet Fertility in Rice

Kamau, Peter K., Sano, Shingo, Takami, Tsuneaki, Matsushima, Ryo, Maekawa, Masahiko, Sakamoto, Wataru

Plant and Cell Physiology 56 ( 5 ) 977 - 91 2015

Language：English Publishing type：Research paper (scientific journal)

Chloroplasts are not generated de novo but proliferate from a pre-existing population of plastids present in meristematic cells. Chloroplast division is executed by the co-ordinated action of at least two molecular machineries: internal machinery located on the stromal side of the inner envelope membrane and external machinery located on the cytosolic side of the outer envelope membrane. To date, molecular studies of chloroplast division in higher plants have been limited to several species such as Arabidopsis. To elucidate chloroplast division in rice, we performed forward genetics and isolated a mutant displaying large chloroplasts among an ethyl methanesulfonate (EMS)-mutagenized Oryza sativa spp japonica Nipponbare population. Using a map-based approach, this mutation, termed giant chloroplast (gic), was allocated in a gene that encodes a protein that is homologous to Paralog of ARC6 (PARC6), which is known to play a role in chloroplast division. GIC is unique in that it has a long C-terminal extension that is not present in other PARC6 homologs. Characterization of gic phenotypes in a rice field showed that gic exhibited defective growth in seed setting, suggesting that the gic mutant negatively affects the reproductive stage. This report is the first describing a chloroplast division mutant in monocotyledons and its effect on plant development.

DOI： 10.1093/pcp/pcv024

Vegetative and Sperm Cell-Specific Aquaporins of Arabidopsis Highlight the Vacuolar Equipment of Pollen and Contribute to Plant Reproduction International journal

Wudick, Michael M., Doan-Trung Luu, Tournaire-Roux, Colette, Sakamoto, Wataru, Maurel, Christophe

Plant Physiology 164 ( 4 ) 1697 - 706 2014

Language：English Publishing type：Research paper (scientific journal)

The water and nutrient status of pollen is crucial to plant reproduction. Pollen grains of Arabidopsis (Arabidopsis thaliana) contain a large vegetative cell and two smaller sperm cells. Pollen grains express AtTIP1;3 and AtTIP5;1, two members of the Tonoplast Intrinsic Protein subfamily of aquaporins. To address the spatial and temporal expression pattern of the two homologs, C-terminal fusions of AtTIP1;3 and AtTIP5;1 with green fluorescent protein and mCherry, respectively, were expressed in transgenic Arabidopsis under the control of their native promoter. Confocal laser scanning microscopy revealed that AtTIP1;3 and AtTIP5;1 are specific for the vacuoles of the vegetative and sperm cells, respectively. The tonoplast localization of AtTIP5;1 was established by reference to fluorescent protein markers for the mitochondria and vacuoles of sperm and vegetative cells and is at variance with the claim that AtTIP5;1 is localized in vegetative cell mitochondria. AtTIP1;3-green fluorescent protein and AtTIP5;1-mCherry showed concomitant expression, from first pollen mitosis up to pollen tube penetration in the ovule, thereby revealing the dynamics of vacuole morphology in maturating and germinating pollen. Transfer DNA insertion mutants for either AtTIP1;3 or AtTIP5;1 showed no apparent growth phenotype and had no significant defect in male transmission of the mutated alleles. By contrast, a double knockout displayed an abnormal rate of barren siliques, this phenotype being more pronounced under limited water or nutrient supply. The overall data indicate that vacuoles of vegetative and sperm cells functionally interact and contribute to male fertility in adverse environmental conditions.

DOI： 10.1104/pp.113.228700

Amyloplast-Localized SUBSTANDARD STARCH GRAIN4 Protein Influences the Size of Starch Grains in Rice Endosperm International journal

Matsushima, Ryo, Maekawa, Masahiko, Kusano, Miyako, Kondo, Hideki, Fujita, Naoko, Kawagoe, Yasushi, Sakamoto, Wataru

Plant Physiology 164 ( 2 ) 623 - 36 2014

Language：English Publishing type：Research paper (scientific journal)

Starch is a biologically and commercially important polymer of glucose and is synthesized to form starch grains (SGs) inside amyloplasts. Cereal endosperm accumulates starch to levels that are more than 90% of the total weight, and most of the intracellular space is occupied by SGs. The size of SGs differs depending on the plant species and is one of the most important factors for industrial applications of starch. However, the molecular machinery that regulates the size of SGs is unknown. In this study, we report a novel rice (Oryza sativa) mutant called substandard starch grain4 (ssg4) that develops enlarged SGs in the endosperm. Enlargement of SGs in ssg4 was also observed in other starch-accumulating tissues such as pollen grains, root caps, and young pericarps. The SSG4 gene was identified by map-based cloning. SSG4 encodes a protein that contains 2,135 amino acid residues and an amino-terminal amyloplast-targeted sequence. SSG4 contains a domain of unknown function490 that is conserved from bacteria to higher plants. Domain of unknown function490-containing proteins with lengths greater than 2,000 amino acid residues are predominant in photosynthetic organisms such as cyanobacteria and higher plants but are minor in proteobacteria. The results of this study suggest that SSG4 is a novel protein that influences the size of SGs. SSG4 will be a useful molecular tool for future starch breeding and biotechnology.

DOI： 10.1104/pp.113.229591

Phosphorylation of photosystem II core proteins prevents undesirable cleavage of D1 and contributes to the fine-tuned repair of photosystem II International journal

Kato, Yusuke, Sakamoto, Wataru

Plant Journal 79 ( 2 ) 312 - 21 2014

Language：English Publishing type：Research paper (scientific journal)

Photosystem II (PSII) is a primary target for light-induced damage in photosynthetic protein complexes. To avoid photoinhibition, chloroplasts have evolved a repair cycle with efficient degradation of the PSII reaction center protein, D1, by the proteases FtsH and Deg. Earlier reports have described that phosphorylated D1 is a poor substrate for proteolysis, suggesting a mechanistic role for protein phosphorylation in PSII quality control, but its precise role remains elusive. STN8, a protein kinase, plays a central role in this phosphorylation process. To elucidate the relationship between phosphorylation of D1 and the protease function we assessed in this study the involvement of STN8, using Arabidopsis thaliana mutants lacking FtsH2 [yellow variegated2 (var2)] and Deg5/Deg8 (deg5 deg8). In support of our presumption we found that phosphorylation of D1 increased more in var2. Furthermore, the coexistence of var2 and stn8 was shown to recover the delay in degradation of D1, resulting in mitigation of the high vulnerability to photoinhibition of var2. Partial D1 cleavage fragments that depended on Deg proteases tended to increase, with concomitant accumulation of reactive oxygen species in the mutants lacking STN8. We inferred that the accelerated degradation of D1 in var2 stn8 presents a tradeoff in that it improved the repair of PSII but simultaneously enhanced oxidative stress. Together, these results suggest that PSII core phosphorylation prevents undesirable cleavage of D1 by Deg proteases, which causes cytotoxicity, thereby balancing efficient linear electron flow and photo-oxidative damage. We propose that PSII core phosphorylation contributes to fine-tuned degradation of D1.

DOI： 10.1111/tpj.12562

An EAR-Dependent Regulatory Module Promotes Male Germ Cell Division and Sperm Fertility in Arabidopsis International journal

Borg, Michael, Rutley, Nicholas, Kagale, Sateesh, Hamamura, Yuki, Gherghinoiu, Mihai, Kumar, Sanjeev, Sari, Ugur, Esparza-Franco, Manuel A., Sakamoto, Wataru, Rozwadowski, Kevin, Higashiyama, Tetsuya, Twella, David

Plant Cell 26 ( 5 ) 2098 - 2113 2014

Language：English Publishing type：Research paper (scientific journal)

The production of the sperm cells in angiosperms requires coordination of cell division and cell differentiation. In Arabidopsis thaliana, the germline-specific MYB protein DUO1 integrates these processes, but the regulatory hierarchy in which DUO1 functions is unknown. Here, we identify an essential role for two germline-specific DUO1 target genes, DAZ1 and DAZ2, which encode EAR motif-containing C2H2-type zinc finger proteins. We show that DAZ1/DAZ2 are required for germ cell division and for the proper accumulation of mitotic cyclins. Importantly, DAZ1/DAZ2 are sufficient to promote G2- to M-phase transition and germ cell division in the absence of DUO1. DAZ1/DAZ2 are also required for DUO1-dependent cell differentiation and are essential for gamete fusion at fertilization. We demonstrate that the two EAR motifs in DAZ1/DAZ2 mediate their function in the male germline and are required for transcriptional repression and for physical interaction with the corepressor TOPLESS. Our findings uncover an essential module in a regulatory hierarchy that drives mitotic transition in male germ cells and implicates gene repression pathways in sperm cell formation and fertility.

DOI： 10.1105/tpc.114.124743

Nucleases in higher plants and their possible involvement in DNA degradation during leaf senescence International journal

Sakamoto, Wataru, Takami, Tsuneaki

Journal of Experimental Botany 65 ( 14 ) 3835 - 43 2014

Language：English Publishing type：Research paper (scientific journal)

During leaf senescence, macromolecules such as proteins and lipids are known to be degraded for redistribution into upper tissues. Similarly, nucleic acids appear to undergo fragmentation or degradation during senescence, but the physiological role of nucleic acid degradation, particularly of genomic DNA degradation, remains unclear. To date, more than a dozen of plant deoxyribonucleases have been reported, whereas it remains to be verified whether any of them degrade DNA during leaf senescence. This review summarizes current knowledge related to the plant nucleases that are induced developmentally or in a tissue-specific manner and are known to degrade DNA biochemically. Of these, several endonucleases (BFN1, CAN1, and CAN2) and an exonuclease (DPD1) in Arabidopsis seem to act in leaf senescence because they were shown to be inducible at the transcript level. This review specifically examines DPD1, which is dual-targeted to chloroplasts and mitochondria. Results show that, among the exonuclease family to which DPD1 belongs, DPD1 expression is extraordinary when estimated using a microarray database. DPD1 is the only example among the nucleases in which DNA degradation has been confirmed in vivo in pollen by mutant analysis. These data imply a significant role of organelle DNA degradation during leaf senescence and implicate DPD1 as a potential target for deciphering nucleotide salvage in plants.

DOI： 10.1093/jxb/eru091

Plant autophagy is responsible for peroxisomal transition and plays an important role in the maintenance of peroxisomal quality International journal

Shibata, Michitaro, Oikawa, Kazusato, Yoshimoto, Kohki, Goto-Yamada, Shino, Mano, Shoji, Yamada, Kenji, Kondo, Maki, Hayashi, Makoto, Sakamoto, Wataru, Ohsumi, Yoshinori, Nishimura, Mikio

Autophagy 10 ( 5 ) 936 - 7 2014

Language：English Publishing type：Research paper (scientific journal)

In photosynthetic cells, a large amount of hydrogen peroxide is produced in peroxisomes through photorespiration, which is a metabolic pathway related to photosynthesis. Hydrogen peroxide, a reactive oxygen species, oxidizes peroxisomal proteins and membrane lipids, resulting in a decrease in peroxisomal quality. We demonstrate that the autophagic system is responsible for the elimination of oxidized peroxisomes in plant. We isolated Arabidopsis mutants that accumulated oxidized peroxisomes, which formed large aggregates. We revealed that these mutants were defective in autophagy-related (ATG) genes and that the aggregated peroxisomes were selectively targeted by the autophagic machinery. These findings suggest that autophagy plays an important role in the quality control of peroxisomes by the selective degradation of oxidized peroxisomes. In addition, the results suggest that autophagy is also responsible for the functional transition of glyoxysomes to leaf peroxisomes.

DOI： 10.4161/auto.28529

Possible compensatory role among chloroplast proteases under excess-light stress condition. International journal

Yusuke Kato, Wataru Sakamoto

Plant signaling & behavior 8 ( 3 ) e23198 2013.3

Language：English Publishing type：Research paper (scientific journal)

The reaction center protein D1 of photosystem II (PSII), known as a primary target of photodamage, is repaired efficiently by the PSII repair cycle, to cope with constant photooxidative damage. Recent studies of Arabidopsis show that the endo-type Deg protease and the exo-type FtsH proteases cooperatively degrade D1 in the PSII repair in vivo. It is particularly interesting that we observed upregulation of Clp and SppA proteases when FtsH was limited in the mutant lacking FtsH2. To examine how the complementary functions of chloroplastic proteases are commonly regulated, we undertook a high-light stress on wild-type Arabidopsis leaves. The result that wild type leaves also showed increased levels of these proteases upon exposure to excessively strong illumination not only revealed the importance of FtsH and Deg in the PSII repair, but also implied cooperation among chloroplastic proteases under chronic stress conditions.

DOI： 10.4161/psb.23198

Possible function of VIPP1 in thylakoids: protection but not formation? International journal

Lingang Zhang, Wataru Sakamoto

Plant signaling & behavior 8 ( 2 ) e22860 2013.2

Language：English Publishing type：Research paper (scientific journal)

VIPP1 protein in photosynthetic organisms is homologous to bacterial PspA, which protects plasma membrane integrity upon stresses. Despite the proposed role of VIPP1 in thylakoid biogenesis, its precise function remains unclear. Recently, our in-depth analysis of Arabidopsis vipp1 mutants revealed VIPP1's involvement in the maintenance of chloroplast envelopes. Chloroplasts in intact vipp1 leaves exhibited spherical balloon-like morphology, which resulted from osmotic stress across envelopes. In fact, observation of VIPP1 fused to green fluorescence protein in vivo revealed that most VIPP1 is localized as a lattice-like macro complex attached along with the envelope. Because of the proposed function in thylakoids, we examined whether vipp1 also exhibited altered morphologies in thylakoids. Results show that thylakoid morphologies were detected irregularly, but vipp1 chloroplasts retained normal-appearing grana stacks. We infer that VIPP1 might influence thylakoids as well as envelopes, but that it is not involved directly in thylakoid membrane formation.

