Performance information

• Characterization of Subcellular Dynamics of Sterol Methyltransferases Clarifies Defective Cell Division in smt2 smt3, a C-24 Ethyl Sterol-Deficient Mutant of Arabidopsis.　　　　　　　　　　　　　　　
  Daisaku Ohta; Ayaka Fuwa; Yuka Yamaroku; Kazuki Isobe; Masatoshi Nakamoto; Atsushi Okazawa; Takumi Ogawa; Kazuo Ebine; Takashi Ueda; Pierre Mercier; Hubert Schaller
  Biomolecules, Volume：14, Number：7, Jul. 2024, ［International magazine］
  An Arabidopsis sterol mutant, smt2 smt3, defective in sterolmethyltransferase2 (SMT2), exhibits severe growth abnormalities. The loss of C-24 ethyl sterols, maintaining the biosynthesis of C-24 methyl sterols and brassinosteroids, suggests specific roles of C-24 ethyl sterols. We characterized the subcellular localizations of fluorescent protein-fused sterol biosynthetic enzymes, such as SMT2-GFP, and found these enzymes in the endoplasmic reticulum during interphase and identified their movement to the division plane during cytokinesis. The mobilization of endoplasmic reticulum-localized SMT2-GFP was independent of the polarized transport of cytokinetic vesicles to the division plane. In smt2 smt3, SMT2-GFP moved to the abnormal division plane, and unclear cell plate ends were surrounded by hazy structures from SMT2-GFP fluorescent signals and unincorporated cellulose debris. Unusual cortical microtubule organization and impaired cytoskeletal function accompanied the failure to determine the cortical division site and division plane formation. These results indicated that both endoplasmic reticulum membrane remodeling and cytokinetic vesicle transport during cytokinesis were impaired, resulting in the defects of cell wall generation. The cell wall integrity was compromised in the daughter cells, preventing the correct determination of the subsequent cell division site. We discuss the possible roles of C-24 ethyl sterols in the interaction between the cytoskeletal network and the plasma membrane.
  English, Scientific journal
  DOI：https://doi.org/10.3390/biom14070868
  DOI ID：10.3390/biom14070868, PubMed ID：39062582, PubMed Central ID：PMC11275053
• A bHLH heterodimer regulates germ cell differentiation in land plant gametophytes.　　　　　　　　　　　　　　　
  Misaki Saito; Ryosuke Momiki; Kazuo Ebine; Yoshihiro Yoshitake; Ryuichi Nishihama; Takuya Miyakawa; Takeshi Nakano; Nobutaka Mitsuda; Takashi Araki; Takayuki Kohchi; Shohei Yamaoka
  Current biology : CB, Sep. 2023, ［International magazine］
  Land plants are a monophyletic group of photosynthetic eukaryotes that diverged from streptophyte algae about 470 million years ago. During both the alternating haploid and diploid stages of the life cycle, land plants form multicellular bodies.1,2,3,4 The haploid multicellular body (gametophyte) produces progenitor cells that give rise to gametes and the reproductive organs.5,6,7,8 In the liverwort Marchantia polymorpha, differentiation of the initial cells of gamete-producing organs (gametangia) from the gametophyte is regulated by MpBONOBO (MpBNB), a member of the basic helix-loop-helix (bHLH) transcription factor subfamily VIIIa. In Arabidopsis thaliana, specification of generative cells in developing male gametophytes (pollen) requires redundant action of BNB1 and BNB2.9 Subfamily XI bHLHs, such as LOTUS JAPONICUS ROOTHAIRLESS LIKE1 (LRL1)/DEFECTIVE REGION OF POLLEN1 (DROP1) and LRL2/DROP2 in A. thaliana and the single LRL/DROP protein MpLRL in M. polymorpha, are the evolutionarily conserved regulators of rooting system development.10 Although the role of LRL1/DROP1 and LRL2/DROP2 in gametogenesis remains unclear, their loss leads to the formation of abnormal pollen devoid of sperm cells.11 Here, we show that BNBs and LRL/DROPs co-localize to gametophytic cell nuclei and form heterodimers. LRL1/DROP1 and LRL2/DROP2 act redundantly to regulate BNB expression for generative cell specification in A. thaliana after asymmetric division of the haploid microspore. MpLRL is required for differentiation of MpBNB-expressing gametangium initial cells in M. polymorpha gametophytes. Our findings suggest that broadly expressed LRL/DROP stabilizes BNB expression, leading to the formation of an evolutionarily conserved bHLH heterodimer, which regulates germ cell differentiation in the haploid gametophyte of land plants.
  English, Scientific journal
  DOI：https://doi.org/10.1016/j.cub.2023.09.020
  DOI ID：10.1016/j.cub.2023.09.020, PubMed ID：37776860
• The SYP123-VAMP727 SNARE complex delivers secondary cell wall components for root hair shank hardening in Arabidopsis.　　　　　　　　　　　　　　　
  Tomoko Hirano; Kazuo Ebine; Takashi Ueda; Takumi Higaki; Takahiro Watanabe-Nakayama; Hiroki Konno; Hisako Takigawa-Imamura; Masa H Sato
  The Plant cell, Sep. 2023, ［International magazine］
  The extended tubular shape of root hairs is established by tip growth and concomitant hardening. Here, we demonstrate that a syntaxin of plants (SYP)123-vesicle-associated membrane protein (VAMP)727-dependent secretion system delivers secondary cell wall components for hardening the subapical zone and shank of Arabidopsis (Arabidopsis thaliana) root hairs. We found increased SYP123 localization at the plasma membrane of the subapical and shank zones compared to the tip region in elongating root hairs. Inhibition of phosphatidylinositol (PtdIns)(3,5)P2 production impaired SYP123 localization at the plasma membrane and SYP123-mediated root hair shank hardening. Moreover, root hair elongation in the syp123 mutant was insensitive to a PtdIns(3,5)P2 synthesis inhibitor. SYP123 interacts with both VAMP721 and VAMP727. syp123 and vamp727 mutants exhibited reduced shank cell wall stiffness due to impaired secondary cell wall component deposition. Based on these results, we conclude that SYP123 is involved in VAMP721-mediated conventional secretion for root hair elongation as well as in VAMP727-mediated secretory functions for the delivery of secondary cell wall components to maintain root hair tubular morphology.
  English, Scientific journal
  DOI：https://doi.org/10.1093/plcell/koad240
  DOI ID：10.1093/plcell/koad240, PubMed ID：37713604
• Membrane trafficking functions of the ANTH/ENTH/VHS domain-containing proteins in plants.　　　　　　　　　　　　　　　
  Yihong Feng; Takuma Hiwatashi; Naoki Minamino; Kazuo Ebine; Takashi Ueda
  FEBS letters, May 2022, ［International magazine］
  Subcellular localization of proteins acting on the endomembrane system is primarily regulated via membrane trafficking. To obtain and maintain the correct protein composition of the plasma membrane and membrane-bound organelles, the loading of selected cargos into transport vesicles is critically regulated at donor compartments by adaptor proteins binding to the donor membrane, the cargo molecules, and the coat-protein complexes, including the clathrin coat. The ANTH/ENTH/VHS domain-containing protein superfamily generally comprises a structurally related ENTH, ANTH, or VHS domain in the N-terminal region and a variable C-terminal region, which is thought to act as an adaptor during transport vesicle formation. This protein family is involved in various plant processes, including pollen tube growth, abiotic stress response, and development. In this review, we provide an overview of the recent findings on ANTH/ENTH/VHS domain-containing proteins in plants.
  English, Scientific journal
  DOI：https://doi.org/10.1002/1873-3468.14368
  DOI ID：10.1002/1873-3468.14368, PubMed ID：35505466
• Phylogenetic distribution and expression pattern analyses identified a divergent basal body assembly protein involved in land plant spermatogenesis　　　　　　　　　　　　　　　
  Shizuka Koshimizu; Naoki Minamino; Tomoaki Nishiyama; Emiko Yoro; Kazuo Ebine; Keiko Sakakibara; Takashi Ueda; Kentaro Yano
  The New phytologist, Volume：236, Number：3, First page：1182, Last page：1196, Jul. 2021, ［International magazine］
  Oogamy is a form of sexual reproduction and evolved independently in animals, fungi, and plants. In streptophyte plants, Charophyceae, Coleochaetophyceae, bryophytes, lycophytes, ferns (monilophytes), and some gymnosperms (Cycads and Ginkgo) utilize spermatozoids as the male gamete. Plant spermatozoids commonly possess characteristic structures such as the spline, which consists of a microtubule array, the multilayered structure (MLS) in which the uppermost layer is continuum of the spline, and multiple flagella. However, the molecular mechanisms underpinning plant spermatogenesis remain to be elucidated. To identify the genes involved in plant spermatogenesis, we performed computational analyses and successfully found deeply divergent BLD10s by combining multiple methods and omics-data. We then validated the functions of candidate genes in the liverwort Marchantia polymorpha and the moss Physcomitrium patens and found that MpBLD10 and PpBLD10 are required for normal basal body and flagella formation. Mpbld10 mutants exhibited defects in remodeling of the cytoplasm and nucleus during spermatozoid formation, thus MpBLD10 should be involved in chromatin reorganization and elimination of the cytoplasm during spermiogenesis. Streptophyte BLD10s are orthologous to BLD10/CEP135 family proteins, which function in basal body assembly, but we found that BLD10s evolved especially fast in land plants and MpBLD10 might obtain additional functions in spermatozoid formation through the fast molecular evolution. This study provides a successful example of combinatorial study from evolutionary and molecular genetic perspectives that elucidated a function of the key protein of the basal body formation that fast evolved in land plants.
  Cold Spring Harbor Laboratory, English, Scientific journal
  DOI：https://doi.org/10.1101/2021.07.25.453666
  DOI ID：10.1101/2021.07.25.453666, PubMed ID：35842793
• Remodeling of organelles and microtubules during spermiogenesis in the liverwort Marchantia polymorpha　　　　　　　　　　　　　　　
  Naoki Minamino; Takuya Norizuki; Shoji Mano; Kazuo Ebine; Takashi Ueda
  Development (Cambridge, England), Volume：149, Number：15, Jul. 2021, ［International magazine］
  Gametogenesis is an essential biological event for sexual reproduction in various organisms. Bryophytes and some other plants employ motile sperms (spermatozoids) as male gametes, which self-locomote to the egg cells to accomplish fertilization. Spermatozoids of bryophytes harbor distinctive morphological characteristics, including the cell body with a helical slender shape and two motile flagella at the anterior edge. During transformation from a spermatid to spermatozoid (spermiogenesis), the shape and cellular contents of spermatids are dynamically reorganized. However, how each organelle is reorganized during plant spermiogenesis remains obscure. In this study, we classified the developmental processes during spermiogenesis in the liverwort Marchantia polymorpha according to cellular and nuclear shapes and flagella development. We then examined the remodeling of microtubules and reorganization of endomembrane organelles during spermiogenesis. The results indicate that the state of post-translational modification of tubulin is dynamically changed during the formation of the flagella and spline, and the plasma membrane and endomembrane organelles are drastically reorganized in a precisely regulated manner during spermiogenesis. These findings are expected to provide useful indexes to classify developmental and subcellular processes of spermiogenesis in bryophytes.
  Cold Spring Harbor Laboratory, English, Scientific journal
  DOI：https://doi.org/10.1101/2021.07.10.451882
  DOI ID：10.1101/2021.07.10.451882, PubMed ID：35924955
• Cargo sorting zones in the trans-Golgi network visualized by super-resolution confocal live imaging microscopy in plants.　　　　　　　　　　　　　　　
