Updated on 2021/10/20

写真a

 
UCHIDA Naoyuki
 
Organization
Center for Gene Research Professor
Graduate School
Graduate School of Science
Title
Professor

Degree 3

  1. 博士(薬学) ( 2004.3   東京大学 ) 

  2. 修士(薬学) ( 2001.3   東京大学 ) 

  3. 学士(薬学) ( 1999.3   東京大学 ) 

Research Interests 8

  1. 多細胞秩序の形成・維持・変化

  2. 細胞間情報伝達

  3. Information Molecules

  4. 受容体

  5. bioactive molecules

  6. stem cell

  7. pluripotency

  8. 植物成長調節

Research Areas 1

  1. Life Science / Plant molecular biology and physiology

Current Research Project and SDGs 2

  1. 植物の組織秩序の理解

  2. 植物の成長調節

Research History 5

  1. Nagoya University   Center for Gene Research   Professor

    2020.4

  2. Nagoya University   Institute of Transformative Bio-Molecules   Designated associate professor

    2013.4 - 2020.3

  3. Nara Institute of Science and Technology   Assistant Professor

    2008.9 - 2013.3

  4. Nara Institute of Science and Technology

    2007.9 - 2008.8

  5. University of California-Davis   博士研究員

    2004.4 - 2007.8

Education 4

  1. The University of Tokyo   Graduate School, Division of Pharmaceutical Sciences

    2001.4 - 2004.3

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    Country: Japan

  2. The University of Tokyo   Graduate School, Division of Pharmaceutical Sciences

    1999.4 - 2001.3

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    Country: Japan

  3. The University of Tokyo   Graduate School, Division of Pharmaceutical Sciences

    1997.4 - 1999.3

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    Country: Japan

  4. The University of Tokyo   Graduate School, Division of Pharmaceutical Sciences

    1995.4 - 1997.3

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    Country: Japan

Professional Memberships 2

  1. THE JAPANESE SOCIETY OF PLANT PHYSIOLOGISTS

  2. THE BOTANICAL SOCIETY OF JAPAN

Committee Memberships 6

  1. 日本植物学会 第84回大会   大会準備委員会 プログラム委員長  

    2020 - 2020.9   

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    Committee type:Academic society

  2. 日本植物生理学会   広報幹事  

    2018.3 - 2020.3   

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    Committee type:Academic society

  3. Journal of Plant Research   Editorial Board  

    2016.1 - 2019.12   

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    Committee type:Academic society

  4. 日本植物学会   電子出版物編集委員会 委員長  

    2016.1 - 2016.12   

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    Committee type:Academic society

  5. 日本植物学会   電子出版物編集委員会 委員  

    2015.1 - 2015.12   

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    Committee type:Academic society

  6. 日本植物学会   広報委員会 ホームページコンテンツ作成グループ委員  

    2014.1 - 2014.12   

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    Committee type:Academic society

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Papers 39

  1. The boundary-expressed EPIDERMAL PATTERNING FACTOR-LIKE2 gene encoding a signaling peptide promotes cotyledon growth during Arabidopsis thaliana embryogenesis. Reviewed International coauthorship

    1) Fujihara R, Uchida N, Tameshige T, Kawamoto N, Hotokezaka Y, Higaki T, Simon R, Torii KU, Tasaka M, Aida M

    Plant Biotechnology   Vol. 38 ( 3 ) page: 317 - 322   2021

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

    DOI: 10.5511/plantbiotechnology.21.0508a

  2. A super-sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair. Reviewed

    Nishimura K, Yamada R, Hagihara S, Iwasaki R, Uchida N, Kamura T, Takahashi K, Torii KU, Fukagawa T.

    Nucleic acids research   Vol. 48 ( 18 ) page: e108   2020.10

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    Publishing type:Research paper (scientific journal)  

    The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes.

    DOI: 10.1093/nar/gkaa748

    PubMed

  3. A Peptide Pair Coordinates Regular Ovule Initiation Patterns with Seed Number and Fruit Size. Reviewed

    Kawamoto N, Del Carpio DP, Hofmann A, Mizuta Y, Kurihara D, Higashiyama T, Uchida N, Torii KU, Colombo L, Groth G, Simon R

    Current biology   Vol. (in press)   2020.9

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

    Ovule development in Arabidopsis thaliana involves pattern formation, which ensures that ovules are regularly arranged in the pistils to reduce competition for nutrients and space. Mechanisms underlying pattern formation in plants, such as phyllotaxis, flower morphogenesis, or lateral root initiation, have been extensively studied, and genes controlling the initiation of ovules have been identified. However, the fundamental patterning mechanism that determines the spacing of ovule anlagen within the placenta remained unexplored. Using natural variation analysis combined with quantitative trait locus analysis, we found that the spacing of ovules in the developing gynoecium and fruits is controlled by two secreted peptides, EPFL2 and EPFL9 (also known as Stomagen), and their receptors from the ERECTA (ER) family that act from the carpel wall and the placental tissue. We found that a signaling pathway controlled by EPFL9 acting from the carpel wall through the LRR-receptor kinases ER, ERL1, and ERL2 promotes fruit growth. Regular spacing of ovules depends on EPFL2 expression in the carpel wall and in the inter-ovule spaces, where it acts through ERL1 and ERL2. Loss of EPFL2 signaling results in shorter gynoecia and fruits and irregular spacing of ovules or even ovule twinning. We propose that the EPFL2 signaling module evolved to control the initiation and regular, equidistant spacing of ovule primordia, which may serve to minimize competition between seeds or facilitate equal resource allocation. Together, EPFL2 and EPFL9 help to coordinate ovule patterning and thereby seed number with gynoecium and fruit growth through a set of shared receptors.

    DOI: 10.1016/j.cub.2020.08.050

    PubMed

  4. Induction of Multichotomous Branching by CLAVATA Peptide in Marchantia polymorpha. Reviewed

    Hirakawa Y, Fujimoto T, Ishida S, Uchida N, Sawa S, Kiyosue T, Ishizaki K, Nishihama R, Kohchi T, Bowman JL

    Current biology   Vol. 30 ( 19 ) page: 3833 - 3840   2020.8

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    Publishing type:Research paper (scientific journal)  

    A key innovation in land plants was the evolution of meristems with stem cells possessing multiple cutting faces (division planes) from which three-dimensional growth is derived in both haploid (gametophyte) and diploid (sporophyte) generations [1-3]. Within each meristem exists a pool of stem cells that must be maintained at a relatively constant size for development to occur appropriately [4-6]. In flowering plants, stem cells of the diploid generation are maintained by CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptide signaling [7, 8]. In the liverwort Marchantia polymorpha, the haploid body undergoes dichotomous branching, an ancestral characteristic of growth derived from the meristem, in which two equivalent body axes are developed via stem cell division, regulated by unknown molecular mechanisms. We show here that in M. polymorpha, treatment with MpCLE2/CLAVATA3 (CLV3) peptide resulted in the accumulation of undifferentiated cells, marked by MpYUC2 expression, in the apical meristem. Removal of MpCLE2 peptide resulted in multichotomous branching from the accumulated cells. Genetic analysis demonstrated that the CLAVATA1 (MpCLV1) receptor, but not the WUSCHEL-related HOMEOBOX (MpWOX) transcription factor, is responsible for MpCLE2 peptide signaling. In the apical meristem, MpCLV1 was expressed broadly in the central region, including the MpYUC2-positive area, whereas MpCLE2 was expressed in a largely complementary manner compared to MpYUC2, suggesting MpCLE2 mediates local cell-to-cell communication. CLV3/CLE peptide, a negative regulator of diploid stem cells in flowering plants, acts as a haploid stem cell-promoting signal in M. polymorpha, implicating a critical role for this pathway in the evolution of body plan in land plants.