DOI： 10.4161/psb.22860

Ion gradients in xylem exudate and guttation fluid related to tissue ion levels along primary leaves of barley International journal

Nagai, Makiko, Ohnishi, Miwa, Uehara, Takeo, Yamagami, Mutsumi, Miura, Eiko, Kamakura, Mai, Kitamura, Akira, Sakaguchi, Shu-Ichi, Sakamoto, Wataru, Shimmen, Teruo, Fukaki, Hidehiro, Reid, Robert J., Furukawa, Akio, Mimura, Tetsuro

Plant Cell and Environment 36 ( 10 ) 1826 - 37 2013

Language：English Publishing type：Research paper (scientific journal)

The concentration of ions in plant cells and tissues is an essential factor in determining physiological function. In the present study, we established that concentration gradients of mobile ions exist in both xylem exudates and tissues within a barley (Hordeum vulgare) primary leaf. For K(+) and NO3 (-) , ion concentrations generally decreased from the leaf base to the tip in both xylem exudates and tissues. Ion gradients were also found for Pi and Cl(-) in the xylem. The hydathode strongly absorbed Pi and re-translocated it to the rest of the plant, whereas Cl(-) was extruded. The ion concentration gradients developed early during leaf growth, increased as the tissue aged and remained under both high and low transpiration conditions. Measurement of the expression profiles of Pi, K(+) and NO3 (-) transporters along the longitudinal axis of the leaf revealed that some transporters are more expressed at the hydathode, but for most transporters, there was no significant variation along the leaf. The mechanisms by which longitudinal ion gradients develop in leaves and their physiological functions are discussed.

DOI： 10.1111/pce.12090

NYC4, the rice ortholog of Arabidopsis THF1, is involved in the degradation of chlorophyll protein complexes during leaf senescence International journal

Yamatani, Hiroshi, Sato, Yutaka, Masuda, Yu, Kato, Yusuke, Morita, Ryouhei, Fukunaga, Kenji, Nagamura, Yoshiaki, Nishimura, Minoru, Sakamoto, Wataru, Tanaka, Ayumi, Kusaba, Makoto

Plant Journal 74 ( 4 ) 652 - 62 2013

Language：English Publishing type：Research paper (scientific journal)

Yellowing/chlorophyll breakdown is a prominent phenomenon in leaf senescence, and is associated with the degradation of chlorophyll - protein complexes. From a rice mutant population generated by ionizing radiation, we isolated nyc4-1, a stay-green mutant with a defect in chlorophyll breakdown during leaf senescence. Using gene mapping, nyc4-1 was found to be linked to two chromosomal regions. We extracted Os07g0558500 as a candidate for NYC4 via gene expression microarray analysis, and concluded from further evidence that disruption of the gene by a translocation-related event causes the nyc4 phenotype. Os07g0558500 is thought to be the ortholog of THF1 in Arabidopsis thaliana. The thf1 mutant leaves show variegation in a light intensity-dependent manner. Surprisingly, the Fv /Fm value remained high in nyc4-1 during the dark incubation, suggesting that photosystem II retained its function. Western blot analysis revealed that, in nyc4-1, the PSII core subunits D1 and D2 were significantly retained during leaf senescence in comparison with wild-type and other non-functional stay-green mutants, including sgr-2, a mutant of the key regulator of chlorophyll degradation SGR. The role of NYC4 in degradation of chlorophyll and chlorophyll - protein complexes during leaf senescence is discussed.

DOI： 10.1111/tpj.12154

Highly Oxidized Peroxisomes Are Selectively Degraded via Autophagy in Arabidopsis International journal

Shibata, Michitaro, Oikawa, Kazusato, Yoshimoto, Kohki, Kondo, Maki, Mano, Shoji, Yamada, Kenji, Hayashi, Makoto, Sakamoto, Wataru, Ohsumi, Yoshinori, Nishimura, Mikio

Plant Cell 25 ( 12 ) 4967 - 83 2013

Language：English Publishing type：Research paper (scientific journal)

The positioning of peroxisomes in a cell is a regulated process that is closely associated with their functions. Using this feature of the peroxisomal positioning as a criterion, we identified three Arabidopsis thaliana mutants (peroxisome unusual positioning1 [peup1], peup2, and peup4) that contain aggregated peroxisomes. We found that the PEUP1, PEUP2, and PEUP4 were identical to Autophagy-related2 (ATG2), ATG18a, and ATG7, respectively, which are involved in the autophagic system. The number of peroxisomes was increased and the peroxisomal proteins were highly accumulated in the peup1 mutant, suggesting that peroxisome degradation by autophagy (pexophagy) is deficient in the peup1 mutant. These aggregated peroxisomes contained high levels of inactive catalase and were more oxidative than those of the wild type, indicating that peroxisome aggregates comprise damaged peroxisomes. In addition, peroxisome aggregation was induced in wild-type plants by exogenous application of hydrogen peroxide. The cat2 mutant also contained peroxisome aggregates. These findings demonstrate that hydrogen peroxide as a result of catalase inactivation is the inducer of peroxisome aggregation. Furthermore, an autophagosome marker, ATG8, frequently colocalized with peroxisome aggregates, indicating that peroxisomes damaged by hydrogen peroxide are selectively degraded by autophagy in the wild type. Our data provide evidence that autophagy is crucial for quality control mechanisms for peroxisomes in Arabidopsis.

DOI： 10.1105/tpc.113.116947

A Phylogenetic Re-evaluation of Morphological Variations of Starch Grains among Poaceae Species Reviewed

Ryo Matsushima, Jun Yamashita, Shungo Kariyama, Takashi Enomoto, Wataru Sakamoto

Journal of Applied Glycoscience 60 ( 1 ) 37 - 44 2013

The lattice-like structure observed by Vipp1-GFP in arabidopsis chloroplasts

Lingang Zhang, Yusuke Kato, Koji Saigo, Ute C. Vothknecht, Wataru Sakamoto

Advanced Topics in Science and Technology in China 394 - 397 2013

Language：English Publishing type：Part of collection (book) Publisher：Springer

Vipp1 (vesicle inducing protein in plastids 1) is proposed to play a role in thylakoid biogenesis. It is closely related to PspA (phage shock protein A), a bacterial protein that is induced under stress conditions. Despite its discovery a decade ago and extensive analysis in cyanobacteria, green algae and higher plants, the precise role of Vipp1 in the process of chloroplast development remains unclear. In this research, we expressed Vipp1 C-terminally fused to GFP (Vipp1-GFP) in Arabidopsis and found that Vipp1 is able to assemble into rod-shaped supercomplexes. Vipp1-GFP can rescue heterotrophic growth of a vipp1 knock-down mutant, suggesting that it complements Vipp1 function. Interestingly, Vipp1-GFP rods always appeared to cross with each other to form a lattice-like structure, which is similar to a scaffold structure formed by PspA in Escherichia coli. Based on these results, we infer that Vipp1 is involved in not only thylakoid biogenesis but chloroplast envelope integrity.

DOI： 10.1007/978-3-642-32034-7_82

A novel link between chloroplast development and stress response lessoned by leaf-variegated mutant

Wataru Sakamoto, Eiko Miura, Yusuke Kato

Advanced Topics in Science and Technology in China 669 - 673 2013

Language：English Publishing type：Part of collection (book) Publisher：Springer

Recessive mutations are known to give rise to cell lineage-type leaf variegation that forms green and white sectors due to arrested chloroplast development. The yellow variegated 2 (var2) mutant in Arabidopsis thaliana has been studied as a typical leaf-variegated mutant whose defect results from the lack of FtsH2 metalloprotease in chloroplasts. To understand physiological properties of variegated sectors, gene expression profiles were investigated between green and white sectors of var2 leaves. Consistent with impaired thylakoid formation, a substantial number of genes related to photosynthesis and chloroplast functions were repressed in white sectors. In addition, many genes were up-regulated in white sectors. Since var2 leaves suffer from photooxidative stress and accumulate high levels of reactive oxygen species (ROS) due to compromised Photosystem II repair, we focused ROS scavenging genes such as Cu/Zn superoxide dismutase 2 (CSD2). Activation of CSD2 was specific to white sectors and appeared to be under the control of copper availability. A lack of thylakoid membranes in white sectors perhaps leads to excess free copper and iron conditions, thus white sectors mimic a copper sufficient condition. We infer that CSD2 acts not only on ROS detoxification but also on copper buffering. Interestingly, an up-regulation of Cu/Zn SOD was commonly observed in various variegated leaves. Our findings thus highlight the crucial role for the control of oxidative stress and free metals in variegated leaf sectors.

DOI： 10.1007/978-3-642-32034-7_145

Variegated Tobacco Leaves Generated by Chloroplast FtsH Suppression: Implication of FtsH Function in the Maintenance of Thylakoid Membranes

Kato, Yusuke, Kouso, Takayoshi, Sakamoto, Wataru

Plant and Cell Physiology 53 ( 2 ) 391 - 404 2012

Language：English Publishing type：Research paper (scientific journal)

Mutants lacking a thylakoid membrane-bound metalloprotease, FtsH, are known to cause leaf variegation in Arabidopsis. However, the effect of reduced FtsH levels on leaf variegation has scarcely been examined in other plants. In this study, we performed RNA interference (RNAi) by which FtsH expression was suppressed in tobacco. The resulting FtsH knock-down tobacco plants showed variegation in their leaves, and a negative correlation between the degree of variegation and the level of FtsH, which supported earlier observations in Arabidopsis. A decrease of NtFtsH2 as well as NtFtsH1 suggested that these are the two major isoforms comprising the FtsH complex in tobacco chloroplasts. The RNAi tobacco lines also showed photoinhibition-vulnerable phenotypes, as evidenced by high-light-sensitive PSII activity and retarded degradation of D1 protein. Interestingly, the formation of variegated sectors during leaf development appeared to differ between Arabidopsis and tobacco. In contrast to the formation of variegation in Arabidopsis, the yellow sectors in FtsH RNAi tobacco emerged from green leaves at a late stage of leaf development. A series of cytological observations implied that thylakoid membranes were dismantled after development had already occurred. Late formation of variegation in FtsH RNAi tobacco suggested that the heteromeric FtsH complex is important for maintaining thylakoid membranes.

DOI： 10.1093/pcp/pcr189

Essential Role of VIPP1 in Chloroplast Envelope Maintenance in Arabidopsis International journal

Zhang, Lingang, Kato, Yusuke, Otters, Stephanie, Vothknecht, Ute C., Sakamoto, Wataru

Plant Cell 24 ( 9 ) 3695 - 707 2012

Language：English Publishing type：Research paper (scientific journal)

VESICLE-INDUCING PROTEIN IN PLASTIDS1 (VIPP1), proposed to play a role in thylakoid biogenesis, is conserved in photosynthetic organisms and is closely related to Phage Shock Protein A (PspA), which is involved in plasma membrane integrity in Escherichia coli. This study showed that chloroplasts/plastids in Arabidopsis thaliana vipp1 knockdown and knockout mutants exhibit a unique morphology, forming balloon-like structures. This altered morphology, as well as lethality of vipp1, was complemented by expression of VIPP1 fused to green fluorescent protein (VIPP1-GFP). Several lines of evidence show that the balloon chloroplasts result from chloroplast swelling related to osmotic stress, implicating VIPP1 in the maintenance of plastid envelopes. In support of this, Arabidopsis VIPP1 rescued defective proton leakage in an E. coli pspA mutant. Microscopy observation of VIPP1-GFP in transgenic Arabidopsis revealed that VIPP1 forms large macrostructures that are integrated into various morphologies along the envelopes. Furthermore, live imaging revealed that VIPP1-GFP is highly mobile when chloroplasts are subjected to osmotic stress. VIPP1-GFP showed dynamic movement in the transparent area of spherical chloroplasts, as the fluorescent molecules formed filament-like structures likely derived from disassembly of the large VIPP1 complex. Collectively, our data demonstrate that VIPP1 is a multifunctional protein in chloroplasts that is critically important for envelope maintenance.

DOI： 10.1105/tpc.112.103606

Mutations defective in ribonucleotide reductase activity interfere with pollen plastid DNA degradation mediated by DPD1 exonuclease International journal

Tang, Lay Yin, Matsushima, Ryo, Sakamoto, Wataru

Plant Journal 70 ( 4 ) 637 - 49 2012

Language：English Publishing type：Research paper (scientific journal)

Organellar DNAs in mitochondria and plastids are present in multiple copies and make up a substantial proportion of total cellular DNA despite their limited genetic capacity. We recently demonstrated that organellar DNA degradation occurs during pollen maturation, mediated by the Mg(2+) -dependent organelle exonuclease DPD1. To further understand organellar DNA degradation, we characterized a distinct mutant (dpd2). In contrast to the dpd1 mutant, which retains both plastid and mitochondrial DNAs, dpd2 showed specific accumulation of plastid DNAs. Multiple abnormalities in vegetative and reproductive tissues of dpd2 were also detected. DPD2 encodes the large subunit of ribonucleotide reductase, an enzyme that functions at the rate-limiting step of de novo nucleotide biosynthesis. We demonstrated that the defects in ribonucleotide reductase indirectly compromise the activity of DPD1 nuclease in plastids, thus supporting a different regulation of organellar DNA degradation in pollen. Several lines of evidence provided here reinforce our previous conclusion that the DPD1 exonuclease plays a central role in organellar DNA degradation, functioning in DNA salvage rather than maternal inheritance during pollen development.

