Faculty Profiles - SAKAMOTO Wataru

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