  Yutaro Shimizu; Junpei Takagi; Emi Ito; Yoko Ito; Kazuo Ebine; Yamato Komatsu; Yumi Goto; Mayuko Sato; Kiminori Toyooka; Takashi Ueda; Kazuo Kurokawa; Tomohiro Uemura; Akihiko Nakano
  Nature communications, Volume：12, Number：1, First page：1901, Last page：1901, Mar. 2021, ［International magazine］
  The trans-Golgi network (TGN) has been known as a key platform to sort and transport proteins to their final destinations in post-Golgi membrane trafficking. However, how the TGN sorts proteins with different destinies still remains elusive. Here, we examined 3D localization and 4D dynamics of TGN-localized proteins of Arabidopsis thaliana that are involved in either secretory or vacuolar trafficking from the TGN, by a multicolor high-speed and high-resolution spinning-disk confocal microscopy approach that we developed. We demonstrate that TGN-localized proteins exhibit spatially and temporally distinct distribution. VAMP721 (R-SNARE), AP (adaptor protein complex)-1, and clathrin which are involved in secretory trafficking compose an exclusive subregion, whereas VAMP727 (R-SNARE) and AP-4 involved in vacuolar trafficking compose another subregion on the same TGN. Based on these findings, we propose that the single TGN has at least two subregions, or "zones", responsible for distinct cargo sorting: the secretory-trafficking zone and the vacuolar-trafficking zone.
  English, Scientific journal
  DOI：https://doi.org/10.1038/s41467-021-22267-0
  DOI ID：10.1038/s41467-021-22267-0, PubMed ID：33772008, PubMed Central ID：PMC7997971
• The SYP123-VAMP727 SNARE complex is involved in the delivery of inner cell wall components to the root hair shank in Arabidopsis
  Tomoko Hirano; Kazuo Ebine; Takashi Ueda; Takumi Higaki; Takahiro Nakayama; Hiroki Konno; Hisako Takigawa-Imamura; Masa H. Sato
  Dec. 2020
  A root hair is a long tubular protrusion from a root hair cell established via tip growth, which is accomplished by the polarized deposition of membranous and cell wall components at the root hair apex accompanied by simultaneous hardening of the shank. The polarized secretion of materials to the root hair apex is well investigated; however, little is known about the deposition of inner cell wall materials at the root hair shank. We have previously reported that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P₂)/ROP10 signaling is required for the regulation of cortical microtubule construction and the deposition of inner cell wall components at the root hair shank during hardening. To unravel the alternate secretion mechanism for delivery of the inner cell wall components to root hair shank, here, we demonstrate that root hair-specific Qa-SNARE, SYP123, localizes to the subapical zone and shank of elongating root hairs in Arabidopsis. SYP123-mediated root hair elongation was inhibited by the FAB1 inhibitor YM201636, and inhibition of PtdIns(3,5)P₂ production impaired the plasma membrane localization of SYP123. We also showed that SYP123 forms a SNARE complex with VAMP727 on the plasma membrane, and syp123 and vamp727 mutants exhibited lower cell wall stiffness in the root hair shank because of impaired deposition of inner cell wall components. These results indicate that SYP123/VAMP727-mediated secretion is involved in the transport of inner cell wall components for hardening of the root hair shank.
  Cold Spring Harbor Laboratory
  DOI：https://doi.org/10.1101/2020.12.28.424500
  DOI ID：10.1101/2020.12.28.424500
• The liverwort oil body is formed by redirection of the secretory pathway.　　　　　　　　　　　　　　　
  Takehiko Kanazawa; Hatsune Morinaka; Kazuo Ebine; Takashi L Shimada; Sakiko Ishida; Naoki Minamino; Katsushi Yamaguchi; Shuji Shigenobu; Takayuki Kohchi; Akihiko Nakano; Takashi Ueda
  Nature communications, Volume：11, Number：1, First page：6152, Last page：6152, Dec. 2020, ［International magazine］
  Eukaryotic cells acquired novel organelles during evolution through mechanisms that remain largely obscure. The existence of the unique oil body compartment is a synapomorphy of liverworts that represents lineage-specific acquisition of this organelle during evolution, although its origin, biogenesis, and physiological function are yet unknown. We find that two paralogous syntaxin-1 homologs in the liverwort Marchantia polymorpha are distinctly targeted to forming cell plates and the oil body, suggesting that these structures share some developmental similarity. Oil body formation is regulated by an ERF/AP2-type transcription factor and loss of the oil body increases M. polymorpha herbivory. These findings highlight a common strategy for the acquisition of organelles with distinct functions in plants, via periodical redirection of the secretory pathway depending on cellular phase transition.
  English, Scientific journal
  DOI：https://doi.org/10.1038/s41467-020-19978-1
  DOI ID：10.1038/s41467-020-19978-1, PubMed ID：33262353, PubMed Central ID：PMC7708844
• Longin R-SNARE is retrieved from the plasma membrane by ANTH domain-containing proteins in Arabidopsis
  Masaru Fujimoto; Kazuo Ebine; Kohji Nishimura; Nobuhiro Tsutsumi; Takashi Ueda
  Proceedings of the National Academy of Sciences, First page：202011152, Last page：202011152, Sep. 2020, [Reviewed]
  The plasma membrane (PM) acts as the interface between intra- and extracellular environments and exhibits a tightly regulated molecular composition. The composition and amount of PM proteins are regulated by balancing endocytic and exocytic trafficking in a cargo-specific manner, according to the demands of specific cellular states and developmental processes. In plant cells, retrieval of membrane proteins from the PM depends largely on clathrin-mediated endocytosis (CME). However, the mechanisms for sorting PM proteins during CME remain ambiguous. In this study, we identified a homologous pair of ANTH domain-containing proteins, PICALM1a and PICALM1b, as adaptor proteins for CME of the secretory vesicle-associated longin-type R-SNARE VAMP72 group. PICALM1 interacted with the SNARE domain of VAMP72 and clathrin at the PM. The loss of function of PICALM1 resulted in faulty retrieval of VAMP72, whereas general endocytosis was not considerably affected by this mutation. The double mutant of PICALM1 exhibited impaired vegetative development, indicating the requirement of VAMP72 recycling for normal plant growth. In the mammalian system, VAMP7, which is homologous to plant VAMP72, is retrieved from the PM via the interaction with a clathrin adaptor HIV Rev-binding protein in the longin domain during CME, which is not functional in the plant system, whereas retrieval of brevin-type R-SNARE members is dependent on a PICALM1 homolog. These results indicate that ANTH domain-containing proteins have evolved to be recruited distinctly for recycling R-SNARE proteins and are critical to eukaryote physiology.
  Proceedings of the National Academy of Sciences, Scientific journal
  DOI：https://doi.org/10.1073/pnas.2011152117
  DOI ID：10.1073/pnas.2011152117, ISSN：0027-8424, eISSN：1091-6490, ORCID：80891293
• Deep Imaging Analysis in VISUAL Reveals the Role of YABBY Genes in Vascular Stem Cell Fate Determination.　　　　　　　　　　　　　　　
  Alif Meem Nurani; Yasuko Ozawa; Tomoyuki Furuya; Yuki Sakamoto; Kazuo Ebine; Sachihiro Matsunaga; Takashi Ueda; Hiroo Fukuda; Yuki Kondo
  Plant & cell physiology, Volume：61, Number：2, First page：255, Last page：264, Feb. 2020, [Reviewed], ［Domestic magazine］
  Stem cells undergo cell division and differentiation to ensure organized tissue development. Because plant cells are immobile, plant stem cells ought to decide their cell fate prior to differentiation, to locate specialized cells in the correct position. In this study, based on a chemical screen, we isolated a novel secondary cell wall indicator BF-170, which binds to lignin and can be used to image in vitro and in situ xylem development. Use of BF-170 to observe the vascular differentiation pattern in the in vitro vascular cell induction system, VISUAL, revealed that adaxial mesophyll cells of cotyledons predominantly generate ectopic xylem cells. Moreover, phloem cells are abundantly produced on the abaxial layer, suggesting the involvement of leaf adaxial-abaxial polarity in determining vascular cell fate. Analysis of abaxial polarity mutants highlighted the role of YAB3, an abaxial cell fate regulator, in suppressing xylem and promoting phloem differentiation on the abaxial domains in VISUAL. Furthermore, YABBY family genes affected in vivo vascular development during the secondary growth. Our results denoted the possibility that such mediators of spatial information contribute to correctly determine the cell fate of vascular stem cells, to conserve the vascular pattern of land plants.
  English
  DOI：https://doi.org/10.1093/pcp/pcaa002
  DOI ID：10.1093/pcp/pcaa002, PubMed ID：31922574
• Enrichment of Phosphatidylinositol 4,5-Bisphosphate in the Extra-Invasive Hyphal Membrane Promotes Colletotrichum Infection of Arabidopsis thaliana.　　　　　　　　　　　　　　　
  Takashi L Shimada; Shigeyuki Betsuyaku; Noriko Inada; Kazuo Ebine; Masaru Fujimoto; Tomohiro Uemura; Yoshitaka Takano; Hiroo Fukuda; Akihiko Nakano; Takashi Ueda
  Plant & cell physiology, Volume：60, Number：7, First page：1514, Last page：1524, Jul. 2019, [Reviewed], ［Domestic magazine］
  Pathogenic fungi from the genus Colletotrichum form invasive hyphae; the hyphae are surrounded by an extra-invasive hyphal membrane (EIHM), which is continuous with the plant plasma membrane. Although the EIHM plays a crucial role as the interface between plant and fungal cells, its precise function during Colletotrichum infection remains elusive. Here, we show that enrichment of phosphoinositides (PIs) has a crucial role in Colletotrichum infection. We observed the localization of PIs in Arabidopsis thaliana cells infected by A. thaliana-adapted Colletotrichum higginsianum (Ch), and found that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] was extremely enriched in the EIHM during Ch infection. We also found that phosphatidylinositol 4-phosphate-5 kinase (PIP5K), which catalyzes production of PI(4,5)P2, also accumulated at the EIHM. The overexpression of PIP5K3 in A. thaliana increased hyphal invasion by Ch. An exocytic factor, EXO84b, was targeted to the EIHM during Ch infection, although endocytic factors such as CLATHRIN LIGHT CHAIN 2 and FLOTILLIN 1 did not. Intriguingly, the interfacial membranes between A. thaliana and powdery mildew- or downy mildew-causing pathogens did not accumulate PI(4,5)P2. These results suggest that Ch could modify the PI(4,5)P2 levels in the EIHM to increase the exocytic membrane/protein supply of the EIHM for successful infection. Our results also suggest that PI(4,5)P2 biosynthesis is a promising target for improved defense against Colletotrichum infection.
  English
  DOI：https://doi.org/10.1093/pcp/pcz058
  DOI ID：10.1093/pcp/pcz058, PubMed ID：30989198
• Transcriptional switch for programmed cell death in pith parenchyma of sorghum stems.　　　　　　　　　　　　　　　
  Masaru Fujimoto; Takashi Sazuka; Yoshihisa Oda; Hiroyuki Kawahigashi; Jianzhong Wu; Hideki Takanashi; Takayuki Ohnishi; Jun-Ichi Yoneda; Motoyuki Ishimori; Hiromi Kajiya-Kanegae; Ken-Ichiro Hibara; Fumiko Ishizuna; Kazuo Ebine; Takashi Ueda; Tsuyoshi Tokunaga; Hiroyoshi Iwata; Takashi Matsumoto; Shigemitsu Kasuga; Jun-Ichi Yonemaru; Nobuhiro Tsutsumi
  Proceedings of the National Academy of Sciences of the United States of America, Volume：115, Number：37, First page：E8783-E8792, Last page：E8792, Sep. 2018, [Reviewed], ［International magazine］
  Pith parenchyma cells store water in various plant organs. These cells are especially important for producing sugar and ethanol from the sugar juice of grass stems. In many plants, the death of pith parenchyma cells reduces their stem water content. Previous studies proposed that a hypothetical D gene might be responsible for the death of stem pith parenchyma cells in Sorghum bicolor, a promising energy grass, although its identity and molecular function are unknown. Here, we identify the D gene and note that it is located on chromosome 6 in agreement with previous predictions. Sorghum varieties with a functional D allele had stems enriched with dry, dead pith parenchyma cells, whereas those with each of six independent nonfunctional D alleles had stems enriched with juicy, living pith parenchyma cells. D expression was spatiotemporally coupled with the appearance of dead, air-filled pith parenchyma cells in sorghum stems. Among D homologs that are present in flowering plants, Arabidopsis ANAC074 also is required for the death of stem pith parenchyma cells. D and ANAC074 encode previously uncharacterized NAC transcription factors and are sufficient to ectopically induce programmed death of Arabidopsis culture cells via the activation of autolytic enzymes. Taken together, these results indicate that D and its Arabidopsis ortholog, ANAC074, are master transcriptional switches that induce programmed death of stem pith parenchyma cells. Thus, targeting the D gene will provide an approach to breeding crops for sugar and ethanol production.