DOI： 10.1111/j.1365-313X.2012.04904.x

Cooperative D1 Degradation in the Photosystem II Repair Mediated by Chloroplastic Proteases in Arabidopsis International journal

Kato, Yusuke, Sun, Xuwu, Zhang, Lixin, Sakamoto, Wataru

Plant Physiology 159 ( 4 ) 1428 - 39 2012

Language：English Publishing type：Research paper (scientific journal)

Light energy constantly damages photosynthetic apparatuses, ultimately causing impaired growth. Particularly, the sessile nature of higher plants has allowed chloroplasts to develop unique mechanisms to alleviate the irreversible inactivation of photosynthesis. Photosystem II (PSII) is known as a primary target of photodamage. Photosynthetic organisms have evolved the so-called PSII repair cycle, in which a reaction center protein, D1, is degraded rapidly in a specific manner. Two proteases that perform processive or endopeptidic degradation, FtsH and Deg, respectively, participate in this cycle. To examine the cooperative D1 degradation by these proteases, we engaged Arabidopsis (Arabidopsis thaliana) mutants lacking FtsH2 (yellow variegated2 [var2]) and Deg5/Deg8 (deg5 deg8) in detecting D1 cleaved fragments. We detected several D1 fragments only under the var2 background, using amino-terminal or carboxyl-terminal specific antibodies of D1. The appearance of these D1 fragments was inhibited by a serine protease inhibitor and by deg5 deg8 mutations. Given the localization of Deg5/Deg8 on the luminal side of thylakoid membranes, we inferred that Deg5/Deg8 cleaves D1 at its luminal loop connecting the transmembrane helices C and D and that the cleaved products of D1 are the substrate for FtsH. These D1 fragments detected in var2 were associated with the PSII monomer, dimer, and partial disassembly complex but not with PSII supercomplexes. It is particularly interesting that another processive protease, Clp, was up-regulated and appeared to be recruited from stroma to the thylakoid membrane in var2, suggesting compensation for FtsH deficiency. Together, our data demonstrate in vivo cooperative degradation of D1, in which Deg cleavage assists FtsH processive degradation under photoinhibitory conditions.

DOI： 10.1104/pp.112.199042

Tissue-specific organelle DNA degradation mediated by DPD1 exonuclease. International journal

Lay Yin Tang, Wataru Sakamoto

Plant signaling & behavior 6 ( 9 ) 1391 - 3 2011.9

Language：English Publishing type：Research paper (scientific journal)

Organelle DNA in plastids and mitochondria is present in multiple copies and undergoes degradation developmentally. For example, organelle DNA that is detectable cytologically using DNA-fluorescent dye disappears during pollen development. Nevertheless, nucleases involved in this degradation process remain unknown. Our recent study identified the organelle nuclease, DPD1, which has Mg2+ -dependent exonuclease activity in vitro. The discovery of DPD1 emerged from Arabidopsis mutant screening and concomitant isolation of dpd1 mutants that retain organelle DNA in mature pollen. DPD1 is conserved only in angiosperms: not in other photosynthetic organisms. Despite these findings, the physiological significance of organelle DNA degradation during pollen development remains unclear because dpd1 exhibits no apparent defects in pollen viability or in the maternal inheritance of organelle DNA. We discuss a possible role of organelle DNA degradation mediated by DPD1, based on a DPD1 expression profile studied using in silico analyses.

Widespread Endogenization of Genome Sequences of Non-Retroviral RNA Viruses into Plant Genomes International journal

Chiba, Sotaro, Kondo, Hideki, Tani, Akio, Saisho, Daisuke, Sakamoto, Wataru, Kanematsu, Satoko, Suzuki, Nobuhiro

Plos Pathogens 7 ( 7 ) e1002146 2011

Language：English Publishing type：Research paper (scientific journal)

Non-retroviral RNA virus sequences (NRVSs) have been found in the chromosomes of vertebrates and fungi, but not plants. Here we report similarly endogenized NRVSs derived from plus-, negative-, and double-stranded RNA viruses in plant chromosomes. These sequences were found by searching public genomic sequence databases, and, importantly, most NRVSs were subsequently detected by direct molecular analyses of plant DNAs. The most widespread NRVSs were related to the coat protein (CP) genes of the family Partitiviridae which have bisegmented dsRNA genomes, and included plant- and fungus-infecting members. The CP of a novel fungal virus (Rosellinia necatrix partitivirus 2, RnPV2) had the greatest sequence similarity to Arabidopsis thaliana ILR2, which is thought to regulate the activities of the phytohormone auxin, indole-3-acetic acid (IAA). Furthermore, partitivirus CP-like sequences much more closely related to plant partitiviruses than to RnPV2 were identified in a wide range of plant species. In addition, the nucleocapsid protein genes of cytorhabdoviruses and varicosaviruses were found in species of over 9 plant families, including Brassicaceae and Solanaceae. A replicase-like sequence of a betaflexivirus was identified in the cucumber genome. The pattern of occurrence of NRVSs and the phylogenetic analyses of NRVSs and related viruses indicate that multiple independent integrations into many plant lineages may have occurred. For example, one of the NRVSs was retained in Ar. thaliana but not in Ar. lyrata or other related Camelina species, whereas another NRVS displayed the reverse pattern. Our study has shown that single- and double-stranded RNA viral sequences are widespread in plant genomes, and shows the potential of genome integrated NRVSs to contribute to resolve unclear phylogenetic relationships of plant species.

DOI： 10.1371/journal.ppat.1002146

A Conserved, Mg2+-Dependent Exonuclease Degrades Organelle DNA during Arabidopsis Pollen Development International journal

Matsushima, Ryo, Tang, Lay Yin, Zhang, Lingang, Yamada, Hiroshi, Twell, David, Sakamoto, Wataru

Plant Cell 23 ( 4 ) 1608 - 24 2011

Language：English Publishing type：Research paper (scientific journal)

In plant cells, mitochondria and plastids contain their own genomes derived from the ancestral bacteria endosymbiont. Despite their limited genetic capacity, these multicopy organelle genomes account for a substantial fraction of total cellular DNA, raising the question of whether organelle DNA quantity is controlled spatially or temporally. In this study, we genetically dissected the organelle DNA decrease in pollen, a phenomenon that appears to be common in most angiosperm species. By staining mature pollen grains with fluorescent DNA dye, we screened Arabidopsis thaliana for mutants in which extrachromosomal DNAs had accumulated. Such a recessive mutant, termed defective in pollen organelle DNA degradation1 (dpd1), showing elevated levels of DNAs in both plastids and mitochondria, was isolated and characterized. DPD1 encodes a protein belonging to the exonuclease family, whose homologs appear to be found in angiosperms. Indeed, DPD1 has Mg²⁺-dependent exonuclease activity when expressed as a fusion protein and when assayed in vitro and is highly active in developing pollen. Consistent with the dpd phenotype, DPD1 is dual-targeted to plastids and mitochondria. Therefore, we provide evidence of active organelle DNA degradation in the angiosperm male gametophyte, primarily independent of maternal inheritance; the biological function of organellar DNA degradation in pollen is currently unclear.

DOI： 10.1105/tpc.111.084012

Highly efficient visual selection of transgenic rice plants using green fluorescent protein or anthocyanin synthetic genes

Saika, Hiroaki, Sakamoto, Wataru, Maekawa, Masahiko, Toki, Seiichi

Plant Biotechnology 28 ( 1 ) 2011

Reactive oxygen species derived from impaired quality control of photosystem II are irrelevant to plasma-membrane NADPH oxidases. International journal

Eiko Miura, Yusuke Kato, Wataru Sakamoto

Plant signaling & behavior 5 ( 3 ) 264 - 6 2010.3

Language：English Publishing type：Research paper (scientific journal)

Protein quality control plays an important role in the photosynthetic apparatus because its components receive excess light energy and are susceptible to photooxidative damage. In chloroplasts, photodamage is targeted to the D1 protein of Photosystem II (PSII). The coordinated PSII repair cycle (PSII disassembly, D1 degradation and synthesis, and PSII reassembly) is necessary to mitigate photoinhibition. A thylakoid protease FtsH, which is formed predominantly as a heteromeric complex with two isoforms of FtsH2 and FtsH5 in Arabidopsis, is the major protease involved in PSII repair. A mutant lacking FtsH2 (termed var2) shows compromised D1 degradation. Furthermore, var2 accumulates high levels of chloroplastic reactive oxygen species (cpROS), reflecting photooxidative stress without functional PSII repair. To examine if the cpROS produced in var2 are connected to a ROS signaling pathway mediated by plasma membrane NADPH oxidase (encoded by AtRbohD or AtRbohF), we generated mutants in which either Rboh gene was inactivated under var2 background. Lack of NADPH oxidases had little or no impact on cpROS accumulation. It seems unlikely that cpROS in var2 activate plasma membrane NADPH oxidases to enhance ROS production and the signaling pathway. Mutants that are defective in PSII repair might be valuable for investigating cpROS and their physiological roles.

A Rapid, Direct Observation Method to Isolate Mutants with Defects in Starch Grain Morphology in Rice

Matsushima, Ryo, Maekawa, Masahiko, Fujita, Naoko, Sakamoto, Wataru

Plant and Cell Physiology 51 ( 5 ) 728 - 41 2010

Language：English Publishing type：Research paper (scientific journal)

Starch forms transparent grains, referred to as starch grains (SGs), in amyloplasts. Despite the simple glucose polymer composition of starch, SGs exhibit different morphologies depending on plant species, especially in the endosperm of the Poaceae family. This study reports a novel method for preparing thin sections of endosperm without chemical fixation or resin embedding that allowed us to visualize subcellular SGs clearly. Using this method, we observed the SG morphologies of >5,000 mutagenized rice seeds and were able to isolate mutants in which SGs were morphologically altered. In five mutants, named ssg (substandard starch grain), increased numbers of small SGs (ssg1-ssg3), enlarged SGs (ssg4) and abnormal interior structures of SGs (ssg5) were observed. Amylopectin chain length distribution analysis and identification of the mutated gene suggested a possible allelic relationship between ssg1, ssg2, ssg3 and the previously isolated amylose-extender (ae) mutants, while ssg4 and ssg5 seemed to be novel mutants. Compared with conventional observation methods, the methods developed here are more effective for obtaining fine images of subcellular SGs and are suitable for the observation of a large number of samples.

DOI： 10.1093/pcp/pcq040

NEW INSIGHTS INTO THE TYPES AND FUNCTION OF PROTEASES IN PLASTIDS International journal

Kato, Yusuke, Sakamoto, Wataru, Jeon, KW

International Review of Cell and Molecular Biology, Vol 280 280 185 - 218 2010

Language：English Publishing type：Research paper (scientific journal)

Plastids change their morphology dynamically in response to environmental conditions and developmental status. Related to these plastid dynamics, the quality and quantity controls of proteins are necessary. Therefore, proteases are important as key regulators in almost all processes during the conversion of plastid types and the maintenance of plastid homeostasis. Recent progress in this field has revealed that various proteases and peptidases act on plastids. Results of the studies indicate that the vast majority of plastid proteases are homologous to prokaryotic ones because plastids are thought to originate from endosymbiosis of ancestral cyanobacteria. Moreover, the diversification of subunits of several plastid proteases has been revealed along with new insights into the functions of these homologues. This review provides basic information related to plastid proteases and the current view of their physiological roles in plastid homeostasis and biogenesis.

DOI： 10.1016/S1937-6448(10)80004-8

The FtsH Protease Heterocomplex in Arabidopsis: Dispensability of Type-B Protease Activity for Proper Chloroplast Development International journal

Zhang, Di, Kato, Yusuke, Zhang, Lingang, Fujimoto, Masaru, Tsutsumi, Nobuhiro, Sodmergen, Sakamoto, Wataru

Plant Cell 22 ( 11 ) 3710 - 25 2010

Language：English Publishing type：Research paper (scientific journal)

FtsH is an ATP-dependent metalloprotease present as a hexameric heterocomplex in thylakoid membranes. Encoded in the Arabidopsis thaliana YELLOW VARIEGATED2 (VAR2) locus, FtsH2 is one isoform among major Type A (FtsH1/5) and Type B (FtsH2/8) isomers. Mutants lacking FtsH2 (var2) and FtsH5 (var1) are characterized by a typical leaf-variegated phenotype. The functional importance of the catalytic center (comprised by the zinc binding domain) in FtsH2 was assessed in this study by generating transgenic plants that ectopically expressed FtsH2(488), a proteolytically inactive version of FtsH2. The resulting amino acid substitution inhibited FtsH protease activity in vivo when introduced into Escherichia coli FtsH. By contrast, expression of FtsH2(488) rescued not only leaf variegation in var2 but also seedling lethality in var2 ftsh8, suggesting that the protease activity of Type B isomers is completely dispensable, which implies that the chloroplastic FtsH complex has protease sites in excess and that they act redundantly rather than coordinately. However, expression of FtsH2(488) did not fully rescue leaf variegation in var1 var2 because the overall FtsH levels were reduced under this background. Applying an inducible promoter to our complementation analysis revealed that rescue of leaf variegation indeed depends on the overall amount of FtsH. Our results elucidate protein activity and its amount as important factors for the function of FtsH heterocomplexes that are composed of multiple isoforms in the thylakoid membrane.