    DOI: 10.1016/j.cub.2020.07.016

    PubMed

  5. Control of proliferation in the haploid meristem by CLE peptide signaling in Marchantia polymorpha Reviewed

    Hirakawa Y, Uchida N, Yamaguchi YL, Tabata R, Ishida S, Ishizaki K, Nishihama R, Kohchi T, Sawa S, Bowman JL

    PLOS Genetics   Vol. 15 ( 3 ) page: e1007997   2019.3

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    Publishing type:Research paper (scientific journal)   Publisher:Public Library of Science (PLoS)  

    DOI: 10.1371/journal.pgen.1007997

    PubMed

  6. Stem cells within the shoot apical meristem: identity, arrangement and communication. Reviewed

    Uchida N, Torii KU

    Cellular and molecular life sciences   Vol. 76 ( 6 ) page: 1067 - 1080   2019.3

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1007/s00018-018-2980-z

    PubMed

  7. Harnessing synthetic chemistry to probe and hijack auxin signaling. Reviewed

    Torii KU, Hagihara S, Uchida N, Takahashi K

    The New phytologist   Vol. 220 ( 2 ) page: 417 - 424   2018.10

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1111/nph.15337

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  8. A Super Strong Engineered Auxin-TIR1 Pair. Reviewed

    Yamada R, Murai K, Uchida N, Takahashi K, Iwasaki R, Tada Y, Kinoshita T, Itami K, Torii KU, Hagihara S

    Plant & cell physiology   Vol. 59 ( 8 ) page: 1538 - 1544   2018.8

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1093/pcp/pcy127

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  9. Rapid and reversible root growth inhibition by TIR1 auxin signalling Reviewed

    Matyáš Fendrych, Maria Akhmanova, Jack Merrin, Matouš Glanc, Shinya Hagihara, Koji Takahashi, Naoyuki Uchida, Keiko U. Torii, Jiří Friml

    Nature Plants   Vol. 4 ( 7 ) page: 1 - 7   2018.6

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Palgrave Macmillan Ltd.  

    The phytohormone auxin is the information carrier in a plethora of developmental and physiological processes in plants1. It has been firmly established that canonical, nuclear auxin signalling acts through regulation of gene transcription2. Here, we combined microfluidics, live imaging, genetic engineering and computational modelling to reanalyse the classical case of root growth inhibition3 by auxin. We show that Arabidopsis roots react to addition and removal of auxin by extremely rapid adaptation of growth rate. This process requires intracellular auxin perception but not transcriptional reprogramming. The formation of the canonical TIR1/AFB–Aux/IAA co-receptor complex is required for the growth regulation, hinting to a novel, non-transcriptional branch of this signalling pathway. Our results challenge the current understanding of root growth regulation by auxin and suggest another, presumably non-transcriptional, signalling output of the canonical auxin pathway.

    DOI: 10.1038/s41477-018-0190-1

    Scopus

    PubMed

  10. Chemical hijacking of auxin signaling with an engineered auxin-TIR1 pair Reviewed

    Naoyuki Uchida, Koji Takahashi, Rie Iwasaki, Ryotaro Yamada, Masahiko Yoshimura, Takaho A Endo, Seisuke Kimura, Hua Zhang, Mika Nomoto, Yasuomi Tada, Toshinori Kinoshita, Kenichiro Itami, Shinya Hagihara, Keiko U. Torii

    Nature Chemical Biology   Vol. 14 ( 3 ) page: 299 - 305   2018.3

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

    The phytohormone auxin indole-3-acetic acid (IAA) regulates nearly all aspects of plant growth and development. Despite substantial progress in our understanding of auxin biology, delineating specific auxin response remains a major challenge. Auxin regulates transcriptional response via its receptors, TIR1 and AFB F-box proteins. Here we report an engineered, orthogonal auxin-TIR1 receptor pair, developed through a bump-and-hole strategy, that triggers auxin signaling without interfering with endogenous auxin or TIR1/AFBs. A synthetic, convex IAA (cvxIAA) hijacked the downstream auxin signaling in vivo both at the transcriptomic level and in specific developmental contexts, only in the presence of a complementary, concave TIR1 (ccvTIR1) receptor. Harnessing the cvxIAA-ccvTIR1 system, we provide conclusive evidence for the role of the TIR1-mediated pathway in auxin-induced seedling acid growth. The cvxIAA-ccvTIR1 system serves as a powerful tool for solving outstanding questions in auxin biology and for precise manipulation of auxin-mediated processes as a controllable switch.

    DOI: 10.1038/nchembio.2555

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  11. ERECTA-family genes coordinate stem cell functions between the epidermal and internal layers of the shoot apical meristem Reviewed

    Yuka Kimura, Masao Tasaka, Keiko U. Torii, Naoyuki Uchida

    Development (Cambridge)   Vol. 145 ( 1 )   2018.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Company of Biologists Ltd  

    The epidermal cell layer and the tissues that lie underneath have different intrinsic functions during plant development. The stem cells within the shoot apical meristem (SAM) that give rise to aerial structures are located in the epidermal and internal tissue layers. However, our understanding of how the functions of these stem cells are coordinated across tissue layers so stem cells can behave as a single population remains limited. WUSCHEL (WUS) functions as a master regulator of stem cell activity. Here, we show that loss of function in the ERECTA (ER)-family receptor kinase genes can rescue the mutant phenotype of wus plants (loss of stem cells), as demonstrated by the reinstated expression of a stem cell marker gene in the SAM epidermis. Localized ER expression in the epidermis can suppress the SAM phenotype caused by loss of ER-family activity. Furthermore, the CLAVATA3- and cytokinin-induced outputs, which contribute to stem cell homeostasis, are dysfunctional in a tissue layer-specific manner in ER-family mutants. Collectively, our findings suggest that the ER family plays a role in the coordination of stem cell behavior between different SAM tissue layers.

    DOI: 10.1242/dev.156380

    Scopus

    PubMed

  12. Discovery of synthetic small molecules that enhance the number of stomata: C-H functionalization chemistry for plant biology Reviewed

    Asraa Ziadi, Naoyuki Uchida, Hiroe Kato, Rina Hisamatsu, Ayato Sato, Shinya Hagihara, Kenichiro Itami, Keiko U. Torii

    CHEMICAL COMMUNICATIONS   Vol. 53 ( 69 ) page: 9632 - 9635   2017.9

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ROYAL SOC CHEMISTRY  

    The increasing climate changes and global warming are leading to colossal agricultural problems such as abatement of food production and quality. As stomatal development is considered to play a key role in crop plant productivity and water-use efficiency, studying stomatal development is useful for understanding the productivity of plant systems for both natural and agricultural systems. Herein, we report the first-in-class synthetic small molecules enhancing the number of stomata in Arabidopsis thaliana that have been discovered by screening of the chemical library and further optimized by the Pd-catalyzed C-H arylation reaction. The present study shows not only huge potential of small molecules to control the cellular and developmental processes of stomata without using genetically modified plants, but also the power of C-H functionalization chemistry to rapidly identify the optimized compounds.