  English, Scientific journal
  DOI：https://doi.org/10.1073/pnas.1807501115
  DOI ID：10.1073/pnas.1807501115, ORCID：50019891, PubMed ID：30150370, PubMed Central ID：PMC6140496, SCOPUS ID：85053035451
• ANTH domain-containing proteins are required for the pollen tube plasma membrane integrity via recycling ANXUR kinases.　　　　　　　　　　　　　　　
  Muro K; Matsuura-Tokita K; Tsukamoto R; Kanaoka MM; Ebine K; Higashiyama T; Nakano A; Ueda T
  Communications biology, Volume：1, Sep. 2018, [Reviewed]
  During plant reproduction, sperm cells are delivered to ovules through growing pollen tubes. This process involves tip-localized receptor kinases regulating integrity and/or guidance of pollen tubes, whose localizations must be strictly regulated. However, the molecular basis for tip-localization of these molecules remains largely elusive. Here we show that a pair of AP180 N-terminal homology domain-containing proteins, PICALM5a and PICALM5b, is responsible for the tip-localization of ANXUR receptor kinases acting in an autocrine signaling pathway required for pollen tube integrity in Arabidopsis thaliana. The picalm5a picalm5b double mutant exhibits reduced fertility, and the double mutant pollen is defective in pollen tube integrity with premature bursts. The tip localization of ANXUR proteins is severely impaired in picalm5a picalm5b pollen tubes, whereas another receptor kinase PRK6 acting in pollen tube guidance is not affected. Based on these results, we propose that PICALM5 proteins serve as specific loading adaptors to recycle ANXUR proteins.
  NATURE PUBLISHING GROUP, English, Scientific journal
  DOI：https://doi.org/10.1038/s42003-018-0158-8
  DOI ID：10.1038/s42003-018-0158-8, eISSN：2399-3642, ORCID：50019969, PubMed ID：30272028, PubMed Central ID：PMC6158268, Web of Science ID：WOS:000461126500152
• RAB GTPases in the Basal Land Plant Marchantia polymorpha
  Naoki Minamino; Takehiko Kanazawa; Atsuko Era; Kazuo Ebine; Akihiko Nakano; Takashi Ueda
  Plant and Cell Physiology, Volume：59, Number：4, First page：845, Last page：856, Apr. 2018, [Reviewed]
  The RAB GTPase is an evolutionarily conserved machinery component of membrane trafficking, which is the fundamental system for cell viability and higher order biological functions. The composition of RAB GTPases in each organism is closely related to the complexity and organization of the membrane trafficking pathway, which has been developed uniquely to realize the organism-specific membrane trafficking system. Comparative genomics has suggested that terrestrialization and/or multicellularization were associated with the expansion of membrane trafficking pathways in green plants, which has yet to be validated in basal land plant lineages. To obtain insight into the diversification of membrane trafficking systems in green plants, we analyzed RAB GTPases encoded in the genome of the liverwort Marchantia polymorpha in a comprehensive manner. We isolated all genes for RAB GTPases in Marchantia and analyzed their expression patterns and subcellular localizations in thallus cells. While a majority of MpRAB GTPases exhibited a ubiquitous expression pattern, specific exceptions were also observed
  MpRAB2b, which contains a sequence similar to an intraflagellar transport protein at the C-terminal region
  and MpRAB23, which has been secondarily lost in angiosperms, were specifically expressed in the male reproductive organ. MpRAB21, which is another RAB GTPase whose homolog is absent in Arabidopsis, exhibited endosomal localization with RAB5 members in Marchantia. These results suggest that Marchantia possesses unique membrane trafficking pathways involving a unique repertoire of RAB GTPases.
  Oxford University Press, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcy027
  DOI ID：10.1093/pcp/pcy027, ISSN：1471-9053, ORCID：50019878, SCOPUS ID：85045524602
• Distinct sets of tethering complexes, SNARE complexes, and Rab GTPases mediate membrane fusion at the vacuole in Arabidopsis
  Kodai Takemoto; Kazuo Ebine; Jana Christin Askani; Falco Krüger; Zaida Andrés Gonzalez; Emi Ito; Tatsuaki Goh; Karin Schumacher; Akihiko Nakano; Takashi Ueda
  Proceedings of the National Academy of Sciences of the United States of America, Volume：115, Number：10, First page：E2457, Last page：E2466, Mar. 2018, [Reviewed]
  Membrane trafficking plays pivotal roles in various cellular activities and higher-order functions of eukaryotes and requires tethering factors to mediate contact between transport intermediates and target membranes. Two evolutionarily conserved tethering complexes, homotypic fusion and protein sorting (HOPS) and class C core vacuole/endosome tethering (CORVET), are known to act in endosomal/vacuolar transport in yeast and animals. Both complexes share a core subcomplex consisting of Vps11, Vps18, Vps16, and Vps33, and in addition to this core, HOPS contains Vps39 and Vps41, whereas CORVET contains Vps3 and Vps8. HOPS and CORVET subunits are also conserved in the model plant Arabidopsis. However, vacuolar trafficking in plants occurs through multiple unique transport pathways, and how these conserved tethering complexes mediate endosomal/vacuolar transport in plants has remained elusive. In this study, we investigated the functions of VPS18, VPS3, and VPS39, which are core complex, CORVET-specific, and HOPS-specific subunits, respectively. Impairment of these tethering proteins resulted in embryonic lethality, distinctly altering vacuolar morphology and perturbing transport of a vacuolar membrane protein. CORVET interacted with canonical RAB5 and a plant-specific R-soluble NSF attachment protein receptor (SNARE), VAMP727, which mediates fusion between endosomes and the vacuole, whereas HOPS interacted with RAB7 and another R-SNARE, VAMP713, which likely mediates homotypic vacuolar fusion. These results indicate that CORVET and HOPS act in distinct vacuolar trafficking pathways in plant cells, unlike those of nonplant systems that involve sequential action of these tethering complexes during vacuolar/lysosomal trafficking. These results highlight a unique diversification of vacuolar/lysosomal transport that arose during plant evolution, using evolutionarily conserved tethering components.
  National Academy of Sciences, English, Scientific journal
  DOI：https://doi.org/10.1073/pnas.1717839115
  DOI ID：10.1073/pnas.1717839115, ISSN：1091-6490, ORCID：50019890, SCOPUS ID：85042903326
• Integration of two RAB5 groups during endosomal transport in plants
  Ito E; Ebine K; Choi S.-W; Ichinose S; Uemura T; Nakano A; Ueda T
  eLife, Volume：7, 2018, [Reviewed]
  DOI：https://doi.org/10.7554/eLife.34064.001
  DOI ID：10.7554/eLife.34064.001, ORCID：50019904, SCOPUS ID：85051951897
• Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily　　　　　　　　　　　　　　　
  Huoi Ung; Purva Karia; Kazuo Ebine; Takashi Ueda; Keiko Yoshioka; Wolfgang Moeder
  PLANT PHYSIOLOGY, Volume：175, Number：1, First page：473, Last page：485, Sep. 2017, [Reviewed]
  The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphate substrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis (Arabidopsis thaliana) encodes three TTMgenes. We have previously reported that AtTTM2 displays pyrophosphatase activity and is involved in pathogen resistance. Here, we report the biochemical activity and biological function of AtTTM1 and diversification of the biological roles between AtTTM1 and 2. Biochemical analyses revealed that AtTTM1 displays pyrophosphatase activity similar to AtTTM2, making them the only TTMs characterized so far to act on a diphosphate substrate. However, knockout mutant analysis showed that AtTTM1 is not involved in pathogen resistance but rather in leaf senescence. AtTTM1 is transcriptionally up-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced and natural senescence. The double mutant of AtTTM1 and AtTTM2 did not show synergistic effects, further indicating the diversification of their biological function. However, promoter swap analyses revealed that they functionally can complement each other, and confocal microscopy revealed that both proteins are tail-anchored proteins that localize to the mitochondrial outer membrane. Additionally, transient overexpression of either gene in Nicotiana benthamiana induced senescence-like cell death upon dark treatment. Taken together, we show that two TTMs display the same biochemical properties but distinct biological functions that are governed by their transcriptional regulation. Moreover, this work reveals a possible connection of immunity-related programmed cell death and senescence through novel mitochondrial tail-anchored proteins.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1104/pp.17.00700
  DOI ID：10.1104/pp.17.00700, ISSN：0032-0889, eISSN：1532-2548, ORCID：50019897, SCOPUS ID：85029233944, Web of Science ID：WOS:000408792200036
• Constitutive activation of plant-specific RAB5 GTPase confers increased resistance against adapted powdery mildew fungus　　　　　　　　　　　　　　　
  Noriko Inada; Kazuo Ebine; Emi Ito; Akihiko Nakano; Takashi Ueda
  PLANT BIOTECHNOLOGY, Volume：34, Number：2, First page：89, Last page：95, Jun. 2017, [Reviewed]
  The obligate biotrophic fungal pathogens that cause powdery mildew disease establish infection in living host cells by modifying host cellular functions, including membrane trafficking. Previously, we reported that two Arabidopsis thaliana RAB5 GTPases, plant-specific ARA6/RABF1 and canonical ARA7/RABF2b, accumulate at the extrahaustorial membrane (EHM), which surrounds the specialized infection hypha called the haustorium. In this study, we examined the role of ARA6 and ARA7, which regulate distinctive endosomal trafficking pathways, in plant-powdery mildew fungus interactions. Although ARA6- and ARA7-related mutants did not exhibit altered susceptibility to the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii, overexpression of constitutively active ARA6, but not constitutively active ARA7, repressed proliferation of G. orontii. The repression of fungal proliferation was associated with accelerated formation of the callosic encasement around the haustorium. Furthermore, microscopic observation revealed an accumulation of the constitutively active form of ARA6, but not active ARA7, at the EHM. These results indicate that plant-specific ARA6 has a specific role in plant-powdery mildew fungus interaction, and manipulation of ARA6 activity could be a novel tool to overcome this plant disease.
  JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY, English, Scientific journal
  DOI：https://doi.org/10.5511/plantbiotechnology.17.0501a
  DOI ID：10.5511/plantbiotechnology.17.0501a, ISSN：1342-4580, ORCID：50019903, SCOPUS ID：85021696796, Web of Science ID：WOS:000404743200003
• Modulation of Plant RAB GTPase-Mediated Membrane Trafficking Pathway at the Interface Between Plants and Obligate Biotrophic Pathogens　　　　　　　　　　　　　　　
  Noriko Inada; Shigeyuki Betsuyaku; Takashi L. Shimada; Kazuo Ebine; Emi Ito; Natsumaro Kutsuna; Seiichiro Hasezawa; Yoshitaka Takano; Hiroo Fukuda; Akihiko Nakano; Takashi Ueda
  PLANT AND CELL PHYSIOLOGY, Volume：57, Number：9, First page：1854, Last page：1864, Sep. 2016, [Reviewed]
  RAB5 is a small GTPase that acts in endosomal trafficking. In addition to canonical RAB5 members that are homologous to animal RAB5, land plants harbor a plant-specific RAB5, the ARA6 group, which regulates trafficking events distinct from canonical RAB5 GTPases. Here, we report that plant RAB5, both canonical and plant-specific members, accumulate at the interface between host plants and biotrophic fungal and oomycete pathogens. Biotrophic fungi and oomycetes colonize living plant tissues by establishing specialized infection hyphae, the haustorium, within host plant cells. We found that Arabidopsis thaliana ARA6/RABF1, a plant-specific RAB5, is localized to the specialized membrane that surrounds the haustorium, the extrahaustorial membrane (EHM), formed by the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii. Whereas the conventional RAB5 ARA7/RABF2b was also localized to the EHM, endosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and RAB5-activating proteins were not, which suggests that the EHM has modified endosomal characteristic. The recruitment of host RAB5 to the EHM was a property shared by the barley-adapted powdery mildew fungus Blumeria graminis f. sp. hordei and the oomycete Hyaloperonospora arabidopsidis, but the extrahyphal membrane surrounding the hypha of the hemibiotrophic fungus Colletotrichum higginsianum at the biotrophic stage was devoid of RAB5. The localization of RAB5 to the EHM appears to correlate with the functionality of the haustorium. Our discovery sheds light on a novel relationship between plant RAB5 and obligate biotrophic pathogens.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcw107
  DOI ID：10.1093/pcp/pcw107, ISSN：0032-0781, eISSN：1471-9053, ORCID：50019889, SCOPUS ID：84988407826, Web of Science ID：WOS:000384717400005
• Plasmodium Rab5b is secreted to the cytoplasmic face of the tubovesicular network in infected red blood cells together with N-acylated adenylate kinase 2　　　　　　　　　　　　　　　
  Kazuo Ebine; Makoto Hirai; Miako Sakaguchi; Kazuhide Yahata; Osamu Kaneko; Yumiko Saito-Nakano
  MALARIA JOURNAL, Volume：15, Number：1, Jun. 2016, [Reviewed]
  Background: Rab5 GTPase regulates membrane trafficking between the plasma membrane and endosomes and harbours a conserved C-terminal isoprenyl modification that is necessary for membrane recruitment. Plasmodium falciparum encodes three Rab5 isotypes, and one of these, Rab5b (PfRab5b), lacks the C-terminal modification but possesses the N-terminal myristoylation motif. PfRab5b was reported to localize to the parasite periphery. However, the trafficking pathway regulated by PfRab5b is unknown.
  Methods: A complementation analysis of Rab5 isotypes was performed in Plasmodium berghei. A constitutively active PfRab5b mutant was expressed under the regulation of a ligand-dependent destabilization domain (DD)-tag system in P. falciparum. The localization of PfRab5b was evaluated after removing the ligand followed by selective permeabilization of the membrane with different detergents. Furthermore, P. falciparum N-terminally myristoylated adenylate kinase 2 (PfAK2) was co-expressed with PfRab5b, and trafficking of PfAK2 to the parasitophorous vacuole membrane was examined by confocal microscopy.
  Results: PfRab5b complemented the function of PbRab5b, however, the conventional C-terminally isoprenylated Rab5, PbRab5a or PbRab5c, did not. The constitutively active PfRab5b mutant localized to the cytosol of the parasite and the tubovesicular network (TVN), a region that extends from the parasitophorous vacuole membrane (PVM) in infected red blood cells (iRBCs). By removing the DD-ligand, parasite cytosolic PfRab5b signal disappeared and a punctate structure adjacent to the endoplasmic reticulum (ER) and parasite periphery accumulated. The peripheral PfRab5b was sensitive to extracellular proteolysis after treatment with streptolysin O, which selectively permeabilizes the red blood cell plasma membrane, indicating that PfRab5b localized on the iRBC cytoplasmic face of the TVN. Transport of PfAK2 to the PVM was abrogated by overexpression of PfRab5b, and PfAK2 accumulated in the punctate structure together with PfRab5b.
  Conclusion: N-myristoylated Plasmodium Rab5b plays a role that is distinct from that of conventional mammalian Rab5 isotypes. PfRab5b localizes to a compartment close to the ER, translocated to the lumen of the organelle, and co-localizes with PfAK2. PfRab5b and PfAK2 are then transported to the TVN, and PfRab5b localizes on the iRBC cytoplasmic face of TVN. These data demonstrate that PfRab5b is transported from the parasite cytosol to TVN together with N-myristoylated PfAK2 via an uncharacterized membrane-trafficking pathway.
  BIOMED CENTRAL LTD, English, Scientific journal
  DOI：https://doi.org/10.1186/s12936-016-1377-4
  DOI ID：10.1186/s12936-016-1377-4, ISSN：1475-2875, ORCID：50019887, SCOPUS ID：84975815488, Web of Science ID：WOS:000378829700001
• AtNHX5 and AtNHX6 Are Required for the Subcellular Localization of the SNARE Complex That Mediates the Trafficking of Seed Storage Proteins in Arabidopsis　　　　　　　　　　　　　　　
  Xuexia Wu; Kazuo Ebine; Takashi Ueda; Quan-Sheng Qiu
  PLOS ONE, Volume：11, Number：3, Mar. 2016, [Reviewed]
  The SNARE complex composed of VAMP727, SYP22, VTI11 and SYP51 is critical for protein trafficking and PSV biogenesis in Arabidopsis. This SNARE complex directs the fusion between the prevacuolar compartment (PVC) and the vacuole, and thus mediates protein trafficking to the vacuole. In this study, we examined the role of AtNHX5 and AtNHX6 in regulating this SNARE complex and its function in protein trafficking. We found that AtNHX5 and AtNHX6 were required for seed production, protein trafficking and PSV biogenesis. We further found that the nhx5 nhx6 syp22 triple mutant showed severe defects in seedling growth and seed development. The triple mutant had short siliques and reduced seed sets, but larger seeds. In addition, the triple mutant had numerous smaller protein storage vacuoles (PSVs) and accumulated precursors of the seed storage proteins in seeds. The PVC localization of SYP22 and VAMP727 was repressed in nhx5 nhx6, while a significant amount of SYP22 and VAMP727 was trapped in the Golgi or TGN in nhx5 nhx6. AtNHX5 and AtNHX6 were co-localized with SYP22 and VAMP727. Three conserved acidic residues, D164, E188, and D193 in AtNHX5 and D165, E189, and D194 in AtNHX6, were essential for the transport of the storage proteins, indicating the importance of exchange activity in protein transport. AtNHX5 or AtNHX6 did not interact physically with the SNARE complex. Taken together, AtNHX5 and AtNHX6 are required for the PVC localization of the SNARE complex and hence its function in protein transport. AtNHX5 and AtNHX6 may regulate the subcellular localization of the SNARE complex by their transport activity.
  PUBLIC LIBRARY SCIENCE, English, Scientific journal
  DOI：https://doi.org/10.1371/journal.pone.0151658
  DOI ID：10.1371/journal.pone.0151658, ISSN：1932-6203, ORCID：50019899, SCOPUS ID：84978151448, Web of Science ID：WOS:000372580300106
• Roles of membrane trafficking in plant cell wall dynamics　　　　　　　　　　　　　　　
  Kazuo Ebine; Takashi Ueda
  Frontiers in Plant Science, Volume：6, Number：OCTOBER, Oct. 2015, [Reviewed]
  The cell wall is one of the characteristic components of plant cells. The cell wall composition differs among cell types and is modified in response to various environmental conditions. To properly generate and modify the cell wall, many proteins are transported to the plasma membrane or extracellular space through membrane trafficking, which is one of the key protein transport mechanisms in eukaryotic cells. Given the diverse composition and functions of the cell wall in plants, the transport of the cell wall components and proteins that are involved in cell wall-related events could be specialized for each cell type, i.e., the machinery for cell wall biogenesis, modification, and maintenance could be transported via different trafficking pathways. In this review, we summarize the recent progress in the current understanding of the roles and mechanisms of membrane trafficking in plant cells and focus on the biogenesis and regulation of the cell wall.
  FRONTIERS MEDIA SA, English
  DOI：https://doi.org/10.3389/fpls.2015.00878
  DOI ID：10.3389/fpls.2015.00878, ISSN：1664-462X, ORCID：50019879, SCOPUS ID：84947445699, Web of Science ID：WOS:000364344600001
• Integral roles of membrane trafficking in biogenesis and modulation of the plant cell wall(The plant cell wall as an information processing system)　　　　　　　　　　　　　　　
  Ebine Kazuo; Ueda Takashi
  Regulation of Plant Growth & Development, Volume：50, Number：1, First page：43, Last page：49, 2015
  <p>The cell wall is a characteristic component of plant cells, whose structure and composition vary depending on the cell type, developmental stage, and environmental condition. For construction and proper modulation of the cell wall, a vast number of proteins and polysaccharides should be transported to the plasma membrane and/or the extracellular space correctly. The delivery of these materials depends on the membrane trafficking system, a key mechanism of intracellular transport connecting single-membrane bounded organelles and the extracellular space. Although molecular mechanisms of membrane trafficking are largely conserved in eukaryotic lineages, plants could have developed unique trafficking mechanisms to construct the cell wall and maintain its integrity, given the plant-specific nature of the cell wall. In this review, we summarize recent progress in the field of membrane trafficking in plant cells, especially focusing on the cell wall.</p>
  一般社団法人 植物化学調節学会, Japanese
  DOI：https://doi.org/10.18978/jscrp.50.1_43
  DOI ID：10.18978/jscrp.50.1_43, ISSN：1346-5406, CiNii Articles ID：110009969798, CiNii Books ID：AA11550064
• Plant Vacuolar Trafficking Occurs through Distinctly Regulated Pathways　　　　　　　　　　　　　　　
  Kazuo Ebine; Takeshi Inoue; Jun Ito; Emi Ito; Tomohiro Uemura; Tatsuaki Goh; Hiroshi Abe; Ken Sato; Akihiko Nakano; Takashi Ueda
  CURRENT BIOLOGY, Volume：24, Number：12, First page：1375, Last page：1382, Jun. 2014, [Reviewed]
  The multifunctional vacuole is the largest organelle in plant cells, and many proteins are transported to and stored in this organelle; thus, the vacuole has great physiological and agronomical importance. However, the molecular mechanism and regulation of plant vacuolar traffic remain largely unknown. In this study, we demonstrate that multiple vacuolar trafficking pathways operate in plants. RAB5 and RAB7 are evolutionarily conserved subfamilies of Rab GTPase [1-3], whose animal and yeast counterparts regulate vacuolar/endosomal trafficking in a sequential manner [4-7]. Functional analyses of a putative activating complex for RAB7 indicated that this complex is responsible for maturation from RAB5- to RAB7-positive endosomes in plant cells. Moreover, these machinery components are recruited to a more complex trafficking network. Mutations in RAB5 and RAB7 conferred counteracting effects on the vti11 mutant. Furthermore, impairment of RAB5- and RAB7-dependent pathways differentially affected the transport of distinctive cargos. These results indicate that plants have developed a complex vacuolar transport system distinct from that of nonplant systems by assigning evolutionarily conserved machinery to unique trafficking pathways. These pathways provide a fundamental basis for plant development at the cellular and higher-ordered levels.
