Updated on 2024/10/07

写真a

 
NISHIMURA, Yoshiki
 
Affiliation
Faculty of Education and Integrated Arts and Sciences, School of Education
Job title
Associate Professor

Research Experience

  • 2024.04
    -
    Now

    Waseda University   TWIns   Affiliate Professor

  • 2008.09
    -
    2024.03

    2008.9- Kyoto University, Dep. of Botany, Assistant Professor   -

  • 2006.07
    -
     

    2006.7-2008.8 University of Tokyo, IMCB, Assistant Professor   -

  • 2003.04
    -
     

    2003.4-2006.6 Cornell University (USA), postdoctoral fellow   -

  • 2001.04
    -
     

    2001.4-2003.3 University of Tokyo, postdoctoral fellow   -

Professional Memberships

  •  
     
     

    The Japanese Society of Plant Morphology

  •  
     
     

    The Japanese Society of Plant Physiologists

  •  
     
     

    The Botanical Society of Japan

Research Areas

  • Molecular biology / Morphology and anatomical structure / Plant molecular biology and physiology

Research Interests

  • endosymbiotic theory

  • DNA replication, repair

  • optical tweezers

  • nucleoid

  • mitochondrial DNA

  • cpDNA

▼display all

Awards

  • 平瀬賞

    2017   日本植物形態学会  

  • 奨励賞

    2009   日本植物学会   葉緑体母性遺伝の分子機構の探究

  • Francis Goelet Distinguished Postdoctoral Fellowship Award

    2003   Boyce Thompson Institute for Plant Research  

  • 奨励賞

    2000   日本植物形態学会  

  • 科学技術省

    2000   フジ産経グループ  

Media Coverage

  • 葉緑体のDNA折りたたむ「クリップ」の仕組み発見 京大チ ーム

    Newspaper, magazine

    毎日新聞  

    2021.05

  • 葉緑体の分裂、連続観察 京大グループ

    Newspaper, magazine

    Author: Other  

    読売新聞  

    2018.06

  • 「京都大学、葉緑体の染色体分離の瞬間をとらえた-葉緑体核様体の柔軟なネット ワーク構造を解明-

    Internet

    日経バイオテクOnline  

    2018.05

  • 京大など、葉緑体増殖の基礎的仕組みを解明

    Newspaper, magazine

    日本経済新聞  

    2017.05

  • Holliday junction discovery in chloroplasts.

    Internet

    Scientific inquirer  

    2017.05

  • Disentangling chloroplast genetics, Scientists isolate a critical gene for plant health.

    Internet

    Science Daily  

    2017.05

  • 母性遺伝のしくみを解明

    共同通信  

    2006.01

  • ミトコンドリアDNA の母性遺伝機構、粘菌・脊椎動物で共通—東大が解明

    Newspaper, magazine

    日刊工業新聞  

    2006.01

▼display all

 

Papers

  • Plastid Nucleoids: Insights into Their Shape and Dynamics

    Yoshiki Nishimura

    Plant And Cell Physiology    2024.05

    DOI

    Scopus

    4
    Citation
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  • Chloroplast biogenesis involves spatial coordination of nuclear and organellar gene expression in Chlamydomonas

    Yi Sun, Shiva Bakhtiari, Melissa Valente-Paterno, Yanxia Wu, Yoshiki Nishimura, Weike Shen, Christopher Law, James Dhaliwal, Daniel Dai, Khanh Huy Bui, William Zerges

    Plant Physiology   196 ( 1 ) 112 - 123  2024.05  [Refereed]

     View Summary

    Abstract

    The localization of translation can direct the polypeptide product to the proper intracellular compartment. Our results reveal translation by cytosolic ribosomes on a domain of the chloroplast envelope in the unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii). We show that this envelope domain of isolated chloroplasts retains translationally active ribosomes and mRNAs encoding chloroplast proteins. This domain is aligned with localized translation by chloroplast ribosomes in the translation zone, a chloroplast compartment where photosystem subunits encoded by the plastid genome are synthesized and assembled. Roles of localized translation in directing newly synthesized subunits of photosynthesis complexes to discrete regions within the chloroplast for their assembly are suggested by differences in localization on the chloroplast of mRNAs encoding either subunit of the light-harvesting complex II or the small subunit of Rubisco. Transcription of the chloroplast genome is spatially coordinated with translation, as revealed by our demonstration of a subpopulation of transcriptionally active chloroplast nucleoids at the translation zone. We propose that the expression of chloroplast proteins by the nuclear-cytosolic and organellar genetic systems is organized in spatially aligned subcompartments of the cytoplasm and chloroplast to facilitate the biogenesis of the photosynthetic complexes.

    DOI

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    1
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    (Scopus)
  • A conserved RWP-RK transcription factor VSR1 controls gametic differentiation in volvocine algae

    Sa Geng, Takashi Hamaji, Patrick J Ferris, Minglu Gao, Yoshiki Nishimura, James Umen

    Proc Natl Acad Sci USA   120 ( 29 ) e2305099120  2023.07  [Refereed]

    DOI

  • Three genomes in the algal genus Volvox reveal the fate of a haploid sex-determining region after a transition to homothallism.

    Kayoko Yamamoto, Takashi Hamaji, Hiroko Kawai-Toyooka, Ryo Matsuzaki, Fumio Takahashi, Yoshiki Nishimura, Masanobu Kawachi, Hideki Noguchi, Yohei Minakuchi, James G Umen, Atsushi Toyoda, Hisayoshi Nozaki

    Proceedings of the National Academy of Sciences of the United States of America   118 ( 21 )  2021.05  [International journal]

     View Summary

    Transitions between separate sexes (dioecy) and other mating systems are common across eukaryotes. Here, we study a change in a haploid dioecious green algal species with male- and female-determining chromosomes (U and V). The genus Volvox is an oogamous (with large, immotile female gametes and small, motile male gametes) and includes both heterothallic species (with distinct male and female genotypes, associated with a mating-type system that prevents fusion of gametes of the same sex) and homothallic species (bisexual, with the ability to self-fertilize). We date the origin of an expanded sex-determining region (SDR) in Volvox to at least 75 Mya, suggesting that homothallism represents a breakdown of dioecy (heterothallism). We investigated the involvement of the SDR of the U and V chromosomes in this transition. Using de novo whole-genome sequences, we identified a heteromorphic SDR of ca 1 Mbp in male and female genotypes of the heterothallic species Volvox reticuliferus and a homologous region (SDLR) in the closely related homothallic species Volvox africanus, which retained several different hallmark features of an SDR. The V. africanus SDLR includes a large region resembling the female SDR of the presumptive heterothallic ancestor, whereas most genes from the male SDR are absent. However, we found a multicopy array of the male-determining gene, MID, in a different genomic location from the SDLR. Thus, in V. africanus, an ancestrally female genotype may have acquired MID and thereby gained male traits.

    DOI PubMed

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    21
    Citation
    (Scopus)
  • HBD1 protein with a tandem repeat of two HMG-box domains is a DNA clip to organize chloroplast nucleoids in Chlamydomonas reinhardtii

    Mari Takusagawa, Yusuke Kobayashi, Yoichiro Fukao, Kumi Hidaka, Masayuki Endo, Hiroshi Sugiyama, Takashi Hamaji, Yoshinobu Kato, Isamu Miyakawa, Osami Misumi, Toshiharu Shikanai, Yoshiki Nishimura

    Proceedings of the National Academy of Sciences   118 ( 20 ) e2021053118 - e2021053118  2021.05  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA–protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga <italic>Chlamydomonas reinhardtii</italic> identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of the <italic>HBD1</italic> gene by CRISPR-Cas9–mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeast <italic>Δabf2</italic> mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids.

    DOI

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    9
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  • The Pentatricopeptide Repeat Protein PGR3 Is Required for the Translation of petL and ndhG by Binding Their 5′ UTRs

    Haruka Higashi, Yoshinobu Kato, Tomoya Fujita, Shintaro Iwasaki, Masayuki Nakamura, Yoshiki Nishimura, Mizuki Takenaka, Toshiharu Shikanai

    Plant and Cell Physiology    2021.01

     View Summary

    <title>Abstract</title>
    PGR3 is a P-class pentatricopeptide repeat (PPR) protein required for the stabilization of petL operon RNA and the translation of the petL gene in plastids. Irrespective of its important roles in plastids, key questions have remained unanswered, including how PGR3 protein promotes translation and which plastid mRNA PGR3 activates the translation. Here, we show that PGR3 facilitates the translation from ndhG, in addition to petL, through binding to their 5′ untranslated regions (UTRs). Ribosome profiling and RNA sequencing in pgr3 mutants revealed that translation from petL and ndhG was specifically suppressed. Harnessing small RNA fragments protected by PPR proteins in vivo, we probed the PGR3 recruitment to the 5′ UTRs of petL and ndhG. The putative PGR3-bound RNA segments per se repress the translation possibly with a strong secondary structure and thereby block ribosomes’ access. However, the PGR3 binding antagonizes the effects and facilitates the protein synthesis from petL and ndhG in vitro. The prediction of the 3-dimensional structure of PGR3 suggests that the 26th PPR motif plays important roles in target RNA binding. Our data show the specificity of a plastidic RNA-binding protein and provide a mechanistic insight into translational control.

    DOI

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    8
    Citation
    (Scopus)
  • Holliday Junction Resolvase MOC1 Maintains Plastid and Mitochondrial Genome Integrity in Algae and Bryophytes.

    Yusuke Kobayashi, Masaki Odahara, Yasuhiko Sekine, Takashi Hamaji, Sumire Fujiwara, Yoshiki Nishimura, Shin-Ya Miyagishima

    Plant physiology   184 ( 4 ) 1870 - 1883  2020.12  [International journal]

     View Summary

    When DNA double-strand breaks occur, four-stranded DNA structures called Holliday junctions (HJs) form during homologous recombination. Because HJs connect homologous DNA by a covalent link, resolution of HJ is crucial to terminate homologous recombination and segregate the pair of DNA molecules faithfully. We recently identified Monokaryotic Chloroplast1 (MOC1) as a plastid DNA HJ resolvase in algae and plants. Although Cruciform cutting endonuclease1 (CCE1) was identified as a mitochondrial DNA HJ resolvase in yeasts, homologs or other mitochondrial HJ resolvases have not been identified in other eukaryotes. Here, we demonstrate that MOC1 depletion in the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens induced ectopic recombination between short dispersed repeats in ptDNA. In addition, MOC1 depletion disorganized thylakoid membranes in plastids. In some land plant lineages, such as the moss P. patens, a liverwort and a fern, MOC1 dually targeted to plastids and mitochondria. Moreover, mitochondrial targeting of MOC1 was also predicted in charophyte algae and some land plant species. Besides causing instability of plastid DNA, MOC1 depletion in P. patens induced short dispersed repeat-mediated ectopic recombination in mitochondrial DNA and disorganized cristae in mitochondria. Similar phenotypes in plastids and mitochondria were previously observed in mutants of plastid-targeted (RECA2) and mitochondrion-targeted (RECA1) recombinases, respectively. These results suggest that MOC1 functions in the double-strand break repair in which a recombinase generates HJs and MOC1 resolves HJs in mitochondria of some lineages of algae and plants as well as in plastids in algae and plants.

