Faculty Profiles - SAKAMOTO Wataru

Papers - SAKAMOTO Wataru

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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

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

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

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

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