Updated on 2025/06/18

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写真a

 
Daisuke Maruyama
 
Organization
Graduate School of Nanobioscience Department of Life and Environmental System Science Associate Professor
School of Science Department of Science
Title
Associate Professor
Homepage
https://kaken.nii.ac.jp/d/r/80724111.ja.html
Profile

シロイヌナズナを使った被子植物生殖の細胞生物学。

細胞核の融合、助細胞の不活性化と多花粉管拒否、多精拒否、内部形質膜崩壊など生殖独自の細胞現象を広く解析中。

2020~2022年度　学術変革領域(B) 植物生殖改変：https://www.remod-reprod.com

研究室HP　https://www.arabi-embryology.com 

External link

Degree

  • 博士（理学） ( 名古屋大学 )

Research Interests

  • nuclear fusion

  • Pollen tube

  • Inner Vegetative Plasma Membrane

  • Cell fusion

  • Central cell

  • Egg cell

  • Synergid cell

Research Areas

  • Life Science / Plant molecular biology and physiology

  • Life Science / Cell biology

Education

  • Nagoya University   Graduate School of Science   Department of Biological Science

    2004.4 - 2010.3

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  • Nagoya University   School of Science   Division of Biological Science

    2000.4 - 2004.3

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Research History

  • Graduate School of Nanobioscience, Yokohama City University   Kihara Institute for Biological Research   Associate Professor

    2022.4

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  • International College of Arts and Sciences Life and Environmental Science, Yokohama City University   Kihara Institute for Biological Research   Assistant Professor

    2016.4 - 2022.3

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  • Nagoya University   Institute of Transformative Bio-Molecules   Designated Assistant Professor

    2014.4 - 2016.3

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  • Nagoya University   Institute of Transformative Bio-Molecules   Research Fellow of the Japan Society for the Promotion of Science

    2013.4 - 2014.3

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  • Nagoya University   Division of Biological Research, Graduate School of Science   Research Fellow of the Japan Society for the Promotion of Science

    2011.4 - 2013.3

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  • Nagoya University   Division of Biological Research, Graduate School of Science   GCOE Pre-fellow

    2010.4 - 2011.3

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Professional Memberships

  • THE JAPANESE SOCIETY OF PLANT MORPHOLOGY

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  • THE JAPANESE SOCIETY OF PLANT PHYSIOLOGISTS

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  • THE BOTANICAL SOCIETY OF JAPAN

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  • JAPAN SOCIETY FOR CELL BIOLOGY

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  • THE MOLECULAR BIOLOGY SOCIETY OF JAPAN

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Papers

  • The peri-germ cell membrane: poorly characterized but key interface for plant reproduction

    Naoya Sugi, Andrea R. M. Calhau, Nathanaël M. A. Jacquier, Marina Millan-Blanquez, Jörg D. Becker, Kevin Begcy, Frédéric Berger, Cécile Bousquet-Antonelli, Daniel Bouyer, Giampiero Cai, Alice Y. Cheung, Sílvia Coimbra, Philipp Denninger, Thomas Dresselhaus, José A. Feijó, John E. Fowler, Danny Geelen, Ueli Grossniklaus, Tetsuya Higashiyama, David Honys, Tomoko Igawa, Gwyneth Ingram, Yvon Jaillais, Mark A. Johnson, Mariko Kato, Miki Kawachi, Tomokazu Kawashima, Yu-Jin Kim, Hong-Ju Li, Sébastien Mongrand, Kazuki Motomura, Shiori Nagahara, Kohdai P. Nakajima, Brad Nelms, Li-Jia Qu, Arp Schnittger, Stefan Scholten, Stefanie Sprunck, Meng-Xiang Sun, David Twell, Dolf Weijers, Wei-Cai Yang, Daisuke Maruyama, Thomas Widiez

    Nature Plants   2024.10

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

    DOI： 10.1038/s41477-024-01818-5

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    Other Link： https://www.nature.com/articles/s41477-024-01818-5

  • Letter to the Editor: Blue Light Irradiation Induces Pollen Tube Rupture in Various Flowering Plants.

    Naoya Sugi, Daichi Susaki, Yoko Mizuta, Tetsu Kinoshita, Daisuke Maruyama

    Plant & cell physiology   2024.2

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    Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cold Spring Harbor Laboratory  

    Abstract

    Pollen tubes exhibit one of the fastest apical growth rates among plant cells. Maintaining the proper balance between turgor pressure and cell wall synthesis at the pollen tube tip is crucial for this rapid growth, and any disruption can result in pollen tube rupture. In our study, we reveal that exposure to short-wavelength visible light, specifically blue light, induces pollen tube rupture. The frequency of pollen tube rupture increases in an intensity-dependent manner. Additionally, we observed Ca<sup>2+</sup>influx after blue light irradiation, accompanying with either pollen tube rupture or a temporary halt in elongation. These findings offer insights into the interplay between pollen tube integrity maintenance and Ca<sup>2+</sup>influx at the pollen tube tip, presenting a novel and efficient method to control pollen tube burst.

    Subject Areas

    (1) growth and development

    (11) new methodology

    DOI： 10.1093/pcp/pcae018

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  • Microtubules ensure transport of vegetative nuclei and sperm cells by fine-tuning their home positions

    Kazuki Motomura, Haruna Tsuchi, Marin Komojiri, Ayumi Matsumoto, Naoya Sugi, Daichi Susaki, Atsushi Takeda, Tetsu Kinoshita, Daisuke Maruyama

    2024.2

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    DOI： 10.1101/2024.01.31.578224

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  • Exploring novel polytubey reproduction pathways utilizing cumulative genetic tools. Invited Reviewed

    Naoya Sugi, Daisuke Maruyama

    Plant and Cell Physiology   2023.3

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    Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

    Abstract

    In the anthers and ovaries of flowers, pollen grains and embryo sacs are produced with uniform cell compositions. This stable gametogenesis enables elaborate interactions between male and female gametophytes after pollination, forming the highly successful sexual reproduction system in flowering plants. As most ovules are fertilized with a single pollen tube, the resulting genome set in the embryo and endosperm is determined in a single pattern by independent fertilization of the egg cell and central cell by two sperm cells. However, if ovules receive four sperm cells from two pollen tubes, the expected options for genome sets in the developing seeds would more than double. In wild-type Arabidopsis thaliana plants, around 5% of ovules receive two pollen tubes. Recent studies have elucidated the abnormal fertilization in supernumerary pollen tubes and sperm cells related to polytubey, polyspermy, heterofertilization, and fertilization recovery. Analyses of model plants have begun to uncover the mechanisms underlying this new pollen tube biology. Here, we review unusual fertilization phenomena and propose several breeding applications for flowering plants. These arguments contribute to the remodeling of plant reproduction, a challenging concept that alters typical plant fertilization by utilizing the current genetic toolbox.