    DOI PubMed

    Scopus

    8
    Citation
    (Scopus)
  • A non-photosynthetic green alga illuminates the reductive evolution of plastid electron transport systems

    Motoki Kayama, Jun-Feng Chen, Takashi Nakada, Yoshiki Nishimura, Toshiharu Shikanai, Tomonori Azuma, Hideaki Miyashita, Shinichi Takaichi, Yuichiro Kashiyama, Ryoma Kamikawa

    BMC Biology   18 ( 1 )  2020.12

     View Summary

    <title>Abstract</title>
    <sec>
    <title>Background</title>
    Plastid electron transport systems are essential not only for photosynthesis but also for dissipating excess reducing power and sinking excess electrons generated by various redox reactions. Although numerous organisms with plastids have lost their photoautotrophic lifestyles, there is a spectrum of known functions of remnant plastids in non-photosynthetic algal/plant lineages; some of non-photosynthetic plastids still retain diverse metabolic pathways involving redox reactions while others, such as apicoplasts of apicomplexan parasites, possess highly reduced sets of functions. However, little is known about underlying mechanisms for redox homeostasis in functionally versatile non-photosynthetic plastids and thus about the reductive evolution of plastid electron transport systems.


    </sec>
    <sec>
    <title>Results</title>
    Here we demonstrated that the central component for plastid electron transport systems, plastoquinone/plastoquinol pool, is still retained in a novel strain of an obligate heterotrophic green alga lacking the photosynthesis-related thylakoid membrane complexes. Microscopic and genome analyses revealed that the Volvocales green alga, chlamydomonad sp. strain NrCl902, has non-photosynthetic plastids and a plastid DNA that carries no genes for the photosynthetic electron transport system. Transcriptome-based in silico prediction of the metabolic map followed by liquid chromatography analyses demonstrated carotenoid and plastoquinol synthesis, but no trace of chlorophyll pigments in the non-photosynthetic green alga. Transient RNA interference knockdown leads to suppression of plastoquinone/plastoquinol synthesis. The alga appears to possess genes for an electron sink system mediated by plastid terminal oxidase, plastoquinone/plastoquinol, and type II NADH dehydrogenase. Other non-photosynthetic algae/land plants also possess key genes for this system, suggesting a broad distribution of an electron sink system in non-photosynthetic plastids.


    </sec>
    <sec>
    <title>Conclusion</title>
    The plastoquinone/plastoquinol pool and thus the involved electron transport systems reported herein might be retained for redox homeostasis and might represent an intermediate step towards a more reduced set of the electron transport system in many non-photosynthetic plastids. Our findings illuminate a broadly distributed but previously hidden step of reductive evolution of plastid electron transport systems after the loss of photosynthesis.


    </sec>

    DOI

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    6
    Citation
    (Scopus)
  • Step-wise elimination of α-mitochondrial nucleoids and mitochondrial structure as a basis for the strict uniparental inheritance in Cryptococcus neoformans

    Yoshiki Nishimura, Toshiharu Shikanai, Susumu Kawamoto, Akio Toh-E

    Scientific report   10 ( 1 ) 2468 - 2468  2020.02  [Refereed]

    Authorship:Lead author, Corresponding author

    DOI

    Scopus

    11
    Citation
    (Scopus)
  • Isolation and characterization of Chlamydomonas autophagy-related mutants in nutrient-deficient conditions.

    Kajikawa M, Yamauchi M, Shinkawa H, Tanaka M, Hatano K, Nishimura Y, Kato M, Fukuzawa H

    Plant & cell physiology    2018.10  [Refereed]

    DOI PubMed

    Scopus

    39
    Citation
    (Scopus)
  • Chloroplast nucleoids as a transformable network revealed by live imaging with a microfluidic device

    Yoshitaka Kamimura, Hitomi Tanaka, Yusuke Kobayashi, Toshiharu Shikanai, Yoshiki Nishimura

    Communications Biology   1 ( 47 ) 1 - 7  2018.05  [Refereed]  [International journal]

    Authorship:Last author, Corresponding author

    DOI

    Scopus

    13
    Citation
    (Scopus)
  • Identification of Holliday junction resolvases crucial for the chloroplast nucleoid morphology and segregation

    Yusuke Kobayashi, Osami Misumi, Yoshiki Nishimura

    PLANT MORPHOLOGY   30 ( 1 ) 73 - 81  2018

    Authorship:Last author, Corresponding author

    DOI

  • Finding Holliday Junction Resolvases: A Crucial Factor for Chloroplast Nucleoid Segregation

    Kobayashi Y, Misumi O, Nishimura Y

    Cytologia   82 ( 5 ) 465 - 466  2017.12  [Refereed]  [Invited]

    Authorship:Last author, Corresponding author

    DOI

    Scopus

  • Gene Regulatory Networks for the Haploid-to-Diploid Transition of Chlamydomonas reinhardtii

    Sunjoo Joo, Yoshiki Nishimura, Evan Cronmiller, Ran Ha Hong, Thamali Kariyawasam, Ming Hsiu Wang, Nai Chun Shao, Saif-El-Din El Akkad, Takamasa Suzuki, Tetsuya Higashiyama, Eonseon Jin, Jae-Hyeok Lee

    PLANT PHYSIOLOGY   175 ( 1 ) 314 - 332  2017.09  [Refereed]

     View Summary

    The sexual cycle of the unicellular Chlamydomonas reinhardtii culminates in the formation of diploid zygotes that differentiate into dormant spores that eventually undergo meiosis. Mating between gametes induces rapid cell wall shedding via the enzyme g-lysin; cell fusion is followed by heterodimerization of sex-specific homeobox transcription factors, GSM1 and GSP1, and initiation of zygote-specific gene expression. To investigate the genetic underpinnings of the zygote developmental pathway, we performed comparative transcriptome analysis of both pre- and post-fertilization samples. We identified 253 transcripts specifically enriched in early zygotes, 82% of which were not up-regulated in gsp1 null zygotes. We also found that the GSM1/GSP1 heterodimer negatively regulates the vegetative wall program at the posttranscriptional level, enabling prompt transition from vegetative wall to zygotic wall assembly. Annotation of the g-lysin-induced and early zygote genes reveals distinct vegetative and zygotic wall programs, supported by concerted up-regulation of genes encoding cell wall-modifying enzymes and proteins involved in nucleotide-sugar metabolism. The haploid-to-diploid transition in Chlamydomonas is masterfully controlled by the GSM1/GSP1 heterodimer, translating fertilization and gamete coalescence into a bona fide differentiation program. The fertilization-triggered integration of genes required to make related, but structurally and functionally distinct organelles-the vegetative versus zygote cell wall-presents a likely scenario for the evolution of complex developmental gene regulatory networks.

    DOI

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    33
    Citation
    (Scopus)
  • Holliday junction resolvases mediate chloroplast nucleoid segregation

    Yusuke Kobayashi, Osami Misumi, Masaki Odahara, Kota Ishibashi, Masafumi Hirono, Kumi Hidaka, Masayuki Endo, Hiroshi Sugiyama, Hiroshi Iwasaki, Tsuneyoshi Kuroiwa, Toshiharu Shikanai, Yoshiki Nishimura

    SCIENCE   356 ( 6338 ) 631 - 634  2017.05  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    Holliday junctions, four-stranded DNA structures formed during homologous recombination, are disentangled by resolvases that have been found in prokaryotes and eukaryotes but not in plant organelles. Here, we identify monokaryotic chloroplast 1 (MOC1) as a Holliday junction resolvase in chloroplasts by analyzing a green alga Chlamydomonas reinhardtii mutant defective in chloroplast nucleoid (DNA-protein complex) segregation. MOC1 is structurally similar to a bacterial Holliday junction resolvase, resistance to ultraviolet (Ruv) C, and genetically conserved among green plants. Reduced or no expression of MOC1 in Arabidopsis thaliana leads to growth defects and aberrant chloroplast nucleoid segregation. In vitro biochemical analysis and high-speed atomic force microscopic analysis revealed that A. thaliana MOC 1 (AtMOC1) binds and cleaves the core of Holliday junctions symmetrically. MOC1 may mediate chloroplast nucleoid segregation in green plants by resolving Holliday junctions.

    DOI

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    38
    Citation
    (Scopus)
  • Dynamic Interplay between Nucleoid Segregation and Genome Integrity in Chlamydomonas Chloroplasts

    Masaki Odahara, Yusuke Kobayashi, Toshiharu Shikanai, Yoshiki Nishimura

    PLANT PHYSIOLOGY   172 ( 4 ) 2337 - 2346  2016.12  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    The chloroplast (cp) genome is organized as nucleoids that are dispersed throughout the cp stroma. Previously, a cp homolog of bacterial recombinase RecA (cpRECA) was shown to be involved in the maintenance of cp genome integrity by repairing damaged chloroplast DNA and by suppressing aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. Here, overexpression and knockdown analysis of cpRECA in the green alga Chlamydomonas reinhardtii revealed that cpRECA was involved in cp nucleoid dynamics as well as having a role in maintaining cp genome integrity. Overexpression of cpRECA tagged with yellow fluorescent protein or hemagglutinin resulted in the formation of giant filamentous structures that colocalized exclusively to chloroplast DNA and cpRECA localized to cp nucleoids in a heterogenous manner. Knockdown of cpRECA led to a significant reduction in cp nucleoid number that was accompanied by nucleoid enlargement. This phenotype resembled those of gyrase inhibitor-treated cells and monokaryotic chloroplast mutant cells and suggested that cpRECA was involved in organizing cp nucleoid dynamics. The cp genome also was destabilized by induced recombination between short dispersed repeats in cpRECA-knockdown cells and gyrase inhibitor-treated cells. Taken together, these results suggest that cpRECA and gyrase are both involved in nucleoid dynamics and the maintenance of genome integrity and that the mechanisms underlying these processes may be intimately related in C. reinhardtii cps.

    DOI PubMed

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    6
    Citation
    (Scopus)
  • PCR-based Assay for Genome Integrity after Methyl Methanesulfonate Damage in Physcomitrella patens.

    Masaki Odahara, Takayuki Inouye, Yoshiki Nishimura, Yasuhiko Sekine

    bio-protocol   6 ( 19 ) e1954  2016.05  [Refereed]

    DOI

  • Sequence of the Gonium pectorale Mating Locus Reveals a Complex and Dynamic History of Changes in Volvocine Algal Mating Haplotypes

    Takashi Hamaji, Yuko Mogi, Patrick J. Ferris, Toshiyuki Mori, Shinya Miyagishima, Yukihiro Kabeya, Yoshiki Nishimura, Atsushi Toyoda, Hideki Noguchi, Asao Fujiyama, Bradley J. S. C. Olson, Tara N. Marriage, Ichiro Nishii, James G. Umen, Hisayoshi Nozaki

    G3-GENES GENOMES GENETICS   6 ( 5 ) 1179 - 1189  2016.05  [Refereed]

     View Summary

    Sex-determining regions (SDRs) or mating-type (MT) loci in two sequenced volvocine algal species, Chlamydomonas reinhardtii and Volvox carteri, exhibit major differences in size, structure, gene content, and gametolog differentiation. Understanding the origin of these differences requires investigation of MT loci from related species. Here, we determined the sequences of the minus and plus MT haplotypes of the isogamous 16-celled volvocine alga, Gonium pectorale, which is more closely related to the multicellular V. carteri than to C. reinhardtii. Compared to C. reinhardtii MT, G. pectorale MT is moderately larger in size, and has a less complex structure, with only two major syntenic blocs of collinear gametologs. However, the gametolog content of G. pectorale MT has more overlap with that of V. carteri MT than with C. reinhardtii MT, while the allelic divergence between gametologs in G. pectorale is even lower than that in C. reinhardtii. Three key sex-related genes are conserved in G. pectorale MT: GpMID and GpMTD1 in MT-, and GpFUS1 in MT+. GpFUS1 protein exhibited specific localization at the plus-gametic mating structure, indicating a conserved function in fertilization. Our results suggest that the G. pectorale-V. carteri common ancestral MT experienced at least one major reformation after the split from C. reinhardtii, and that the V. carteri ancestral MT underwent a subsequent expansion and loss of recombination after the divergence from G. pectorale. These data begin to polarize important changes that occurred in volvocine MT loci, and highlight the potential for discontinuous and dynamic evolution in SDRs.