    DOI: 10.1039/c7cc04526c

    Web of Science

    PubMed

  13. Mechanisms and Strategies Shaping Plant Peptide Hormones Reviewed

    Yuki Hirakawa, Keiko U. Torii, Naoyuki Uchida

    PLANT AND CELL PHYSIOLOGY   Vol. 58 ( 8 ) page: 1313 - 1318   2017.8

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    Plant genomes encode a variety of short peptides acting as signaling molecules. Since the discovery of tomato systemin, a myriad of peptide signals, ranging in size, structure and modifications, have been found in plants. Moreover, new peptides are still being identified. Surprisingly, non-plant organisms, especially pathogens, also produce peptides which exert hormonal activities against host plants by hijacking their endogenous reception systems. In this review, we focus on short secretory peptides ranging from five to 20 amino acids. We first summarize recent advances in understanding relationships between the bioactivities and structures of plant peptide hormones. Subsequently, we introduce the topic of peptides produced by non-plant organisms. Lastly, we describe artificial peptides synthesized in laboratories, which possess intriguing bioactive properties beyond those of natural peptide hormones.

    DOI: 10.1093/pcp/pcx069

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  14. ERECTA-family receptor kinase genes redundantly prevent premature progression of secondary growth in the Arabidopsis hypocotyl Reviewed

    Shuka Ikematsu, Masao Tasaka, Keiko U. Torii, Naoyuki Uchida

    NEW PHYTOLOGIST   Vol. 213 ( 4 ) page: 1697 - 1709   2017.3

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

    Secondary growth is driven by continuous cell proliferation and differentiation of the cambium that acts as vascular stem cells, producing xylem and phloem to expand vascular tissues laterally. During secondary growth of hypocotyls in Arabidopsis thaliana, the xylem undergoes a drastic phase transition from a parenchyma-producing phase to a fiber-producing phase at the appropriate time. However, it remains to be fully elucidated how progression of secondary growth is properly controlled.
    We focused on phenotypes of hypocotyl vasculatures caused by double mutation in ERECTA (ER) and ER-LIKE1 (ERL1) receptor-kinase genes to elucidate their roles in secondary growth.
    ER and ERL1 redundantly suppressed excessive radial growth of the hypocotyl vasculature during secondary growth. ER and ERL1 also prevented premature initiation of the fiber differentiation process mediated by the NAC SECONDARY WALL THICKENING PROMOTING FACTORs in the hypocotyl xylem. Upon floral transition, the hypocotyl xylem gained a competency to respond to GA in a BREVIPEDICELLUS-dependent manner, which was a prerequisite for fiber differentiation. However, even after the floral transition, ER and ERL1 prevented precocious initiation of the GA-mediated fiber formation.
    Collectively, our findings reveal that ER and ERL1 redundantly prevent premature progression of sequential events in secondary growth.

    DOI: 10.1111/nph.14335

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    PubMed

  15. Cryptic bioactivity capacitated by synthetic hybrid plant peptides Reviewed

    Yuki Hirakawa, Hidefumi Shinohara, Kai Welke, Stephan Irle, Yoshikatsu Matsubayashi, Keiko U. Torii, Naoyuki Uchida

    NATURE COMMUNICATIONS   Vol. 8   page: 14318   2017.2

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    Evolution often diversifies a peptide hormone family into multiple subfamilies, which exert distinct activities by exclusive interaction with specific receptors. Here we show that systematic swapping of pre-existing variation in a subfamily of plant CLE peptide hormones leads to a synthetic bifunctional peptide that exerts activities beyond the original subfamily by interacting with multiple receptors. This approach provides new insights into the complexity and specificity of peptide signalling.

    DOI: 10.1038/ncomms14318

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  16. Stem development through vascular tissues: EPFL-ERECTA family signaling that bounces in and out of phloem Reviewed

    Toshiaki Tameshige, Shuka Ikematsu, Keiko U. Torii, Naoyuki Uchida

    JOURNAL OF EXPERIMENTAL BOTANY   Vol. 68 ( 1 ) page: 45 - 53   2017.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    Plant cells communicate with each other using a variety of signaling molecules. Recent studies have revealed that various types of secreted peptides, as well as phytohormones known since long ago, mediate cell-cell communication in diverse contexts of plant life. These peptides affect cellular activities, such as proliferation and cell fate decisions, through their perception by cell surface receptors located on the plasma membrane of target cells. ERECTA (ER), an Arabidopsis thaliana receptor kinase gene, was first identified as a stem growth regulator, and since then an increasing number of studies have shown that ER is involved in a wide range of developmental and physiological processes. In particular, molecular functions of ER have been extensively studied in stomatal patterning. Furthermore, the importance of ER signaling in vascular tissues of inflorescence stems, especially in phloem cells, has recently been highlighted. In this review article, first we briefly summarize the history of ER research including studies on stomatal development, then introduce ER functions in vascular tissues, and discuss its interactions with phytohormones and other receptor kinase signaling pathways. Future questions and challenges will also be addressed.

    DOI: 10.1093/jxb/erw447

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  17. A Secreted Peptide and Its Receptors Shape the Auxin Response Pattern and Leaf Margin Morphogenesis Reviewed

    Toshiaki Tameshige, Satoshi Okamoto, Jin Suk Lee, Mitsuhiro Aida, Masao Tasaka, Keiko U. Torii, Naoyuki Uchida

    CURRENT BIOLOGY   Vol. 26 ( 18 ) page: 2478 - 2485   2016.9

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

    Secreted peptides mediate intercellular communication [1, 2]. Several secreted peptides in the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family regulate morphogenesis of tissues, such as stomata and inflorescences in plants [3-15]. The biological functions of other EPFL family members remain unknown. Here, we show that the EPFL2 gene is required for growth of leaf teeth. EPFL2 peptide physically interacts with ERECTA (ER) family receptor-kinases and, accordingly, the attenuation of ER family activities leads to formation of toothless leaves. During the tooth growth process, responses to the phytohormone auxin are maintained at tips of the teeth to promote their growth [16-19]. In the growing tooth tip of epfl2 and multiple er family mutants, the auxin response becomes broader. Conversely, overexpression of EPFL2 diminishes the auxin response, indicating that the EPFL2 signal restricts the auxin response to the tooth tip. Interestingly, the tip-specific auxin response in turn organizes characteristic expression patterns of ER family and EPFL2 by enhancing ER family expression at the tip while eliminating the EPFL2 expression from the tip. Our findings identify the novel ligand-receptor pairs promoting the tooth growth, and further reveal a feedback circuit between the peptide-receptor system and auxin response as a mechanism for maintaining proper auxin maxima during leaf margin morphogenesis.

    DOI: 10.1016/j.cub.2016.07.014

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  18. Efficient In Planta Detection and Dissection of De Novo Mutation Events in the Arabidopsis thaliana Disease Resistance Gene UNI Reviewed

    Tomohiko Ogawa, Akiko Mori, Kadunari Igari, Miyo Terao Morita, Masao Tasaka, Naoyuki Uchida

    PLANT AND CELL PHYSIOLOGY   Vol. 57 ( 6 ) page: 1123 - 1132   2016.6

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    Plants possess disease resistance (R) proteins encoded by R genes, and each R protein recognizes a specific pathogen factor(s) for immunity. Interestingly, a remarkably high degree of polymorphisms in R genes, which are traces of past mutation events during evolution, suggest the rapid diversification of R genes. However, little is known about molecular aspects that facilitate the rapid change of R genes because of the lack of tools that enable us to monitor de novo R gene mutations efficiently in an experimentally feasible time scale, especially in living plants. Here we introduce a model assay system that enables efficient in planta detection of de novo mutation events in the Arabidopsis thaliana R gene UNI in one generation. The uni-1D mutant harbors a gain-of-function allele of the UNI gene. uni-1D heterozygous individuals originally exhibit dwarfism with abnormally short stems. However, interestingly, morphologically normal stems sometimes emerge spontaneously from the uni-1D plants, and the morphologically reverted tissues carry additional de novo mutations in the UNI gene. Strikingly, under an extreme condition, almost half of the examined population shows the reversion phenomenon. By taking advantage of this phenomenon, we demonstrate that the reversion frequency is remarkably sensitive to a variety of fluctuations in DNA stability, underlying a mutable tendency of the UNI gene. We also reveal that activities of the salicylic acid pathway and DNA damage sensor pathway are involved in the reversion phenomenon. Thus, we provide an experimentally feasible model tool to explore factors and conditions that significantly affect the R gene mutation phenomenon.