    DOI： 10.1093/pcp/pcad021

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  • Removal of the endoplasma membrane upon sperm cell activation after pollen tube discharge Reviewed

    Naoya Sugi, Rie Izumi, Shun Tomomi, Daichi Susaki, Tetsu Kinoshita, Daisuke Maruyama

    Frontiers in Plant Science   14   1116289   2023.1

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    Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

    In pollen and pollen tubes, immotile sperm cells are enclosed by an inner vegetative plasma membrane (IVPM), a single endomembrane originating from the vegetative-cell plasma membrane. It is widely believed that sperm cells must be removed from the IVPM prior to gamete associations and fusions; however, details of the timing and morphological changes upon IVPM dissociation remain elusive. Here, we report a rapid IVPM breakdown immediately before double fertilization in Arabidopsis thaliana. The IVPM was stably observed in coiling pollen tubes when pollen tube discharge was prevented using lorelei mutant ovules. In contrast, a semi-in vivo fertilization assay in wild-type ovules demonstrated fragmented IVPM around sperm nuclei 1 min after pollen tube discharge. These observations revealed the dynamic alteration of released sperm cells and provided new insights into double fertilization in flowering plants. With a summary of recent findings on IVPM lipid composition, we discussed the possible physiological signals controlling IVPM breakdown.

    DOI： 10.3389/fpls.2023.1116289

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  • Transcriptome analyses uncover reliance of endosperm gene expression on <i>Arabidopsis</i> embryonic development International journal

    Yilin Zhang, Daisuke Maruyama, Erika Toda, Atsuko Kinoshita, Takashi Okamoto, Nobutaka Mitsuda, Hironori Takasaki, Masaru Ohme Takagi

    FEBS Letters   2023.1

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

    Endosperm-embryo development in flowering plants is regulated coordinately by signal exchange during seed development. However, such a reciprocal control mechanism has not been clearly identified. In this study, we identified an endosperm-specific gene, LBD35, expressed in an embryonic development-dependent manner, by a comparative transcriptome and cytological analyses of double-fertilized and single-fertilized seeds prepared by using the kokopelli mutant, which frequently induces single fertilization events. Transcriptome analysis using LBD35 as a marker of the central cell fertilization event identified that 141 genes, including 31 genes for small cysteine-rich peptides, are expressed in a double fertilization-dependent manner. Our results reveal possible embryonic signals that regulate endosperm gene expression and provide a practicable method to identify genes involved in the communication during endosperm-embryo development.

    DOI： 10.1002/1873-3468.14570

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  • 植物細胞の分化運命の制御と可塑性

    丸山 大輔, 水多 陽子, 山岡 尚平

    植物科学の最前線   14 ( A )   1 - 2   2023

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    Language：Japanese   Publisher：公益社団法人 日本植物学会  

    DOI： 10.24480/bsj-review.14a1.00236

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  • F-actin regulates the polarized secretion of pollen tube attractants in Arabidopsis synergid cells Reviewed

    Daichi Susaki, Rie Izumi, Takao Oi, Hidenori Takeuchi, Ji Min Shin, Naoya Sugi, Tetsu Kinoshita, Tetsuya Higashiyama, Tomokazu Kawashima, Daisuke Maruyama

    The Plant Cell   2022.12

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    Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

    Abstract

    Pollen tube attraction is a key event of sexual reproduction in flowering plants. In the ovule, two synergid cells neighboring the egg cell control pollen tube arrival via the active secretion of attractant peptides such as AtLURE1 and XIUQIU from the filiform apparatus facing toward the micropyle. Distinctive cell polarity together with longitudinal F-actin and microtubules are hallmarks of the synergid cell in various species, though the functions of these cellular structures are unclear. In this study we used genetic and pharmacological approaches to indicate the roles of cytoskeletal components in filiform apparatus formation and pollen tube guidance in Arabidopsis thaliana. Genetic inhibition of microtubule formation reduced invaginations of the plasma membrane but did not abolish micropylar AtLURE1.2 accumulation. By contrast, the expression of a dominant-negative form of ACTIN8 induced disorganization of the filiform apparatus and loss of polar AtLURE1.2 distribution toward the filiform apparatus. Interestingly, after pollen tube reception, F-actin became unclear for a few hours in the persistent synergid cell, which may be involved in pausing and resuming pollen tube attraction during early polytubey block. Our data suggest that F-actin plays a central role in maintaining cell polarity and in mediating male–female communication in the synergid cell.

    DOI： 10.1093/plcell/koac371

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    Plant Morphology   34   69 - 76   2022.12

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  • Possible molecular mechanisms of persistent pollen tube growth without de novo transcription Reviewed

    Kazuki Motomura, Naoya Sugi, Atsushi Takeda, Shohei Yamaoka, Daisuke Maruyama

    Frontiers in Plant Science   13   2022.11

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    Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

    The vegetative cell nucleus proceeds ahead of a pair of sperm cells located beneath the pollen tube tip during germination. The tip-localized vegetative nucleus had been considered to play a pivotal role in the control of directional pollen tube growth and double fertilization. However, we recently reported the female-targeting behavior of pollen tubes from mutant plants, of which the vegetative nucleus and sperm nuclei were artificially immotile. We showed that the apical region of the mutant pollen tubes became physiologically enucleated after the first callose plug formation, indicating the autonomously growing nature of pollen tubes without the vegetative nucleus and sperm cells. Thus, in this study, we further analyzed another Arabidopsis thaliana mutant producing physiologically enucleated pollen tubes and discussed the mechanism by which a pollen tube can grow without de novo transcription from the vegetative nucleus. We propose several possible molecular mechanisms for persistent pollen tube growth, such as the contribution of transcripts before and immediately after germination and the use of persistent transcripts, which may be important for a competitive race among pollen tubes.

    DOI： 10.3389/fpls.2022.1020306

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  • Elongation of Siliques Without Pollination 3 Regulates Nutrient Flow Necessary for Embryogenesis. Reviewed

    Hironori Takasaki, Miho Ikeda, Reika Hasegawa, Zhang Yilin, Shingo Sakamoto, Daisuke Maruyama, Nobutaka Mitsuda, Tetsu Kinoshita, Masaru Ohme-Takagi

    Plant & cell physiology   2022.10

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

    Apomixis, defined as the transfer of maternal germplasm to offspring without fertilization, enables the fixation of F1-useful traits, providing advantages in crop breeding. However, most apomictic plants require pollination to produce endosperm. Endosperm is essential for embryogenesis and its development is suppressed until fertilization. We show that expression of a chimeric repressor of the Elongation of Siliques without Pollination 3 (ESP3) gene (Pro35S:ESP3-SRDX) induces ovule enlargement without fertilization in Arabidopsis thaliana. The ESP3 gene encodes a protein similar to the FWA homeodomain transcription factor containing a StAR-related lipid-transfer (START) domain. However, ESP3 lacks the homeobox-encoding region. Genes related to the cell cycle and sugar metabolism were upregulated in unfertilized Pro35S:ESP3-SRDX ovules similar as in fertilized seeds, while those related to autophagy were downregulated similar to fertilized seeds. Unfertilized Pro35S:ESP3-SRDX ovules partially nourished embryos when only the egg was fertilized, accumulating hexoses without central cell proliferation. ESP3 may regulate nutrient flow during seed development, and ESP3-SRDX could be a useful tool for complete apomixis that does not require pseudo-fertilization.