    DOI PubMed

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    23
    Citation
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  • C-Terminal Region of Sulfite Reductase Is Important to Localize to Chloroplast Nucleoids in Land Plants

    Yusuke Kobayashi, Takuto Otani, Kota Ishibashi, Toshiharu Shikanai, Yoshiki Nishimura

    GENOME BIOLOGY AND EVOLUTION   8 ( 5 ) 1459 - 1466  2016.05  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    Chloroplast (cp) DNA is compacted into cpDNA-protein complexes, called cp nucleoids. An abundant and extensively studied component of cp nucleoids is the bifunctional protein sulfite reductase (SiR). The preconceived role of SiR as the core cp nucleoid protein, however, is becoming less likely because of the recent findings that SiRs do not associate with cp nucleoids in some plant species, such as Zea mays and Arabidopsis thaliana. To address this discrepancy, we have performed a detailed phylogenetic analysis of SiRs, which shows that cp nucleoid-type SiRs share conserved C-terminally encoded peptides (CEPs). The CEPs are likely to form a bacterial ribbon helix helix DNA-binding motif, implying a potential role in attaching SiRs onto cp nucleoids. A proof-of-concept experiment was conducted by fusing the nonnucleoid-type SiR from A. thaliana (AtSiR) with the CEP from the cp nucleoid-type SiR of Phaseolus yulgaris. The addition of the CEP drastically altered the intra-cp localization of AtSiR to cp nucleoids. Our analysis supports the possible functions of CEPs in determining the localization of SiRs to cp nucleoids and illuminates a possible evolutionary scenario for SiR as a cp nucleoid protein.

    DOI PubMed

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    6
    Citation
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  • A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha

    Christian R. Boehm, Minoru Ueda, Yoshiki Nishimura, Toshiharu Shikanai, Jim Haseloff

    PLANT AND CELL PHYSIOLOGY   57 ( 2 ) 291 - 299  2016.02  [Refereed]

     View Summary

    Recently, the liverwort Marchantia polymorpha has received increasing attention as a basal plant model for multicellular studies. Its ease of handling, well-characterized plastome and proven protocols for biolistic plastid transformation qualify M. polymorpha as an attractive platform to study the evolution of chloroplasts during the transition from water to land. In addition, chloroplasts of M. polymorpha provide a convenient test-bed for the characterization of genetic elements involved in plastid gene expression due to the absence of mechanisms for RNA editing. While reporter genes have proven valuable to the qualitative and quantitative study of gene expression in chloroplasts, expression of green fluorescent protein (GFP) in chloroplasts of M. polymorpha has proven problematic. We report the design of a codon-optimized gfp varian, mturq2cp, which allowed successful expression of a cyan fluorescent protein under control of the tobacco psbA promoter from the chloroplast genome of M. polymorpha. We demonstrate the utility of mturq2cp in (i) early screening for transplastomic events following biolistic transformation of M. polymorpha spores; (ii) visualization of stromules as elements of plastid structure in Marchantia; and (iii) quantitative microscopy for the analysis of promoter activity.

    DOI

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    17
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  • Eukaryotic Components Remodeled Chloroplast Nucleoid Organization during the Green Plant Evolution

    Yusuke Kobayashi, Mari Takusagawa, Naomi Harada, Yoichiro Fukao, Shohei Yamaoka, Takayuki Kohchi, Koichi Hori, Hiroyuki Ohta, Toshiharu Shikanai, Yoshiki Nishimura

    GENOME BIOLOGY AND EVOLUTION   8 ( 1 ) 1 - 16  2016.01  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    Chloroplast (cp) DNA is thought to originate from the ancestral endosymbiont genome and is compacted to form nucleoprotein complexes, cp nucleoids. The structure of cp nucleoids is ubiquitously observed in diverse plants from unicellular algae to flowering plants and is believed to be a multifunctional platform for various processes, including cpDNA replication, repair/recombination, transcription, and inheritance. Despite its fundamental functions, the protein composition for cp nucleoids in flowering plants was suggested to be divergent from those of bacteria and algae, but the evolutionary process remains elusive. In this research, we aimed to reveal the evolutionary history of cp nucleoid organization by analyzing the key organisms representing the three evolutionary stages of eukaryotic phototrophs: the chlorophyte alga Chlamydomonas reinhardtii, the charophyte alga Klebsormidium flaccidum, and the most basal land plant Marchantia polymorpha. To clarify the core cp nucleoid proteins in C. reinhardtii, we performed an LC-MS/MS analysis using highly purified cp nucleoid fractions and identified a novel SAP domain-containing protein with a eukaryotic origin as a constitutive core component. Then, homologous genes for cp nucleoid proteins were searched for in C. reinhardtii, K. flaccidum, and M. polymorpha using the genome databases, and their intracellular localizations and DNA binding activities were investigated by cell biological/biochemical analyses. Based on these results, we propose a model that recurrent modification of cp nucleoid organization by eukaryotic factors originally related to chromatin organization might have been the driving force for the diversification of cp nucleoids since the early stage of green plant evolution.

    DOI PubMed

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  • RECA plays a dual role in the maintenance of chloroplast genome stability in Physcomitrella patens

    Masaki Odahara, Takayuki Inouye, Yoshiki Nishimura, Yasuhiko Sekine

    PLANT JOURNAL   84 ( 3 ) 516 - 526  2015.11  [Refereed]

     View Summary

    Chloroplast DNA (cpDNA) encodes essential genes for chloroplast functions, including photosynthesis. Homologous recombination occurs frequently in cpDNA; however, its significance and underlying mechanism remain poorly understood. In this study, we analyzed the role of a nuclear-encoded chloroplast-localized homolog of RecA recombinase, which is a key factor in homologous recombination in bacteria, in the moss Physcomitrella patens. Complete knockout (KO) of the P. patens chloroplast RecA homolog RECA2 caused a modest growth defect and conferred sensitivity to methyl methanesulfonate and UV. The KO mutant exhibited low recovery of cpDNA from methyl methanesulfonate damage, suggesting that RECA2 knockout impairs repair of damaged cpDNA. The RECA2 KO mutant also exhibited reduced cpDNA copy number and an elevated level of cpDNA molecule resulting from aberrant recombination between short dispersed repeats (13-63 bp), indicating that the RECA2 KO chloroplast genome was destabilized. Taken together, these data suggest a dual role for RECA2 in the maintenance of chloroplast genome stability: RECA2 suppresses aberrant recombination between short dispersed repeats and promotes repair of damaged DNA.

    DOI PubMed

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    19
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  • Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha

    Kimitsune Ishizaki, Ryuichi Nishihama, Minoru Ueda, Keisuke Inoue, Sakiko Ishida, Yoshiki Nishimura, Toshiharu Shikanai, Takayuki Kohchi

    PLOS ONE   10 ( 9 ) e0138876  2015.09  [Refereed]

     View Summary

    We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3'-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3xCitrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3xFLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.

    DOI PubMed

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    177
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  • Algae Sense Exact Temperatures: Small Heat Shock Proteins Are Expressed at the Survival Threshold Temperature in Cyanidioschyzon merolae and Chlamydomonas reinhardtii

    Yusuke Kobayashi, Naomi Harada, Yoshiki Nishimura, Takafumi Saito, Mami Nakamura, Takayuki Fujiwara, Tsuneyoshi Kuroiwa, Osami Misumi

    GENOME BIOLOGY AND EVOLUTION   6 ( 10 ) 2731 - 2740  2014.10  [Refereed]

     View Summary

    The primitive red alga Cyanidioschyzon merolae inhabits acidic hot springs and shows robust resistance to heat shock treatments up to 63 A degrees C. Microarray analysis was performed to identify the key genes underlying the high temperature tolerance of this organism. Among the upregulated genes that were identified, we focused on two small heat shock proteins (sHSPs) that belong to a unique class of HSP families. These two genes are located side by side in an inverted repeat orientation on the same chromosome and share a promoter. These two genes were simultaneously and rapidly upregulated in response to heat shock treatment (&gt; 1,000-fold more than the control). Interestingly, upregulation appeared to be triggered not by a difference in temperatures, but rather by the absolute temperature. Similar sHSP structural genes have been reported in the green alga Chlamydomonas reinhardtii, but the threshold temperature for the expression of these sHSP-encoding genes in Ch. reinhardtii was different from the threshold temperature for the expression of the sHSP genes from Cy. merolae. These results indicate the possible importance of an absolute temperature sensing system in the evolution and tolerance of high-temperature conditions among unicellular microalgae.

    DOI PubMed

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    60
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  • chlB Requirement for Chlorophyll Biosynthesis under Short Photoperiod in Marchantia polymorpha L.

    Minoru Ueda, Ayumi Tanaka, Kazuhiko Sugimoto, Toshiharu Shikanai, Yoshiki Nishimura

    GENOME BIOLOGY AND EVOLUTION   6 ( 3 ) 620 - 628  2014  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    Chlorophylls (Chls) play pivotal roles in energy absorption and transduction and also in charge separation in reaction centers in all photosynthetic organisms. In Chl biosynthesis steps, only a step for the enzymatic reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is mediated by both nuclear- and chloroplast-encoded genes in land plants. Many plants encode the genes for light-dependent Pchlide reductase (LPOR) and light-independent Pchlide reductase (DPOR) in the nucleus and chloroplast genome, respectively. During the diversification of land plants, the reduction step of Pchlide to Chlide has become solely dependent on LPOR, and the genes for DPOR have been lost from chloroplast genome. It remains unclear why DPOR persists in some land plants, how they were eliminated from chloroplast genomes during the diversification of land plants, and under what environmental conditions DPOR was required. We demonstrate that DPOR is functional in liverwort (Marchantia polymorpha L.) and plays an important role in Chl biosynthesis. Having established a plastid transformation system in liverwort, we disrupted chlB, which encodes a subunit of DPOR in the M. polymorpha chloroplast genome. Morphological and Chl content analysis of a chlB mutant grown under different photoperiods revealed that DPOR is particularly required for Chl biosynthesis under short-day conditions. Our findings suggest that an environmental condition in the form of photoperiod is an important factor that determines the loss or retention of chloroplast-encoded genes mediating Pchlide reduction to Chlide.