DOI： 10.1105/tpc.110.079202

Comparative transcriptome analysis of green/white variegated sectors in Arabidopsis yellow variegated2: responses to oxidative and other stresses in white sectors International journal

Miura, Eiko, Kato, Yusuke, Sakamoto, Wataru

Journal of Experimental Botany 61 ( 9 ) 2433 - 45 2010

Language：English Publishing type：Research paper (scientific journal)

The yellow variegated2 (var2) mutant in Arabidopsis thaliana has been studied as a typical leaf-variegated mutant whose defect results from the lack of FtsH2 metalloprotease in chloroplasts. The var2 green sectors suffer from photo-oxidative stress and accumulate high levels of reactive oxygen species (ROS) because of compromised Photosystem II repair. This study investigated and compared microarray-based expression profiles of green and white sectors of var2 leaves. Results suggest that ROS that accumulate in chloroplasts of var2 green sectors do not cause much significant change in the transcriptional profile related to ROS signalling and scavenging. By contrast, transcriptome in the white sectors apparently differs from those in the green sectors and wild type. Numerous genes related to photosynthesis and chloroplast functions were repressed in the white sectors. Furthermore, many genes related to oxidative stress were up-regulated. Among them, ROS scavenging genes were specifically examined, such as Cu/Zn superoxide dismutase 2 (CSD2), that were apparently up-regulated in white but not in the green sectors. Up-regulation of CSD2 appears to be partly attributable to the lack of a microRNA (miR398) in the white sectors. It was concluded that the white sectors exhibit a response to oxidative and other stresses, including CSD2 up-regulation, which might be commonly found in tissues with abnormal chloroplast differentiation.

DOI： 10.1093/jxb/erq075

Identification and Characterization of High Molecular Weight Complexes Formed by Matrix AAA Proteases and Prohibitins in Mitochondria of Arabidopsis thaliana International journal

Piechota, Janusz, Kolodziejczak, Marta, Juszczak, Ilona, Sakamoto, Wataru, Janska, Hanna

Journal of Biological Chemistry 285 ( 17 ) 12512 - 21 2010

Language：English Publishing type：Research paper (scientific journal)

We identify and characterize two matrix (m)-AAA proteases (AtFtsH3 and AtFtsH10) present in the mitochondria of Arabidopsis thaliana. AtFtsH3 is the predominant protease in leaves of wild type plants. Both proteases assemble with prohibitins (PHBs) into high molecular weight complexes (approximately 2 MDa), similarly to their yeast counterparts. A smaller PHB complex (approximately 1 MDa), without the m-AAA proteases, was also detected. Unlike in yeast, stable prohibitin-independent high molecular weight assemblies of m-AAA proteases could not be identified in A. thaliana. AtFtsH3 and AtFtsH10 form at least two types of m-AAA-PHB complexes in wild type plants. The one type contains PHBs and AtFtsH3, and the second one is composed of PHBs and both AtFtsH3 and AtFtsH10. Complexes composed of PHBs and AtFtsH10 were found in an Arabidopsis mutant lacking AtFtsH3 (ftsh3). Thus, both AtFtsH3 and AtFtsH10 may form hetero- and homo-oligomeric complexes with prohibitins. The increased level of AtFtsH10 observed in ftsh3 suggests that functions of the homo- and hetero-oligomeric complexes containing AtFtsH3 can be at least partially substituted by AtFtsH10 homo-oligomers. The steady-state level of the AtFtsH10 transcripts did not change in ftsh3 compared with wild type plants, but we found that almost twice more of the AtFtsH10 transcripts were associated with polysomes in ftsh3. Based on this result, we assume that the AtFtsH10 protein is synthesized at a higher rate in the ftsh3 mutant. Our results provide the first data on the composition of m-AAA and PHB complexes in plant mitochondria and suggest that the abundance of m-AAA proteases is regulated not only at the transcriptional but also at the translational level.

DOI： 10.1074/jbc.M109.063644

Arrested Differentiation of Proplastids into Chloroplasts in Variegated Leaves Characterized by Plastid Ultrastructure and Nucleoid Morphology

Sakamoto, Wataru, Uno, Yasuyuki, Zhang, Quan, Miura, Eiko, Kato, Yusuke, Sodmergen

Plant and Cell Physiology 50 ( 12 ) 2069 - 83 2009

Language：English Publishing type：Research paper (scientific journal)

Leaf variegation is seen in many ornamental plants and is often caused by a cell-lineage type formation of white sectors lacking functional chloroplasts. A mutant showing such leaf variegation is viable and is therefore suitable for studying chloroplast development. In this study, the formation of white sectors was temporally investigated in the Arabidopsis leaf-variegated mutant var2. Green sectors were found to emerge from white sectors after the formation of the first true leaf. Transmission electron microscopic examination of plastid ultrastructures confirmed that the peripheral zone in the var2 shoot meristem contained proplastids but lacked developing chloroplasts that were normally detected in wild type. These data suggest that chloroplast development proceeds very slowly in var2 variegated leaves. A notable feature in var2 is that the plastids in white sectors contain remarkable globular vacuolated membranes and prolamellar body-like structures. Although defective plastids were hardly observed in shoot meristems, they began to accumulate during early leaf development. Consistent with these observations, large plastid nucleoids detected in white sectors by DNA-specific fluorescent dyes were characteristic of those found in proplastids and were clearly distinguished from those in chloroplasts. These results strongly imply that in white sectors, differentiation of plastids into chloroplasts is arrested at the early stage of thylakoid development. Interestingly, large plastid nucleoids were detected in variegated sectors from species other than Arabidopsis. Thus, plastids in variegated leaves appear to share a common feature and represent a novel plastid type.

DOI： 10.1093/pcp/pcp127

Dynamic behavior of organellar DNAs during male gametophyte development in higher plants

Sakamoto, Wataru, Matsushita, Ryo

Genes & Genetic Systems 84 ( 6 ) 487 - 487 2009

Leaf-variegated tobacco plants generated by knocking-down FtsH gene

Kouso, Takayoshi, Kato, Yusuke, Sakamoto, Wataru

Genes & Genetic Systems 84 ( 6 ) 471 - 471 2009

Localization and expression of serine racemase in Arabidopsis thaliana International journal

Sugimoto, Manabu, Sakamoto, Wataru, Fujitani, Yoshiyuki

Amino Acids 36 ( 3 ) 587 - 90 2009

Language：English Publishing type：Research paper (scientific journal)

Arabidopsis plants transformed by promoter of A. thaliana serine racemase fused with beta-glucuronidase (GUS) reporter gene showed strong GUS staining in elongating and developing cells such as tip regions of primary and lateral roots, developing leaves, and shoot meristems. RT-PCR and digital northern hybridization showed that expression of the serine racemase gene was not induced by L- and D-serine, light irradiation, biotic and abiotic stresses.

DOI： 10.1007/s00726-008-0112-z

The lack of mitochondrial AtFtsH4 protease alters Arabidopsis leaf morphology at the late stage of rosette development under short-day photoperiod International journal

Gibala, Marta, Kicia, Marta, Sakamoto, Wataru, Gola, Edyta M., Kubrakiewicz, Janusz, Smakowska, Elwira, Janska, Hanna

Plant Journal 59 ( 5 ) 685 - 99 2009

Language：English Publishing type：Research paper (scientific journal)

AtFtsH4 is one of four inner membrane-bound mitochondrial ATP-dependent metalloproteases in Arabidopsis thaliana, called AAA proteases, whose catalytic site is exposed to the intermembrane space. In the present study, we used a reverse-genetic approach to investigate the physiological role of the AtFtsH4 protease. We found that loss of AtFtsH4 did not significantly affect Arabidopsis growth under optimal conditions (long days); however, severe morphological and developmental abnormalities in late rosette development occurred under short-day conditions. The asymmetric shape and irregular serration of expanding leaf blades were the most striking features of the ftsh4 mutant phenotype. The severe abnormal morphology of the leaf blades was accompanied by ultrastructural changes in mitochondria and chloroplasts. These abnormalities correlated with elevated levels of reactive oxygen species and carbonylated mitochondrial proteins. We found that two classes of molecular chaperones, Hsp70 and prohibitins, were over-expressed in ftsh4 mutants during late vegetative growth under both short- and long-day conditions. Taken together, our data indicate that lack of AtFtsH4 results in impairment of organelle development and Arabidopsis leaf morphology under short-day conditions.

DOI： 10.1111/j.1365-313X.2009.03907.x

Visualization of Plastids in Pollen Grains: Involvement of FtsZ1in Pollen Plastid Division

Tang, Lay Yin, Nagata, Noriko, Matsushima, Ryo, Chen, Yuling, Yoshioka, Yasushi, Sakamoto, Wataru

Plant and Cell Physiology 50 ( 4 ) 904 - 8 2009

Language：English Publishing type：Research paper (scientific journal)

Visualizing organelles in living cells is a powerful method to analyze their intrinsic mechanisms. Easy observation of chlorophyll facilitates the study of the underlying mechanisms in chloroplasts, but not in other plastid types. Here, we constructed a transgenic plant enabling visualization of plastids in pollen grains. Combination of a plastid-targeted fluorescent protein with a pollen-specific promoter allowed us to observe the precise number, size and morphology of plastids in pollen grains of the wild type and the ftsZ1 mutant, whose responsible gene plays a central role in chloroplast division. The transgenic material presented in this work is useful for studying the division mechanism of pollen plastids.

DOI： 10.1093/pcp/pcp042

Protein Quality Control in Chloroplasts: A Current Model of D1 Protein Degradation in the Photosystem II Repair Cycle International journal

Kato, Yusuke, Sakamoto, Wataru

Journal of Biochemistry 146 ( 4 ) 463 - 9 2009

Language：English Publishing type：Research paper (scientific journal)

The chloroplast originated from endosymbiosis of photosynthetic bacteria. Thus, mechanisms essential for chloroplast biogenesis/homeostasis (protein synthesis, import from cytosol, assembly, and degradation) are predominantly governed by prokaryotic systems. Among these, the quality control system is crucial, because light energy constantly damages photosynthetic proteins and excessive light often limits plant growth by irreversibly inactivating the photosynthetic apparatuses. Here, we overview prokaryotic proteases (FtsH and Deg) which are two enzymes that play critical roles in this system. We particularly focus on Photosystem II (PSII) in thylakoid membranes, which is composed of more than 20 subunits. Among the subunits is one of the intrinsic reaction centre proteins (D1) which is considered to be the target of photodamage. Its rapid and specific turnover suggests that photodamaged D1 is degraded by these proteases and replaced with a de novo synthesized one in a system which is termed the PSII repair cycle. We discuss a current model of D1 degradation which is executed by a concerted action of particular FtsH and Deg isoforms.

DOI： 10.1093/jb/mvp073

The Variegated Mutants Lacking Chloroplastic FtsHs Are Defective in D1 Degradation and Accumulate Reactive Oxygen Species International journal

Kato, Yusuke, Miura, Eiko, Ido, Kunio, Ifuku, Kentaro, Sakamoto, Wataru

Plant Physiology 151 ( 4 ) 1790 - 801 2009

Language：English Publishing type：Research paper (scientific journal)

In the photosynthetic apparatus, a major target of photodamage is the D1 reaction center protein of photosystem II (PSII). Photosynthetic organisms have developed a PSII repair cycle in which photodamaged D1 is selectively degraded. A thylakoid membrane-bound metalloprotease, FtsH, was shown to play a critical role in this process. Here, the effect of FtsHs in D1 degradation was investigated in Arabidopsis (Arabidopsis thaliana) mutants lacking FtsH2 (yellow variegated2 [var2]) or FtsH5 (var1). Because these mutants are characterized by variegated leaves that sometimes complicate biochemical studies, we employed another mutation, fu-gaeri1 (fug1), that suppresses leaf variegation in var1 and var2 to examine D1 degradation. Two-dimensional blue native PAGE showed that var2 has less PSII supercomplex and more PSII intermediate lacking CP43, termed RC47, than the wild type under normal growth light. Moreover, our histochemical and quantitative analyses revealed that chloroplasts in var2 accumulate significant levels of reactive oxygen species, such as superoxide radical and hydrogen peroxide. These results indicate that the lack of FtsH2 leads to impaired D1 degradation at the step of RC47 formation in PSII repair and to photooxidative stress even under nonphotoinhibitory conditions. Our in vivo D1 degradation assays, carried out by nonvariegated var2 fug1 and var1 fug1 leaves, demonstrated that D1 degradation was impaired in different light conditions. Taken together, our results suggest the important role of chloroplastic FtsHs, which was not precisely examined in vivo. Attenuated D1 degradation in the nonvariegated mutants also suggests that leaf variegation seems to be independent of the PSII repair.