    DOI： 10.1093/pcp/pcac151

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  • Persistent directional growth capability in Arabidopsis thaliana pollen tubes after nuclear elimination from the apex Reviewed International journal

    Kazuki Motomura, Hidenori Takeuchi, Michitaka Notaguchi, Haruna Tsuchi, Atsushi Takeda, Tetsu Kinoshita, Tetsuya Higashiyama, Daisuke Maruyama

    Nature Communications   12 ( 1 )   2331 - 2331   2021.4

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    Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

    <title>Abstract</title>During the double fertilization process, pollen tubes deliver two sperm cells to an ovule containing the female gametes. In the pollen tube, the vegetative nucleus and sperm cells move together to the apical region where the vegetative nucleus is thought to play a crucial role in controlling the direction and growth of the pollen tube. Here, we report the generation of pollen tubes in <italic>Arabidopsis thaliana</italic> whose vegetative nucleus and sperm cells are isolated and sealed by callose plugs in the basal region due to apical transport defects induced by mutations in the WPP domain-interacting tail-anchored proteins (WITs) and sperm cell-specific expression of a dominant mutant of the CALLOSE SYNTHASE 3 protein. Through pollen-tube guidance assays, we show that the physiologically anuclear mutant pollen tubes maintain the ability to grow and enter ovules. Our findings provide insight into the sperm cell delivery mechanism and illustrate the independence of the tip-localized vegetative nucleus from directional growth control of the pollen tube.

    DOI： 10.1038/s41467-021-22661-8

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    Other Link： http://www.nature.com/articles/s41467-021-22661-8

  • Dynamics of the cell fate specifications during female gametophyte development in Arabidopsis Reviewed

    Daichi Susaki, Takamasa Suzuki, Daisuke Maruyama, Minako Ueda, Tetsuya Higashiyama, Daisuke Kurihara

    PLOS BIOLOGY   19 ( 3 )   2021.3

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    DOI： 10.1371/journal.pbio.3001123

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  • ARP2/3-independent WAVE/SCAR pathway and class XI myosin control sperm nuclear migration in flowering plants Reviewed

    Mohammad Foteh Ali, Umma Fatema, Xiongbo Peng, Samuel W. Hacker, Daisuke Maruyama, Meng-Xiang Sun, Tomokazu Kawashima

    Proceedings of the National Academy of Sciences   117 ( 51 )   32757 - 32763   2020.12

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    Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

    After eukaryotic fertilization, gamete nuclei migrate to fuse parental genomes in order to initiate development of the next generation. In most animals, microtubules control female and male pronuclear migration in the zygote. Flowering plants, on the other hand, have evolved actin filament (F-actin)-based sperm nuclear migration systems for karyogamy. Flowering plants have also evolved a unique double-fertilization process: two female gametophytic cells, the egg and central cells, are each fertilized by a sperm cell. The molecular and cellular mechanisms of how flowering plants utilize and control F-actin for double-fertilization events are largely unknown. Using confocal microscopy live-cell imaging with a combination of pharmacological and genetic approaches, we identified factors involved in F-actin dynamics and sperm nuclear migration in<italic>Arabidopsis thaliana</italic>(<italic>Arabidopsis</italic>) and<italic>Nicotiana tabacum</italic>(tobacco). We demonstrate that the F-actin regulator, SCAR2, but not the ARP2/3 protein complex, controls the coordinated active F-actin movement. These results imply that an ARP2/3-independent WAVE/SCAR-signaling pathway regulates F-actin dynamics in female gametophytic cells for fertilization. We also identify that the class XI myosin XI-G controls active F-actin movement in the<italic>Arabidopsis</italic>central cell. XI-G is not a simple transporter, moving cargos along F-actin, but can generate forces that control the dynamic movement of F-actin for fertilization. Our results provide insights into the mechanisms that control gamete nuclear migration and reveal regulatory pathways for dynamic F-actin movement in flowering plants.

    DOI： 10.1073/pnas.2015550117

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    Other Link： https://syndication.highwire.org/content/doi/10.1073/pnas.2015550117

  • The nuclear envelope protein KAKU4 determines the migration order of the vegetative nucleus and sperm cells in pollen tubes. Reviewed International journal

    Chieko Goto, Kentaro Tamura, Satsuki Nishimaki, Daisuke Maruyama, Ikuko Hara-Nishimura

    Journal of experimental botany   71 ( 20 )   6273 - 6281   2020.10

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    A putative component protein of the nuclear lamina, KAKU4, modulates nuclear morphology in Arabidopsis thaliana seedlings but its physiological significance is unknown. KAKU4 was highly expressed in mature pollen grains, each of which has a vegetative cell and two sperm cells. KAKU4 protein was highly abundant on the envelopes of vegetative nuclei (VNs) and less abundant on the envelopes of sperm cell nuclei (SCNs) in pollen grains and elongating pollen tubes. VNs are irregularly shaped in wild-type pollen. However, KAKU4 deficiency caused them to become more spherical. After a pollen grain germinates, the VN and sperm cells enter and move along the pollen tube. In the wild type, the VN preceded the SCNs in more than 90% of the pollen tubes, whereas in kaku4 mutants, the VN preceded the SCNs in only about half of the pollen tubes. kaku4 pollen was less competitive for fertilization than wild-type pollen after pollination. These results lead us to hypothesize that the nuclear shape in vegetative cells of pollen grains affects the orderly migration of the VN and sperm cells in pollen tubes.

    DOI： 10.1093/jxb/eraa367

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  • Arabidopsis GEX1 Is a Nuclear Membrane Protein of Gametes Required for Nuclear Fusion During Reproduction Reviewed

    Shuh-ichi Nishikawa, Yuki Yamaguchi, Chiharu Suzuki, Ayaka Yabe, Yuzuru Sato, Daisuke Kurihara, Yoshikatsu Sato, Daichi Susaki, Tetsuya Higashiyama, Daisuke Maruyama

    Frontiers in Plant Science   11   2020.10

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    Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

    DOI： 10.3389/fpls.2020.548032

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  • Fertilization-Coupled Sperm Nuclear Fusion Is Required for Normal Endosperm Nuclear Proliferation. Reviewed

    Daisuke Maruyama, Tetsuya Higashiyama, Toshiya Endo, Shuh-Ichi Nishikawa

    Plant & cell physiology   61 ( 1 )   29 - 40   2020.1

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    Angiosperms exhibit double fertilization, a process in which one of the sperm cells released from the pollen tube fertilizes the egg, while the other sperm cell fertilizes the central cell, giving rise to the embryo and endosperm, respectively. We have previously reported two polar nuclear fusion-defective double knockout mutants of Arabidopsis thaliana immunoglobulin binding protein (BiP), a molecular chaperone of the heat shock protein 70 (Hsp70) localized in the endoplasmic reticulum (ER), (bip1 bip2) and its partner ER-resident J-proteins, ERdj3A and P58IPK (erdj3a p58ipk). These mutants are defective in the fusion of outer nuclear membrane and exhibit characteristic seed developmental defects after fertilization with wild-type pollen, which are accompanied by aberrant endosperm nuclear proliferation. In this study, we used time-lapse live-cell imaging analysis to determine the cause of aberrant endosperm nuclear division in these mutant seeds. We found that the central cell of bip1 bip2 or erdj3a p58ipk double mutant female gametophytes was also defective in sperm nuclear fusion at fertilization. Sperm nuclear fusion was achieved after the onset of the first endosperm nuclear division. However, division of the condensed sperm nucleus resulted in aberrant endosperm nuclear divisions and delayed expression of paternally derived genes. By contrast, the other double knockout mutant, erdj3b p58ipk, which is defective in the fusion of inner membrane of polar nuclei but does not show aberrant endosperm nuclear proliferation, was not defective in sperm nuclear fusion at fertilization. We thus propose that premitotic sperm nuclear fusion in the central cell is critical for normal endosperm nuclear proliferation.