    DOI PubMed

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    19
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  • Distribution of the Sex-Determining Gene MID and Molecular Correspondence of Mating Types within the Isogamous Genus Gonium (Volvocales, Chlorophyta)

    Takashi Hamaji, Patrick J. Ferris, Ichiro Nishii, Yoshiki Nishimura, Hisayoshi Nozaki

    PLoS ONE   8 ( 5 ) e64385  2013.05  [Refereed]

     View Summary

    Background:Isogamous organisms lack obvious cytological differences in the gametes of the two complementary mating types. Consequently, it is difficult to ascertain which of the two mating types are homologous when comparing related but sexual isolated strains or species. The colonial volvocalean algal genus Gonium consists of such isogamous organisms with heterothallic mating types designated arbitrarily as plus or minus in addition to homothallic strains. Homologous molecular markers among lineages may provide an "objective" framework to assign heterothallic mating types.Methodology/Principal Findings:Using degenerate primers designed based on previously reported MID orthologs, the "master regulator" of mating types/sexes in the colonial Volvocales, MID homologs were identified and their presence/absence was examined in nine strains of four species of Gonium. Only one of the two complementary mating types in each of the four heterothallic species has a MID homolog. In addition to heterothallic strains, a homothallic strain of G. multicoccum has MID. Molecular evolutionary analysis suggests that MID of this homothallic strain retains functional constraint comparable to that of the heterothallic strains.Conclusion/Significance:We coordinated mating genotypes based on presence or absence of a MID homolog, respectively, in heterothallic species. This scheme should be applicable to heterothallic species of other isogamous colonial Volvocales including Pandorina and Yamagishiella. Homothallism emerged polyphyletically in the colonial Volvocales, although its mechanism remains unknown. Our identification of a MID homolog for a homothallic strain of G. multicoccum suggests a MID-dependent mechanism is involved in the sexual developmental program of this homothallic species. © 2013 Hamaji et al.

    DOI PubMed J-GLOBAL

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    16
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  • Subfunctionalization of Sigma Factors during the Evolution of Land Plants Based on Mutant Analysis of Liverwort (Marchantia polymorpha L.) MpSIG1

    Minoru Ueda, Tsuneaki Takami, Lianwei Peng, Kimitsune Ishizaki, Takayuki Kohchi, Toshiharu Shikanai, Yoshiki Nishimura

    GENOME BIOLOGY AND EVOLUTION   5 ( 10 ) 1836 - 1848  2013  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    Sigma factor is a subunit of plastid-encoded RNA polymerase that regulates the transcription of plastid-encoded genes by recognizing a set of promoters. Sigma factors have increased in copy number and have diversified during the evolution of land plants, but details of this process remain unknown. Liverworts represent the basal group of embryophytes and are expected to retain the ancestral features of land plants. In liverwort (Marchantia polymorpha L.), we isolated and characterized a T-DNA-tagged mutant (Mpsig1) of sigma factor 1 (MpSIG1). The mutant did not show any visible phenotypes, implying that MpSIG1 function is redundant with that of other sigma factors. However, quantitative reverse-transcription polymerase chain reaction and RNA gel blot analysis revealed that genes related to photosynthesis were downregulated, resulting in the minor reduction of some protein complexes. The transcript levels of genes clustered in the petL, psaA, psbB, psbK, and psbE operons of liverwort were lower than those in the wild type, a result similar to that in the SIG1 defective mutant in rice (Oryza sativa). Overexpression analysis revealed primitive functional divergence between the SIG1 and SIG2 proteins in bryophytes, whereas these proteins still retain functional redundancy. We also discovered that the predominant sigma factor for ndhF mRNA expression has been diversified in liverwort, Arabidopsis (Arabidopsis thaliana), and rice. Our study shows the ancestral function of SIG1 and the process of functional partitioning (subfunctionalization) of sigma factors during the evolution of land plants.

    DOI PubMed J-GLOBAL

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    13
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  • Composition and physiological function of the chloroplast NADH dehydrogenase-like complex in Marchantia polymorpha

    Minoru Ueda, Tetsuki Kuniyoshi, Hiroshi Yamamoto, Kazuhiko Sugimoto, Kimitsune Ishizaki, Takayuki Kohchi, Yoshiki Nishimura, Toshiharu Shikanai

    PLANT JOURNAL   72 ( 4 ) 683 - 693  2012.11  [Refereed]

     View Summary

    The chloroplast NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport and chloro-respiration and consists of five sub-omplexes, which in angiosperms further associate with photosystem I (PSI) to form a super-complex. In Marchantia polymorpha, 11 plastid-encoded subunits and all the nuclear-encoded subunits of the A, B, membrane and ferredoxin-binding sub-complexes are conserved. However, it is unlikely that the genome of this liverwort encodes Lhca5 and Lhca6, both of which mediate NDHPSI super-complex formation. It is also unlikely that the subunits of the lumen sub-complex, PnsL1L4, are encoded by the genome. Consistent with this in silico prediction, the results of blue-native gel electrophoresis showed that NDH subunits were detected in a protein complex with lower molecular mass in Marchantia than the NDHPSI super-complex in Arabidopsis. Using the plastid transformation technique, we knocked out the ndhB gene in Marchantia. Although the wild-type genome copies were completely segregated out, the Delta ndhB lines grew like the wild-type photoautotrophically. A post-illumination transient increase in chlorophyll fluorescence, which reflects NDH activity in vivo in angiosperms, was absent in the thalli of the Delta ndhB lines. In ruptured chloroplasts, antimycin A-insensitive, and ferredoxin-dependent plastoquinone reduction was impaired, suggesting that chloroplast NDH mediates similar electron transport in Marchantia and Arabidopsis, despite its possible difference in structure. As in angiosperms, linear electron transport was not strongly affected in the Delta ndhB lines. However, the plastoquinone pool was slightly more reduced at low light intensity, suggesting that chloroplast NDH functions in redox balancing of the inter system, especially under low light conditions.

    DOI

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  • Gsp1 Triggers the Sexual Developmental Program Including Inheritance of Chloroplast DNA and Mitochondrial DNA in Chlamydomonas reinhardtii

    Yoshiki Nishimura, Toshiharu Shikanai, Soichi Nakamura, Maki Kawai-Yamada, Hirofumi Uchimiya

    PLANT CELL   24 ( 6 ) 2401 - 2414  2012.06  [Refereed]

    Authorship:Lead author

     View Summary

    The isogamous green alga Chlamydomonas reinhardtii has emerged as a premier model for studying the genetic regulation of fertilization and sexual development. A key regulator is known to be a homeoprotein gene, GAMETE-SPECIFIC PLUS1 (GSP1), which triggers the zygotic program. In this study, we isolated a mutant, biparental31 (bp31), which lacks GSP1. bp31 mt+ gametes fuse normally to form zygotes, but the sexual development of the resulting diploid cell is arrested and pellicle/zygospore/tetrad formation is abolished. The uniparental inheritance of chloroplast (cp) and mitochondrial (mt) DNA (cytoplasmic inheritance) was also impaired. bp31 has a deletion of similar to 60 kb on chromosome 2, including GSP1. The mutant phenotype was not rescued by transformation with GSP1 alone but could be rescued by the cotransformation with GSP1 and another gene, INOSITOL MONOPHOSPHATASE-LIKE1, which is involved in various cellular processes, including the phosphatidylinositol signaling pathway. This study confirms the importance of Gsp1 in mediating the zygotic program, including the uniparental inheritance of cp/mtDNA. Moreover, the results also suggest a role for inositol metabolism in the sexual developmental program.

    DOI PubMed J-GLOBAL

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  • Exploring the mystery of uniparental inheritance using the green algae, Chlamydomonas reinhardtii

      34 ( 5 ) 453 - 459  2012

    Authorship:Lead author, Corresponding author

    CiNii

  • Molecular mechanism of maternal inheritance and sexual differentiation

    Yoshiki Nishimura

    PLANT MORPHOLOGY   23 ( 1 ) 11 - 16  2011  [Refereed]

    Authorship:Lead author, Corresponding author

     View Summary

    Chloroplast (cp) and mitochondrial (mt) genomes are inherited almost exclusively from one parent in diverse taxa of plants and animals. Uniparental inheritance of mt/cp genomes was long thought to be the passive outcome of the fact that eggs contain multiple numbers of organelles whereas the contributions from male gametes are limited. However, the process is likely to be more dynamic because uniparental inheritance occurs in organisms that produce gametes of identical sizes (isogamous). In Chlamydomonas reinhardtii, uniparental inheritance of cp/mt genomes is achieved by a series of mating type-controlled events that actively eliminate mating type minus (mt-) cpDNA within 60 min after mating. How Chlamydomonas selectively degrades mt- cpDNA has long fascinated researchers and is the subject of this review.

    DOI CiNii

  • Molecular mechanism of cytoplasmic inheritance — 101 years of challenges —

    Yoshiki Nishimura

    PLANT MORPHOLOGY   23 ( 1 ) 1 - 2  2011  [Refereed]

    Authorship:Lead author, Corresponding author

     View Summary

    A symposium entitled "Molecular mechanism of cytoplasmic inheritance — 101 years of challenges —"was held at the 74th meeting of the Botanic Society of Japan in 2010 to commemorate 101th anniversary of the discovery of cytoplasmic inheritance. This symposium was co-supported by Journal of Plant Research and Japanese Society of Plant Morphology. Researchers studying various eukaryotes including slime molds, green algae, higher plants, and animals were invited to create an opportunity to overview the history and frontiers of researches in this field and to discuss the molecular mechanisms and significance of cytoplasmic inheritance during the evolution of eukaryotes.

    DOI CiNii

  • Differential Replication of Two Chloroplast Genome Forms in Heteroplasmic Chlamydomonas reinhardtii Gametes Contributes to Alternative Inheritance Patterns

    Yoshiki Nishimura, David B. Stern

    GENETICS   185 ( 4 ) 1167 - U61  2010.08  [Refereed]

    Authorship:Lead author

     View Summary

    Two mechanisms for chloroplast DNA replication have been revealed through the study of an unusual heteroplasmic strain of the green alga Chlamydomonas reinhardtii. Heteroplasmy is a state in which more than one genome type occurs in a mitochondrion or chloroplast. The Chlamydomonas strain spa19 bears two distinct chloroplast genomes, termed PS+ and PS- PS+ genomes predominate and are stably maintained in vegetative cells, despite their lack of known replication origins. In sexual crosses with spa19 as the mating type plus parent, however, PS+ genomes are transmitted in only similar to 25% of tetrads, whereas the PS- genomes are faithfully inherited in all progeny. In this research, we have explored the mechanism underlying this biased uniparental inheritance. We show that the relative reduction and dilution of PS+ vs. PS- genomes takes place during gametogenesis. Bromodeoxyuridine labeling, followed by immunoprecipitation and PCR, was used to compare replication activities of PS+ and PS- genomes. We found that the replication of PS+ genomes is specifically suppressed during gametogenesis and germination of zygospores, a phenomenon that also was observed when spa19 cells were treated with rifampicin, an inhibitor of the chloroplast RNA polymerase. Furthermore, when bromodeoxyuridine incorporation was compared at 11 sites within the chloroplast genome between vegetative cells, gametes, and rifampicin-treated cells by quantitative PCR, we found that incorporation was often reduced at the same sites in gametes that were also sensitive to rifampicin treatment. We conclude that a transcription-mediated form of DNA replication priming, which may be downregulated during gametogenesis, is indispensable for robust maintenance of PS+ genomes. These results highlight the potential for chloroplast genome copy number regulation through alternative replication strategies.