    DOI: 10.1093/pcp/pcw060

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  19. Impact of erecta mutation on leaf serration differs between Arabidopsis accessions Reviewed

    Toshiaki Tameshige, Satoshi Okamoto, Masao Tasaka, Keiko U. Torii, Naoyuki Uchida

    PLANT SIGNALING & BEHAVIOR   Vol. 11 ( 12 ) page: e1261231   2016

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:TAYLOR & FRANCIS INC  

    Serrations or teeth of plant leaves are a morphological trait regulated genetically and environmentally. Very recently, it has been reported that the receptor kinases encoded by three ERECTA (ER)-family genes, ER, ER-LIKE1 (ERL1) and ERL2, redundantly play a role in tooth growth in Arabidopsis thaliana. In the report, Columbia (Col) accession was used for analyses, where none of the signal mutant of the ER-family genes exhibited serration defects. The toothless, smooth leaf margin phenotype was evident only when two out of the three ER-family genes were lost. Interestingly, it has been widely recognized that the Arabidopsis accession Landsberg erecta (L.er), which carries a loss-of-function mutation in ER, develops round leaves with smaller leaf teeth. Here, we show that the functional ER transgene promotes the tooth growth in L. er to the level of Col, indicating that the er mutation in L. er is likely responsible for the reduced growth of leaf teeth. This suggests that er single mutation affects tooth growth in a different manner between Col and L. er backgrounds, though the molecular basis for this background-dependent effect remains to be addressed.

    DOI: 10.1080/15592324.2016.1261231

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  20. Cell walls as a stage for intercellular communication regulating shoot meristem development Reviewed

    Toshiaki Tameshige, Yuki Hirakawa, Keiko U. Torii, Naoyuki Uchida

    FRONTIERS IN PLANT SCIENCE   Vol. 6   page: 324   2015.5

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:FRONTIERS RESEARCH FOUNDATION  

    Aboveground organs of plants are ultimately derived/generated from the shoot apical meristem (SAM), which is a proliferative tissue located at the apex of the stem. The SAM contains a population of stem cells that provide new cells for organ/tissue formation. The SAM is composed of distinct cell layers and zones with different properties. Primordia of lateral organs develop at the periphery of the SAM. The shoot apex is a dynamic and complex tissue, and as such intercellular communications among cells, layers and zones play significant roles in the coordination of cell proliferation, growth and differentiation to achieve elaborate morphogenesis. Recent findings have highlighted the importance of a number of signaling molecules acting in the cell wall space for the intercellular communication, including classic phytohormones and secretory peptides. Moreover, accumulating evidence has revealed that cell wall properties and their modifying enzymes modulate hormone actions. In this review, we outline how behaviors of signaling molecules and changes of cell wall properties are integrated for the shoot meristem regulation.

    DOI: 10.3389/fpls.2015.00324

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  21. Transcriptional, Posttranscriptional, and Posttranslational Regulation of SHOOT MERISTEMLESS Gene Expression in Arabidopsis Determines Gene Function in the Shoot Apex Reviewed

    Jose Antonio Aguilar-Martinez, Naoyuki Uchida, Brad Townsley, Donnelly Ann West, Andrea Yanez, Nafeesa Lynn, Seisuke Kimura, Neelima Sinha

    PLANT PHYSIOLOGY   Vol. 167 ( 2 ) page: 424 - 442   2015.2

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC PLANT BIOLOGISTS  

    The activity of SHOOTMERISTEMLESS (STM) is required for the functioning of the shoot apical meristem (SAM). STM is expressed in the SAM but is down-regulated at the site of leaf initiation. STM is also required for the formation of compound leaves. However, how the activity of STM is regulated at the transcriptional, posttranscriptional, and posttranslational levels is poorly understood. We previously found two conserved noncoding sequences in the promoters of STM-like genes across angiosperms, the K-box and the RB-box. Here, we characterize the function of the RB-box in Arabidopsis (Arabidopsis thaliana). The RB-box, along with the K-box, regulates the expression of STM in leaf sinuses, which are areas on the leaf blade with meristematic potential. The RB-box also contributes to restrict STM expression to the SAM. We identified FAR1-RELATED SEQUENCES-RELATED FACTOR1 (FRF1) as a binding factor to the RB-box region. FRF1 is an uncharacterized member of a subfamily of four truncated proteins related to the FAR1-RELATED SEQUENCES factors. Internal deletion analysis of the STM promoter identified a region required to repress the expression of STM in hypocotyls. Expression of STM in leaf primordia under the control of the JAGGED promoter produced plants with partially undifferentiated leaves. We further found that the ELK domain has a role in the posttranslational regulation of STM by affecting the nuclear localization of STM.

    DOI: 10.1104/pp.114.248625

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  22. Identification of EMS-induced causal mutations in Arabidopsis thaliana by next-generation sequencing Reviewed

    Naoyuki Uchida, Tomoaki Sakamoto, Masao Tasaka, Tetsuya Kurata

    Methods in Molecular Biology   Vol. 1062   page: 259 - 270   2014

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    Emerging next-generation sequencing (NGS) technologies are powerful tools for the identification of causal mutations underlying phenotypes of interest in Arabidopsis thaliana. Based on a methodology termed bulked segregant analysis (BSA), whole-genome sequencing data are derived from pooled F2 segregants after crossing a mutant to a different polymorphic accession and are analyzed for single nucleotide polymorphisms (SNPs). Then, a genome region spanning the causal mutation site is narrowed down by linkage analysis of SNPs in the accessions used to produce the F1 generation. Next, candidate SNPs for the causative mutation are extracted by filtering the linked SNPs using multiple appropriate criteria. Effects of each candidate SNP on the function of the corresponding gene are evaluated to identify the causal mutation, and its validity is then confirmed by independent criteria. This chapter describes the identification by NGS analysis of causal recessive mutations derived from EMS mutagenesis. © 2014 Springer Science+Business Media New York.

    DOI: 10.1007/978-1-62703-580-4_14

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  23. Regulation of plant vascular stem cells by endodermis-derived EPFL-family peptide hormones and phloem-expressed ERECTA-family receptor kinases Reviewed

    Naoyuki Uchida, Masao Tasaka

    JOURNAL OF EXPERIMENTAL BOTANY   Vol. 64 ( 17 ) page: 5335 - 5343   2013.12

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    Plant vasculatures are complex tissues consisting of (pro)cambium, phloem, and xylem. The (pro) cambium serves as vascular stem cells that produce all vascular cells. The Arabidopsis ERECTA (ER) receptor kinase is known to regulate the architecture of inflorescence stems. It was recently reported that the er mutation enhances a vascular phenotype induced by a mutation of TDR/PXY, which plays a significant role in procambial proliferation, suggesting that ER participates in vascular development. However, detailed molecular mechanisms of the ER-dependent vascular regulation are largely unknown. Here, this work found that ER and its paralogue, ER-LIKE1, were redundantly involved in procambial development of inflorescence stems. Interestingly, their activity in the phloem was sufficient for vascular regulation. Furthermore, two endodermis-derived peptide hormones, EPFL4 and EPFL6, were redundantly involved in such regulation. It has been previously reported that EPFL4 and EPFL6 act as ligands of phloem-expressed ER for stem elongation. Therefore, these findings indicate that cell-cell communication between the endodermis and the phloem plays an important role in procambial development as well as stem elongation. Interestingly, similar EPFL-ER modules control two distinct developmental events by slightly changing their components: the EPFL4/6-ER module for stem elongation and the EPFL4/6-ER/ERL1 module for vascular development.