  CELL PRESS, English, Scientific journal
  DOI：https://doi.org/10.1016/j.cub.2014.05.004
  DOI ID：10.1016/j.cub.2014.05.004, ISSN：0960-9822, eISSN：1879-0445, ORCID：50019886, SCOPUS ID：84902684556, Web of Science ID：WOS:000337648200028
• Interactomics of Qa-SNARE in Arabidopsis thaliana　　　　　　　　　　　　　　　
  Masayuki Fujiwara; Tomohiro Uemura; Kazuo Ebine; Yuka Nishimori; Takashi Ueda; Akihiko Nakano; Masa H. Sato; Yoichiro Fukao
  PLANT AND CELL PHYSIOLOGY, Volume：55, Number：4, First page：781, Last page：789, Apr. 2014, [Reviewed]
  Membrane trafficking in plants is involved in cellular development and the adaptation to various environmental changes. SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins mediate the fusion between vesicles and organelles to facilitate transport cargo proteins in cells. To characterize further the SNARE protein networks in cells, we carried out interactome analysis of SNARE proteins using 12 transgenic Arabidopsis thaliana plants expressing green fluorescent protein (GFP)-tagged Qa-SNAREs (SYP111, SYP121, SYP122, SYP123, SYP132, SYP21, SYP22, SYP31, SYP32, SYP41, SYP42 and SYP43). Microsomal fractions were prepared from each transgenic root, and subjected to immunoprecipitation (IP) using micromagnetic beads coupled to anti-GFP antibodies. To identify Qa-SNARE-interacting proteins, all immunoprecipitated products were then subjected to mass spectrometric (IP-MS) analysis. The IP-MS data revealed not only known interactions of SNARE proteins, but also unknown interactions. The IP-MS results were next categorized by gene ontology analysis. The data revealed that categories of cellular component organization, the cytoskeleton and endosome were enriched in the SYP2, SYP3 and SYP4 groups. In contrast, transporter activity was classified specifically in the SYP132 group. We also identified a novel interaction between SYP22 and VAMP711, which was validated using co-localization analysis with confocal microscopy and IP. Additional novel SNARE-interacting proteins play roles in vesicle transport and lignin biosynthesis, and were identified as membrane microdomain-related proteins. We propose that Qa-SNARE interactomics is useful for understanding SNARE interactions across the whole cell.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcu038
  DOI ID：10.1093/pcp/pcu038, ISSN：0032-0781, eISSN：1471-9053, ORCID：50019902, SCOPUS ID：84898905936, Web of Science ID：WOS:000334679500012
• Erratum: Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions (Nature (2008) 456 (962-966) DOI:10.1038/nature07409)
  Dhonukshe P; Tanaka H; Goh T; Ebine K; M{\"a}h{\"o}nen A.P; Prasad K; Blilou I; Geldner N; Xu J; Uemura T; Chory J; Ueda T; Nakano A; Scheres B; Friml J
  Nature, Volume：511, Number：7509, First page：370, 2014, [Reviewed]
  DOI：https://doi.org/10.1038/nature13549
  DOI ID：10.1038/nature13549, ORCID：50019884, SCOPUS ID：84904409041
• RABA Members Act in Distinct Steps of Subcellular Trafficking of the FLAGELLIN SENSING2 Receptor　　　　　　　　　　　　　　　
  Seung-won Choi; Takayuki Tamaki; Kazuo Ebine; Tomohiro Uemura; Takashi Ueda; Akihiko Nakano
  PLANT CELL, Volume：25, Number：3, First page：1174, Last page：1187, Mar. 2013, [Reviewed]
  Cell surface proteins play critical roles in the perception of environmental stimuli at the plasma membrane (PM) and ensuing signal transduction. Intracellular localization of such proteins must be strictly regulated, which requires elaborate integration of exocytic and endocytic trafficking pathways. Subcellular localization of Arabidopsis thaliana FLAGELLIN SENSING2 (FLS2), a receptor that recognizes bacterial flagellin, also depends on membrane trafficking. However, our understanding about the mechanisms involved is still limited. In this study, we visualized ligand-induced endocytosis of FLS2 using green fluorescent protein (GFP)-tagged FLS2 expressed in Nicotiana benthamiana. Upon treatment with the flg22 peptide, internalized FLS2-GFP from the PM was transported to a compartment with properties intermediate between the trans-Golgi network (TGN) and the multivesicular endosome. This compartment gradually discarded the TGN characteristics as it continued along the trafficking pathway. We further found that FLS2 endocytosis involves distinct RABA/RAB11 subgroups at different steps. Moreover, we demonstrated that transport of de novo-synthesized FLS2 to the PM also involves a distinct RABA/RAB11 subgroup. Our results demonstrate the complex regulatory system for properly localizing FLS2 and functional differentiation in RABA members in endo- and exocytosis.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1105/tpc.112.108803
  DOI ID：10.1105/tpc.112.108803, ISSN：1040-4651, ORCID：50019905, SCOPUS ID：84876758349, Web of Science ID：WOS:000318157100029
• Flowering Time Modulation by a Vacuolar SNARE via FLOWERING LOCUS C in Arabidopsis thaliana　　　　　　　　　　　　　　　
  Kazuo Ebine; Tomohiro Uemura; Akihiko Nakano; Takashi Ueda
  PLOS ONE, Volume：7, Number：7, Jul. 2012, [Reviewed]
  The transition of plant growth from vegetative to reproductive phases is one of the most important and dramatic events during the plant life cycle. In Arabidopsis thaliana, flowering promotion involves at least four genetically defined regulatory pathways, including the photoperiod-dependent, vernalization-dependent, gibberellin-dependent, and autonomous promotion pathways. Among these regulatory pathways, the vernalization-dependent and autonomous pathways are integrated by the expression of FLOWERING LOCUS C (FLC), a negative regulator of flowering; however, the upstream regulation of this locus has not been fully understood. The SYP22 gene encodes a vacuolar SNARE protein that acts in vacuolar and endocytic trafficking pathways. Loss of SYP22 function was reported to lead to late flowering in A. thaliana plants, but the mechanism has remained completely unknown. In this study, we demonstrated that the late flowering phenotype of syp22 was due to elevated expression of FLC caused by impairment of the autonomous pathway. In addition, we investigated the DOC1/BIG pathway, which is also suggested to regulate vacuolar/endosomal trafficking. We found that elevated levels of FLC transcripts accumulated in the doc1-1 mutant, and that syp22 phenotypes were exaggerated with a double syp22 doc1-1 mutation. We further demonstrated that the elevated expression of FLC was suppressed by ara6-1, a mutation in the gene encoding plant-unique Rab GTPase involved in endosomal trafficking. Our results indicated that vacuolar and/or endocytic trafficking is involved in the FLC regulation of flowering time in A. thaliana.
  PUBLIC LIBRARY SCIENCE, English, Scientific journal
  DOI：https://doi.org/10.1371/journal.pone.0042239
  DOI ID：10.1371/journal.pone.0042239, ISSN：1932-6203, ORCID：50019901, SCOPUS ID：84864411840, Web of Science ID：WOS:000306950200187
• Statistical organelle dissection of Arabidopsis guard cells using image database LIPS　　　　　　　　　　　　　　　
  Takumi Higaki; Natsumaro Kutsuna; Yoichiroh Hosokawa; Kae Akita; Kazuo Ebine; Takashi Ueda; Noriaki Kondo; Seiichiro Hasezawa
  SCIENTIFIC REPORTS, Volume：2, May 2012, [Reviewed]
  To comprehensively grasp cell biological events in plant stomatal movement, we have captured microscopic images of guard cells with various organelles markers. The 28,530 serial optical sections of 930 pairs of Arabidopsis guard cells have been released as a new image database, named Live Images of Plant Stomata (LIPS). We visualized the average organellar distributions in guard cells using probabilistic mapping and image clustering techniques. The results indicated that actin microfilaments and endoplasmic reticulum (ER) are mainly localized to the dorsal side and connection regions of guard cells. Subtractive images of open and closed stomata showed distribution changes in intracellular structures, including the ER, during stomatal movement. Time-lapse imaging showed that similar ER distribution changes occurred during stomatal opening induced by light irradiation or femtosecond laser shots on neighboring epidermal cells, indicating that our image analysis approach has identified a novel ER relocation in stomatal opening.
  NATURE PUBLISHING GROUP, English, Scientific journal
  DOI：https://doi.org/10.1038/srep00405
  DOI ID：10.1038/srep00405, ISSN：2045-2322, ORCID：50019906, SCOPUS ID：84861145038, Web of Science ID：WOS:000304044100002
• Qa-SNAREs localized to the trans-Golgi network regulate multiple transport pathways and extracellular disease resistance in plants　　　　　　　　　　　　　　　
  Tomohiro Uemura; Hyeran Kim; Chieko Saito; Kazuo Ebine; Takashi Ueda; Paul Schulze-Lefert; Akihiko Nakano
  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Volume：109, Number：5, First page：1784, Last page：1789, Jan. 2012, [Reviewed]
  In all eukaryotic cells, a membrane-trafficking system connects the post-Golgi organelles, such as the trans-Golgi network (TGN), endosomes, vacuoles, and the plasma membrane. This complex network plays critical roles in several higher-order functions in multicellular organisms. The TGN, one of the important organelles for protein transport in the post-Golgi network, functions as a sorting station, where cargo proteins are directed to the appropriate post-Golgi compartments. Unlike its roles in animal and yeast cells, the TGN has also been reported to function like early endosomal compartments in plant cells. However, the physiological roles of the TGN functions in plants are not understood. Here, we report a study of the SYP4 group (SYP41, SYP42, and SYP43), which represents the plant orthologs of the Tlg2/syntaxin16 Qa-SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) that localizes on the TGN in yeast and animal cells. The SYP4 group regulates the secretory and vacuolar transport pathways in the post-Golgi network and maintains the morphology of the Golgi apparatus and TGN. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance responses to a fungal pathogen. We also reveal a plant cell-specific higher-order role of the SYP4 group in the protection of chloroplasts from salicylic acid-dependent biotic stress.
  NATL ACAD SCIENCES, English, Scientific journal
  DOI：https://doi.org/10.1073/pnas.1115146109
  DOI ID：10.1073/pnas.1115146109, ISSN：0027-8424, ORCID：50019880, SCOPUS ID：84863129073, Web of Science ID：WOS:000299731400085
• Endosomal trafficking pathway regulated by ARA6, a RAB5 GTPase unique to plants　　　　　　　　　　　　　　　
  Kazuo Ebine; Naoto Miyakawa; Masaru Fujimoto; Tomohiro Uemura; Akihiko Nakano; Takashi Ueda
  Small GTPases, Volume：3, Number：1, 2012, [Reviewed]
  Lineage-specific expansion, followed by functional diversification of key components that act in membrane trafficking, is thought to contribute to lineage-specific diversification of organelles and membrane trafficking pathways. Indeed, recent comparative genomic studies have indicated that specific expansion of RAB and SNARE molecules occurred independently in various eukaryotic lineages over evolutionary history. However, experimental verification of this notion is difficult, because detailed functional analyses of RAB and SNARE proteins uniquely acquired by specific lineages are essential to understanding how new membrane trafficking pathways may have evolved. Recently, we found that a plant-specific RAB GTPase, ARA6, and a plant-unique R-SNARE, VAMP727, mediate a trafficking pathway from endosomes to the plasma membrane in Arabidopsis thaliana. Although a similar endosomal trafficking pathway was also reported in animals, the molecular machineries acting in these trafficking systems differ between animals and plants. Thus, trafficking pathways from endosomes to the plasma membrane appear to have been acquired independently in animal and plant systems. We further demonstrated that the ARA6-mediated trafficking pathway is required for the proper salt-stress response of A. thaliana. These results indicate that acquisition of a new membrane trafficking pathway may be associated with maximization of the fitness of each organism in a lineage-specific manner. © 2012 Landes Bioscience.