    DOI PubMed J-GLOBAL

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  • Principal component and hierarchical clustering analysis of metabolites in destructive weeds; polygonaceous plants

    Atsuko Miyagi, Hideyuki Takahashi, Kentaro Takahara, Takayuki Hirabayashi, Yoshiki Nishimura, Takafumi Tezuka, Maki Kawai-Yamada, Hirofumi Uchimiya

    METABOLOMICS   6 ( 1 ) 146 - 155  2010.03  [Refereed]

     View Summary

    Comprehensive analysis of metabolites using capillary electrophoresis-mass spectrometry was carried out in harmful weeds belonging to Polygonaceae. A principal component analysis revealed clear distinctions among eight Rumex species and Fallopia japonica. Hierarchical clustering data showed that respective metabolites can be grouped due to species differences. There was a positive relationship between oxalate and citrate, oxalate and ascorbate, and oxalate and glutamine. The amount of oxalate per leaf fresh weight was not affected by increased concentrations of exogenously supplied nutrients from Hoagland&apos;s formulation in one of the most destructive weeds R. obtusifolius. The oxalate accumulation in this plant is independent of external nutrient level, where nutrient-rich environments apparently stimulate internal constituents such as amino acids and other metabolites.

    DOI

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    59
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  • Uniparental inheritance of cpDNA and the genetic control of sexual differentiation in Chlamydomonas reinhardtii

    Yoshiki Nishimura

    JOURNAL OF PLANT RESEARCH   123 ( 2 ) 149 - 162  2010.03  [Refereed]

    Authorship:Lead author, Corresponding author

     View Summary

    An intriguing feature of most eukaryotes is that chloroplast (cp) and mitochondrial (mt) genomes are inherited almost exclusively from one parent. Uniparental inheritance of cp/mt genomes was long thought to be a passive outcome, based on the fact that eggs contain multiple numbers of organelles, while male gametes contribute, at best, only a few cp/mtDNA. However, the process is likely to be more dynamic because uniparental inheritance occurs in organisms that produce gametes of identical sizes (isogamous). In Chlamydomonas reinhardtii, the uniparental inheritance of cp/mt genomes is achieved by a series of mating type-controlled events that actively eliminate the mating type minus (mt-) cpDNA. The method by which Chlamydomonas selectively degrades mt- cpDNA has long fascinated researchers, and is the subject of this review.

    DOI PubMed CiNii J-GLOBAL

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    21
    Citation
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  • [Maturation and longevity of mRNA in chloroplasts: the functions of PPR proteins in RNA editing and the molecular mechanisms of RNA stability control in chloroplasts].

    Y. Nishimura, T. Shikanai

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   54 ( 16 Suppl ) 2098 - 2101  2009.12

    Authorship:Lead author

     View Summary

    Nishimura Y, Shikanai T, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 2009, vol. 54, no. 16 Suppl, pp. 2098-2101, 2009

    PubMed CiNii

  • Genome analysis and its significance in four unicellular algae, Cyanidioschyzon merolae, Ostreococcus tauri, Chlamydomonas reinhardtii, and Thalassiosira pseudonana (vol 121, pg 3, 2008)

    Osami Misumi, Yamato Yoshida, Keiji Nishida, Takayuki Fujiwara, Takayuki Sakajiri, Syunsuke Hirooka, Yoshiki Nishimura, Tsuneyoshi Kuroiwa

    JOURNAL OF PLANT RESEARCH   121 ( 2 ) 251 - 251  2008.03  [Refereed]

    DOI

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  • Genome analysis and its significance in four unicellular algae, Cyanidioshyzon merolae, Ostreococcus tauri, Chlamydomonas reinhardtii, and Thalassiosira pseudonana

    Osami Misumi, Yamato Yoshida, Keiji Nishida, Takayuki Fujiwara, Takayuki Sakajiri, Syunsuke Hirooka, Yoshiki Nishimura, Tsuneyoshi Kuroiwa

    JOURNAL OF PLANT RESEARCH   121 ( 1 ) 3 - 17  2008.01  [Refereed]

     View Summary

    Algae play a more important role than land plants in the maintenance of the global environment and productivity. Progress in genome analyses of these organisms means that we can now obtain information on algal genomes, global annotation and gene expression. The full genome information for several algae has already been analyzed. Whole genomes of the red alga Cyanidioshyzon merolae, the green algae Ostreococcus tauri and Chlamydomonas reinhardtii, and the diatom Thalassiosira pseudonana have been sequenced. Genome composition and the features of cells among the four algae were compared. Each alga maintains basic genes as photosynthetic eukaryotes and possesses additional gene groups to represent their particular characteristics. This review discusses and introduces the latest research that makes the best use of the particular features of each organism and the significance of genome analysis to study biological phenomena. In particular, examples of post-genome studies of organelle multiplication in C. merolae based on analyzed genome information are presented.

    DOI J-GLOBAL

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  • Maternal inheritance of chloroplast DNA: active destruction of male DNA

    Y. Nishimura

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.   50 ( 14 Suppl ) 1845 - 1846  2005.11

     View Summary

    Nishimura Y, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 2005, vol. 50, no. 14 Suppl, pp. 1845-1846, 2005

    PubMed

  • Antisense transcript and RNA processing alterations suppress instability of polyadenylated mRNA in chlarnydornonas chloroplasts

    Y Nishimura, EA Kikis, SL Zimmer, Y Komine, DB Stern

    PLANT CELL   16 ( 11 ) 2849 - 2869  2004.11  [Refereed]

    Authorship:Lead author

     View Summary

    In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain Delta26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. A collection of photosynthetic revertants was obtained from Delta26pAtE, and gel blot hybridizations revealed RNA processing alterations in the majority of these suppressor of polyadenylation (spa) strains, resulting in a failure to expose the atpB mRNA 3' poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PS+, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PS-, was required for viability but could not produce stable atpB transcripts. Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PS+ genome stabilizes atpB mRNA by generating an atpB antisense transcript, which attenuates the degradation of the polyadenylated form. The accumulation of double-stranded RNA was confirmed by insensitivity of atpB mRNA from PS+ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain Delta26, in which atpB mRNA is unstable because of the lack of a 3' stem-loop structure. In this context, when a 121-nucleotide segment of atpB antisense RNA was expressed from an ectopic site, an elevated accumulation of atpB mRNA resulted. Finally, when spa19 was placed in a genetic background in which expression of the chloroplast exoribonuclease polynucleotide phosphorylase was diminished, the PS+ genome and the antisense transcript were no longer required for photosynthesis. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 3'--&gt;5' exonuclease activity, a phenomenon that may occur naturally in the symmetrically transcribed and densely packed chloroplast genome.

    DOI PubMed J-GLOBAL

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    42
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  • Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D

    M Matsuzaki, O Misumi, T Shin-I, S Maruyama, M Takahara, SY Miyagishima, T Mori, K Nishida, F Yagisawa, K Nishida, Y Yoshida, Y Nishimura, S Nakao, T Kobayashi, Y Momoyama, T Higashiyama, A Minoda, M Sano, H Nomoto, K Oishi, H Hayashi, F Ohta, S Nishizaka, S Haga, S Miura, T Morishita, Y Kabeya, K Terasawa, Y Suzuki, Y Ishii, S Asakawa, H Takano, N Ohta, H Kuroiwa, K Tanaka, N Shimizu, S Sugano, N Sato, H Nozaki, N Ogasawara, Y Kohara, T Kuroiwa

    NATURE   428 ( 6983 ) 653 - 657  2004.04  [Refereed]

     View Summary

    Small, compact genomes of ultrasmall unicellular algae provide information on the basic and essential genes that support the lives of photosynthetic eukaryotes, including higher plants(1,2). Here we report the 16,520,305-base-pair sequence of the 20 chromosomes of the unicellular red alga Cyanidioschyzon merolae 10D as the first complete algal genome. We identified 5,331 genes in total, of which at least 86.3% were expressed. Unique characteristics of this genomic structure include: a lack of introns in all but 26 genes; only three copies of ribosomal DNA units that maintain the nucleolus; and two dynamin genes that are involved only in the division of mitochondria and plastids. The conserved mosaic origin of Calvin cycle enzymes in this red alga and in green plants supports the hypothesis of the existence of single primary plastid endosymbiosis. The lack of a myosin gene, in addition to the unexpressed actin gene, suggests a simpler system of cytokinesis. These results indicate that the C. merolae genome provides a model system with a simple gene composition for studying the origin, evolution and fundamental mechanisms of eukaryotic cells.

    DOI PubMed J-GLOBAL

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    894
    Citation
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  • Pelargonium embryogenesis: cytological investigations of organelles in early embryogenesis from the egg to the two-celled embryo

    H Kuroiwa, Y Nishimura, T Higashiyama, T Kuroiwa

    SEXUAL PLANT REPRODUCTION   15 ( 1 ) 1 - 12  2002.06  [Refereed]

     View Summary

    Changes in the distribution of organelles and organelle-DNA in Pelargonium zonale from the mature egg cell stage to the first zygotic division during the early stages of embryogenesis were investigated using electron microscopy and fluorescence microscopy. The mature egg is a large, polarized bulbous-shaped cell, tapering toward its micropylar end. The wide chalazal region has a large nucleus that is surrounded by cytoplasm containing many giant mitochondria and large amyloplasts. The mitochondria contain a large amount of mitochondrial DNA and appear as long stretched rods or complex rings, sometimes consisting of several concentric or half-concentric circles in sections. The time from pollination to cell fusion is approximately 6-9 h and it is 20-24 h until the first zygotic division. The changes in the zygote and its organelles preparatory to division occur in 3 stages. At stage 1 (6-9 h after pollination), cell fusion occurs and the zygote begins to elongate. Many vacuoles of varying size appear surrounding the nucleus. At stage 2 (9-15 h), the zygote nucleus migrates to a central position in the cell and the mitochondria form a single ring that becomes either irregularly crushed or appears as long thin strings. Amyloplasts exhibit a gradual decrease in the number of starch grains. At stage 3 (15-20 h), the vacuoles disappear, except for a few that remain in the micropylar region, and cell size decreases. Mitochondria become short, fine strings or small rings. Amyloplasts with starch grains are no longer observed, but are transformed into large proplastids. Following the first division of the zygote, approximately equal-sized apical and basal cells are formed. Short rod-shaped or small ring-shaped mitochondria are randomly distributed near the nucleus of the apical cell, whereas mitochondria in the basal cell are long and rod-shaped. In the electron microscope, two types of plastids can be distinguished: dark oval plastids originating from the sperm cell, which are observed in both the apical and basal cell, and others with a less dense, amorphous matrix, believed to originate from egg amyloplasts, which are unevenly distributed in the micropylar region of the basal cell. Fluorometry using a video-intensified microscope photon counting system reveals that, correlated with changes in mitochondrial morphology, DNA amount within the mitochondrion decreases linearly during these stages.