    DOI： 10.1093/pcp/pcz158

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  • KAKU4-mediated deformation of the vegetative nucleus controls its precedent migration over sperm cells in pollen tubes

    Chieko Goto, Kentaro Tamura, Satsuki Nishimaki, Naoki Yanagisawa, Kumi Matsuura-Tokita, Tetsuya Higashiyama, Daisuke Maruyama, Ikuko Hara-Nishimura

    2019.9

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    Publisher：Cold Spring Harbor Laboratory  

    Abstract

    A putative nuclear lamina protein, KAKU4, modulates nuclear morphology inArabidopsis thalianaseedlings but its physiological significance is unknown.KAKU4was strongly expressed in mature pollen grains, each of which has a vegetative cell and two sperm cells. KAKU4 protein was highly abundant on the envelopes of vegetative nuclei (VNs) and less abundant on the envelopes of sperm cell nuclei (SCNs) in pollen grains and elongating pollen tubes. VN is irregularly shaped in wild-type pollen. However,KAKU4deficiency caused it to become more spherical. These results suggest that the dense accumulation of KAKU4 is responsible for the irregular shape of the VNs. After a pollen grain germinates, the VN and SCNs migrate to the tip of the pollen tube. In the wild type, the VN preceded the SCNs in 91–93% of the pollen tubes, whereas inkaku4mutants, the VN trailed the SCNs in 39–58% of the pollen tubes.kaku4pollen was less competitive than wild-type pollen after pollination, although it had an ability to fertilize. Taken together, our results suggest that controlling the nuclear shape in vegetative cells of pollen grains byKAKU4ensures the orderly migration of the VN and sperm cells in pollen tubes.

    Highlight

    The nuclear envelope protein KAKU4 is involved in controlling the migration order of vegetative nuclei and sperm cells in pollen tubes, affecting the competitive ability of pollen for fertilization.

    DOI： 10.1101/774489

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  • A pharmacological study of Arabidopsis cell fusion between the persistent synergid and endosperm Reviewed

    Kazuki Motomura, Tomokazu Kawashima, Frédéric Berger, Tetsu Kinoshita, Tetsuya Higashiyama, Daisuke Maruyama

    Journal of Cell Science   131 ( 2 )   jcs204123   2018

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    Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Company of Biologists Ltd  

    DOI： 10.1242/jcs.204123

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  • Spatiotemporal deep imaging of syncytium induced by the soybean cyst nematode Heterodera glycines Reviewed

    Mina Ohtsu, Yoshikatsu Sato, Daisuke Kurihara, Takuya Suzaki, Masayoshi Kawaguchi, Daisuke Maruyama, Tetsuya Higashiyama

    PROTOPLASMA   254 ( 6 )   2107 - 2115   2017.11

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

    DOI： 10.1007/s00709-017-1105-0

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  • Fluorescent Labeling of the Cyst Nematode Heterodera glycines in Deep-Tissue Live Imaging Reviewed

    Mina Ohtsu, Daisuke Kurihara, Yoshikatsu Sato, Takuya Suzaki, Masayoshi Kawaguchi, Daisuke Maruyama, Tetsuya Higashiyama

    CYTOLOGIA   82 ( 3 )   251 - 259   2017.6

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

    DOI： 10.1508/cytologia.82.251

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  • RETINOBLASTOMA RELATED1 mediates germline entry in Arabidopsis Reviewed

    Xin'Ai Zhao, Jonathan Bramsiepe, Matthias Van Durme, Shinichiro Komaki, Maria Ada Prusicki, Daisuke Maruyama, Joachim Forner, Anna Medzihradszky, Erik Wijnker, Hirofumi Harashima, You Lu, Anja Schmidt, Daniela Guthorl, Rosa Sahun Logrono, Yonsheng Guan, Gaetan Pochon, Ueli Grossniklaus, Thomas Laux, Tetsuya Higashiyama, Jan U. Lohmann, Moritz K. Nowack, Arp Schnittger

    SCIENCE   356 ( 6336 )   396 - +   2017.4

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

    DOI： 10.1126/science.aaf6532

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  • Live-cell imaging of F-actin dynamics during fertilization in Arabidopsis thaliana Reviewed

    Daichi Susaki, Daisuke Maruyama, Ramesh Yelagandula, Frederic Berger, Tomokazu Kawashima

    Methods in Molecular Biology   1669   47 - 54   2017

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    Language：English   Publishing type：Part of collection (book)   Publisher：Humana Press Inc.  

    DOI： 10.1007/978-1-4939-7286-9_4

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  • Cell fusion and nuclear fusion in plants Reviewed

    Daisuke Maruyama, Mina Ohtsu, Tetsuya Higashiyama

    SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY   60   127 - 135   2016.12

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    Authorship：Lead author,　Corresponding author   Language：English  

    DOI： 10.1016/j.semcdb.2016.07.024

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  • The end of temptation: the elimination of persistent synergid cell identity Reviewed

    Daisuke Maruyama, Tetsuya Higashiyama

    CURRENT OPINION IN PLANT BIOLOGY   34   122 - 126   2016.12

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    Authorship：Lead author   Language：English  

    DOI： 10.1016/j.pbi.2016.10.011

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  • Pollen tube contents initiate ovule enlargement and enhance seed coat development without fertilization Reviewed

    Ryushiro D. Kasahara, Michitaka Notaguchi, Shiori Nagahara, Takamasa Suzuki, Daichi Susaki, Yujiro Honma, Daisuke Maruyama, Tetsuya Higashiyama

    SCIENCE ADVANCES   2 ( 10 )   e1600554   2016.10

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

    DOI： 10.1126/sciadv.1600554

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  • The AMOR Arabinogalactan Sugar Chain Induces Pollen-Tube Competency to Respond to Ovular Guidance. Reviewed International journal

    Akane G Mizukami, Rie Inatsugi, Jiao Jiao, Toshihisa Kotake, Keiko Kuwata, Kento Ootani, Satohiro Okuda, Subramanian Sankaranarayanan, Yoshikatsu Sato, Daisuke Maruyama, Hiroaki Iwai, Estelle Garénaux, Chihiro Sato, Ken Kitajima, Yoichi Tsumuraya, Hitoshi Mori, Junichiro Yamaguchi, Kenichiro Itami, Narie Sasaki, Tetsuya Higashiyama

    Current biology : CB   26 ( 8 )   1091 - 7   2016.4

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    Precise directional control of pollen-tube growth by pistil tissue is critical for successful fertilization of flowering plants [1-3]. Ovular attractant peptides, which are secreted from two synergid cells on the side of the egg cell, have been identified [4-6]. Emerging evidence suggests that the ovular directional cue is not sufficient for successful guidance but that competency control by the pistil is critical for the response of pollen tubes to the attraction signal [1, 3, 7]. However, the female molecule for this competency induction has not been reported. Here we report that ovular methyl-glucuronosyl arabinogalactan (AMOR) induces competency of the pollen tube to respond to ovular attractant LURE peptides in Torenia fournieri. We developed a method for assaying the response capability of a pollen tube by micromanipulating an ovule. Using this method, we showed that pollen tubes growing through a cut style acquired a response capability in the medium by receiving a sufficient amount of a factor derived from mature ovules of Torenia. This factor, named AMOR, was identified as an arabinogalactan polysaccharide, the terminal 4-O-methyl-glucuronosyl residue of which was necessary for its activity. Moreover, a chemically synthesized disaccharide, the β isomer of methyl-glucuronosyl galactose (4-Me-GlcA-β-(1→6)-Gal), showed AMOR activity. No specific sugar-chain structure of plant extracellular matrix has been identified as a bioactive molecule involved in intercellular communication. We suggest that the AMOR sugar chain in the ovary renders the pollen tube competent to the chemotropic response prior to final guidance by LURE peptides.