  Taylor and Francis Inc., English, Scientific journal
  DOI：https://doi.org/10.4161/sgtp.3.1.18299
  DOI ID：10.4161/sgtp.3.1.18299, ISSN：2154-1256, ORCID：50019900, PubMed ID：22710734, SCOPUS ID：84859092534
• Dynamic behavior of clathrin in Arabidopsis thaliana unveiled by live imaging　　　　　　　　　　　　　　　
  Emi Ito; Masaru Fujimoto; Kazuo Ebine; Tomohiro Uemura; Takashi Ueda; Akihiko Nakano
  PLANT JOURNAL, Volume：69, Number：2, First page：204, Last page：216, Jan. 2012, [Reviewed]
  Clathrin-coated vesicles (CCV) are necessary for selective transport events, including receptor-mediated endocytosis on the plasma membrane and cargo molecule sorting in the trans-Golgi network (TGN). Components involved in CCV formation include clathrin heavy and light chains and several adaptor proteins that are conserved among plants. Clathrin-dependent endocytosis has been shown to play an integral part in plant endocytosis. However, little information is known about clathrin dynamics in living plant cells. In this study, we have visualized clathrin in Arabidopsis thaliana by tagging clathrin light chain with green fluorescent protein (CLCGFP). Quantitative evaluations of colocalization demonstrate that the majority of CLCGFP is localized to the TGN, and a minor population is associated with multivesicular endosomes and the Golgi trans-cisternae. Live imaging further demonstrated the presence of highly dynamic clathrin-positive tubules and vesicles, which appeared to mediate interactions between the TGNs. CLCGFP is also targeted to cell plates and the plasma membrane. Although CLCGFP colocalizes with a dynamin isoform at the plasma membrane, these proteins exhibit distinct distributions at newly forming cell plates. This finding indicates independent functions of CLC (clathrin light chains) and dynamin during the formation of cell plates. We have also found that brefeldin A and wortmannin treatment causes distinctly different alterations in the dynamics and distribution of clathrin-coated domains at the plasma membrane. This could account for the different effects of these drugs on plant endocytosis.
  WILEY-BLACKWELL, English, Scientific journal
  DOI：https://doi.org/10.1111/j.1365-313X.2011.04782.x
  DOI ID：10.1111/j.1365-313X.2011.04782.x, ISSN：0960-7412, ORCID：50019892, SCOPUS ID：84855671917, Web of Science ID：WOS:000298874300002
• The occurrence of 'bulbs', a complex configuration of the vacuolar membrane, is affected by mutations of vacuolar SNARE and phospholipase in Arabidopsis　　　　　　　　　　　　　　　
  Chieko Saito; Tomohiro Uemura; Chie Awai; Motoki Tominaga; Kazuo Ebine; Jun Ito; Takashi Ueda; Hiroshi Abe; Miyo Terao Morita; Masao Tasaka; Akihiko Nakano
  PLANT JOURNAL, Volume：68, Number：1, First page：64, Last page：73, Oct. 2011, [Reviewed]
  The plant vacuole fulfills a variety of functions, and is essential for plant growth and development. We previously identified complex and mobile structures on the continuous vacuolar membrane, which we refer to as 'bulbs'. To ascertain their biological significance and function, we searched for markers associated with bulbs, and mutants that show abnormalities with respect to bulbs. We observed bulb-like structures after expression of non-membranous proteins as well as the functional soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) molecules VAM3 and VTI11. Bulbs are formed in more tissues than previously reported, including flowering organs, suspension culture cells, endodermal cells in the flowering stem, and at very early stages of seed germination. Using existing and newly developed marker lines, we found that the frequency of bulb occurrence is significantly decreased in multiple shoot gravitropism (sgr) mutants, which are known to have a defect in vacuolar membrane properties in endodermal cells. Based on results with new marker lines, which enabled us to observe the process of bulb biogenesis, and analysis of the phenotypes of these mutants, we propose multiple mechanisms for bulb formation, one of which may be that used for formation of transvacuolar strands.
  WILEY-BLACKWELL, English, Scientific journal
  DOI：https://doi.org/10.1111/j.1365-313X.2011.04665.x
  DOI ID：10.1111/j.1365-313X.2011.04665.x, ISSN：0960-7412, ORCID：50019883, SCOPUS ID：80053575143, Web of Science ID：WOS:000295395900006
• Genetic identification of Arabidopsis RID2 as an essential factor involved in pre-rRNA processing　　　　　　　　　　　　　　　
  Iwai Ohbayashi; Mineko Konishi; Kazuo Ebine; Munetaka Sugiyama
  PLANT JOURNAL, Volume：67, Number：1, First page：49, Last page：60, Jul. 2011, [Reviewed]
  A temperature-sensitive mutant of Arabidopsis, root initiation defective 2-1 (rid2-1), is characterized by peculiar defects in callus formation. To gain insights into the requirements for the reactivation of cell division, we analyzed this mutant and isolated the gene responsible, RID2. The phenotypes of rid2-1 in tissue culture and in seedlings indicated that the rid2 mutation has various (acute and non-acute) inhibitory effects on different aspects of cell proliferation. This suggests that the RID2 function is not directly involved in every cycle of cell division, but is related to 'vitality', supporting cell proliferation. The rid2-1 mutation was shown to cause nucleolar vacuolation and excessive accumulation of various intermediates of pre-rRNA processing. Positional cloning of the RID2 gene revealed that it encodes an evolutionarily conserved methyltransferase-like protein, which was found to localize in the nucleus, with accumulation being most evident in the nucleolus. It can be inferred from these findings that RID2 contributes to the nucleolar activity for pre-rRNA processing, probably through some methylation reaction.
  WILEY-BLACKWELL, English, Scientific journal
  DOI：https://doi.org/10.1111/j.1365-313X.2011.04574.x
  DOI ID：10.1111/j.1365-313X.2011.04574.x, ISSN：0960-7412, eISSN：1365-313X, ORCID：50019894, SCOPUS ID：79959717714, Web of Science ID：WOS:000292104700005
• A membrane trafficking pathway regulated by the plant-specific RAB GTPase ARA6　　　　　　　　　　　　　　　
  Kazuo Ebine; Masaru Fujimoto; Yusuke Okatani; Tomoaki Nishiyama; Tatsuaki Goh; Emi Ito; Tomoko Dainobu; Aiko Nishitani; Tomohiro Uemura; Masa H. Sato; Hans Thordal-Christensen; Nobuhiro Tsutsumi; Akihiko Nakano; Takashi Ueda
  NATURE CELL BIOLOGY, Volume：13, Number：7, First page：853, Last page：U279, Jul. 2011, [Reviewed]
  Endosomal trafficking plays an integral role in various eukaryotic cell activities and serves as a basis for higher-order functions in multicellular organisms. An understanding of the importance of endosomal trafficking in plants is rapidly developing(1,2), but its molecular mechanism is mostly unknown. Several key regulators of endosomal trafficking, including RAB5, which regulates diverse endocytic events in animal cells(3,4), are highly conserved. However, the identification of lineage-specific regulators in eukaryotes indicates that endosomal trafficking is diversified according to distinct body plans and life styles. In addition to orthologues of metazoan RAB5, landplants possess a unique RAB5 molecule, which is one of the most prominent features of plant RAB GTPase organization(5,6). Plants have also evolved a unique repertoire of SNAREs, the most distinctive of which are diverse VAMP7-related longins, including plant-unique VAMP72 derivatives(7). Here, we demonstrate that a plant-unique RAB5 protein, ARA6, acts in an endosomal trafficking pathway in Arabidopsis thaliana. ARA6 modulates the assembly of a distinct SNARE complex from conventional RAB5, and has a functional role in the salinity stress response. Our results indicate that plants possess a unique endosomal trafficking network and provide the first indication of a functional link between a specific RAB and a specific SNARE complex in plants.
  NATURE PUBLISHING GROUP, English, Scientific journal
  DOI：https://doi.org/10.1038/ncb2270
  DOI ID：10.1038/ncb2270, ISSN：1465-7392, eISSN：1476-4679, ORCID：50019885, SCOPUS ID：79959939840, Web of Science ID：WOS:000292305700016
• Vacuolar/pre-vacuolar compartment Qa-SNAREs VAM3/SYP22 and PEP12/SYP21 have interchangeable functions in Arabidopsis　　　　　　　　　　　　　　　
  Tomohiro Uemura; Miyo Terao Morita; Kazuo Ebine; Yusuke Okatani; Daisuke Yano; Chieko Saito; Takashi Ueda; Akihiko Nakano
  PLANT JOURNAL, Volume：64, Number：5, First page：864, Last page：873, Dec. 2010, [Reviewed]
  P>SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) mediate specific membrane fusion between transport vesicles or organelles and target membranes. VAM3/SYP22 and PEP12/SYP21 are Qa-SNAREs that act in the vacuolar transport pathway of Arabidopsis thaliana, and are localized predominantly on the vacuolar membrane and the pre-vacuolar compartment (PVC), respectively. Previous studies have shown that loss-of-function mutants of VAM3/SYP22 or PEP12/SYP21 showed male gametophytic lethality, suggesting that VAM3/SYP22 and PEP12/SYP21 possess different, non-redundant functions. We have re-evaluated the effects of mutations in these genes using T-DNA insertion mutants in the Columbia accession. We found that a mutation in VAM3/SYP22 (vam3-1) caused pleiotropic abnormalities, including semi-dwarfism and wavy leaves. In contrast, a loss-of-function mutant of PEP12/SYP21 (pep12) showed no apparent abnormal phenotype. We also found that the double vam3-1 pep12 mutant had severely reduced fertilization competence, although male and female gametophytes (vam3-1-pep12-) maintained the ability to fertilize. Moreover, promoter swapping analysis revealed that expression of a GFP-PEP12/SYP21 fusion under the control of the VAM3/SYP22 promoter suppressed all phenotypes of the vam3-1 mutant. These results indicate that the functions of VAM3/SYP22 and PEP12/SYP21 were redundant and interchangeable.
  WILEY-BLACKWELL PUBLISHING, INC, English, Scientific journal
  DOI：https://doi.org/10.1111/j.1365-313X.2010.04372.x
  DOI ID：10.1111/j.1365-313X.2010.04372.x, ISSN：0960-7412, ORCID：50019907, SCOPUS ID：78649573452, Web of Science ID：WOS:000284592400013
• Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis　　　　　　　　　　　　　　　
  Yohann Boutte; Marcia Frescatada-Rosa; Shuzhen Men; Cheung-Ming Chow; Kazuo Ebine; Anna Gustavsson; Lenore Johansson; Takashi Ueda; Ian Moore; Gerd Juergens; Markus Grebe
  EMBO JOURNAL, Volume：29, Number：3, First page：546, Last page：558, Feb. 2010, [Reviewed]
  Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin-and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mislocalizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis. The EMBO Journal (2010) 29, 546-558. doi:10.1038/emboj.2009.363; Published online 3 December 2009
  NATURE PUBLISHING GROUP, English, Scientific journal
  DOI：https://doi.org/10.1038/emboj.2009.363
  DOI ID：10.1038/emboj.2009.363, ISSN：0261-4189, ORCID：50019893, SCOPUS ID：76349116068, Web of Science ID：WOS:000274233400004
• Application of Lifeact Reveals F-Actin Dynamics in Arabidopsis thaliana and the Liverwort, Marchantia polymorpha　　　　　　　　　　　　　　　
  Atsuko Era; Motoki Tominaga; Kazuo Ebine; Chie Awai; Chieko Saito; Kimitsune Ishizaki; Katsuyuki T. Yamato; Takayuki Kohchi; Akihiko Nakano; Takashi Ueda
  PLANT AND CELL PHYSIOLOGY, Volume：50, Number：6, First page：1041, Last page：1048, Jun. 2009, [Reviewed]
  Actin plays fundamental roles in a wide array of plant functions, including cell division, cytoplasmic streaming, cell morphogenesis and organelle motility. Imaging the actin cytoskeleton in living cells is a powerful methodology for studying these important phenomena. Several useful probes for live imaging of filamentous actin (F-actin) have been developed, but new versatile probes are still needed. Here, we report the application of a new probe called Lifeact for visualizing F-actin in plant cells. Lifeact is a short peptide comprising 17 amino acids that was derived from yeast Abp140p. We used a LifeactVenus fusion protein for staining F-actin in Arabidopsis thaliana and were able to observe dynamic rearrangements of the actin meshwork in root hair cells. We also used LifeactVenus to visualize the actin cytoskeleton in the liverwort Marchantia polymorpha; this revealed unique and dynamic F-actin motility in liverwort cells. Our results suggest that Lifeact could be a useful tool for studying the actin cytoskeleton in a wide range of plant lineages.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcp055
  DOI ID：10.1093/pcp/pcp055, ISSN：0032-0781, ORCID：50019888, SCOPUS ID：67649369329, Web of Science ID：WOS:000266967600004
• Unique mechanism of plant endocytic/vacuolar transport pathways　　　　　　　　　　　　　　　
  Kazuo Ebine; Takashi Ueda
  JOURNAL OF PLANT RESEARCH, Volume：122, Number：1, First page：21, Last page：30, Jan. 2009, [Reviewed]
  The post-Golgi traffic network in plant cells is highly complex, which is correlated with the large number of genes related to this function. RABs and SNAREs are key regulators of tethering and fusion of transport vesicles to target membranes, and the numbers of these regulators have also expanded in plant lineages. In addition to this increase in the net number of genes, plants also seem to have evolved new gene families tailored to fulfill plant-unique functions. In this article, we summarize recent progress in studies on plant-unique RABs and SNAREs functioning in post-Golgi trafficking, with a special focus on the endocytic pathway.