    DOI J-GLOBAL

    Scopus

    10
    Citation
    (Scopus)
  • An mt(+) gamete-specific nuclease that targets mt(-) chloroplasts during sexual reproduction in C-reinhardtii

    Y Nishimura, O Misumi, K Kato, N Inada, T Higashiyama, Y Momoyama, T Kuroiwa

    GENES & DEVELOPMENT   16 ( 9 ) 1116 - 1128  2002.05  [Refereed]

    Authorship:Lead author, Corresponding author

     View Summary

    Although the active digestion of mating-type minus (mt(-)) chloroplast DNA (cpDNA) in young zygotes is considered to be the basis for the uniparental inheritance of cpDNA in Chlamydomonas reinhardtii, little is known about the underlying molecular mechanism. One model of active digestion proposes that nucleases are either synthesized or activated to digest mt(-) cpDNA. We used a native-PAGE/in gelo assay to investigate nuclease activities in chloroplasts from young zygotes, and identified a novel Ca2+-dependent nuclease activity. The timing of activation (similar to60-90 min after mating) and the localization of the nuclease activity (in mt(-) chloroplasts) coincided with the active digestion of mt(-) cpDNA. Furthermore, the activity of the nuclease was coregulated with the maturation of mating-type plus (mt(+)) gametes, which would enable the efficient digestion of mt(-) cpDNA. Based on these observations, we propose that the nuclease (designated as (M) under bart(+)-specific (DN) under bar ase, MDN) is a developmentally controlled nuclease that is activated in mt(+) gametes and participates in the destruction of mt(-) cpDNA in young zygotes, thereby ensuring uniparental inheritance of chloroplast traits.

    DOI PubMed J-GLOBAL

    Scopus

    66
    Citation
    (Scopus)
  • Pollen tube attraction by the synergid cell

    T Higashiyama, S Yabe, N Sasaki, Y Nishimura, S Miyagishima, H Kuroiwa, T Kuroiwa

    SCIENCE   293 ( 5534 ) 1480 - 1483  2001.08  [Refereed]

     View Summary

    In flowering plants, guidance of the pollen tube to the embryo sac (the haploid female gametophyte) is critical for successful fertilization. The target embryo sac may attract the pollen tube as the final step of guidance in the pistil. We show by laser Ceti ablation that two synergid cells adjacent to the egg cell attract the pollen tube. A single synergid cell was sufficient to generate an attraction signal, and two cells enhanced it. After fertilization, the embryo sac no longer attracts the pollen tube, despite the persistence of one synergid cell. This cessation of attraction might be involved in blocking polyspermy.

    DOI PubMed J-GLOBAL

    Scopus

    334
    Citation
    (Scopus)
  • Effects of chloroplast DNA content on the cell proliferation and aging in Chlamydomonas reinhardtii

    O Misumi, Y Nishimura, T Kuroiwa

    JOURNAL OF PLANT RESEARCH   114 ( 1114 ) 125 - 131  2001.06  [Refereed]

     View Summary

    Chlamydomonas is an unicellular green alga that contains one cup-shaped chloroplast with about 60 copies of cpDNA. Chloroplasts (cp) multiply in the cytoplasm of the plant cell by binary division, with multiple copies of cpDNA transmitted and maintained in successive generations. The effect of cpDNA copy number on cell proliferation and aging was investigated using a C. reinhardtii moc mutant, which has an undispersed cp-nucleoid and unequal segregation of cpDNA during cell division. When the mother cell divided into four daughters, one moc daughter cell chloroplast contained about 60 copies of cpDNA, and the chloroplasts in the three other daughter cells contained the 4-7 copies of cpDNA. In liquid medium, the number of moc cells at the period of stationary phase was about one-third that of the wild type. To observe the process of proliferation and aging in the mother cell, we used solid medium. Three out of four moc cell spores were preferentially degenerated 60 days after cell transfer. To confirm this, wild-type and moc mother cells containing four daughter cells were treated with novobiocin to inhibit cpDNA replication. Cell degeneration increased only in the moc strain following novobiocin introduction. In total, our results suggest that cells possessing smaller amounts of cpDNA degenerate and age more rapidly.

    DOI

    Scopus

    5
    Citation
    (Scopus)
  • The chloroplast clpP gene, encoding a proteolytic subunit of ATP-dependent protease, is indispensable for chloroplast development in tobacco

    T Shikanai, K Shimizu, K Ueda, Y Nishimura, T Kuroiwa, T Hashimoto

    PLANT AND CELL PHYSIOLOGY   42 ( 3 ) 264 - 273  2001.03  [Refereed]

     View Summary

    ClpP is a proteolytic subunit of the ATP-dependent Clp protease, which is found in chloroplasts in higher plants. Proteolytic subunits are encoded both by the chloroplast gene, clpP, and a nuclear multi gene family. We insertionally disrupted clpP by chloroplast transformation in tobacco. However, complete segregation was impossible, indicating that the chloroplast-encoded clpP gene has an indispensable function for cell survival, In the heteroplasmic clpP disruptant, the leaf surface was rough by clumping, and the lateral leaf expansion was irregularly arrested, which led to an asymmetric, slender leaf shape, Chloroplasts consisted of two populations: chloroplasts that were similar to the wild type, and small chloroplasts that emitted high chi fluorescence. Ultrastructural analysis of chloroplast development suggested that clpP disruption also induced swelling of the thylakoid lumen in the meristem plastids and inhibition of etioplast development in the dark. Tn mature leaves, thylakoid membranes of the smaller chloroplast population consisted exclusively of large stacks of tightly appressed membranes. These results indicate that chloroplast-encoded ClpP is involved in multiple processes of chloroplast development, including a housekeeping function that is indispensable for cell survival.

    DOI PubMed CiNii J-GLOBAL

    Scopus

    147
    Citation
    (Scopus)
  • Effects of chloroplast DNA content on the cell proliferation and aging in Chlamydomonas reinhardtii

    O. Misumi, Y. Nishimura, T. Kuroiwa

    Journal of Plant Research   114 ( 1114 ) 125 - 131  2001  [Refereed]

    DOI CiNii

  • Characterization of novel cytokinesis-defective mutants of Chlamydomonas reinhardtii

    O. Misumi, Y. Nishimura, T. Kuroiwa

    Cytologia   65 ( 4 ) 429 - 434  2000  [Refereed]

     View Summary

    We isolated 2 independent cytokinesis-defective mutants, inc-1 and inc-2, of the unicellular green alga Chlamydomonas reinhardtii through DNA insertional mutagenesis. Characterization of the mutants, using fluorescence microscopy and microfluorometry, revealed that progression of furrowing begins in inc cells after nuclear division. Unlike wild-type cells, however, the cleavage furrow of the dividing cells disappears before cytokinesis. Successive nuclear divisions with incomplete cytokinesis produce multinucleate cells. Additionally, chloroplast autofluorescence revealed that the chloroplast fails to divide throughout the process of cytokinesis in the mutant cells. We also examined the number of chloroplast nucleoids (cp-nucleoids) and chloroplast DNA (cpDNA) content per chloroplast as indicators of cell cycle. We observed cp-nucleoids through DAPI staining and measured the intensity of DAPI fluorescence with a video-intensified microscope photon-counting system (VIMPCS). Both the number of cp-nucleoids and cpDNA content per chloroplast increased under incomplete cytokinesis in inc cells. The inc mutants represent novel cytokinesis-defective mutants of C. reinhardtii.

    DOI CiNii J-GLOBAL

    Scopus

    1
    Citation
    (Scopus)
  • Comparative analysis of DNA synthesis activity in plastid-nuclei and mitochondrial-nuclei simultaneously isolated from cultured tobacco cells

    A Sakai, T Suzuki, N Nagata, N Sasaki, Y Miyazawa, C Saito, N Inada, Y Nishimura, T Kuroiwa

    PLANT SCIENCE   140 ( 1 ) 9 - 19  1999.01  [Refereed]

     View Summary

    Plastid-nuclei (plastid-nucleoids) and mitochondrial-nuclei (mitochondrial-nucleoids) were simultaneously isolated from cultured tobacco cells (Nicotiana tabacum L., line BY-2), and their activity synthesizing DNA in vitro was examined. The isolated plastid- and mitochondrial-nuclei incorporated about 20 and 50 pmol of dCTP, respectively, into DNA per microgram of template DNA during a 60-min incubation. The DNA synthetic activity of the two organelle-nuclei exhibited similar responses to various inhibitors; it was resistant to aphidicolin and sensitive to N-ethylmaleimide and high concentrations of ddCTP, which are all characteristics of gamma-like DNA polymerase. The responses of the DNA synthetic activity in the two organelle-nuclei to pH and divalent- and monovalent-metal cation concentrations were also similar. Moreover, an in situ DNA polymerase assay following SDS-polyacrylamide gel electrophoresis revealed that DNA. polymerases with an apparent molecular mass of 116 kDa were present in both the isolated plastid- and mitochondrial-nuclei, and that the two 116-kDa DNA polymerases were quite similar in terms of their sensitivity to various inhibitors, optimum assay conditions, and template preferences. These results indicate that DNA replication in plastids and mitochondria may be conducted by DNA polymerases that have quite similar characteristics. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

    DOI

    Scopus

    12
    Citation
    (Scopus)
  • Isolation and phenotypic characterization of Chlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

    O Misumi, L Suzuki, Y Nishimura, A Sakai, S Kawano, H Kuroiwa, T Kuroiwa

    PROTOPLASMA   209 ( 3-4 ) 273 - 282  1999  [Refereed]

     View Summary

    Each wild-type Chlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we named moc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed that moc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in the moc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2-8 copies) of cpDNA, However, most individual moc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of the moc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure of moc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells and moc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novel moc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.

    DOI

    Scopus

    27
    Citation
    (Scopus)
  • Active digestion of mt+derived chloroplast DNA in individual zygotes of Chlamydomonas reinhardtii revealed by the Optical tweezers.