    DOI： 10.1016/j.cub.2016.02.040

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  • Termination mechanism of the pollen tube attraction in Arabidopsis thaliana Invited Reviewed

    Daisuke MARUYAMA, Tetsuya HIGASHIYAMA

    Plant Morphology   28 ( 1 )   43 - 47   2016.4

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    Authorship：Lead author   Language：Japanese  

    DOI： 10.5685/plmorphol.28.43

    J-GLOBAL

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  • Fertilization-independent Cell-fusion between the Synergid and Central Cell in the Polycomb Mutant Reviewed

    Kazuki Motomura, Frederic Berger, Tomokazu Kawashima, Tetsu Kinoshita, Tetsuya Higashiyama, Daisuke Maruyama

    CELL STRUCTURE AND FUNCTION   41 ( 2 )   121 - 125   2016

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    DOI： 10.1247/csf.16010

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  • Selective nuclear elimination in multinucleate cells Reviewed

    Daisuke Maruyama, Tomokazu Kawashima, Tetsuya Higashiyama

    ONCOTARGET   6 ( 31 )   30447 - 30448   2015.10

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    DOI： 10.18632/oncotarget.5450

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  • Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism Reviewed

    Daisuke Maruyama, Ronny Voelz, Hidenori Takeuchi, Toshiyuki Mori, Tomoko Igawa, Daisuke Kurihara, Tomokazu Kawashima, Minako Ueda, Masaki Ito, Masaaki Umeda, Shuh-ichi Nishikawa, Rita Gross-Hardt, Tetsuya Higashiyama

    CELL   161 ( 4 )   907 - 918   2015.5

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    DOI： 10.1016/j.cell.2015.03.018

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  • BiP3 supports the early stages of female gametogenesis in the absence of BiP1 and BiP2 in Arabidopsis thaliana. Reviewed

    Maruyama D, Endo T, Nishikawa S

    Plant signaling & behavior   10 ( 7 )   e1035853   2015

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  • Different Sets of ER-Resident J-Proteins Regulate Distinct Polar Nuclear-Membrane Fusion Events in Arabidopsis thaliana Reviewed

    Daisuke Maruyama, Masaya Yamamoto, Toshiya Endo, Shuh-ichi Nishikawa

    PLANT AND CELL PHYSIOLOGY   55 ( 11 )   1937 - 1944   2014.11

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    DOI： 10.1093/pcp/pcu120

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  • Dynamic F-actin movement is essential for fertilization in Arabidopsis thaliana Reviewed

    Tomokazu Kawashima, Daisuke Maruyama, Murat Shagirov, Jing Li, Yuki Hamamura, Ramesh Yelagandula, Yusuke Toyama, Frederic Berger

    ELIFE   3   2014.10

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    DOI： 10.7554/eLife.04501

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  • Multiple BiP Genes of Arabidopsis thaliana are Required for Male Gametogenesis and Pollen Competitiveness Reviewed

    Daisuke Maruyama, Tomoyuki Sugiyama, Toshiya Endo, Shuh-ichi Nishikawa

    PLANT AND CELL PHYSIOLOGY   55 ( 4 )   801 - 810   2014.4

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    DOI： 10.1093/pcp/pcu018

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  • Independent Control by Each Female Gamete Prevents the Attraction of Multiple Pollen Tubes Reviewed

    Daisuke Maruyama, Yuki Hamamura, Hidenori Takeuchi, Daichi Susaki, Moe Nishimaki, Daisuke Kurihara, Ryushiro D. Kasahara, Tetsuya Higashiyama

    DEVELOPMENTAL CELL   25 ( 3 )   317 - 323   2013.5

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    Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

    DOI： 10.1016/j.devcel.2013.03.013

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  • Fertilization recovery system is dependent on the number of pollen grains for efficient reproduction in plants Reviewed

    Ryushiro D. Kasahara, Daisuke Maruyama, Tetsuya Higashiyama

    Plant Signaling and Behavior   8 ( 4 )   e236901 - e236904   2013.4

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

    DOI： 10.4161/psb.23690

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  • Fertilization Recovery after Defective Sperm Cell Release in Arabidopsis Reviewed

    Ryushiro D. Kasahara, Daisuke Maruyama, Yuki Hamamura, Takashi Sakakibara, David Twell, Tetsuya Higashiyama

    CURRENT BIOLOGY   22 ( 12 )   1084 - 1089   2012.6

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

    DOI： 10.1016/j.cub.2012.03.069

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  • BiP-mediated polar nuclei fusion is essential for the regulation of endosperm nuclei proliferation in Arabidopsis thaliana Reviewed

    Daisuke Maruyama, Toshiya Endo, Shuh-ichi Nishikawa

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   107 ( 4 )   1684 - 1689   2010.1

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    DOI： 10.1073/pnas.0905795107

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  • Arabidopsis thaliana Has a Set of J Proteins in the Endoplasmic Reticulum that are Conserved from Yeast to Animals and Plants Reviewed

    Masaya Yamamoto, Daisuke Maruyama, Toshiya Endo, Shuh-ichi Nishikawa

    PLANT AND CELL PHYSIOLOGY   49 ( 10 )   1547 - 1562   2008.10

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    DOI： 10.1093/pcp/pcn119

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  • Reproduction defects of the T-DNA mutants of endoplasmic reticulum Hsp70 in Arabidopsis thaliana

    Daisuke Maruyama, Toshiya Endo, Shuh-ichi Nishikawa

    PLANT AND CELL PHYSIOLOGY   48   S153 - S153   2007

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  • Functional analysis of J proteins in the endoplasmic reticulum of Arabidopsis thaliana Reviewed

    Masaya Yamamoto, Daisuke Maruyama, Toshiya Endo, Shuh-ichi Nishikawa

    PLANT AND CELL PHYSIOLOGY   48   S154 - S154   2007

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  • Callose (beta-1,3 glucan) is essential for Arabidopsis pollen wall patterning, but not tube growth Reviewed

    Shuh-ichi Nishikawa, Gregory M. Zinkl, Robert J. Swanson, Daisuke Maruyama, Daphne Preuss

    BMC PLANT BIOLOGY   5   22   2005.10

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    DOI： 10.1186/1471-2229-5-22

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MISC

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Presentations

  • 花粉発生過程のライブイメージングと分裂誘導による雄原細胞の分化機構の解明

    永原史織, 丸山大輔, 山岡尚平, 水多陽子

    第65回 日本植物生理学会年会  2024.3 

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    Event date： 2024.3

    Language：Japanese   Presentation type：Oral presentation (general)  