  SPRINGER TOKYO, English, Scientific journal
  DOI：https://doi.org/10.1007/s10265-008-0200-x
  DOI ID：10.1007/s10265-008-0200-x, ISSN：0918-9440, ORCID：50019882, SCOPUS ID：58149333152, Web of Science ID：WOS:000262241300002
• Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions　　　　　　　　　　　　　　　
  Pankaj Dhonukshe; Hirokazu Tanaka; Tatsuaki Goh; Kazuo Ebine; Ari Pekka Mahonen; Kalika Prasad; Ikram Blilou; Niko Geldner; Jian Xu; Tomohiro Uemura; Joanne Chory; Takashi Ueda; Akihiko Nakano; Ben Scheres; Jiri Friml
  NATURE, Volume：456, Number：7224, First page：962, Last page：U75, Dec. 2008, [Reviewed]
  Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication - a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family(1) are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution- dependent development including embryo patterning, organogenesis and tropisms(2-7). Polar PIN localization determines the direction of intercellular auxin flow(8), yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis- dependent mechanism of PIN polarity generation and analyse its developmental implications. Real- time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non- polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway- associated root- forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two- step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis- dependent polarity of individual cells and auxin distribution- dependent cell fate establishment for multicellular patterning.
  NATURE PUBLISHING GROUP, English, Scientific journal
  DOI：https://doi.org/10.1038/nature07409
  DOI ID：10.1038/nature07409, ISSN：0028-0836, ORCID：50019895, SCOPUS ID：56349106623, Web of Science ID：WOS:000261768300048
• A SNARE Complex Unique to Seed Plants Is Required for Protein Storage Vacuole Biogenesis and Seed Development of Arabidopsis thaliana　　　　　　　　　　　　　　　
  Kazuo Ebine; Yusuke Okatani; Tomohiro Uemura; Tatsuaki Goh; Keiko Shoda; Mitsuru Niihama; Miyo Terao Morita; Christoph Spitzer; Marisa S. Otegui; Akihiko Nakano; Takashi Ueda
  PLANT CELL, Volume：20, Number：11, First page：3006, Last page：3021, Nov. 2008, [Reviewed]
  The SNARE complex is a key regulator of vesicular traffic, executing membrane fusion between transport vesicles or organelles and target membranes. A functional SNARE complex consists of four coiled-coil helical bundles, three of which are supplied by Q-SNAREs and another from an R-SNARE. Arabidopsis thaliana VAMP727 is an R-SNARE, with homologs only in seed plants. We have found that VAMP727 colocalizes with SYP22/VAM3, a Q- SNARE, on a subpopulation of prevacuolar compartments/endosomes closely associated with the vacuolar membrane. Genetic and biochemical analyses, including examination of a synergistic interaction of vamp727 and syp22 mutations, histological examination of protein localization, and coimmunoprecipitation from Arabidopsis lysates indicate that VAMP727 forms a complex with SYP22, VTI11, and SYP51 and that this complex plays a crucial role in vacuolar transport, seed maturation, and vacuole biogenesis. We suggest that the VAMP727 complex mediates the membrane fusion between the prevacuolar compartment and the vacuole and that this process has evolved as an essential step for seed development.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1105/tpc.107.057711
  DOI ID：10.1105/tpc.107.057711, ISSN：1040-4651, ORCID：50019896, SCOPUS ID：58149332213, Web of Science ID：WOS:000262131500009
• VPS9a, the common activator for two distinct types of Rab5 GTPases, is essential for the development of Arabidopsis thaliana　　　　　　　　　　　　　　　
  Tatsuaki Goh; Wakana Uchida; Satoko Arakawa; Emi Ito; Tomoko Dainobu; Kazuo Ebine; Masaki Takeuchi; Ken Sato; Takashi Ueda; Akihiko Nakano
  PLANT CELL, Volume：19, Number：11, First page：3504, Last page：3515, Nov. 2007, [Reviewed]
  Rab5, a subfamily of Rab GTPases, regulates a variety of endosomal functions as a molecular switch. Arabidopsis thaliana has two different types of Rab5-member GTPases: conventional type, ARA7 and RHA1, and a plant-specific type, ARA6. We found that only one guanine nucleotide exchange factor (GEF), named VPS9a, can activate all Rab5 members to GTP-bound forms in vitro in spite of their diverged structures. In the vps9a-1 mutant, whose GEF activity is completely lost, embryogenesis was arrested at the torpedo stage. Green fluorescent protein (GFP)-ARA7 and ARA6-GFP were diffused in cytosol like GDP-fixed mutants of Rab5 in vps9a-1, indicating that both types of GTPase are regulated by VPS9a. In the leaky vps9a-2 mutant, elongation of the primary root was severely affected. Overexpression of the GTP-fixed form of ARA7 suppressed the vps9a-2 mutation, but overexpression of ARA6 had no apparent effects. These results indicate that the two types of plant Rab5 members are functionally differentiated, even though they are regulated by the same activator, VPS9a.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1105/tpc.107.053876
  DOI ID：10.1105/tpc.107.053876, ISSN：1040-4651, ORCID：50019898, SCOPUS ID：37849024683, Web of Science ID：WOS:000252268700016
• The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5　　　　　　　　　　　　　　　
  Thomas J. Haas; Marek K. Sliwinski; Dana E. Martinez; Mary Preuss; Kazuo Ebine; Takashi Ueda; Erik Nielsen; Greg Odorizzi; Marisa S. Otegui
  PLANT CELL, Volume：19, Number：4, First page：1295, Last page：1312, Apr. 2007, [Reviewed]
  In yeast and mammals, the AAA ATPase Vps4p/SKD1 (for Vacuolar protein sorting 4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1) is required for the endosomal sorting of secretory and endocytic cargo. We identified a VPS4/SKD1 homolog in Arabidopsis thaliana, which localizes to the cytoplasm and to multivesicular endosomes. In addition, green fluorescent protein - SKD1 colocalizes on multivesicular bodies with fluorescent fusion protein endosomal Rab GTPases, such as ARA6/RabF1, RHA1/RabF2a, and ARA7/RabF2b, and with the endocytic marker FM4-64. The expression of SKD1(E232Q), an ATPase-deficient version of SKD1, induces alterations in the endosomal system of tobacco (Nicotiana tabacum) Bright Yellow 2 cells and ultimately leads to cell death. The inducible expression of SKD1E232Q in Arabidopsis resulted in enlarged endosomes with a reduced number of internal vesicles. In a yeast two-hybrid screen using Arabidopsis SKD1 as bait, we isolated a putative homolog of mammalian LYST-INTERACTING PROTEIN5 (LIP5)/SKD1 BINDING PROTEIN1 and yeast Vta1p (for Vps twenty associated 1 protein). Arabidopsis LIP5 acts as a positive regulator of SKD1 by increasing fourfold to fivefold its in vitro ATPase activity. We isolated a knockout homozygous Arabidopsis mutant line with a T-DNA insertion in LIP5. lip5 plants are viable and show no phenotypic alterations under normal growth conditions, suggesting that basal SKD1 ATPase activity is sufficient for plant development and growth.
  AMER SOC PLANT BIOLOGISTS, English
  DOI：https://doi.org/10.1105/tpc.106.049346
  DOI ID：10.1105/tpc.106.049346, ISSN：1040-4651, ORCID：50019881, SCOPUS ID：34250643053, Web of Science ID：WOS:000246802200015
• Myxomycetes collected at the Tokiwamatsu Imperial Villa, Tokyo　　　　　　　　　　　　　　　
  Yamamoto Yukinori; Hagiwara Hiromitsu; Degawa Yousuke; Kawakami Shin-ichi; Inaba Shigeki; Kamono Akiko; Tanahashi Masahiko; Ebine Kazuo
  Miscellaneous reports of the National Park for Nature Study, Volume：36, First page：31, Last page：65, Mar. 2005
  Myxomycetes collected at the Tokiwamatsu Imperial Villa, Tokyo Pref., Japan were examined by field work and moist chamber culture. As a result, 56 taxa were enumerated. Among them 11 taxa (Clastoderma debaryanum van imperatorium, Dictydiaethalium ferrugineum, Licea kleistobolus, L pedicellata (Arcyria affinis), Diderma microsporum, Didymium chrysosporum, Physarum leucophaeum, P. melleum f. luteum, P. sulphureum and Macbrideola martini were new to Tokyo Pref. (Bonin Islands are excluded). As a total, 210 myxomycetes become known from Tokyo Pref, and 25 taxa are illustrated to clarify the concept of each taxon. Both lists of the myxomycetes found in Tokyo Pref. and the corticolous myxomycetes collected from living trees in this study are added.