    Nishimura, Y, Misumi, O, Matsunaga, S, Higashiyama. T, Yokota, A, Kuroiwa, T

    Proc. Natl. Acad. Sci. USA   96   12577 - 12582  1999  [Refereed]

  • Comparative analysis of DNA synthesis activity in plastid-nuclei and mitochondrial-nuclei simultaneously isolated from cultured tobacco cells

    A Sakai, T Suzuki, N Nagata, N Sasaki, Y Miyazawa, C Saito, N Inada, Y Nishimura, T Kuroiwa

    PLANT SCIENCE   140 ( 1 ) 9 - 19  1999.01  [Refereed]

     View Summary

    Plastid-nuclei (plastid-nucleoids) and mitochondrial-nuclei (mitochondrial-nucleoids) were simultaneously isolated from cultured tobacco cells (Nicotiana tabacum L., line BY-2), and their activity synthesizing DNA in vitro was examined. The isolated plastid- and mitochondrial-nuclei incorporated about 20 and 50 pmol of dCTP, respectively, into DNA per microgram of template DNA during a 60-min incubation. The DNA synthetic activity of the two organelle-nuclei exhibited similar responses to various inhibitors; it was resistant to aphidicolin and sensitive to N-ethylmaleimide and high concentrations of ddCTP, which are all characteristics of gamma-like DNA polymerase. The responses of the DNA synthetic activity in the two organelle-nuclei to pH and divalent- and monovalent-metal cation concentrations were also similar. Moreover, an in situ DNA polymerase assay following SDS-polyacrylamide gel electrophoresis revealed that DNA. polymerases with an apparent molecular mass of 116 kDa were present in both the isolated plastid- and mitochondrial-nuclei, and that the two 116-kDa DNA polymerases were quite similar in terms of their sensitivity to various inhibitors, optimum assay conditions, and template preferences. These results indicate that DNA replication in plastids and mitochondria may be conducted by DNA polymerases that have quite similar characteristics. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

    DOI DOI2

    Scopus

    12
    Citation
    (Scopus)
  • The biparental transmission of the mitochondrial genome in Chlamydomonas reinhardtii visualized in living cells

    Y Nishimura, T Higashiyama, L Suzuki, O Misumi, T Kuroiwa

    EUROPEAN JOURNAL OF CELL BIOLOGY   77 ( 2 ) 124 - 133  1998.10  [Refereed]

     View Summary

    In the isogamous green alga Chlamydomonas reinhardtii, the chloroplast genome is transmitted from the mt(+) parent, while the mitochondrial genes are believed to be inherited from the mt(-) parent. Chloroplast nucleoids have been visualized by DAPI (4,6-diamidino-2-phenylindole) staining, and the preferential digestion of the mt chloroplast nucleoids has been observed in young zygotes. However, the mitochondrial nucleoids have never been visualized, and their behavior is only deduced from genetic and biochemical studies.
    We discovered that the mitochondrial and chloroplast genomes can be visualized simultaneously in living cells, using the fluorescent dye SYBR GreenI. The ability to visualize the mitochondrial and chloroplast genome in vivo permits the direct observation of the number, distribution and behavior of the chloroplast and mitochondrial nucleoids in young zygotes. Using this method, the biparental transmission of the mitochondrial genome was revealed.

    DOI

  • The biparental transmission of the mitochondrial genome in Chlamydomonas reinhardtii visualized in living cells

    Y Nishimura, T Higashiyama, L Suzuki, O Misumi, T Kuroiwa

    EUROPEAN JOURNAL OF CELL BIOLOGY   77 ( 2 ) 124 - 133  1998.10  [Refereed]

     View Summary

    In the isogamous green alga Chlamydomonas reinhardtii, the chloroplast genome is transmitted from the mt(+) parent, while the mitochondrial genes are believed to be inherited from the mt(-) parent. Chloroplast nucleoids have been visualized by DAPI (4,6-diamidino-2-phenylindole) staining, and the preferential digestion of the mt chloroplast nucleoids has been observed in young zygotes. However, the mitochondrial nucleoids have never been visualized, and their behavior is only deduced from genetic and biochemical studies.
    We discovered that the mitochondrial and chloroplast genomes can be visualized simultaneously in living cells, using the fluorescent dye SYBR GreenI. The ability to visualize the mitochondrial and chloroplast genome in vivo permits the direct observation of the number, distribution and behavior of the chloroplast and mitochondrial nucleoids in young zygotes. Using this method, the biparental transmission of the mitochondrial genome was revealed.

    PubMed

  • Optical isolation of individual mitochondria of Physarum polycephalum for PCR analysis

    T Kuroiwa, K Ishibashi, H Takano, T Higashiyama, N Sasaki, Y Nishimura, S Matsunaga

    PROTOPLASMA   194 ( 3-4 ) 275 - 279  1996  [Refereed]

     View Summary

    We attempted to amplify a specific region of mitochondrial DNA (mtDNA) using the polymerase chain reaction (PCR) from fewer than ten mitochondria isolated individually by microdissection or use of an optical tweezer. We selected preliminarily isolated mitochondria from Physarum polycephalum as the model materials and tried to amplify the mtDNA region corresponding to the specific mitochondrial plasmid of this true slime mould. For separation of a few mitochondria from the mitochondrial population, we initially used a destruction method in which excluded mitochondria were disrupted by a UV laser. However, mtDNA was still amplified, although weakly, from mitochondria that had been destroyed by the UV laser. Therefore, we used an optical tweezer to trap individual mitochondria and separate them from the others. The required number of mitochondria were separated from the mitochondrial suspension through a narrow canal of isolation buffer and used directly for PCR amplification. The results showed that the mtDNA could be amplified from at least 9 mitochondria trapped by the optical tweezer.

    DOI

    Scopus

    11
    Citation
    (Scopus)

▼display all

Presentations

  • Sequencing the mating type locus of the isogamous colonial Gonium pectorale

    15th International Conference on the Cell & Molecular Biology of Chlamydomonas 

    Presentation date: 2013.06

  • RecAホモログによる葉緑体ゲノム安定性の維持

    第54回日本植物生理学会年会  日本植物生理学会

    Presentation date: 2013.03

  • 緑藻クラミドモナスの生殖とオルガネラ遺伝をつなぐ遺伝子を探る

    第54回日本植物生理学会年会  日本植物生理学会

    Presentation date: 2013.03

  • 母性遺伝を操る生殖プログラムの構造

    日本植物学会第76回大会  日本植物学会

    Presentation date: 2012.09

  • 単細胞緑藻クラミドモナスにおいて細胞質遺伝はGsp1によって制御される

    日本植物形態学会第24回大会  日本植物形態学会

    Presentation date: 2012.09

  • Gsp1 triggers a sexual developmental program including the cytoplasmic inheritance in Chlamydomonas reinhardtii

    15th International conference on the cell & molecular biology of Chlamydomonas 

    Presentation date: 2012.06

▼display all

Research Projects

  • Genomic regulation of intracellular symbiotic organelles: from technological innovation to understanding and application of life phenomena

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2024.04
    -
    2029.03
     

  • Elucidation of the regulatory mechanism of chloroplast nucleoid with DNA supercoils as the core

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    Project Year :

    2021.04
    -
    2024.03
     

  • Exploring the molecular mechanism of cell cycle-mediated solid&#8211;liquid phase transition of chloroplast nucleoids

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2021.09
    -
    2023.03
     

  • Harnessing the uniparental inheritance of chloroplasts

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

    Project Year :

    2019.04
    -
    2021.03
     

  • Hacking the sexual program of green algae by functional polyamides

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)

    Project Year :

    2018.06
    -
    2021.03
     

    Nishimura Yoshiki

     View Summary

    Functional polyamide is an artificial chemical that binds directly to a specific target sequence. The target sequence can be arbitrarily designed and applied to suppress the expression of target genes. Using this technology, we analyzed ZYRE, a cis-element of the zygote specific genes of the unicellular green alga Chlamydomonas. The first barrier in this research was the improvement of cell permeability, which was solved by the addition of Arginine and TAMRA. RT-qPCR analysis confirmed the repression of zygote specific gene expression by polyamide treatment targeting ZYRE, supporting the importance of ZYRE in the zygote-specific gene expression in Chlamydomonas.

  • Exploring the dynamism of chloroplastic chromosomes, nucleoids

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    Project Year :

    2018.04
    -
    2021.03
     

    Nishimura Yoshiki

     View Summary

    Compaction of bulky DNA is a universal issue for all DNA-based life forms. Both Chloroplasts and mitochondria maintain their own multi-copy genomes organized as nucleoids, but the mechanism of DNA compaction remains obscure. Here, we discovered a chloroplast nucleoid protein (HBD1) that is highly similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p) in terms of possessing two DNA-binding high mobility group box (HMG-box) domains. Our analyses of HBD1 based on DNA origami/atomic force microscopy showed that HBD1 is capable of compacting DNA by introducing U-turns and cross-strand bridges with the two HMG-box domains, indicating that proteins with two HMG-box domains could compact DNA in both mitochondrial and chloroplast nucleoids.

  • Harnessing chloroplast inheritance

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

    Project Year :

    2017.04
    -
    2019.03
     

  • Deciphering the dynamics of chloroplast nucleoid network

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

    Project Year :

    2016.04
    -
    2019.03
     

    Nishimura Yoshiki

     View Summary

    Chloroplast (cp) DNA and the gene expression machinery are the fundamental system for the photosynthesis and biogenesis of chloroplasts. CpDNA molecules are packaged by multiple number of proteins into a minute globular structure called cp nucleoids. Cp nucleoids are the platform for multiple processes including DNA replication, repair, transcription, and inheritance. In this research, the process of chloroplast nucleoid division was visualized by our microfluidic device-based live imaging technology. Furthermore, chloroplast-localized Holliday junction resolvase was identified as a critical factor for the division of cp nucleoids.

  • The molecular mechanism of uniparental inheraitance of mtDNA analyzed by combining the optical tweezers and microfluidic device.

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

    Project Year :

    2014.04
    -
    2017.03
     

    Nishimura Yoshiki

     View Summary

    Living organisms consist of diverse tissues, cells and organelles, which can be compared to a complex society with various groups and individuals. To decipher the characteristics of individual cells and connections between them, it would be necessary to establish technologies to isolate individual cells for further molecular analyses. For this, we aimed to combine the optical tweezers and microfluidic devises. With this technique, the molecular mechanism of uniparental inheritance of mtDNA was analyzed using Cryptococcus neoformans as a model system, which revealed the active elimination of α-mtDNA in the early developing zygotes that lead to the uniparental inheritance of mtDNA.

  • Evolutional strategy for the establishment of angiosperm-type chloroplast NDH

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

    Project Year :

    2010.04
    -
    2013.03
     

    SHIKANAI TOSHIHARU, TSUGEKI Ryuuji, NISHIMURA Yoshiki

     View Summary

    We discovered that a novel protein, NdhS is necessary for the high-affinity binding of chloroplast NDH with ferredoxin. Chloroplast NDH is not NAD(P)H dehydrogenase but antimycin A-resistant ferredoxin-dependent plastoquinone reductase (FQR). We also clarified the assembly process of subcomplex A of chloroplast NDH in Arabidopsis with the discovery of several assembly factors specifically required for this process. We further discovered that in Marchantia chloroplast NDH does not form the supercomplex with photosystem I and is involved in the redox homeostasis in chloroplasts at low light intensity.

  • 母性遺伝の分子プログラム解読に挑む

    日本学術振興会  科学研究費助成事業 若手研究(A)

    Project Year :

    2011
     
     
     

    西村 芳樹

  • Exploring the molecular mechanism of maternal inheritance

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

    Project Year :

    2007
    -
    2009
     

    NISHIMURA Yoshiki

     View Summary

    Uniparental inheritance of mitochondrial (mt) and chloroplast (cp) DNA is a phenomenon common to diverse taxa of plants and animals. Uniparental inheritance was long thought to be a passive outcome, based on the fact that eggs contain multiple numbers of organelles, while the contributions from male gametes are limited. However, it is likely to be more dynamic process, including the active digestion of male mt/cpDNA. In this project, Chlamydomonas mutants defective in uniparental inheritance of cpDNA were obtained and analyzed, which revealed that a homeotic gene that regulate the sexual development is also a key factor for uniparental inheritance.