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  • シロイヌナズナの新規精細胞単離系の確立

    小橋瑞香, 杉直也, 須崎大地, 元村一基, 海老根一生, 木下哲, 丸山大輔

    第65回 日本植物生理学会年会  2024.3 

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    Event date： 2024.3

    Language：Japanese   Presentation type：Poster presentation  

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  • 青色光照射による効率的な花粉管破裂誘導法の開発

    杉直也, 須崎大地, 水多陽子, 木下 哲, 丸山大輔

    第65回 日本植物生理学会年会  2024.3 

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    Event date： 2024.3

    Language：Japanese   Presentation type：Oral presentation (general)  

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  • 卵細胞特異的な精細胞活性化因子EC1のアミロイド凝集に関する組織学的解析

    御調日向子, 永原史織, 後藤友美, 大井崇生, 武内秀憲, 豊岡公徳, 木下哲, 須崎大地, 丸山大輔

    第65回 日本植物生理学会年会  2024.3 

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    Event date： 2024.3

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  • 卵細胞外の斑点状構造はどのように形成されるか

    須崎大地, 大井崇生, 御調日向子, 武内秀憲, 永原史織, 杉直也, 木下哲, 丸山大輔

    第65回 日本植物生理学会年会  2024.3 

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    Event date： 2024.3

    Language：Japanese   Presentation type：Oral presentation (general)  

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  • 細胞融合を通じた植物長個体覚醒の理解に向けて

    丸山 大輔, 大津 美奈, 野田口 理孝, 須崎 大地, 太田 かおる, 木下 哲

    日本植物学会第87回大会  2023.9 

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    Event date： 2023.9

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  • 受精時の精核融合の完了は胚発生の開始に必須ではない

    西川周一, 高木祐理, 高松優菜, 松本光梨, 植田美那子, 丸山大輔

    日本植物学会第87回大会  2023.9 

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  • 助細胞における花粉管誘引シグナルの極性分泌ダイナミクスの解析

    永原史織, 丸山大輔, 東山哲也, 水多陽子, 武内秀憲

    日本植物学会第87回大会  2023.9 

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    Language：Japanese   Presentation type：Oral presentation (general)  

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  • 卵細胞外の斑点状構造はどのように形成されるか

    須崎大地, 大井崇生, 武内秀憲, 永原史織, 御調日向子, 杉直也, 木下哲, 丸山大輔

    日本植物学会第87回大会  2023.9 

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  • 重複受精過程で見られるCa2+誘導性の選択的生体膜崩壊メカニズムの解析

    杉直也, 泉理恵, 須崎大地, 海老根一生, 木下哲, 丸山 大輔

    日本植物学会第87回大会  2023.9 

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  • 植物の重複受精過程で観察される精細胞を包む単膜の選択的崩壊機構の解析

    杉 直也, 泉 理恵, 須崎 大地, 海老根 一生, 木下 哲, 丸山 大輔

    第75回 日本細胞生物学会大会  2023.6 

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  • 植物における細胞融合因子の同定と解析

    丸山大輔, 須﨑大地, 太田かおる, 木下哲

    第75回 日本細胞生物学会大会  2023.6 

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  • Analysis of a key factor regulating cell fusion between early endosperm and persistent synergid.

    Daisuke Maruyama

    The 33rd International Conference on Arabidopsis Research  2023.6 

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  • An importunate lover goes to DEAD-End: a cell fusion changes the destination of second sperm cell pair and avoids polyspermy. Invited

    Daisuke Maruyama

    The 27th International Congress on Sexual Plant Reproduction, 2024  2024.7 

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  • 二本目の花粉管誘引を利用する多精拒否機構の解明

    丸山大輔

    日本細胞生物学会  2016.6 

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  • 受精依存的に発現する シロイヌナズナの細胞融合因子の同定 Invited

    丸山大輔

    日本分子生物学会 第44回年会  2021.12 

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  • アクチン繊維に依存した 花粉管誘引物質の分泌制御の解析

    丸山大輔, 泉理恵, 武内秀憲, 永原史織, 須﨑大地, 河島友和, 東山哲也, 木下哲

    第85回日本植物学会  2021.9 

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  • シロイヌナズナの生殖に伴う非配偶子融合の意義 Invited

    丸山 大輔, 須﨑 大地, 太田 かおる, 木下 哲

    日本動物学会第91回大会  2021.9 

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  • 死を回避した助細胞は新たな運命を手に入れられるだろうか？

    丸山大輔

    植物生殖改変ワークショップ  2021.3 

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  • 植物受精の動態をライブイメージングで観察する Invited

    丸山 大輔

    エビデントランチョンセミナー  2023.3 

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  • シロイヌナズナの非配偶子性の 細胞融合の意義を理解する

    丸山 大輔

    日本植物学会第86回大会  2022.9 

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Awards

  • Hirase Award

    2021.9   Persistent directional growth capability in Arabidopsis thaliana pollen tubes after nuclear elimination from the apex

    Kazuki Motomura, Hidenori Takeuchi, Michitaka Notaguchi, Haruna Tsuchi, Atsushi Takeda, Tetsu Kinoshita, Tetsuya Higashiyama, Daisuke Maruyama

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  • BSJ Encouragement Prize

    2018.9   The Botanical Society of Japan  

    Daisuke MARUYAMA

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  • The Young Scientists’ Prize

    2016.4   Ministry of education science sports and culture of japan  

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  • Ishida Award

    2015.11  

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  • Hirase Award

    2015.9   The Japanese Society of Plant Morphology   Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism.

    Maruyama, D. Volz, R. Takeuchi, H. Mori, T. Igawa, T. Kurihara, D. Kawashima, T. Ueda, M. Ito, M. Umeda, M. Nishikawa, S. Gross-Hardt, R. Higashiyama, T

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  • Best Poster Award

    2015.8   Termination of the pollen tube attraction caused by rapid elimination of the synergid cell through a cell-fusion mechanism.

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  • Young Scientist Award for Best Presentation

    2015.7   Japan Society for Cell Biology  

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Research Projects

  • 助細胞が誘導する新奇の受粉非依存的胚珠肥大現象の解析と応用

    Grant number：23K17375  2023.6 - 2026.3

    日本学術振興会  科学研究費助成事業  挑戦的研究(開拓)

    丸山 大輔, 野田口 理孝

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    Grant amount：\26000000 （ Direct Cost: \20000000 、 Indirect Cost：\6000000 ）

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  • Remodeling Plant Reproduction System by Cell Fate Manipulations.