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  24. ERECTA-Family Receptor Kinases Regulate Stem Cell Homeostasis via Buffering its Cytokinin Responsiveness in the Shoot Apical Meristem Reviewed

    Naoyuki Uchida, Masanori Shimada, Masao Tasaka

    PLANT AND CELL PHYSIOLOGY   Vol. 54 ( 3 ) page: 343 - 351   2013.3

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    Shoot apical meristems (SAMs), which are maintained at the tips of stems, are indeterminate structures and sources of stem cells from which all aerial organs are ultimately derived. Although mechanisms that regulate the homeostasis of the stem cells have been extensively investigated, identification of further unknown regulators should provide better understanding of the regulation. Here, we report that members of the Arabidopsis ERECTA (ER) receptor kinase family redundantly play a significant role in the regulation of stem cell homeostasis. In wild-type seedlings, the expression of WUSCHEL (WUS), a central regulator of the stem cell population, is stimulated by cytokinin. Interestingly, however, the SAM morphology and the expression of CLAVATA3 (CLV3), which is expressed in stem cells and therefore serves as a stem cell marker, are relatively stable against cytokinin treatment regardless of increased WUS expression. These findings indicate the presence of a mechanism to buffer stem cell homeostasis against an increase in cytokinin. Mutant seedlings lacking all ER-family members, which are expressed in the SAM, show an increase in the stem cell population and also the up-regulation of a cytokinin-responsive gene in the SAM. In this mutant, WUS expression is stimulated by cytokinin treatment as efficiently as in wild-type plants. However, in contrast to wild-type plants, SAM morphology and CLV3 expression respond drastically to cytokinin treatment, suggesting that the buffering mechanism to maintain stem cell homeostasis against an increase in cytokinin is severely impaired in this mutant. We suggest that the ER family regulates stem cell homeostasis via buffering its cytokinin responsiveness in the SAM.

    DOI: 10.1093/pcp/pcs109

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  25. Biological role of the two overlapping poly(A)-binding protein interacting motifs 2 (PAM2) of eukaryotic releasing factor eRF3 in mRNA decay Reviewed

    Masanori Osawa, Nao Hosoda, Tamiji Nakanishi, Naoyuki Uchida, Tomomi Kimura, Shunsuke Imai, Asako Machiyama, Toshiaki Katada, Shin-ichi Hoshino, Ichio Shimada

    RNA-A PUBLICATION OF THE RNA SOCIETY   Vol. 18 ( 11 ) page: 1957 - 1967   2012.11

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    Eukaryotic releasing factor GSPT/eRF3 mediates translation termination-coupled mRNA decay via interaction with a cytosolic poly(A)-binding protein (PABPC1). A region of eRF3 containing two overlapping PAM2 (PABPC1-interacting motif 2) motifs is assumed to bind to the PABC domain of PABPC1, on the poly(A) tail of mRNA. PAM2 motifs are also found in the major deadenylases Caf1-Ccr4 and Pan2-Pan3, whose activities are enhanced upon PABPC1 binding to these motifs. Their deadenylase activities are regulated by eRF3, in which two overlapping PAM2 motifs competitively prevent interaction with PABPC1. However, it is unclear how these overlapping motifs recognize PABC and regulate deadenylase activity in a translation termination-coupled manner. We used a dominant-negative approach to demonstrate that the N-terminal PAM2 motif is critical for eRF3 binding to PABPC1 and that both motifs are required for function. Isothermal titration calorimetry (ITC) and NMR analyses revealed that the interaction is in equilibrium between the two PAM2-PABC complexes, where only one of the two overlapping PAM2 motifs is PABC-bound and the other is PABC-unbound and partially accessible to the other PABC. Based on these results, we proposed a biological role for the overlapping PAM2 motifs in the regulation of deadenylase accessibility to PABPC1 at the 39 end of poly(A).

    DOI: 10.1261/rna.035311.112

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  26. Regulation of inflorescence architecture by intertissue layer ligand-receptor communication between endodermis and phloem Reviewed

    Naoyuki Uchida, Jin Suk Lee, Robin J. Horst, Hung-Hsueh Lai, Ryoko Kajita, Tatsuo Kakimoto, Masao Tasaka, Keiko U. Torii

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 109 ( 16 ) page: 6337 - 6342   2012.4

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    Multicellular organisms achieve final body shape and size by coordinating cell proliferation, expansion, and differentiation. Loss of function in the Arabidopsis ERECTA (ER) receptor-kinase gene confers characteristic compact inflorescence architecture, but its underlying signaling pathways remain unknown. Here we report that the expression of ER in the phloem is sufficient to rescue compact er inflorescences. We further identified two EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) secreted peptide genes, EPFL4 and EPFL6/CHALLAH (CHAL), as redundant, upstream components of ER-mediated inflorescence growth. The expression of EPFL4 or EPFL6 in the endodermis, a layer adjacent to phloem, is sufficient to rescue the er-like inflorescence of epfl4 epfl6 plants. EPFL4 and EPFL6 physically associate with ER in planta. Finally, transcriptome analysis of er and epfl4 epfl6 revealed a potential downstream component as well as a role for plant hormones in EPFL4/6- and ER-mediated inflorescence growth. Our results suggest that inter-cell layer communication between the endodermis and phloem mediated by peptide ligands and a receptor kinase coordinates proper inflorescence architecture in Arabidopsis.

    DOI: 10.1073/pnas.1117537109

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  27. Modulation of the balance between stem cell proliferation and consumption by ERECTA-family genes Reviewed

    Naoyuki Uchida, Masanori Shimada, Masao Tasaka

    Plant Signaling and Behavior   Vol. 7 ( 11 ) page: 1506 - 1508   2012

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

    Stem cells in the vegetative shoot apical meristem proliferate to produce more stem cells (self-renewal) and are simultaneously consumed to form leaf promordia. Therefore, to keep a stable number of stem cells, regulation of the balance between their proliferation and consumption is important. Recently we reported that stem cell population is increased in mutant plants lacking the entire ERECTA (ER) receptor kinase family. Here we describe that loss of function of the entire ER-family causes a decrease in leaf number in spite of the increase in stem cell population. This suggests that stem cell consumption might be decreased in the mutant, and this could be one of reasons why stem cell population appears to be increased. This situation is in sharp contrast to clv3 mutant, which also shows an increase in stem cell population but does not show a decrease in leaf production. We briefly discuss differences between the er-family mutant and the clv3 mutant. © 2012 Landes Bioscience.

    DOI: 10.4161/psb.22080

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  28. Regulation of NB-LRR-type UNI and its related signaling pathway: Signaling crosstalk and methodology for quick identification of related factors Reviewed

    Naoyuki Uchida, Masao Tasaka

    Plant Signaling and Behavior   Vol. 6 ( 8 ) page: 1219 - 1222   2011.8

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    Activation of NB-LRR-related UNI proteins by uni-1D mutation, a gain-of-function mutation of the UNI gene, induces some pathogenesis-related responses and also affects morphology through modulation of meristem activities. In a recent study we reported that the uni-1D phenotypes require cooperative action of ERECTA (ER) receptor kinase family members in UNI-expressing cells, suggesting that an intracellular signaling crosstalk between ER-family-dependent and UNI-triggered signaling pathways plays a significant role in the phenotypes. Further we recently succeeded in the establishment of a methodology for rapid identification of factors involved in the UNI function. EMS-induced causal mutations that suppress the uni- 1D phenotypes could be identified using whole-genome-sequencing technologies with much less labor compared with the conventional map-based cloning method that is generally time-consuming and labor-intensive. Thus it would be now possible to intensively identify factors that play significant roles in regulation of UNI proteins and/or UNI-related signaling pathways. © 2011 Landes Bioscience.