  • 単一細胞の顕微解析による細胞質遺伝機構の分子形態学的研究

    日本学術振興会  科学研究費助成事業 特別研究員奨励費

    Project Year :

    2001
    -
    2003
     

    西村 芳樹

     View Summary

    細胞内小器官であるミトコンドリアや葉緑体は、各々細胞核とは異なる独自のDNAを保持しており、それらはヒトを含む多くの動植物において母性遺伝する。従来、この現象は雌雄の配偶子の大きさの違いに基づき、確率論的に説明されてきた。しかし全く同型の配偶子により生殖を行う単細胞緑藻クラミドモナス(Chlamydomonas reinhardtii)においても葉緑体の母性遺伝が観察されたことから(Sager,1954)、より積極的な母性遺伝制御機構の存在が示唆されてきた。1999年、我々はこのクラミドモナスを用い、雄葉緑体DNAが接合後90分以内に積極的かつ完全に分解されることを示した(Nishimura et al.,1999 PNAS)。そして今回、さらに雄葉緑体DNAの分解に関わる酵素の解析を行った。
    我々はまず、接合子より無傷葉緑体を新規に開発したエアーブラシ法によって単離し、その中に含まれるDNA分解酵素活性の変化を、接合後の時間を追って観察した。DNA分解酵素の検出には、非変性条件で酵素活性を分析できるNative-PAGE in gel assay法を採用した。その結果、接合後90分後までに活性がピークに達する分子量140kDaの新規DNA分解酵素活性が検出された。さらに、葉緑体内のデンプン蓄積量が違う雌雄の配偶子を接合させることにより、接合子から雌雄の葉緑体をそれぞれ密度勾配遠心法で選択的に取り出す方法を開発した。この方法を用いて雌雄の葉緑体におけるDNA分解酵素活性の変化を比較したところ、DNA分解酵素活性の上昇は、父方の葉緑体でのみ起きることが明らかとなった。今回発見されたDNA分解酵素活性の上昇のタイミング、及び局在は、雄葉緑体DNAの分解と正確に一致するものであり、これが母性遺伝に関与する可能性が強く示唆されることとなった。
    次に、我々は様々な生活環の細胞におけるDNA分解酵素活性の比較を行った。その結果、このDNA分解酵素活性は、配偶子誘導とともに雌の細胞でのみ誘導されることが明らかになった。以上の結果より、我々は今回発見された新規DNA分解酵素を雌配偶子特異的DNase (Mating type minus gamete specific DNase : MDN)と命名し、このMDNに基づく新しい母性遺伝機構のモデルを提唱した(Nishimura et al.,2002 Genes & Dev.)。

  • 顕微操作による細胞質遺伝の機構に関する分子形態学的研究

    日本学術振興会  科学研究費助成事業 特別研究員奨励費

    Project Year :

    1998
    -
    2000
     

    西村 芳樹

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Misc

  • Dynamic motion of chloroplast nucleoids captured by the microfluidic system

    Yoshitaka Kamimura, Yusuke Kobayashi, Yoshiki Nishimura

    Cytologia   85 ( 3 ) 177 - 178  2020.09

    Rapid communication, short report, research note, etc. (scientific journal)  

    DOI

  • Plastoquinone-mediated electron transport in a non-photosynthetic plastid of the heterotrophic green alga Hyalogonium sp.

    Motoki Kayama, Jun-Feng Chen, Takashi Nakada, Yoshiki Nishimura, Toshiharu Shikanai, Hideaki Miyashita, Yuichiro Kashiyama, Ryoma Kamikawa

    VIII ECOP-ISOP 2019    2019.07  [Refereed]

  • オートファジーに依存した緑藻Chlamydomonas reinhardtiiの生存と油脂蓄積

    梶川 昌孝, 山内 万里花, 新川 はるか, 田中 学, 幡野 恭子, 西村 芳樹, 加藤 美砂子, 福澤 秀哉

    藻類   67 ( 1 ) 70  2019.03

    Research paper, summary (national, other academic conference)  

  • ペンタトリコペプチドリピートタンパク質PGR3は葉緑体コードNDHサブユニットであるndhGの翻訳を制御する

    東遥香, 加藤義宣, 藤田智也, 藤田智也, 水戸麻理, 岩崎信太郎, 岩崎信太郎, 西村芳樹, 竹中瑞樹, 鹿内利治

    日本植物生理学会年会(Web)   60th  2019

    J-GLOBAL

  • 緑色植物における葉緑体DNAリガーゼのバクテリア型から真核生物型への機能移譲

    浜地貴志, 小林優介, 山岡尚平, 鹿内利治, 西村芳樹

    日本植物学会大会研究発表記録   83rd  2019

    J-GLOBAL

  • 母性遺伝メカニズムの解明に向けたクラミドモナス有性生殖のゲノム編集アプローチ

    浜地貴志, 山岡尚平, 鹿内利治, 西村芳樹

    日本植物学会大会研究発表記録   82nd   256  2018.09

    J-GLOBAL

  • ゲノム編集の時代における「緑の酵母」クラミドモナスの復権

    浜地貴志, 西村芳樹

    生物工学会誌   96 ( 8 ) 475  2018.08

    J-GLOBAL

  • オートファジーを欠損させた緑藻における窒素欠乏応答異常

    梶川昌孝, 山内万里香, 新川はるか, 田中学, 幡野恭子, 西村芳樹, 加藤美砂子, 福澤秀哉

    第59回日本植物生理学会年会要旨集    2018.03

    Research paper, summary (national, other academic conference)  

  • オートファジーを欠損した緑藻における窒素欠乏応答異常

    梶川昌孝, 山内万里花, 新川はるか, 田中学, 幡野恭子, 西村芳樹, 奥公秀, 阪井康能, 加藤美砂子, 加藤美砂子, 福澤秀哉

    日本植物学会大会研究発表記録   81st   137  2017.09

    J-GLOBAL

  • 葉緑体ゲノムの分配はHollidayジャンクション解離酵素MOC1により保障される

    小林 優介, 三角 修己, 西村 芳樹

    ライフサイエンス 新着論文レビュー    2017.05  [Invited]

  • 葉緑体核様体コア因子の多様性と進化

    小林優介, 田草川真理, 田草川真理, 原田尚実, 深尾陽一朗, 深尾陽一朗, 山岡尚平, 河内孝之, 堀孝一, 太田啓之, 太田啓之, 鹿内利治, 西村芳樹

    日本植物学会大会研究発表記録   79th   165  2015.09

    J-GLOBAL

  • Identification of a gene which is in the intermediate stage of gene loss from chloroplast genome in Marchantia polymorpha

    Ueda, M, Tanaka, A, Shikanai, T, Nishimura, Y

    日本分子生物学会、第35回年会、於 神戸ポートアイランド、2013年12月    2013  [Refereed]

  • ゼニゴケ葉緑体RNAポリメラーゼシグマ因子(MpSIG1)の機能解析から明らかとなった陸上植物進化におけるシグマ因子の機能分化

    上田 実, 高見 常明, Peng Lianwei, 鹿内 利治, 西村 芳樹

    日本植物生理学会シンポジウム、第52回年会、於 京都産業大学、2012年3月    2013  [Refereed]

  • Maintenance of chloroplast genome stability by a homolog of RecA

    Masaki Odahara, Takayuki Inouye, Yoshiki Nishimura, Yasuhiko Sekine

    GENES & GENETIC SYSTEMS   87 ( 6 ) 406 - 406  2012.12

    Research paper, summary (international conference)  

  • Chloroplast genetic engineering technology in Marchantia polymorpha(L.).

    Minoru Ueda, Tetsuki Kuniyoshi, Hiroshi Yamamoto, Kazuhiko Sugimoto, Yoshiki Nishimura, Toshiharu Shikanai

    Marchantia Workshop 2012    2012

  • ゼニゴケ葉緑体RNAポリメラーゼシグマ因子(Mpsig1)変異体の解析から明らかとなった陸上植物シグマ因子の機能分化

    上田 実, 高見 常明, Peng Lianwei, 石崎 公庸, 河内 孝之, 鹿内 利治, 西村 芳樹

    日本分子生物学会、第35回年会、於 パシフィコ横浜、2011年12月    2011  [Refereed]

  • Development of a novel selectable marker for liverwort (Marchantia polymorpha).

    Ueda M, Ishizaki K, Yamato K. T, Kohchi T, Shikanai T, Nishimura Y

    Marchantia workshop 2010, March 11-12, 2010, Kyoto, Japan    2010  [Refereed]

  • 新規ゼニゴケ核ゲノム形質転換選抜用マーカーの開発

    上田 実, 石崎 公庸, 大和 勝幸, 河内 孝之, 鹿内 利治, 西村 芳樹

    日本植物生理学会、 第51回年会、於 熊本大学、2010年3月    2010  [Refereed]

    DOI

  • Antisense transcript suppress instability of polyadenylated mRNA in Chlamydomonas chloroplasts

    Yoshiki Nishimura, Hirofumi Uchimiya, David B. Stern

    PLANT AND CELL PHYSIOLOGY   48   S37 - S37  2007

    Research paper, summary (international conference)  

  • 葉緑体の増殖とmultiple FtsZ ringの形成について

    黒岩晴子, 森稔幸, 宮城島進也, 高原学, 西村芳樹, 黒岩常祥

    日本植物学会大会研究発表記録   66th   167  2002.09

    J-GLOBAL

  • Molecular mechanism of maternal inheritance of chloroplast in Chlamydomonas - a maternal nuclease that targets paternal chloroplasts

    Y Nishimura, O Misumi, T Higashiyama, T Kuroiwa

    PLANT AND CELL PHYSIOLOGY   43   S82 - S82  2002

    Research paper, summary (international conference)  

  • MOLECULAR MECHANISM OF CYTOPLASMIC INHERITANCE REVEALED BY ANALYZING A SINGLE CELL :

    NISHIMURA Yoshiki, KUROIWA Tuneyoshi

    Plant and cell physiology   42   s25  2001

    CiNii

  • Preferential destruction of male derived chloroplast DNA in a single zygote of Chlamydomonas reinhardtii revealed with the Optical Tweezer

    NISHIMURA Yoshiki, MISUMI Osami, MATSUNAGA Sachihiro, HIGASHIYAMA Tetsuya, YOKOTA Akiho, KUROSAWA Tsuneyoshi

      35   177 - 177  2000.05

    CiNii

  • UNIPARENTAL DEGRADATION OF CHLOROPLAST GENOME IN A SINGLE ZYGOTE OF CHLAMYDOMONAS REVEALED WITH THE OPTICAL TWEEZERS :

    NISHIMURA Yoshiki, HIGASHIYAMA Tetsuya, MISUMI Osami, YOKOTA Akiho, KUROIWA Tsuneyoshi

    Plant and cell physiology   41   s102  2000

    CiNii

  • ACTIVE DEGRADATION OF PATERNAL CHLOROPLAST GENOME IN SINGLE ZYGOTE REVEALED WITH OPTICAL TWEEZERS.

    NISHIMURA Yoshiki, HIGASHIYAMA Tetsuya, MISUMI Osami, KUROIWA Tuneyoshi, YOKOYA Akiho

      40   s28 - s28  1999.03

    CiNii

  • 顕微操作によるChlamydomonas reinhardtiiの細胞質遺伝機構の分子生物学的解析

    西村 芳樹, 東山 哲也, 松永 幸大, 鈴木 玲奈, 酒井 敦, 高野 博嘉, 河野 重行, 黒岩 常祥

    日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan   61   315 - 315  1997.09

    CiNii

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Syllabus

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Sub-affiliation

  • Faculty of Science and Engineering   Graduate School of Advanced Science and Engineering