DOI： 10.1104/pp.109.146589

Activation of the heterotrimeric G protein alpha-subunit GPA1 suppresses the ftsh-mediated inhibition of chloroplast development in Arabidopsis International journal

Zhang, Lingang, Wei, Qing, Wu, Wenjuan, Cheng, Yuxiang, Hu, Guangzhen, Hu, Fenhong, Sun, Yi, Zhu, Ying, Sakamoto, Wataru, Huang, Jirong

Plant Journal 58 ( 6 ) 1041 - 53 2009

Language：English Publishing type：Research paper (scientific journal)

Heterotrimeric G protein knock-out mutants have no phenotypic defect in chloroplast development, and the connection between the G protein signaling pathway and chloroplast development has only been inferred from pharmaceutical evidence. Thus, whether G protein signaling plays a role in chloroplast development remains an open question. Here, we present genetic evidence, using the leaf-variegated mutant thylakoid formation 1 (thf1), indicating that inactivation or activation of the endogenous G protein alpha-subunit (GPA1) affects chloroplast development, as does the ectopic expression of the constitutively active Galpha-subunit (cGPA1). Molecular biological and genetic analyses showed that FtsH complexes, which are composed of type-A (FtsH1/FtsH5) and type-B (FtsH2/FtsH8) subunits, are required for cGPA1-promoted chloroplast development in thf1. Furthermore, the ectopic expression of cGPA1 rescues the leaf variegation of ftsh2. Consistent with this finding, microarray analysis shows that ectopic expression of cGPA1 partially corrects mis-regulated gene expression in thf1. This overlooked function of G proteins provides new insight into our understanding of the integrative signaling network, which dynamically regulates chloroplast development and function in response to both intracellular and extracellular signals.

DOI： 10.1111/j.1365-313X.2009.03843.x

Plant mitochondrial rhomboid, AtRBL12, has different substrate specificity from its yeast counterpart International journal

Kmiec-Wisniewska, Beata, Krumpe, Katrin, Urantowka, Adam, Sakamoto, Wataru, Pratje, Elke, Janska, Hanna

Plant Molecular Biology 68 ( 1-2 ) 159 - 71 2008

Language：English Publishing type：Research paper (scientific journal)

Rhomboid proteins comprise a class of serine proteases that are conserved in all kingdoms of organisms. They contain six or seven transmembrane helices and control a wide range of cellular functions and developmental processes by intramembrane proteolysis. This paper provides experimental evidence for the existence of rhomboid proteases in plant mitochondria and chloroplasts. Among 15 putative rhomboid-like proteins in Arabidopsis thaliana, we selected five predicted as mitochondrially targeted. For these proteins we performed the GFP transient assay, and identified two homologues, AtRBL11 (At5g25752) and AtRBL12 (At1g18600) to be targeted into plastids and mitochondria, respectively. Phylogenetic analysis reveals that AtRBL12 or AtRBL11 have only one clear orthologue in plant species with completely sequenced genomes. Complementation of the yeast lacking a functional copy of mitochondrial rhomboid with AtRBL12 indicates that this plant protease, in contrast to the human orthologue, does not recognize the yeast substrates, cytochrome c peroxidase (Ccp1) or dynamin-like GTPase (Mgm1). In agreement with this, we did not observe processing of Mgm1 when labeled precursor of this protein was incubated in vitro with Arabidopsis mitochondrial extract. Our results imply that plant mitochondrial rhomboids function in a specific manner and thus differ from their yeast and mammal counterparts.

DOI： 10.1007/s11103-008-9359-8

Influence of chloroplastic photo-oxidative stress on mitochondrial alternative oxidase capacity and respiratory properties: A case study with arabidopsis yellow variegated 2

Yoshida, Keisuke, Watanabe, Chihiro, Kato, Yusuke, Sakamoto, Wataru, Noguchi, Ko

Plant and Cell Physiology 49 ( 4 ) 592 - 603 2008

Language：English Publishing type：Research paper (scientific journal)

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy-wasteful cyanide (CN)-resistant respiration. Although it has been demonstrated that leaf AOX is up-regulated under high-light (HL) conditions, the in vivo mechanism of AOX up-regulation by light is still unknown. In the present study, we examined whether the photo-oxidative stress in the chloroplast modulates mitochondrial respiratory properties, especially the AOX capacity, using Arabidopsis leaf-variegated mutant yellow variegated 2 (var2) and exposing plants to HL. var2 mutants lack FtsH2 metalloprotease required for the repair of damaged PSII. Indeed, var2-1 suffered from photo-oxidative stress even before the HL treatments. While the activities of tricarboxylic acid cycle enzymes and cytochrome c oxidase in var2-1 were almost identical to those in the wild type, the amount of AOX protein and the CN-resistant respiration rate were higher in var2-1. Real-time PCR analysis revealed that HL treatment induced the expression of some energy-dissipating respiratory genes, including AOX1a, NDB2 and UCP5, more strongly in var2-1. Western blotting using var2-1 leaf extracts specific to green or white sectors, containing functional or non-functional photosynthetic apparatus, respectively, revealed that more AOX protein was induced in the green sectors by the HL treatment. These results indicate that photo-oxidative stress by excess light is involved in the regulation of respiratory gene expression and the modulation of respiratory properties, especially the AOX up-regulation.

DOI： 10.1093/pcp/pcn031

Mitochondrial dynamics in plant male gametophyte visualized by fluorescent live imaging

Matsushima, Ryo, Hamamura, Yuki, Higashiyama, Tetsuya, Arimura, Shin-ichi, Sodmergen, Tsutsumi, Nobuhiro, Sakamoto, Wataru

Plant and Cell Physiology 49 ( 7 ) 1074 - 83 2008

Language：English Publishing type：Research paper (scientific journal)

Visualization of organelles in living cells is a powerful method for studying their dynamic behavior. Here we attempted to visualize mitochondria in angiosperm male gametophyte (pollen grain from Arabidopsis thaliana) that are composed of one vegetative cell (VC) and two sperm cells (SCs). Combination of mitochondria-targeted fluorescent proteins with VC- or SC-specific expression allowed us to observe the precise number and dynamic behavior of mitochondria in the respective cell types. Furthermore, live imaging of SC mitochondria during double fertilization confirmed previous observations, demonstrated by electron microscopy in other species, that sperm mitochondria enter into the egg and central cells. We also attempted to visualize mutant mitochondria that were elongated due to a defect in mitochondrial division. This mutant phenotype was indeed detectable in VC mitochondria of a heterozygous F(1) plant, suggesting active mitochondrial division in male gametophyte. Finally, we performed mutant screening and isolated a putative mitochondrial protein transport mutant whose phenotype was detectable only in haploid cells. The transgenic materials presented in this work are useful not only for live imaging but also for studying mitochondrial functions by mutant analysis.

DOI： 10.1093/pcp/pcn084

Arabidopsis ELONGATED MITOCHONDRIA1 is required for localization of DYNAMIN-RELATED PROTEIN3A to mitochondrial fission sites International journal

Arimura, Shin-ichi, Fujimoto, Masaru, Doniwa, Yoko, Kadoya, Naoki, Nakazono, Mikio, Sakamoto, Wataru, Tsutsumi, Nobuhiro

Plant Cell 20 ( 6 ) 1555 - 66 2008

Language：English Publishing type：Research paper (scientific journal)

Mitochondrial fission is achieved partially by the activity of self-assembling dynamin-related proteins (DRPs) in diverse organisms. Mitochondrial fission in Arabidopsis thaliana is mediated by DRP3A and DRP3B, but the other genes and molecular mechanisms involved have yet to be elucidated. To identify these genes, we screened and analyzed Arabidopsis mutants with longer and fewer mitochondria than those of the wild type. ELM1 was found to be responsible for the phenotype of elongated mitochondria. This phenotype was also observed in drp3a plants. EST and genomic sequences similar to ELM1 were found in seed plants but not in other eukaryotes. ELM1:green fluorescent protein (GFP) was found to surround mitochondria, and ELM1 interacts with both DPR3A and DRP3B. In the elm1 mutant, DRP3A:GFP was observed in the cytosol, whereas in wild-type Arabidopsis, DRP3A:GFP localized to the ends and constricted sites of mitochondria. These results collectively suggest that mitochondrial fission in Arabidopsis is mediated by the plant-specific factor ELM1, which is required for the relocalization of DRP3A (and possibly also DRP3B) from the cytosol to mitochondrial fission sites.

DOI： 10.1105/tpc.108.058578

Functional characterization of key structural genes in rice flavonoid biosynthesis International journal

Shih, Chun Hat, Chu, Hung, Tang, Lee Kwan, Sakamoto, Wataru, Maekawa, Masahiko, Chu, Ivan K., Wang, Mingfu, Lo, Clive

Planta 228 ( 6 ) 1043 - 54 2008

Language：English Publishing type：Research paper (scientific journal)

Rice is a model system for monocot but the molecular features of rice flavonoid biosynthesis have not been extensively characterized. Rice structural gene homologs encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) were identified by homology searches. Unique differential expression of OsF3H, OsDFR, and OsANS1 controlled by the Pl(w) locus, which contains the R/B-type regulatory genes OSB1 and OSB2, was demonstrated during light-induced anthocyanin accumulation in T65-Plw seedlings. Previously, F3H genes were often considered as early genes co-regulated with CHS and CHI genes in other plants. In selected non-pigmented rice lines, OSB2 is not expressed following illumination while their expressed OSB1sequences all contain the same nucleotide change leading to the T(64) M substitution within the conserved N-terminal interacting domain. Furthermore, the biochemical roles of the expressed rice structural genes (OsCHS1, OsCHI, OsF3H, and OsF3'H) were established in planta for the first time by complementation in the appropriate Arabidopsis transparent testa mutants. Using yeast two-hybrid analysis, OsCHS1 was demonstrated to interact physically with OsF3H, OsF3'H, OsDFR, and OsANS1, suggesting the existence of a macromolecular complex for anthocyanin biosynthesis in rice. Finally, flavones were identified as the major flavonoid class in the non-pigmented T65 seedlings in which the single-copy OsF3H gene was not expressed. Competition between flavone and anthocyanin pathways was evidenced by the significant reduction of tricin accumulation in the T65-Plw seedlings.

DOI： 10.1007/s00425-008-0806-1

The model plant Medicago truncatula exhibits biparental plastid inheritance

Matsushima, Ryo, Hu, Yingchun, Toyoda, Kazuhiro, Sodmergen, Sakamoto, Wataru

Plant and Cell Physiology 49 ( 1 ) 81 - 91 2008

Language：English Publishing type：Research paper (scientific journal)

The plastid, which originated from the endosymbiosis of a cyanobacterium, contains its own plastid DNA (ptDNA) that exhibits a unique mode of inheritance. Approximately 80% of angiosperms show maternal inheritance, whereas the remainder exhibit biparental inheritance of ptDNA. Here we studied ptDNA inheritance in the model legume, Medicago truncatula. Cytological analysis of mature pollen with DNA-specific fluorescent dyes suggested that M. truncatula is one of the few model plants potentially showing biparental inheritance of ptDNA. We further examined pollen by electron microscopy and revealed that the generative cell (a mother of sperm cells) indeed has many DNA-containing plastids. To confirm biparental inheritance genetically, we crossed two ecotypes (Jemalong A17 and A20), and the transmission mode of ptDNA was investigated by a PCR-assisted polymorphism. Consistent with the cytological observations, the majority of F(1) plants possessed ptDNAs from both parents. Interestingly, cotyledons of F(1) plants tended to retain a biparental ptDNA population, while later emergent leaves tended to be uniparental with either one of the parental plastid genotypes. Biparental transmission was obvious in the F(2) population, in which all plants showed homoplasmy with either a paternal or a maternal plastid genotype. Collectively, these data demonstrated that M. truncatula is biparental for ptDNA transmission and thus can be an excellent model to study plastid genetics in angiosperms.

DOI： 10.1093/pcp/pcm170

The BnALMT1 Protein That is an Aluminum-Activated Malate Transporter is Localized in the Plasma Membrane. International journal

Ayalew Ligaba, Maki Katsuhara, Wataru Sakamoto, Hideaki Matsumoto

Plant signaling & behavior 2 ( 4 ) 255 - 7 2007.7

Language：English Publishing type：Research paper (scientific journal)

We have previously reported that Al-induces citrate and malate efflux from P-sufficient and P-deficient plants of rape (Brassica napus L.) and that P-deficiency alone could not induce this response. Further investigation showed that the transcript of two genes designated BnALMT1 and BnALMT2 is accumulated in roots by Al-treatment. Transgenic tobacco cells (Nicotiana tabacum) and Xenopus laevis oocytes expressing the BnALMT1 and BnALMT2 proteins released more malate than control cells in the presence of Al, indicating that the BnALMT genes encode an Al-activated malate transporter. The transgenic tobacco cells exposed to toxic level of Al grew better than control cells indicating that the genes can enhance Al-resistance of plant cells. In this study we showed the subcellular localization of BnALMT1 fused to the green fluoresce protein (GFP). The BnALMT1:: GFP construct was transiently expressed in protoplasts prepared from Arabidopsis leaves using the polyethylene glycol (PEG) method. The result showed that the BnALMT1 protein is localized in the plasma membrane. This provides further evidence that the BnALMT proteins facilitate the transport of malate across the plasma membrane (PM).

The balance between chloroplast protein synthesis and degradation as an important factor of chloroplast biogenesis and maintenance

Sakamoto, W.