    DOI: 10.4161/psb.6.8.16181

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  29. Arabidopsis ERECTA-Family Receptor Kinases Mediate Morphological Alterations Stimulated by Activation of NB-LRR-Type UNI Proteins Reviewed

    Naoyuki Uchida, Kadunari Igari, Naomi L. Bogenschutz, Keiko U. Torii, Masao Tasaka

    PLANT AND CELL PHYSIOLOGY   Vol. 52 ( 5 ) page: 804 - 814   2011.5

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    Shoot apical meristems (SAMs), which maintain stem cells at the tips of stems, and axillary meristems (AMs), which arise at leaf axils for branch formation, play significant roles in the establishment of plant architecture. Previously, we showed that, in Arabidopsis thaliana, activation of NB-LRR (nucleotide-binding site-leucine-rich repeat)-type UNI proteins affects plant morphology through modulation of the regulation of meristems. However, information about genes involved in the processes was still lacking. Here, we report that ERECTA (ER) receptor kinase family members cooperatively mediate the morphological alterations that are stimulated by activation of UNI proteins. uni-1D is a gain-of-function mutation in the UNI gene and uni-1D mutants exhibit early termination of inflorescence stem growth and also formation of extra AMs at leaf axils. The former defect involves modulation of the SAM activity and is suppressed by er mutation. Though the AM phenotype is not affected by a single er mutation, it is suppressed by simultaneous mutations of ER-family members. It was previously shown that trans-zeatin (tZ)-type cytokinins were involved in the morphological phenotypes of uni-1D mutants and that expression of CYP735A2, which is essential for biosynthesis of tZ-type cytokinins, was modulated in uni-1D mutants. We show that this modulation of CYP735A2 expression requires activities of ER-family members. Moreover, the ER activity in UNI-expressing cells contributes to all morphological phenotypes of uni-1D mutants, suggesting that a cross-talk between ER-family-dependent and UNI-triggered signaling pathways plays a significant role in the morphological alterations observed in uni-1D mutants.

    DOI: 10.1093/pcp/pcr032

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  30. Identification of EMS-Induced Causal Mutations in a Non-Reference Arabidopsis thaliana Accession by Whole Genome Sequencing Reviewed

    Naoyuki Uchida, Tomoaki Sakamoto, Tetsuya Kurata, Masao Tasaka

    PLANT AND CELL PHYSIOLOGY   Vol. 52 ( 4 ) page: 716 - 722   2011.4

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    The most frequently used method to identify mutations induced by a commonly used mutagen, EMS (ethyl methane sulfonate), in Arabidopsis thaliana has been map-based cloning. The first step of this method is crossing a mutant with a plant of another accession as it requires polymorphisms between accessions for linkage analysis. Therefore, to perform the method routinely, it is greatly preferred to use accession combinations between which enough polymorphisms are already known. Further, it requires laborious examination of a large number of F(2) recombinants using many markers to detect each polymorphism. After linkage analysis narrows down the chromosomal region containing the causal mutation, sequencing candidate genes one by one within the region is necessary until the mutation is finally identified. Overall, this method is generally time-consuming and labor intensive, and it becomes harder when multiple loci are involved in phenotypes. A few recent reports showed that causal mutations induced by EMS could be identified by deep-sequencing technologies with less labor compared with the conventional method when mutants were generated in the Arabidopsis reference Columbia background whose genome organization is well known. Here we report that we succeeded in rapid identification of EMS-induced causal mutations in a non-reference accession background, whose whole genome sequence is not publicly available, using one round of whole genome sequencing. Moreover, in our case, we could monitor the causal locus and the transgenic reporter locus simultaneously, implying that this methodology could theoretically be applicable to analyzing even complex traits. We describe the pipeline of this methodology and discuss its characteristics.

    DOI: 10.1093/pcp/pcr029

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  31. Intersections between immune responses and morphological regulation in plants Reviewed

    Naoyuki Uchida, Masao Tasaka

    JOURNAL OF EXPERIMENTAL BOTANY   Vol. 61 ( 10 ) page: 2539 - 2547   2010.6

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    Successful plant pathogens have developed strategies to interfere with the defence mechanisms of their host plants through evolution. Conversely, host plants have evolved systems to counteract pathogen attack. Some pathogens induce pathogenic symptoms on plants that include morphological changes in addition to interference with plant growth. Recent studies, based on molecular biology and genetics using Arabidopsis thaliana, have revealed that factors derived from pathogens can modulate host systems and/or host factors that play important roles in the morphological regulation of host plants. Other reports, meanwhile, have shown that factors known to have roles in plant morphology also function in plant immune responses. Evolutionary conservation of these factors and systems implies that host-pathogen interactions and the evolution they drive have yielded tight links between morphological processes and immune responses. In this review, recent findings about these topics are introduced and discussed.

    DOI: 10.1093/jxb/erq126

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  32. Coordination of leaf development via regulation of KNOX1 genes Reviewed

    Naoyuki Uchida, Seisuke Kimura, Daniel Koenig, Neelima Sinha

    JOURNAL OF PLANT RESEARCH   Vol. 123 ( 1 ) page: 7 - 14   2010.1

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

    Class I KNOTTED1-LIKE HOMEOBOX (KNOX1) genes are expressed in the shoot apical meristem (SAM) to effect its formation and maintenance. KNOX1 genes are also involved in leaf shape control throughout angiosperm evolution. Leaves can be classified as either simple or compound, and KNOX1 expression patterns in leaf primordia are highly correlated with leaf shape; in most simple-leafed species, KNOX1 genes are expressed only in the SAM but not in leaf primordia, while in compound-leafed species they are expressed both in the SAM and leaf primordia. How can KNOX1 expression be maintained to a high degree in the SAM, but simultaneously be so variable in leaves? This dichotomy suggests that the processes of leaf and SAM development have been compartmentalized during evolution. Here, we introduce our findings regarding the regulation of expression of SHOOT MERISTEMLESS, a KNOX1 gene, together with a brief review of KNOX1 genes from an evolutionary viewpoint. We also present our findings regarding another aspect of KNOX1 regulation via a protein-protein interaction network involved in the natural variation in leaf shape. Both aspects of KNOX1 regulation could be utilized for fine-tuning leaf morphology during evolution without affecting the essential function of KNOX genes in the shoot.

    DOI: 10.1007/s10265-009-0248-2

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  33. New perspectives on plant defense responses through modulation of developmental pathways Reviewed

    Kwi-Mi Chung, Kadunari Igari, Naoyuki Uchida, Masao Tasaka

    MOLECULES AND CELLS   Vol. 26 ( 2 ) page: 107 - 112   2008.8

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    Invasion mechanisms of pathogens and counteracting defense mechanisms of plants are highly diverse and perpetually evolving. While most classical studies of plant defense have focused only on defense-specific factor-mediated responses, recent work is beginning to shed light on the involvement of non-stress signal components, especially growth and developmental processes. This shift in focus links plant resistance more closely with growth and development. In this review, we summarize our current understanding of how pathogens manipulate host developmental processes and, conversely, of how plants deploy their developmental processes for self-protection. We conclude by introducing our recent work on UNI, a novel R protein in Arabidopsis which mediates cross-talk between developmental processes and defense responses.