    Grant number：20B306  2020.10 - 2023.3

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

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  • 助細胞から胚への細胞運命転換の誘導を通じた多胚性種子の研究

    Grant number：20H05781  2020.10 - 2023.3

    日本学術振興会  科学研究費助成事業 学術変革領域研究(B)  学術変革領域研究(B)

    丸山 大輔

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    Grant amount：\40950000 （ Direct Cost: \31500000 、 Indirect Cost：\9450000 ）

    被子植物の卵細胞の隣には、花粉管の誘引を担う助細胞が存在する。シロイヌナズナでは受精後に発達する種子の中で、役目を終えた助細胞は胚乳と細胞融合することで消滅する（助細胞胚乳融合）。ところが、われわれが分離した助細胞の細胞融合が起こらないシロイヌナズナのctl17変異体では、発達中の種子にある助細胞が胚のそばで伸長していた。本研究では受精後のプログラム細胞死の過程を阻害し、かつ胚形成遺伝子を過剰発現されることで、助細胞が胚へと細胞運命転換して多胚種子が得られるのではないかと考えた。
    2020年度に計画していた多胚種子作製に向けたシロイヌナズナ変異株の準備は新型コロナウイルスによって、遅延が生じたものの、2021年度中に目的通りの植物系統を作出することができた。また、助細胞胚乳融合の鍵因子であるCTL17を未受精の胚珠の助細胞や卵細胞で発現させた異所発現実験の成果も得られた。これらについて2021年、日本動物学会第91回大会のシンポジウムや、日本分子生物学会第44回年会のシンポジウムにて発表した。
    実験に用いる変異体の整備については特に、助細胞核崩壊に必要なエチレンシグナルを欠損するein2変異とctl17変異をともにもつ二重変異体の作出できたことが大きいが、いまのところ、この植物で受精後の種子における助細胞の発達促進効果は観察されていない。また、ein2 ctl17二重変異体については、体細胞胚形成遺伝子であるRKD1を助細胞で異所発現させる遺伝子を導入し、ヘテロ接合体を得た。

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  • Remodeling plant reproduction system by cell fate manipulations.

    Grant number：20H05778  2020.10 - 2023.3

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

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    Grant amount：\15860000 （ Direct Cost: \12200000 、 Indirect Cost：\3660000 ）

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  • 精細胞を覆う機能未知の膜構造の崩壊制御に着目した植物受精メカニズムの解明

    Grant number：20K21432  2020.7 - 2023.3

    日本学術振興会  科学研究費助成事業 挑戦的研究(萌芽)  挑戦的研究(萌芽)

    丸山 大輔

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    Grant amount：\5330000 （ Direct Cost: \4100000 、 Indirect Cost：\1230000 ）

    被子植物の精細胞は花粉管と呼ばれる細い管状構造の内部を通り、胚珠の中にある卵細胞まで届けられることで受精を行う。花粉管伸長の間、精細胞は2つ1組が内部形質膜とよばれる栄養細胞の単膜花粉管栄養核とつながった雄性生殖単位として共に行動する。シロイヌナズナの重複受精のライブイメージングから、内部形質膜は精細胞が放出された瞬間に崩壊して精細胞膜を露出させることがわかった。その生理的な性質と研究の今後の展望についてのまとめを、2022年6月発行の「月刊細胞」に概説した。
    これまでは内部形質膜局在タンパク質を内部形質膜崩壊のレポーターに用いていたが、我々は内部形質膜と精細胞の間に蓄積する多糖分解酵素を蛍光タンパク質でラベルしたものが、内部形質膜崩壊による漏出に伴ったシグナル低下を指標に内部形質膜の完全性を感度良くモニタできるレポーターとして機能することを見出した。これら内部形質膜のレポーター遺伝子を、花粉管発芽直後に精細胞が放出されるanx1 anx2二重変異をもつ植物へと導入し、花粉管内容物の放出と内部形質膜崩壊の関係を調べた。すると、anx1 anx2二重変異によって膨圧の適切な調節を待たずに放出が起きるためか、花粉粒の外側に漏出する花粉管内容物に精細胞が含まれるパターンと含まれないパターンの両方が観察された。興味深いことに、精細胞が花粉粒の外側まで出る場合は速やかに内部形質膜の崩壊が起きるのに対し、精細胞が花粉粒に止まった場合は内部形質膜が安定的に保たれることがわかった。これらの観察から、精細胞の放出にともなう内部形質膜崩壊において、花粉外の環境刺激が重要な役割をはたしていることが示唆された。一方で花粉管の放出時に一過的に発生するといわれる、過酸化水素や細胞内カルシウム濃度上昇は内部形質膜崩壊の決定的なシグナルではないことが示唆された。

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  • 助細胞胚乳融合の変異体を利用したシロイヌナズナ多精拒否機構の研究

    Grant number：20H03280  2020.4 - 2023.3

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    丸山 大輔

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    Grant amount：\17940000 （ Direct Cost: \13800000 、 Indirect Cost：\4140000 ）

    本研究ではシロイヌナズナの受精後に花粉管を受け入れなかった方の助細胞（残存助細胞）が受精した中央細胞（胚乳）と細胞融合する現象である助細胞胚乳融合の発生学的意義を解明するヒントとしてDEAD-End 現象に着目している。DEAD-Endとは二本目の花粉管を受け入れた胚珠特異的に観察される、花粉管内容物が胚乳へと到達する新奇の受精現象である。われわれはDEAD-Endが、助細胞胚乳融合によって胚乳と構造的に一続きとなった残存助細胞に対し、受精後の誘引停止機構を間逃れて到達した2本目の花粉管が内容物を放出することによって起きる現象であるとの仮説を立てている。この仮説を検証するために、これまで順遺伝学的アプローチによるシロイヌナズナの変異体解析から、助細胞胚乳融合に必要なE3ユビキチンリガーゼとしてCTL17を同定した。CTL17の同定と機能解析について2021年度の進展を日本動物学会第91回大会のシンポジウムや、日本分子生物学会第44回年会のシンポジウムにて発表した。
    2020年度では2本目の花粉管誘引率が高いエチレン応答経路の変異体ein3に対してctl17欠損を導入することで、当初の仮説を証明する予定であったが、ein3変異体では、予備実験で用いたein3 eil1二重変異体よりもDEAD-Endを示す胚珠の出現率が低く、成功しなかった。そこで、2021年度ではein3 eil1 ctl17三重変異体の作製を進めるとともに、念のためにein2 ctl17二重変異体の作製も行った。すると、後者の変異体作製の過程で、予想外にもein2変異体において助細胞胚乳融合率の有意な低下がみられた。

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  • Analyses of mechanism regulating periodicity in the callose plug formation

    Grant number：20H05422  2020.4 - 2021.3

    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)  Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

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    Grant amount：\7540000 （ Direct Cost: \5800000 、 Indirect Cost：\1740000 ）

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  • シロイヌナズナの受精領域における配偶子間相互作用の研究

    Grant number：19H04869  2019.4 - 2021.3

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    丸山 大輔

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    Grant amount：\8060000 （ Direct Cost: \6200000 、 Indirect Cost：\1860000 ）