Photosynthesis Research 91 ( 2-3 ) 268 - 268 2007

Chemically induced expression of rice OSB2 under the control of the OsPR1.1 promoter confers increased anthocyanin accumulation in transgenic rice International journal

Kawahigashi, Hiroyuki, Hirose, Sakiko, Iwai, Takayoshi, Ohashi, Yuko, Sakamoto, Wataru, Maekawa, Masahiko, Ohkawa, Yasunobu

Journal of Agricultural and Food Chemistry 55 ( 4 ) 1241 - 7 2007

Language：English Publishing type：Research paper (scientific journal)

Anthocyanin pigmentation provides an excellent system with which to study the regulation of gene expression in higher plants. In this study, OsPR1.1 promoter was isolated and the promoter activity was monitored using a reporter gene OSB2, which encodes a transcription factor for anthocyanin synthesis in rice plants. We introduced PR::OSB2 plasmid into an isogenic Taichung 65, no. 99-962 T-65 CBA B9F5 (T65 CBA), rice line (Oryza sativa L.) and found that the transgenic rice plants exhibited anthocyanin accumulation by the induced expression of OSB2 after chemical treatments with methyl jasmonate (MeJA) and 2,6-dichloroisonicotinic acid (DCINA). The shoots of the PR::OSB2 transgenic rice plants changed color to red after application of the chemicals accompanying with the increased anthocyanin content to approximately 5-fold by MeJA and 2-fold by DCINA, respectively. The anthocyanin accumulation was consistent with the increase of the expression of OSB2 and anthocyanidin synthase (ANS). This color change system could provide a useful and easy way to produce transgenic plants for monitoring of chemicals in the environment.

DOI： 10.1021/jf062339w

Physiological and biochemical dissection of the white sectors of yellow variegated2

Kato, Yusuke, Miura, Eiko, Matushima, Ryo, Sakamoto, Wataru

Plant and Cell Physiology 48 2007

Isolation and characterization of Ty1/copia-like retrotransposons in mung bean (Vigna radiata)

Xiao, Weimin, Su, Yuhui, Sakamoto, Wataru, Sodmergen

Journal of Plant Research 120 ( 2 ) 323 - 8 2007

Language：English Publishing type：Research paper (scientific journal)

Two Ty1/copia-like retrotransposons, RTvr1 and RTvr2, were isolated from mung bean (Vigna radiata (L.) Wilczek) genomic DNA and are the first complete elements of this kind to be reported in this legume. Nucleotide sequence analyses revealed that both elements are AT-rich (60% and 61%, respectively) and are flanked by a target-site duplication of 5 bp. The structures of RTvr1 and RTvr2 are those of typical long terminal repeat retrotransposons. Both transposons were able to produce putative proteins with the domain order of Gag-protease-integrase-reverse transcriptase-RNase H, indicating that RTvr1 and RTvr2 belong to the Ty1/copia-like retrotransposons. Except for a 2,500-bp insertion region in RTvr2, the overall similarity between RTvr1 and RTvr2 is 92%. Dot blots showed that these two retroelements were present at a copy number of 120 per mung bean haploid genome. Multiple sequence alignments showed that the conserved motifs of the aspartic proteases, integrase, reverse transcriptase, and the RNase H in the Ty1/copia-like group all exist in RTvr1 and RTvr2.

DOI： 10.1007/s10265-006-0045-0

Multiple intracellular locations of Lon protease in arabidopsis: Evidence for the localization of AtLon4 to chloroplasts

Ostersetzer, Oren, Kato, Yusuke, Adam, Zach, Sakamoto, Wataru

Plant and Cell Physiology 48 ( 6 ) 881 - 5 2007

Language：English Publishing type：Research paper (scientific journal)

Arabidopsis contains four Lon protease-like proteins (AtLon1-AtLon4), predicted to be localized in different cellular organelles, including mitochondria, peroxisomes and plastids. A notable question is whether Lon is present in chloroplasts, since it is absent from cyanobacteria and thus appears to have been lost during the evolution of photosynthetic organisms. Based on in vivo transient assays, we found that AtLon4 is dually targeted to both mitochondria and chloroplasts. Furthermore, immunoblot analysis localized AtLon4 to the thylakoids. Thus, in spite of its absence from basal photosynthetic organisms, our results suggest the presence of Lon in plant plastids.

DOI： 10.1093/pcp/pcm052

Genetic dissection of leaf variegation in Arabidopsis

Sakamoto, Wataru

Genes & Genetic Systems 82 ( 6 ) 501 - 501 2007

Identification of fug1 and sco1 as a suppressor of leaf variegation in var mutants

Miura, Eiko, Koto, Yusuke, Matsushima, Ryo, Sakamoto, Wataru

Plant and Cell Physiology 48 2007

Distribution mechanism and physiological roles of inorganic ion gradient in a leaf

Nagai, Makiko, Uehara, Takeo, Miura, Eiko, Sakamoto, Wataru, Yamagami, Mutsmui, Fukaki, Hidehiro, Kitamura, Akira, Mimura, Tetsuro

Plant and Cell Physiology 48 2007

Flower and leaf color modification by expression of transcriptional factors in petunia

Umemoto, Naovuki, Fukaya, Noritaka, Takano, Masayo, Sakamoto, Wataru

Plant and Cell Physiology 48 2007

The balance between protein synthesis and degradation in chloroplasts determines leaf variegation in Arabidopsis yellow variegated mutants International journal

Miura, Eiko, Kato, Yusuke, Matsushima, Ryo, Albrecht, Veronica, Laalami, Soumaya, Sakamoto, Wataru

Plant Cell 19 ( 4 ) 1313 - 28 2007

Language：English Publishing type：Research paper (scientific journal)

An Arabidopsis thaliana leaf-variegated mutant yellow variegated2 (var2) results from loss of FtsH2, a major component of the chloroplast FtsH complex. FtsH is an ATP-dependent metalloprotease in thylakoid membranes and degrades several chloroplastic proteins. To understand the role of proteolysis by FtsH and mechanisms leading to leaf variegation, we characterized the second-site recessive mutation fu-gaeri1 (fug1) that suppressed leaf variegation of var2. Map-based cloning and subsequent characterization of the FUG1 locus demonstrated that it encodes a protein homologous to prokaryotic translation initiation factor 2 (cpIF2) located in chloroplasts. We show evidence that cpIF2 indeed functions in chloroplast protein synthesis in vivo. Suppression of leaf variegation by fug1 is observed not only in var2 but also in var1 (lacking FtsH5) and var1 var2. Thus, suppression of leaf variegation caused by loss of FtsHs is most likely attributed to reduced protein synthesis in chloroplasts. This hypothesis was further supported by the observation that another viable mutation in chloroplast translation elongation factor G also suppresses leaf variegation in var2. We propose that the balance between protein synthesis and degradation is one of the determining factors leading to the variegated phenotype in Arabidopsis leaves.

DOI： 10.1105/tpc.106.049270

White leaf sectors in yellow variegated2 are formed by viable cells with undifferentiated plastids International journal

Kato, Yusuke, Miura, Eiko, Matsushima, Ryo, Sakamoto, Wataru

Plant Physiology 144 ( 2 ) 952 - 60 2007

Language：English Publishing type：Research paper (scientific journal)

The yellow variegated2 (var2) is one of the best-characterized Arabidopsis (Arabidopsis thaliana) mutants showing leaf variegation. Leaf variegation of var2 results from the loss of an ATP-dependent metalloprotease, FtsH2, which is a major component of the FtsH heterocomplex in thylakoid membranes. While the functional role of FtsH2 in protein quality control has been extensively studied, the physiological state of plastids in white tissues of the var2 is not well characterized. Here we show that the white tissue in var2 is neither the result of photobleaching nor enhanced senescence. Visualization of plastids by plastid-targeted green fluorescent protein revealed that plastids in the white sector are distinct and have undifferentiated characteristics. The plastids are also distinct in that they contain large nucleoids, a complex structure of plastid DNA and proteins, that are typically found in undifferentiated plastids. Comparative analyses of protein profiles from green and white tissues suggested that the difference was observed in the proteins related to photosynthesis but not due to proteins of other organelles. Thus, cells in the white tissue are viable and their defect is limited to plastid function. The plastid accumulates normal levels of chloroplast transcripts, whereas a substantial repression of nuclear-encoded photosynthetic genes was evident in the white sector. Based upon these results, we inferred that the white sectors in var2 are made by viable cells that have plastids arrested in thylakoid formation. A proposed model to form the variegated sector in var2 is provided.

DOI： 10.1104/pp.107.099002

Relationship between the oxidative stress in chloroplasts and the mitochondrial respiratory properties in yellow variegated 2 mutants

Yoshida, Keisuke, Watanabe, Chihiro, Terashima, Ichiro, Kato, Yusuke, Sakamoto, Wataru, Noguchi, Ko

Plant and Cell Physiology 48 2007

Overexpression and characterization of FtsH protease from Arabidopsis

Amano, T, Sakamoto, W, Shioi, Y

Plant and Cell Physiology 47 2006

Isolation of Arabidopsis mutants with defect in mitochondrial morphology

Arimura, Shin-ichi, Feng Xiaoge, Sakamoto, Wataru, Tsutsumi, Nobuhiro

Genes & Genetic Systems 81 ( 6 ) 451 - 451 2006

Molecular characterization of organelle inheritance in higher plants using model plants

Matsushima, R, Hattori, C, Sodmergen, Sakamoto, W

Plant and Cell Physiology 47 2006

Different amounts of DNA in each mitochondrion in rice root

Takanashi, Hideki, Arimura, Shin-ichi, Sakamoto, Wataru, Tsutsumi, Nobuhiro

Genes & Genetic Systems 81 ( 3 ) 215 - 8 2006

Language：English Publishing type：Research paper (scientific journal)

The DNA content of individual mitochondria in rice root cells was analyzed by fluorescence microscopy. Differences in DNA content were detected between individual mitochondria. Some mitochondria contained no detectable nucleoid (DNA-protein complexes). The percent of mitochondria with DAPI(4',6-Diamidino-2-phenylindole) -stained nucleoids varied over the length of the root (root base, 33%; middle portion of root, 41%; root tip, 91%). The mean amounts of DNA per mitochondrial nucleoid were equivalent to 46.4 kbp in the root base, 52.0 kbp in the middle portion of root and 124.2 kbp in the root tip. The amount of DNA in individual mitochondria and the ratio of mitochondria with visible nucleoids were higher in the root tip than in other parts of the root. The estimated amount of DNA in almost all of the observed mitochondria was smaller than the amount of DNA equivalent to the rice mitochondrial genome size (490 kbp), even in root tip.

DOI： 10.1266/ggs.81.215

Effects of dysfunction in defense systems toward photoinhibition on mitochondrial respiration

Yoshida, K, Sakamoto, W, Shikanai, T, Terashima, I, Noguchi, K

Plant and Cell Physiology 47 2006

Arabidopsis male gametophytic mutants deficient in organellar DNA disappearance

Kubo, M, Matsushima, R, Hattori, C, Sodmergen, Sakamoto, W

Plant and Cell Physiology 47 2006

Characterization of T-DNA insertion mutants for genes encoding mitochondrial FtsHs

Sakamoto, W, Gibala-Litwin, M, Janska, H

Plant and Cell Physiology 47 2006

Protein degradation machineries in plastids International journal

Sakamoto, Wataru

Annual Review of Plant Biology 57 599 - 621 2006

Language：English Publishing type：Research paper (scientific journal)

Plastids undergo drastic morphological and physiological changes under different developmental stages and in response to environmental conditions. A key to accomplishing these transitions and maintaining homeostasis is the quality and quantity control of many plastid proteins by proteases and chaperones. Although a limited number of plastid proteases have been identified by biochemical approaches, recent progress in genome information revealed various plant proteases that are of prokaryotic origin and that are localized in chloroplasts. Of these, ATP-dependent proteases such as Clp, FtsH, and Lon are considered the major enzymes involved in processive degradation (gradual degradation to oligopeptides and amino acids). The basic architecture of plant ATP-dependent proteases is very similar to the architechture of bacterial enzymes, such as those in Escherichia coli, but plastid enzymes apparently have extraordinary numbers of isomers. Recent molecular genetic characterization in Arabidopsis has identified differential roles of these isomers. This review covers what is currently known about the types and function of plastid proteases together with our new observations.

DOI： 10.1146/annurev.arplant.57.032905.105401

[Maternal inheritance of mitochondria in higher plants].

Sodmergen, Wataru Sakamoto

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 50 ( 14 Suppl ) 1795 - 8 2005.11

[Dynamics in mitochondria and chloroplasts: a current overview].

Wataru Sakamoto, Jun-Ichi Hayashi

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 50 ( 14 Suppl ) 1861 - 2 2005.11

[Role of ATP-dependent proteolytic machineries in plastid biogenesis, differentiation, maintenance, and senescence].

