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

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