    本研究ではシロイヌナズナ花粉管放出の直後、重複受精に先立って起こる雌雄の配偶子間相互作用について、卵細胞や助細胞の分泌タンパク質、精細胞の露出などの観点から解析を進めた。助細胞の分泌タンパク質の解析については、助細胞特異的に優性欠損型ACTIN8を発現させたときに花粉管誘引欠損が誘導されることがわかっていた。今年度は花粉管誘引ペプチドであるAtLURE1.2にCitrineを融合したレポーターラインを用いて解析し、AtLURE1.2の極性分泌におけるアクチン繊維形成の重要性を示すことができた。卵細胞の細胞外構造の機能解析については、COPII小胞形成異常を誘導する優性欠損型SAR1を発現させることで、卵細胞のタンパク質を阻害したところ、卵細胞と中央細胞の間に存在するパッチ状の細胞外構造が消失することが示されていた。今年度は優性欠損型SAR1を中央細胞特異的に発現させた。その結果、パッチ状構造の形成に欠損はみられなかったため、卵細胞を覆うパッチ状の細胞外構造の形成は卵細胞の分泌が中心になっていることを明らかにすることができた。これまでの研究で、精細胞を覆う内部形質膜とよばれる単膜系が花粉管内容物の放出直後受精領域で素早く崩壊する様子を観察しており、この膜崩壊が精細胞表面の露出させることで配偶子間相互作用に重要な役割をはたすことを提唱してきた。本年度は同様の崩壊が花粉管を培地上で伸ばしたときに確率的に起きる内容物放出でも観察されることを明らかにした。また、マンニトール溶液添加による浸透圧ショックによって誘導された内容物放出の場合は内部形質膜が安定に維持されることも示した。以上の結果から、内部形質膜の崩壊が花粉管放出と密接につながった生理的現象であることを示すことができた。

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  • 卵細胞被覆構造体の解析を起点とする配偶子融合のメス側因子の探索

    Grant number：17H05846  2017.4 - 2019.3

    文部科学省  新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    丸山大輔

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    Authorship：Principal investigator  Grant type：Competitive

    被子植物の種子形成は、花粉管から放出された2つの精細胞が種子の前駆組織である胚珠へと届けられ、その内部にある卵細胞と中央細胞を重複受精させることで始まる。このとき、卵細胞と中央細胞の間には精細胞を受け入れるための、いわば受精領域とよぶべき場が一過的に出現をするが、現在までに受精領域の役割はほとんど明らかになっていない。われわれは、受精領域がつくられる前の卵細胞と中央細胞の間の連続電子顕微鏡像をFIB-SEMで取得し、180 nm間隔で選択した66枚の画像の三次元像を構築して卵細胞を覆うパッチ状の細胞壁構造の存在を示した。平成30年度ではピッチの密度を90 nmにした計132枚のトレース画像から三次元構築を行い、パッチ状構造の細部の形態をより滑らかに表現することができた。このパッチ状の細胞外構造は卵細胞から特異的に分泌される蛍光タンパク質を用いることで、共焦点顕微鏡によっても観察することができる。そこで卵細胞の分泌を阻害することで、このパッチ状構造の形成も止めることができるのではないかと考えた。この仮説を調べるため、平成30年度ではCOPII小胞形成に必須のSAR1の優性欠損変異体を卵細胞特異的に発現するpEC1.1::SAR1 H74L形質転換体を作製した。この植物では卵細胞から発現する分泌型蛍光タンパク質のシグナルが細胞外領域に局在しなかったことから、未受精胚珠でパッチ状の細胞外領域を欠損している可能性が示唆された。この形質転換体の雌しべに精細胞マーカーラインの花粉を授粉したところ、精細胞は受精領域へと到達せず、受精に失敗することがわかった。したがって、卵細胞の分泌活動が細胞外領域の形成に必須であること、そして、そのパッチ状構造が精細胞放出時における受精領域の瞬間的な形成において、重要な役割をはたすことが示唆された。

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  • Analyses of molecular mechanisms in the synergid-endosperm fusion.

    Grant number：16H06173  2016.4 - 2019.3

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

    Maruyama Daisuke

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    Authorship：Principal investigator  Grant type：Competitive

    Cell-fusion between the persistent synergid and the endosperm (Synergid-Endosperm fusion, SE fusion) appears to be one of the important events in the cessation of pollen tube attraction in fertilized ovule. To elucidate molecular mechanism, we performed a pharmacological analysis and forward genetics.
    Transcriptional or translational Inhibitors severely impaired induction of the SE fusion in fertilized ovules. Frequency of SE fusion also reduced by a treatment of roscovitine, an inhibitor of Cyclin-Dependent Kinases (CDKs). These data indicates an importances of post-fertilization de novo gene expression as well as mitotic entry in the induction of SE fusion.
    In the forward genetics approach, we generated a reporter line that labels mitochondria and nucleus of the synergid cells with different colors, and induced point mutations by an EMS treatment. We isolated eight mutants that frequently showed a defect in SE fusion among 2,201 M1 individuals.

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  • Analysis of polyspermy block system based on multiple pollen tube attraction.

    Grant number：15K14541  2015.4 - 2018.3

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

    Maruyama Daisuke, HIGASHIYAMA Tetsuya, KAWASHIMA Tomokazu, Berger Frederic

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    Authorship：Principal investigator  Grant type：Competitive

    Grant amount：\3900000 （ Direct Cost: \3000000 、 Indirect Cost：\900000 ）

    Zygote of flowering plants prevents multiple fertilizations caused by additional pair of sperm cells from second pollen tube. To examine the mechanism of polyspermy block, we analyzed patterns of released pollen tube contents (PTC) including sperm cells in an Arabidopsis mutant that frequently attracts second pollen tube. Contrary to our expectation, fluorescent signals from the PTC were frequently found in the endosperm but not in the fertilization zone. As this unique phenomenon seems to avoid polyspermy through a rerouting of sperm destination, we further analyzed other mutants that attract multiple pollen tubes and found possible involvement of synergid-endosperm fusion (SE fusion) during early embryogenesis. To prove the relevance of SE fusion in the unusual PTC reception, we tried to isolate mutant plants defective in the SE fusion and finally obtained one.

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  • in vivo花粉管伸長パターンの可視化を基盤とした植物多精拒否の順遺伝学的解析

    Grant number：11J06526  2011 - 2013

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    丸山 大輔

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    Grant amount：\2400000 （ Direct Cost: \2400000 ）

    助細胞-胚乳融合は, 受精後の胚珠において花粉管を受け入れなかった方の助細胞(残存助細胞)が胚乳と融合する現象である. これにより, 分泌前の花粉管誘引物質を含む助細胞に特有の内容物が, 体積の大きい胚乳へと希釈されて助細胞のアイデンティティーは急速に失われて花粉管誘引停止が起こる. また助細胞-胚乳融合の後には, 助細胞核が胚乳核の動かす細胞周期へと強制的に同期させられ, 細胞分裂中に崩壊する. これによって完全に助細胞のアイデンティティーが失われることがわかっている. 以前われわれは花粉管の誘引停止に卵細胞の受精と中央細胞の受精が独立かつ相加的に機能することを示した. そこで, 助細胞-胚乳融合と助細胞核崩壊に重要な役割をはたしていると考えられるエチレンシグナル経路の活性化の2つが, 卵細胞の受精または中央細胞の受精によってどのように制御されているか解析した. 雌性配偶子との細胞融合の成功率が低下した精細胞をつくるkokopelli変異体の花粉を中央細胞のサイトゾルをラベルするpFWA::FWA-GFP形質転換体の雌しべに授粉したところ, 卵細胞ではなく中央細胞の受精が強く助細胞-胚乳融合を誘導することを明らかにした. 一方, エチレンシグナル経路のマーカー遺伝子を導入した形質転換体で同様の実験を行ったところ, エチレンシグナル経路の活性化が卵細胞の受精により誘導されることが示唆された. したがって, 卵細胞と中央細胞の受精が誘導する花粉管誘引停止の実体が, それぞれ助細胞-胚乳融合とエチレンシグナル経路の活性化であることが示唆された.

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