Wataru Sakamoto

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 50 ( 14 Suppl ) 1892 - 5 2005.11

Protein quality control and membrane proteases in chloroplasts

Sakamoto, W

Seikagaku 77 ( 4 ) 2005

FtsH proteases in chloroplasts and cyanobacteria

Adam, Z, Zaltsman, A, Sinvany-Villalobo, G, Sakamoto, W

Physiologia Plantarum 123 ( 4 ) 2005

Isolation and molecular characterization of rbcS in the unicellular green alga Nannochloris bacillaris (Chlorophyta, Trebouxiophyceae)

Yamazaki, T, Yamamoto, M, Sakamoto, W, Kawano, S

Phycological Research 53 ( 1 ) 2005

An Arabidopsis mutant deficient in pollen mitosis II

Kubo, M, Sakamoto, W

Plant and Cell Physiology 46 2005

Quality control of membrane proteins in chloroplasts and its relationship to light adaptation

Sakamoto, W

Plant and Cell Physiology 46 2005

Isolation of mutations that suppress leaf variegation in var2 of Arabidopsis

Miura, E, Sakamoto, W

Plant and Cell Physiology 46 2005

Isolation and characterization of Ty1/copia-like reverse transcriptase sequences from mung bean

Xiao, WM, Sakamoto, W, Sodmergen

Acta Botanica Sinica 46 ( 5 ) 2004

Allelic characterization of the leaf-variegated mutation var2 identifies the conserved amino acid residues of FtsH that are important for ATP hydrolysis and proteolysis International journal

Sakamoto, W, Miura, E, Kaji, Y, Okuno, T, Nishizono, M, Ogura, T

Plant Molecular Biology 56 ( 5 ) 705 - 16 2004

Language：English Publishing type：Research paper (scientific journal)

Arabidopsis var1 and var2 mutants exhibit leaf variegation. VAR1 and VAR2 encode similar FtsH metalloproteases (FtsH5 and FtsH2, respectively). We have previously found many variegated mutants to be allelic to var2. Each mutant was shown to express a different degree of variegation, and the formation of white sectors was enhanced in severely variegated alleles when these alleles were grown at low temperature. VAR1/FtsH5 and VAR2/FtsH2 levels were mutually affected even in the weak alleles, confirming our previous observation that the two proteins form a hetero complex. In this study, the sites of the mutations in these var2 alleles were determined. We isolated eight point mutations. Five alleles resulted in an amino acid substitution. Three of the five amino acid substitutions occurred in Walker A and B motifs of the ATP-binding site, and one occurred in the central pore motif. These mutations were considered to profoundly suppress the ATPase and protease activities. In contrast, one mutation was found in a region that contained no obvious signature motifs, but a neighboring sequence, Gly-Ala-Asp, was highly conserved among the members of the AAA protein family. Site-directed mutagenesis of the corresponding residue in E. coli FtsH indeed showed that this residue is necessary for proper ATP hydrolysis and proteolysis. Based on these results, we propose that the conserved Gly-Ala-Asp motif plays an important role in FtsH activity. Thus, characterization of the var2 alleles could help to identify the physiologically important domain of FtsH.

DOI： 10.1007/s11103-004-4561-9

A mutation of the CRUMPLED LEAF gene that encodes a protein localized in the outer envelope membrane of plastids affects the pattern of cell division, cell differentiation, and plastid division in Arabidopsis International journal

Asano, T, Yoshioka, Y, Kurei, S, Sakamoto, W, Machida, Y

Plant Journal 38 ( 3 ) 448 - 59 2004

Language：English Publishing type：Research paper (scientific journal)

We identified a novel mutation of a nuclear-encoded gene, designated as CRUMPLED LEAF (CRL), of Arabidopsis thaliana that affects the morphogenesis of all plant organs and division of plastids. Histological analysis revealed that planes of cell division were distorted in shoot apical meristems (SAMs), root tips, and embryos in plants that possess the crl mutation. Furthermore, we observed that differentiation patterns of cortex and endodermis cells in inflorescence stems and root endodermis cells were disturbed in the crl mutant. These results suggest that morphological abnormalities observed in the crl mutant were because of aberrant cell division and differentiation. In addition, cells of the crl mutant contained a reduced number of enlarged plastids, indicating that the division of plastids was inhibited in the crl. The CRL gene encodes a novel protein with a molecular mass of 30 kDa that is localized in the plastid envelope. The CRL protein is conserved in various plant species, including a fern, and in cyanobacteria, but not in other organisms. These data suggest that the CRL protein is required for plastid division, and it also plays an important role in cell differentiation and the regulation of the cell division plane in plants. A possible function of the CRL protein is discussed.

DOI： 10.1111/j.1365-313X.2004.02057.x

Isolation of mutants with aberrant mitochondrial morphology from Arabidopsis thaliana

Feng, XG, Arimura, S, Hirano, HY, Sakamoto, W, Tsutsumi, N

Genes & Genetic Systems 79 ( 5 ) 301 - 5 2004

Language：English Publishing type：Research paper (scientific journal)

To identify genes related to plant mitochondrial morphology and dynamics, novel mutants with respect to mitochondrial morphology were isolated from an ethyl methane sulphonate (EMS)-mutated population of Arabidopsis thaliana. Mitochondria were visualized by transforming Arabidopsis with a gene for a fusion protein consisting of GFP and a mitochondria-targeting pre-sequence. From 19,000 M2 populations, 17 mutants were isolated by fluorescent microscopic observations. All mitochondria in these mutants were longer and/or larger than wild-type mitochondria. The approximate chromosomal loci of the mutations of seven mutants that grew well were determined. The mitochondrial phenotypes of six of the mutants were recessive but the mitochondrial phenotype of the seventh mutant was dominant. Chromosomal rough mapping of the seven mutants showed that the mutations occurred at four different loci. At least one of these loci was novel, i.e., it was different from loci of other known mitochondrial morphology mutants of Arabidopsis and different from loci of Arabidopsis homologues of yeast genes related to mitochondrial morphology.

DOI： 10.1266/ggs.79.301

Molecular divergence and characterization of two chloroplast division genes, Ftsz1 and Ftsz2, in the unicellular green alga Nannochloris bacillaris (Chlorophyta)

Koide, T, Yamazaki, T, Yamamoto, M, Fujishita, M, Nomura, H, Moriyama, Y, Sumiya, N, Matsunaga, S, Sakamoto, W, Kawano, S

Journal of Phycology 40 ( 3 ) 2004

Analysis of the CRUMPLED LEAF that is involved in the plastid division, cell division, and cell differentiation in Arabidopsis thaliana Reviewed

Asano T, Yoshioka Y, Kurei S, Sakamoto W, Dmergen S, Fujiwara M, Yoshida S, Machida Y

Plant and Cell Physiology 45 S21 2004

Leaf-variegated mutations and their responsible genes in Arabidopsis thaliana

Sakamoto, W

Genes & Genetic Systems 78 ( 1 ) 1 - 9 2003

Language：English Publishing type：Research paper (scientific journal)

Leaf variegation has long been known as a recessive genetic trait in higher plants. Unlike albino mutants, leaf-variegated mutants are non-lethal and thus enable us to study a novel mechanism of plastid development and maintenance. Variegation results from a defect that makes chloroplast development unstable, since at least part of the tissues gives rise to normal chloroplasts. Despite the fact that leaf-variegated mutants have contributed to the findings of maternal inheritance or have been used as genetic markers, these mutations and the responsible loci have been poorly understood at the molecular level. A comprehensive study of the leaf-variegated mutants is possible in Arabidopsis, since such mutants have been known and the cloning can be at relative ease as a model plant. Here I summarize recent progress on characterization of the Arabidopsis leaf-variegated mutants. Detailed analysis of the responsible loci revealed that variegation is caused by a defect in various metabolic pathways related to organelle functions. Thus, studies on these genes provide us with novel redundant mechanisms by which heteroplasmic organelles such as plastids and mitochondria can survive from an environmental stress.

Coordinated regulation and complex formation of YELLOW VARIEGATED1 and YELLOW VARIEGATED2, chloroplastic FtsH metalloproteases involved in the repair cycle of photosystem II in Arabidopsis thylakoid membranes International journal

Sakamoto, W, Zaltsman, A, Adam, Z, Takahashi, Y

Plant Cell 15 ( 12 ) 2843 - 55 2003

Language：English Publishing type：Research paper (scientific journal)

Arabidopsis yellow variegated1 (VAR1) and VAR2 are separate loci that encode similar chloroplast FtsH proteases. To date, FtsH is the best-characterized protease in thylakoid membranes involved in the turnover of photosynthetic protein complexes. It comprises a protein family that is encoded by 12 different nuclear genes in Arabidopsis. We show here that nine FtsH proteins are located in the chloroplasts. Mutations in either VAR1 or VAR2 cause typical leaf variegation and sensitivity to photoinhibition. By contrast, none of these phenotypes was observed in T-DNA insertion mutants in other ftsH genes (ftsh1, ftsh6, and ftsh8) closely related to VAR1 and VAR2. This finding suggests that VAR1 and VAR2 play a predominant role in the photosystem II repair cycle in thylakoid membranes. By generating VAR1- and VAR2-specific antibodies, we found that loss of either VAR1 or VAR2 results in the decreased accumulation of the other. Thus, the genetic nonredundancy between VAR1 and VAR2 could be attributed to their coordinated regulation at the protein level. These observations led us to examine whether VAR1 and VAR2 form a complex. Sucrose density gradient and gel filtration analyses revealed a complex of approximately 400 to 450 kD, probably representing a hexamer. Furthermore, VAR1 and VAR2 were shown to coprecipitate by immunoprecipitation using VAR1- and VAR2-specific antibodies. The majority of VAR1 appears to exist as heterocomplexes with VAR2, whereas VAR2 may be present as homocomplexes as well. Based on these results, we conclude that VAR1 and VAR2 are the major components of an FtsH complex involved in the repair of photodamaged proteins in thylakoid membranes.

DOI： 10.1105/tpc.017319

[Leaf-variegated mutants in higher plants as tools for studying a novel function in plastids and mitochondria].

Wataru Sakamoto

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 47 ( 12 Suppl ) 1771 - 6 2002.9

Reduction in amounts of mitochondrial DNA in the sperm cells as a mechanism for maternal inheritance in Hordeum vulgare

Sodmergen, Zhang, QA, Zhang, YT, Sakamoto, W, Kuroiwa, T

Planta 216 ( 2 ) 2002

The VAR1 locus of Arabidopsis encodes a chloroplastic FtsH and is responsible for leaf variegation in the mutant alleles International journal

Sakamoto, W, Tamura, T, Hanba-Tomita, Y, Murata, M

Genes To Cells 7 ( 8 ) 769 - 80 2002

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: A leaf-variegated mutation var1 of Arabidopsis results in the development of abnormal plastids and the formation of a green/white sector. Genetic analysis of the var1 mutant indicated that it acts synergistically with another mutation var2, suggesting that the two genes are relevant. The VAR2 locus has been shown to encode a chloroplastic FtsH, an ATP-dependent protease which is possibly involved in the degradation of thylakoid proteins and plastid development. RESULTS: In this study we show that the VAR1 locus encodes a chloroplastic FtsH protein homologous to VAR2. VAR1 contains a conserved motif for ATPase and a metalloprotease characteristic to FtsH proteins, and is targeted into chloroplasts. A VAR1-fusion protein synthesized in vitro exhibited ATPase activity and partial metalloprotease activity. The maximum yield of photochemistry, measured by chlorophyll fluorescence, showed that the var1 mutants were sensitive to photoinhibitory light exposure at 800 micro mol/m2/s. CONCLUSION: VAR1 and VAR2 comprise an FtsH small gene family together with other FtsH genes in Arabidopsis. VAR1 as well as VAR2 may play an important role in degrading photodamaged subunits in photosystem II. Loss of VAR1 and VAR2 perhaps impairs the photoprotection mechanism and thylakoid development, causing leaf variegation as a consequence.

DOI： 10.1046/j.1365-2443.2002.00558.x

Functional divergence of the TFL1-like gene family in Arabidopsis revealed by characterization of a novel homologue

Mimida, N, Goto, K, Kobayashi, Y, Araki, T, Ahn, JH, Weigel, D, Murata, M, Motoyoshi, F, Sakamoto, W

Genes To Cells 6 ( 4 ) 2001

The Purple leaf (Pl) locus of rice: the Pl(w) allele has a complex organization and includes two genes encoding basic helix-loop-helix proteins involved in anthocyanin biosynthesis

Sakamoto, W, Ohmori, T, Kageyama, K, Miyazaki, C, Saito, A, Murata, M, Noda, K, Maekawa, M

Plant and Cell Physiology 42 ( 9 ) 2001

The YELLOW VARIEGATED (VAR2) locus encodes a homologue of FtsH, an ATP-dependent protease in Arabidopsis

Takechi, K, Sodmergen, Murata, M, Motoyoshi, F, Sakamoto, W

Plant and Cell Physiology 41 ( 12 ) 2000

Mitochondrial localization of AtOXA1, an Arabidopsis homologue of yeast Oxa1p involved in the insertion and assembly of protein complexes in mitochondrial inner membrane

Sakamoto, W, Spielewoy, N, Bonnard, G, Murata, M, Wintz, H

Plant and Cell Physiology 41 ( 10 ) 2000

イネ完熟種子糊粉層におけるアントシアニン生合成に係わる転写制御遺伝子のトランジェントアッセイ系

前川雅彦, 景山圭祐, 力石和英, 宇都木繁子, 野田和彦, 坂本亘

育種学研究 2 ( 4 ) 181 - 187 2000

Language：Japanese Publisher：日本育種学会

イネにおけるアントシアニン生合成の転写制御遺伝子の発現解析に必要なトランジェントアッセイ系を, 常時材料を準備できるイネ完熟種子糊粉層で確立した. まず, 岡大資生研紫米 (RIB-PI) の完熟種子を用いて糊粉層を露出するための吸水時間と温度を検討した. 露出できた糊粉層にトウモロコシのアントシアニン生合成の転写制御遺伝子, ClとB-Peruをパーティクルガンで導入したところ, アントシアニンのスポットが観察されたので, 両遺伝子を用いたトランジェントアッセイ系について至適条件を検討した. その結果, 28℃ で4時間吸水させて露出した糊粉層でアントシアニンのスポット数が最大になった. また, 導入するプラスミドの量は, ClとBPeruがともに5μgかClが5μgでB-Peruが10μgのときに, アントシアニンのスポット数が多くなった. 以上の結果から, イネの完熟種子糊粉層を用いたトランジェントアッセイとして, 28℃ で4時間吸水させて糊粉層を露出させ, C1とB-Pmをともに5μgずつ導入することによって, 安定的にアントシアニンを誘導できることが明らかとなった.