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  34. Mechanism of mRNA deadenylation: evidence for a molecular interplay between translation termination factor eRF3 and mRNA deadenylases Reviewed

    Yuji Funakoshi, Yusuke Doi, Nao Hosoda, Naoyuki Uchida, Masanori Osawa, Ichio Shimada, Masafumi Tsujimoto, Tsutomu Suzuki, Toshiaki Katada, Shin-ichi Hoshino

    GENES & DEVELOPMENT   Vol. 21 ( 23 ) page: 3135 - 3148   2007.12

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    In eukaryotes, shortening of the T-poly(A) tail is the rate-limiting step in the degradation of most mRNAs, and two major mRNA deadenylase complexes-Caf1-Ccr4 and Pan2-Pan3-play central roles in this process, referred to as deadenylation. However, the molecular mechanism triggering deadenylation remains elusive. Previously, we demonstrated that eukaryotic releasing factor eRF3 mediates deadenylation and decay of mRNA in a manner coupled to translation termination. Here, we report the mechanism of mRNA deadenylation. The eRF3-mediated deadenylation is catalyzed by both Caf1-Ccr4 and Pan2-Pan3. interestingly, translation termination complexes eRF1-eRF3, Pan2-Pan3, and Caf1-Ccr4 competitively interact with polyadenylate-binding protein PABPC1. In each complex, eRF3, Pan3, and Tob, respectively, mediate PABPC1 binding, and a combination of a PAM2 motif and a PABC domain is commonly utilized for their contacts. A translation-dependent exchange of eRF1-eRF3 for the deadenylase occurs on PABPC1, Consequently, PABPC1 binding leads to the activation of Pan2-Pan3 and Caf1-Ccr4. From these results, we suggest a mechanism of mRNA deadenylation by Pan2-Pan3 and Caf1-Ccr4 in cooperation with eRF3 and PABPC1.

    DOI: 10.1101/gad.1597707

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  35. Regulation of SHOOT MERISTEMLESS genes via an upstream-conserved noncoding sequence coordinates leaf development Reviewed

    Naoyuki Uchida, Brad Townsley, Kook-Hyun Chung, Neelima Sinha

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 104 ( 40 ) page: 15953 - 15958   2007.10

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    The indeterminate shoot apical meristem of plants is characterized by the expression of the Class 1 KNOTTED1-LIKE HOMEOBOX (KNOX1) genes. KNOX1 genes have been implicated in the acquisition and/or maintenance of meristematic fate. One of the earliest indicators of a switch in fate from indeterminate meristem to determinate leaf primordium is the down-regulation of KNOX1 genes orthologous to SHOOT MERISTEMLESS (STM) in Arabidopsis (hereafter called STM genes) in the initiating primordial. In simple leafed plants, this down-regulation persists during leaf formation. In compound leafed plants, however, KNOX1 gene expression is reestablished later in the developing primordia, creating an indeterminate environment for leaflet formation. Despite this knowledge, most aspects of how STM gene expression is regulated remain largely unknown. Here, we identify two evolutionarily conserved noncoding sequences within the 5' upstream region of STM genes in both simple and compound leafed species across monocots and dicots. We show that one of these elements is involved in the regulation of the persistent repression and/or the reestablishment of STM expression in the developing leaves but is not involved in the initial down-regulation in the initiating primordia. We also show evidence that this regulation is developmentally significant for leaf formation in the pathway involving ASYMMETRIC LEAVES1/2 (AS1/2) gene expression; these genes are known to function in leaf development. Together, these findings reveal a regulatory point of leaf development mediated through a conserved, noncoding sequence in STM genes.

    DOI: 10.1073/pnas.0707577104

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  36. Identification of a human cytoplasmic poly(A) nuclease complex stimulated by poly(A)-binding protein Reviewed

    N Uchida, S Hoshino, T Katada

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 279 ( 2 ) page: 1383 - 1391   2004.1

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    The poly( A) tail shortening in mRNA, called deadenylation, is the first rate-limiting step in eukaryotic mRNA turnover, and the polyadenylate-binding protein ( PABP) appears to be involved in the regulation of this step. However, the precise role of PABP remains largely unknown in higher eukaryotes. Here we identified and characterized a human PABP-dependent poly( A) nuclease (hPAN) complex consisting of catalytic hPan2 and regulatory hPan3 subunits. hPan2 has intrinsically a 3' to 5' exoribonuclease activity and requires Mg2+ for the enzyme activity. On the other hand, hPan3 interacts with PABP to simulate hPan2 nuclease activity. Interestingly, the hPAN nuclease complex has a higher substrate specificity to poly( A) RNA upon its association with PABP. Consistent with the roles of hPan2 and hPan3 in mRNA decay, the two subunits exhibit cytoplasmic co-localization. Thus, the human PAN complex is a poly(A)-specific exoribonuclease that is stimulated by PABP in the cytoplasm.

    DOI: 10.1074/jbc.M309125200

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  37. Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation Reviewed

    N Hosoda, T Kobayashi, N Uchida, Y Funakoshi, Y Kikuchi, S Hoshino, T Katada

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 278 ( 40 ) page: 38287 - 38291   2003.10

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    Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the dead-enylation of the poly(A) tail at the 3' end. However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated. Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae. The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP. When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged. The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening. Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon. These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.

    DOI: 10.1074/jbc.C300300200

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  38. [Regulation of mRNA stability in eukaryotic cells]. Reviewed

    Uchida N, Hoshino S, Katada T, Shyu AB

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   Vol. 48 ( 11 Suppl ) page: 1488 - 1495   2003.8

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  39. A novel role of the mammalian GSPT/eRF3 associating with poly(A)-binding protein in cap/poly(A)-dependent translation Reviewed

    N Uchida, S Hoshino, H Imataka, N Sonenberg, T Katada

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 277 ( 52 ) page: 50286 - 50292   2002.12

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    The mammalian GSPT, which consists of amino-terminal (N) and carboxyl-terminal (C) domains, functions as the eukaryotic releasing factor 3 (eRF3) by interacting with eRF1 in translation termination. This function requires only the C-domain that is homologous to the elongation factor (EF) 1alpha, while the N-domain interacts with polyadenylate-binding protein (PABP), which binds the poly(A) tail of mRNA and associates with the eukaryotic initiation factor (eIF) 4G. Here we describe a novel role of GSPT in translation. We first determined an amino acid sequence required for the PABP interaction in the N-domain. Inhibition of this interaction significantly attenuated translation of capped/poly(A)-tailed mRNA not only in an in vitro translation system but also in living cells. There was a PABP-dependent linkage between the termination factor complex eRF1-GSPT and the initiation factor eIF4G associating with 5' cap through eIF4E. Although the inhibition of the GSPT-PABP interaction did not affect the de novo formation of an 80 S ribosomal initiation complex, it appears to suppress the subsequent recycle of ribosome. These results indicate that GSPT/eRF3 plays an important role in translation cycle through the interaction with PABP, in addition to mediating the termination with eRF1.

    DOI: 10.1074/jbc.M203029200

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MISC 10

  1. Synthetic Pair of Modified Auxin and Its Receptor for Freehand Manipulation of Auxin Responses : New Approach for Freehand Manipulation of Plant Hormone Responses

    高橋 宏二, 萩原 伸也, 鳥居 啓子, 打田 直行

    化学と生物 : 日本農芸化学会会誌 : 生命・食・環境   Vol. 57 ( 2 ) page: 80 - 87   2019.2

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    Language:Japanese   Publisher:日本農芸化学会 ; 1962-  

    CiNii Article

  2. 凸凹戦略でデザインした人工オーキシン・受容体ペアにより植物のオーキシンシグナル経路をハイジャックする Invited Reviewed

    打田 直行, 高橋 宏二, 萩原 伸也, 鳥居 啓子

    ライフサイエンス新着論文レビュー     2018

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    DOI: 10.7875/first.author.2018.016

  3. 植物の葉をギザギザにする物質EPFL2の発見

    爲重才覚, 打田直行, 鳥居啓子

    植調   Vol. 51 ( 4 ) page: 110‐114   2017.7

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    J-GLOBAL

  4. 植物の形を調節するペプチドホルモン

    平川有宇樹, 池松朱夏, 打田直行, 鳥居啓子

    現代化学   ( 543 ) page: 26‐29   2016.6

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    J-GLOBAL

  5. 植物が茎を伸ばす仕組みで働くスイッチの発見 植物の背丈を人為的に操る技術につながるか?