  国立科学博物館, Japanese
  ISSN：0385-759X, CiNii Articles ID：110004709621, CiNii Books ID：AN00103849

• シロイヌナズナ根毛側面形成に関与する膜交通系の解明　　　　　　　　　　　　　　　
  佐藤雅彦; 海老根一生; 上田貴志; 檜垣匠; 中山隆宏; 紺野宏記; 今村寿子; 平野朋子
  Volume：87th, 2023
  J-Global ID：202302234121968143
• Analysis of PICALM2 and pollen-specific VAMP72 members in Arabidopsis　　　　　　　　　　　　　　　
  海老根一生; 海老根一生; 藤本優; 室啓太; 武内秀憲; 野澤彰; 澤崎達也; 東山哲也; 上田貴志; 上田貴志
  日本植物生理学会年会(Web), Volume：64th, 2023
  J-Global ID：202302248876760406
• コケ配偶体と花粉における生殖細胞分化の理解と制御に向けて　　　　　　　　　　　　　　　
  山岡尚平; 海老根一生; 海老根一生; 齊藤美咲; 樅木亮介; 吉竹良洋; 上田貴志; 上田貴志; 光田展隆; 西浜竜一; 西浜竜一; 荒木崇; 河内孝之
  Volume：86th, 2022
  J-Global ID：202302230962734914
• SYP123-VAMP727 is involved in the secretion of the inner cell wall components to the root hair shank in Arabidopsis　　　　　　　　　　　　　　　
  佐藤雅彦; 海老根一生; 上田貴志; 檜垣匠; 紺野宏記; 中山隆宏; 平野朋子
  日本植物生理学会年会(Web), Volume：63rd, 2022
  J-Global ID：202202216741771060
• A bHLH transcription factor complex required for land plant germ cell differentiation　　　　　　　　　　　　　　　
  山岡尚平; 齊藤美咲; 樅木亮介; 吉竹良洋; 光田展隆; 海老根一生; 海老根一生; 西浜竜一; 西浜竜一; 荒木崇; 河内孝之
  日本農芸化学会大会講演要旨集(Web), Volume：2022, 2022
  ISSN：2186-7976, J-Global ID：202202210808349888
• Analysis of GH17 members regulating generative cell engulfment by the vegetative cell in Arabidopsis　　　　　　　　　　　　　　　
  海老根一生; 海老根一生; 山岡尚平; 上田貴志; 上田貴志
  日本植物生理学会年会(Web), Volume：62nd, 2021
  J-Global ID：202102282523407154
• シロイヌナズナの雄原細胞形成に関わるGH17メンバーの解析　　　　　　　　　　　　　　　
  海老根一生; 海老根一生; 山岡尚平; 上田貴志; 上田貴志
  Volume：84th, 2020
  J-Global ID：202302278743534156
• Analysis of GH17 members regulating engulfment of the generative cell into the vegetative cell in Arabidopsis　　　　　　　　　　　　　　　
  海老根一生; 海老根一生; 山岡尚平; 上田貴志; 上田貴志
  日本植物生理学会年会(Web), Volume：61st, 2020
  J-Global ID：202002220586226291
• シロイヌナズナにおけるHOPS/CORVET complexの解析
  竹元廣大; 海老根一生; 郷達明; 井藤純; 中野明彦; 中野明彦; 上田貴志
  日本植物学会大会研究発表記録, Volume：79th, First page：163, 01 Sep. 2015
  Japanese
  J-Global ID：201502219286823388
• 学部生に伝える研究最前線　　　　　　　　　　　　　　　
  海老根 一生; 上田 貴志; 牧島 一夫; 中澤 知洋; 寺坂 尚絃; 菅 裕明
  Volume：46, Number：3, First page：7, Last page：9, Sep. 2014
  植物の液胞へ物質を運ぶ仕組みを解明／宇宙最強の磁石天体は、磁力で変形していた／1つの遺伝子から2つのペプチドを同時に合成
  Japanese
  CiNii Articles ID：120005477796, CiNii Books ID：AA11719620
• Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions (vol 456, pg 962, 2008)　　　　　　　　　　　　　　　
  Pankaj Dhonukshe; Hirokazu Tanaka; Tatsuaki Goh; Kazuo Ebine; Ari Pekka Maehoenen; Kalika Prasad; Ikram Blilou; Niko Geldner; Jian Xu; Tomohiro Uemura; Joanne Chory; Takashi Ueda; Akihiko Nakano; Ben Scheres; Jiri Friml
  NATURE, Volume：511, Number：7509, Jul. 2014
  NATURE PUBLISHING GROUP, English, Others
  DOI：https://doi.org/10.1038/nature13549
  DOI ID：10.1038/nature13549, ISSN：0028-0836, eISSN：1476-4687, Web of Science ID：WOS:000338992200043
• 植物の液胞輸送経路の解析
  海老根一生; 井上丈司; 井藤純; 伊藤瑛海; 植村知博; 郷達明; 安部弘; 佐藤健; 中野明彦; 上田貴志
  日本植物生理学会年会要旨集, Volume：55th, First page：210, 11 Mar. 2014
  Japanese
  J-Global ID：201402294643094628
• ゼニゴケを用いた植物特異的膜交通経路の研究
  恵良美子; 海老根一生; 石崎公庸; 白川一; 西浜竜一; 河内孝之; 中野明彦; 上田貴志
  Volume：77th, First page：140, 20 Aug. 2013
  Japanese
  J-Global ID：201302256106492459
• 液胞膜の融合を制御する2つのSNARE複合体の解析
  海老根一生; 藤本優; 郷達明; 井藤純; 齋藤知恵子; 植村知博; 中野明彦; 上田貴志
  日本植物学会大会研究発表記録, Volume：75th, First page：136, 10 Sep. 2011
  Japanese
  J-Global ID：201102253421274334
• 研究ニュース　　　　　　　　　　　　　　　
  丸山 真一朗; 野崎 久義; 井出 哲; 吉岡 孝高; 五神 真; 海老根 一生; 上田 貴志; 若林 憲一
  Volume：43, Number：3, First page：10, Last page：14, Sep. 2011
  ミドリムシの祖先は「アカムシ」だった？／東北沖巨大地震はどのような現象だったのか？／励起子ボース・アインシュタイン凝縮の実現／細胞内の交通網はどのように整備されたのか／緑藻の光の好き嫌いを決める細胞内シグナル
  Japanese
  CiNii Articles ID：120003367893, CiNii Books ID：AA11719620
• エンドソーム/液胞膜の融合を制御するR‐SNAREの解析
  海老根一生; 藤本優; 郷達明; 井藤純; 植村知博; 中野明彦; 上田貴志
  日本植物学会大会研究発表記録, Volume：73rd, First page：143, 17 Sep. 2009
  Japanese
  J-Global ID：200902285989713112
• 種子植物特異的R‐SNAREであるVAMP727の機能解析　　　　　　　　　　　　　　　
  海老根一生; 岡谷祐哉; 郷達明; 井藤純; 植村知博; 中野明彦; 上田貴志
  日本植物生理学会年会要旨集, Volume：50th, First page：317, 16 Mar. 2009
  Japanese
  DOI：https://doi.org/10.14841/jspp.2009.0.0811.0
  DOI ID：10.14841/jspp.2009.0.0811.0, J-Global ID：200902298415758368
• 植物のRab5メンバーの動態と活性化制御機構の解析　　　　　　　　　　　　　　　
  郷 達明; 海老根 一生; 植村 知博; 上田 貴志; 中野 明彦
  Volume：16, Number：2, First page：169, Last page：170, 05 Oct. 2007
  Japanese
  ISSN：1342-2634, CiNii Articles ID：10019824105, CiNii Books ID：AN1055579X
• Functional analysis of AtVamp727 in Arabidopsis thaliana　　　　　　　　　　　　　　　
  Yusuke Okatani; Kazuo Ebine; Tomoko Dainobu; Tatsuaki Goh; Tomohiro Uemura; Akihiko Nakano; Takashi Ueda
  PLANT AND CELL PHYSIOLOGY, Volume：48, First page：S205, Last page：S205, 2007
  OXFORD UNIV PRESS, English, Summary international conference
  ISSN：0032-0781, Web of Science ID：WOS:000245922701298
• Endocytosis regulates flowering of Arabidopsis thaliana　　　　　　　　　　　　　　　
  Kazuo Ebine; Akihiko Nakano; Takashi Ueda
  PLANT AND CELL PHYSIOLOGY, Volume：48, First page：S114, Last page：S114, 2007
  OXFORD UNIV PRESS, English, Summary international conference
  ISSN：0032-0781, Web of Science ID：WOS:000245922700453
• Endocytosis regulates flowering of Arabidopsis thaliana　　　　　　　　　　　　　　　
  EBINE Kazuo; NAKANO Akihiko; UEDA Takashi
  Volume：119, First page：141, Last page：141, 01 Dec. 2006
  English
  ISSN：0918-9440, CiNii Articles ID：10019309816, CiNii Books ID：AA10900218

• The Japanese Society of Plant Physiologists
• The Botanical Society of Japan

• Analysis of plant vacuolar trasnport system regulated by RAB5　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 01 Apr. 2021 - 31 Mar. 2024
  National Institute for Basic Biology
  Grant amount(Total)：4160000, Direct funding：3200000, Indirect funding：960000
  Grant number：21K06222
• 様々なタイプのオートファジーによる植物の高次機能発現　　　　　　　　　　　　　　　
  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), 28 Jun. 2019 - 31 Mar. 2024
  Meiji University
  Grant amount(Total)：66820000, Direct funding：51400000, Indirect funding：15420000
  Grant number：19H05713
• Analysis of membrane trafficking mechanisms regulating male gametogenesis in plant　　　　　　　　　　　　　　　
  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), 01 Apr. 2019 - 31 Mar. 2021
  National Institute for Basic Biology
  Grant amount(Total)：8580000, Direct funding：6600000, Indirect funding：1980000
  Grant number：19H04872
• Analysis of vacuolar transport mechanisms in plant cells　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), 01 Apr. 2018 - 31 Mar. 2021
  Ebine Kazuo, National Institute for Basic Biology
  Grant amount(Total)：4290000, Direct funding：3300000, Indirect funding：990000
  The vacuole is the largest and multifunctional organelle in plant cells, and many proteins are transported to and stored in this organelle through membrane traffic pathways. We analyzed detail molecular mechanisms of transport pathways to the vacuole, especially focus on plant-unique vacuolar transport pathway. We found that at least two plant-unique vacuolar transport pathways exist in plant cells, and one of this pathway contributes to the stress response of plant.
  Grant number：18K06303
• Analysis of membrane trafficking mechanisms regulating gametophyte formation in plant　　　　　　　　　　　　　　　
  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), 01 Apr. 2017 - 31 Mar. 2019
  National Institute for Basic Biology
  Grant amount(Total)：9360000, Direct funding：7200000, Indirect funding：2160000
  Grant number：17H05850
• Analysis of molecular mechanisms of vacuolar membrane fusion regulated by SNARE proteins in plant cells　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B), Grant-in-Aid for Young Scientists (B), 01 Apr. 2015 - 31 Mar. 2017
  EBINE Kazuo
  Grant amount(Total)：4160000, Direct funding：3200000, Indirect funding：960000
  The multifunctional vacuole is the largest organelle in plant cells, to and in which many proteins and other components are transported and stored. Three vacuolar transport pathways exist in Arabidopsis cells, but detail mechanisms of these pathways are still unclear. The analysis of transport mechanism of VHP1 to the vacuole suggests that RAB5-dependent RAB7-AP3-independent pathway, which is one of the plant unique pathways, is related to the transport pathway from ER to the vacuole. SNARE proteins are conserved key molecules regulating membrane fusion in membrane trafficking pathways. Vacuolar transport also involves a set of SNARE proteins, which mainly reside on the vacuolar membrane. Vacuolar SNAREs have also been shown to be required for vacuolar morphogenesis. VAM3/SYP22/SGR3 is a Qa-SNARE localizing on the vacuolar membrane. By the analysis of sgr3-1 mutant, alpha-SNAP was isolated as a novel regulator of vacuolar dynamics in plant.
  Grant number：15K18551
• Functions and molecular mechanisms of membrane trafficking in biogenesis and functions of cell wall　　　　　　　　　　　　　　　
  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), 28 Jun. 2012 - 31 Mar. 2017
  UEDA Takashi; UEMURA Tomohiro; FUJIMOTO Masaru; EBINE Kazuo
  Grant amount(Total)：47190000, Direct funding：36300000, Indirect funding：10890000
  We analyzed mechanisms of cell wall construction and cell wall functions with various techniques, including super-resolution microscopy, biochemistry, and genetics. We succeeded in fine-resolution imaging of transport of the cellulose synthase complex, which is distinctly dependent on PI3K and PI4K. We also analyzed RABA/RAB11 functions in endocytosis of FLS2 to demonstrate functional diversification of RABA/RAB11 members. Furthermore, we demonstrated that the plant vacuolar transport pathway is distinctly regulated from yeasts and animals, which plays a pivotal role in cell wall integrity. Evolutionary cell biological analyses of the membrane trafficking system using the liverwort, Marchantia polymorpha, is also currently underway.
  Grant number：24114003
• アピコンプレクサＲａｂ５Ｂと陸上植物ＡＲＡ６が制御する小胞輸送経路の解析　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows, Grant-in-Aid for JSPS Fellows, 2011 - 31 Mar. 2014
  National Institute of Infectious Diseases
  Grant amount(Total)：2400000, Direct funding：2400000
  Grant number：11J08803
• エンドサイトーシスによる植物の高次形態形成制御の解析　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows, Grant-in-Aid for JSPS Fellows, 2007 - 2009
  The University of Tokyo
  Grant amount(Total)：2700000, Direct funding：2700000
  Grant number：07J05010