DOI： 10.1270/jsbbr.2.181

The strange evolutionary history of plant mitochondrial tRNAs and their aminoacyl-tRNA synthetases

Small, I, Akashi, K, Chapron, A, Dietrich, A, Duchene, AM, Lancelin, D, Marechal-Drouard, L, Menand, B, Mireau, H, Moudden, Y, Ovesna, J, Peeters, N, Sakamoto, W, Souciet, G, Wintz, H

Journal of Heredity 90 ( 3 ) 1999

In situ RNA hybridization using Technovit resin in Arabidopsis thaliana

Takechi, K, Sakamoto, W, Katsuhara, M, Murata, M, Motoyoshi, F

Plant Molecular Biology Reporter 17 ( 1 ) 1999

TERMINAL FLOWER 1-like genes in Brassica species

Mimida, N, Sakamoto, W, Murata, M, Motoyoshi, F

Plant Science 142 ( 2 ) 1999

Characterization of a flower-specific gene encoding a putative myrosinase binding protein in Arabidopsis thaliana

Takechi, K, Sakamoto, W, Utsugi, S, Murata, M, Motoyoshi, F

Plant and Cell Physiology 40 ( 12 ) 1999

A single gene of chloroplast origin codes for mitochondrial and chloroplastic methionyl-tRNA synthetase in Arabidopsis thaliana

Menand, B, Marechal-Drouard, L, Sakamoto, W, Dietrich, A, Wintz, H

Proceedings of the National Academy of Sciences of the United States of America 95 ( 18 ) 1998

Mix 'n' match - Plant mitochondrial tRNAS and their aminoacyl-tRNA synthetases

Small, I, Akashi, K, Chapron, A, Dietrich, A, Duchene, AM, Lancelin, D, Marechal-Drouard, L, Menand, B, Mireau, H, Moudden, Y, Ovesna, J, Peeters, N, Sakamoto, W, Souciet, G, Wintz, H, Moller, IM, Gardestrom, P, Glimelius, K, Glaser, E

Plant Mitochondria: From Gene To Function 1998

Arabidopsis thaliana vegetative storage protein (VSP) genes: gene organization and tissue-specific expression

Utsugi, S, Sakamoto, W, Murata, M, Motoyoshi, F

Plant Molecular Biology 38 ( 4 ) 1998

Isolation of an Arabidopsis thaliana cDNA by complementation of a yeast abc1 deletion mutant deficient in complex III respiratory activity

Cardazzo, B, Hamel, P, Sakamoto, W, Wintz, H, Dujardin, G

Gene 221 ( 1 ) 1998

Cloning and molecular analysis of the Arabidopsis gene Terminal Flower 1

Ohshima, S, Murata, M, Sakamoto, W, Ogura, Y, Motoyoshi, F

Molecular & General Genetics 254 ( 2 ) 1997

An unusual mitochondrial atp9-rpl16 cotranscript found in the maternal distorted leaf mutant of Arabidopsis thaliana: Implication of GUG as an initiation codon in plant mitochondria

Sakamoto, W, Tan, SH, Murata, M, Motoyoshi, F

Plant and Cell Physiology 38 ( 8 ) s55 1997

Functional complementation of an oxa1(-) yeast mutation identifies an Arabidopsis thaliana cDNA involved in the assembly of respiratory complexes

Hamel, P, Sakamoto, W, Wintz, H, Dujardin, G

Plant Journal 12 ( 6 ) 1997

Putative phospholipid hydroperoxide glutathione peroxidase gene from Arabidopsis thaliana induced by oxidative stress

Sugimoto, M, Sakamoto, W

Genes & Genetic Systems 72 ( 5 ) 311 - 316 1997

Language：English Publishing type：Research paper (scientific journal) Publisher：The Genetics Society of Japan

An Arabidopsis cDNA encoding putative phospholipid hydroperoxide glutathione peroxidase (PHGPX) was cloned and sequenced. The cDNA comprised 803 bp and included an open reading frame which encodes a polypeptide of 169 amino acid residues. The deduced amino acid sequence showed about 80 and 50% homology with plant putative PHGPXs and mammalian PHGPXs, respectively. Southern blot analysis suggested that the putative PHGPX gene was a single-copy gene. The expression profile of the putative PHGPX in Arabidopsis under NaCl and Al/Fe treatments, which generate oxidative stress, was analyzed. Northern blot analysis revealed that the Arabidopsis putative PHGPX mRNA levels were increased about 3 and 4.5 times after exposure to NaCl and Al/Fe, respectively. These results suggest that the putative PHGPX gene is induced by oxidative stress in Arabidopsis.<br>

DOI： 10.1266/ggs.72.311

Other Link： https://jlc.jst.go.jp/DN/JALC/00084483256?from=CiNii

Isolation and characterization of cDNA clones corresponding to the genes expressed preferentially in floral organs of Arabidopsis thaliana

Utsugi, S, Sakamoto, W, Ogura, Y, Murata, M, Motoyoshi, F

Plant Molecular Biology 32 ( 4 ) 1996

Altered mitochondrial gene expression in a maternal distorted leaf mutant of Arabidopsis induced by chloroplast mutator

Sakamoto, W, Kondo, H, Murata, M, Motoyoshi, F

Plant Cell 8 ( 8 ) 1996

PLANT-MITOCHONDRIA - FROM GENE TO FUNCTION

SAKAMOTO, W

Breeding Science 45 ( 3 ) 407 - 407 1995

FUNCTION OF THE CHLAMYDOMONAS-REINHARDTII PETD 5' UNTRANSLATED REGION IN REGULATING THE ACCUMULATION OF SUBUNIT-IV OF THE CYTOCHROME B(6)/F COMPLEX

SAKAMOTO, W, CHEN, XM, KINDLE, KL, STERN, DB

Plant Journal 6 ( 4 ) 503 - 512 1994

PETD MESSENGER-RNA MATURATION IN CHLAMYDOMONAS-REINHARDTII CHLOROPLASTS - ROLE OF 5' ENDONUCLEOLYTIC PROCESSING

SAKAMOTO, W, STURM, NR, KINDLE, KL, STERN, DB

Molecular and Cellular Biology 14 ( 9 ) 6180 - 6186 1994

THE PETD GENE IS TRANSCRIBED BY FUNCTIONALLY REDUNDANT PROMOTERS IN CHLAMYDOMONAS-REINHARDTII CHLOROPLASTS

STURM, NR, KURAS, R, BUSCHLEN, S, SAKAMOTO, W, KINDLE, KL, STERN, DB, WOLLMAN, FA

Molecular and Cellular Biology 14 ( 9 ) 1994

RESTRICTION FRAGMENTS HOMOLOGOUS TO MITOCHONDRIAL PLASMID-LIKE DNAS ARE LOCATED WITHIN LIMITED CHROMOSOMAL REGIONS ON THE RICE NUCLEAR GENOME

KANAZAWA, A, KISHIMOTO, N, SAKAMOTO, W, OHSAWA, R, UKAI, Y, TSUTSUMI, N, HIRAI, A, SAITO, A

Theoretical and Applied Genetics 87 ( 5 ) 1993

INVIVO ANALYSIS OF CHLAMYDOMONAS CHLOROPLAST PETD GENE-EXPRESSION USING STABLE TRANSFORMATION OF BETA-GLUCURONIDASE TRANSLATIONAL FUSIONS

SAKAMOTO, W, KINDLE, KL, STERN, DB

Proceedings of the National Academy of Sciences of the United States of America 90 ( 2 ) 497 - 501 1993

DISTRIBUTION AND QUANTITATIVE VARIATION OF MITOCHONDRIAL PLASMID-LIKE DNAS IN CULTIVATED RICE (ORYZA-SATIVA L)

KANAZAWA, A, SAKAMOTO, W, NAKAGAHRA, M, KADOWAKI, K, TSUTSUMI, N, TANO, S

Japanese Journal of Genetics 67 ( 4 ) 309 - 319 1992

Language：English Publishing type：Research paper (scientific journal) Publisher：The Genetics Society of Japan

Distribution of four kinds of mitochondrial plasmid-like DNAs, Bl, B2, B3 and B4 was investigated by Southern analysis using 85 accessions of cultivated rice (Oryza sativa L.). The frequencies of these molecules found in cultivars differed from each other. The mitochondrial genome were categorized into nine types according to their presence or absence. They were found mostly in ecospecies Indica, a few in Javanica, and not found in Japonica. The result indicated the polymorphic and monomorphic patterns of the mitochondrial genomic organization within Indica and Japonica cultivars, respectively. Quantitative variation was found among four kinds of mitochondiral plasmid-like DNAs, suggesting that these molecules were unequally distributed or replicated during the process of mitochondrial division.<br>

DOI： 10.1266/jjg.67.309

Other Link： https://jlc.jst.go.jp/DN/JALC/00079223454?from=CiNii

RFLP ANALYSIS OF NUCLEAR DNAS HOMOLOGOUS WITH MITOCHONDRIAL PLASMID-LIKE DNAS IN CULTIVATED RICE

SAKAMOTO, W, KADOWAKI, K, KISHIMOTO, N, YANO, M, SAITO, A, TANO, S

Theoretical and Applied Genetics 82 ( 2 ) 179 - 184 1991

LINKAGE ANALYSIS OF THE NUCLEAR HOMOLOGS OF MITOCHONDRIAL PLASMID-LIKE DNAS IN RICE

KANAZAWA, A, SAKAMOTO, W, KISHIMOTO, N, YANO, M, TSUTSUMI, N, SAITO, A, TANO, S

Japanese Journal of Genetics 66 ( 5 ) 597 - 607 1991

Language：English Publishing type：Research paper (scientific journal) Publisher：The Genetics Society of Japan

A genetical study on the nucleotide sequences of the nuclear DNAs which share homology with rice mitochondrial plasmid-like DNAs, B1, B2, B3 and B4 was carried out. Restriction fragments of the nuclear DNAs hybridized with these plasmid-like DNAs showed polymorphisms in their length between Indica and Japonica rice cultivars. The hybridized signals found specifically in Indica or Japonica cultivars segregated in the F₂ population derived from a cross between these two subspecies. The observed ratio of the nuclear homologues in the F₂ population demonstrated that they were transmitted according to the Mendelian inheritance. The co-segregation of homologues was examined and the linkage was detected between the B1-nuclear homologue of Japonica and the B4-nuclear homologue of Indica, and also between the nuclear homologues of B2 and B3 of Indica. The linkage between the B1-nuclear homologue of Japonica and the B4-nuclear homologue of Indica was conserved in the different rice cultivars.<br>

DOI： 10.1266/jjg.66.597

Other Link： http://agriknowledge.affrc.go.jp/RN/2010472559

GENETIC-ANALYSIS OF NUCLEAR DNAS HOMOLOGOUS TO SMALL MITOCHONDRIAL PLASMID-LIKE DNAS IN CULTIVATED RICE Reviewed

W SAKAMOTO, KI KADOWAKI, N KISHIMOTO, M YANO, A SAITO, S TANO

RICE GENETICS II 365 - 372 1991

ANALYSIS OF MITOCHONDRIAL DNAS FROM ORYZA-GLABERRIMA AND ITS CYTOPLASMIC SUBSTITUTED LINE FOR ORYZA-SATIVA ASSOCIATED WITH CYTOPLASMIC MALE-STERILITY

SAKAMOTO, W, KADOWAKI, K, TANO, S, YABUNO, T

Japanese Journal of Genetics 65 ( 1 ) 1 - 6 1990

Language：English Publishing type：Research paper (scientific journal) Publisher：The Genetics Society of Japan

It was described previously that the combination of the cytoplasm of a Japanese variety of Oryza sativa and the nucleus of O. glaberrima produced by successive crossings led to male sterility. In the present study, the mitochondrial DNAs (mtDNAs) were isolated from this cytoplasmic male-sterile (CMS) strain and its maintainer strain, and were analyzed with restriction enzymes and by agarose gel electrophoresis. Differences in the restriction pattern were observed between the CMS strain having sativa cytoplasm and its maintainer strain having glaberrima cytoplasm. The plasmid-like DNAs (B1 and B2), which are often observed in mitochondria of CMS strains derived from O. sativa, could not be detected by Southern hybridization analysis as well as gel electrophoresis. It was suggested that the plasmid-like DNAs were not associated with the cytoplasmic male sterility. However, the homologous sequences to B1 and B2 were detected in the nuclear genomes of all species tested.<br>

DOI： 10.1266/jjg.65.1

NUCLEOTIDE-SEQUENCE OF THE CYTOCHROME-OXIDASE SUBUNIT-I GENE FROM RICE MITOCHONDRIA

K KADOWAKI, T SUZUKI, S KAZAMA, T OHFUCHI, W SAKAMOTO

NUCLEIC ACIDS RESEARCH 17 ( 18 ) 7519 - 7519 1989.9

ANALYSIS OF HOMOLOGY OF SMALL PLASMID-LIKE MITOCHONDRIAL DNAS IN THE DIFFERENT CYTOPLASMIC MALE STERILE STRAINS IN RICE

SAKAMOTO, W, MOMOSE, M, TSUTSUMI, N, TANO, S, YAMAGUCHI, H

Japanese Journal of Genetics 64 ( 1 ) 1989

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