    打田直行

    化学と生物   Vol. 51 ( 9 ) page: 588 - 589   2013.9

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    Language:Japanese   Publishing type:Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

    DOI: 10.1271/kagakutoseibutsu.51.588

    J-GLOBAL

  6. 花序の形態の制御で働くEPFL型ペプチドホルモンとその受容体

    打田直行

    植物の生長調節   Vol. 48 ( 1 ) page: 67 - 72   2013.5

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    Language:Japanese   Publishing type:Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

    DOI: 10.18978/jscrp.48.1_67

    J-GLOBAL

  7. Regulation of mRNA stability in eukaryotic cells

    打田直行, 星野真一, 堅田利明, SHYU A-B

    蛋白質 核酸 酵素   Vol. 48 ( 11 ) page: 1488 - 1495   2003.8

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    J-GLOBAL

  8. GSPTファミリーによるmRNA分解制御機構の解析

    星野真一, 細田直, 小林哲夫, 打田直行, 堅田利明

    生化学   Vol. 74 ( 8 ) page: 699   2002.8

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    J-GLOBAL

  9. 動物細胞の翻訳過程においてGSPT/eRF3とPABPの相互作用がもつ新しい役割

    打田直行, 星野真一, 堅田利明

    生化学   Vol. 74 ( 8 ) page: 1009   2002.8

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    J-GLOBAL

  10. A novel G protein family involved in translation termination and mRNA stability

    T Katada, Y Araki, N Hosoda, T Kobayashi, N Uchida, S Hoshino

    JAPANESE JOURNAL OF PHARMACOLOGY   Vol. 88   page: 48P - 48P   2002

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

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KAKENHI (Grants-in-Aid for Scientific Research) 21

  1. 新陳代謝して細胞が入れ替わり続ける組織の恒常性を保つための新規シグナル経路の開拓

    Grant number:21H02503  2021.4 - 2024.3

    打田 直行

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    Authorship:Principal investigator 

    Grant amount:\17160000 ( Direct Cost: \13200000 、 Indirect Cost:\3960000 )

  2. Multi-layered regulatory system of plant resilience under fluctuating environment

    Grant number:20H05905  2020.11 - 2025.3

    Grant-in-Aid for Transformative Research Areas (A)

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    Authorship:Coinvestigator(s) 

  3. 土相・水相・気相の三相をまたぐ不規則な環境変動に対するレジリエンス機構

    Grant number:20H05912  2020.11 - 2025.3

    学術変革領域研究(A)

    芦苅 基行

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    Authorship:Coinvestigator(s) 

    植物にとって部分冠水による低酸素状態や完全冠水による無酸素状態は死に直結する深刻なストレスである。植物は数分から数日の幅で変動する冠水の長さや強度(程度?)に応じて、多段階に通気組織の発達や茎葉伸長を適切に調節する柔軟な応答機構(段階的なステージゲート応答機構)を持ち合わせている。しかし、植物が環境ストレスの時間や強度をどこでどのように受容し複数の応答からどれを選択し発動するのか?は不明である。本課題では、植物の酸素や水分のストレス感受機構と段階的なステージゲート応答の最適な選択機構を明らかにし、植物の冠水に対するレジリエンス機構の解明を目指す。

  4. 植物における機能未知の分泌型ペプチド性シグナル群の機能解明を目指した萌芽的戦略

    Grant number:20K21422  2020.7 - 2022.3

    挑戦的研究(萌芽)

    打田 直行

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    Authorship:Principal investigator 

    Grant amount:\6370000 ( Direct Cost: \4900000 、 Indirect Cost:\1470000 )

    多細胞生物の発生や環境応答などに関わる内在性の新シグナル分子が1つ発見され、その機能に迫る最初のきっかけさえ報告されると、それを皮切りにその関連事象の解明が世界中で始まる。新重要シグナルの発見は、往々にしてそれ以前までの制御モデルの改訂や大きな見直しにつながることから、その分野の発展の大きな節目となるなど、新シグナル分子の機能の発見1つ1つが新しい研究展開・研究転換の萌芽的起点となる。しかし、その最初のきっかけの発見が最も難しい壁である。そこで本研究では、独自に整備する変異体ライブラリーを活用することで、機能未知の植物のペプチド性の分泌シグナル群の機能解明を行う。

  5. 周期的に起こる器官老化と個体最後の変調である個体老化の新タイミング制御経路の開拓

    Grant number:20H05409  2020.4 - 2022.3

    新学術領域研究(研究領域提案型)

    打田 直行

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    Authorship:Principal investigator 

    Grant amount:\9100000 ( Direct Cost: \7000000 、 Indirect Cost:\2100000 )

    植物は葉など側生器官を周期的に作り続け成長する。葉は光合成産物を体中に送り出すが、光合成能が低下した古い葉から順に枯れ、この現象は「葉の老化」と呼ばれる。老化過程で高分子化合物が分解され生じる小分子類は新たな器官の発生や成長に再利用される。すなわち、順に生まれる器官が順に老化し(器官の老化)、その過程で生じる物質を再利用して新たな器官がまた順に生み出される。この「器官の老化と新生」を繰り返す周期の中で成長を続ける植物体にとって、その周期的な一連の発生事象を断ち切る個体最後の変調が「個体の枯死の開始」すなわち「個体全体の老化」である。これら植物の老化開始タイミングの制御の未解明課題に斬り込む。

  6. 従来の想定に無かった全く新しい茎頂幹細胞維持機構と多能性獲得機構の研究

    Grant number:20H04883  2020.4 - 2022.3

    新学術領域研究(研究領域提案型)

    打田 直行

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    Authorship:Principal investigator 

    Grant amount:\11700000 ( Direct Cost: \9000000 、 Indirect Cost:\2700000 )

    多くの植物の地上部の全てを生み出す源は、茎の先端に位置する茎頂分裂組織の内部に一生を通じて多能性を持ったまま維持される茎頂幹細胞である。この茎頂幹細胞は、植物の永続的で旺盛な生命力の根源の大きな1つとして、植物研究者の興味を魅き続けてきた。本提案では、この茎頂幹細胞の「維持」と「多能性の獲得」のそれぞれの仕組みに関して、遺伝学とケミカルバイオロジーに基づく独自の知見を踏まえて、従来の想定になかった全く新しい観点の提唱と理解を目的とした研究を実施する。

  7. 篩部で中継される成長・環境応答シグナルの解析

    Grant number:18H04777  2018.4 - 2020.3

    新学術領域研究(研究領域提案型)

    打田 直行

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    Authorship:Principal investigator 

    Grant amount:\13000000 ( Direct Cost: \10000000 、 Indirect Cost:\3000000 )

    多細胞生物である高等植物が全身的に協調した成長を達成するためのシグナルの実体や作用メカニズムには不明な点が多い。茎の協調的な伸長の制御では、機能冗長的な2つの分泌型ペプチドEPFL4とEPFL6が、その受容体のERECTA (ER)によって篩部伴細胞で受容されると、篩部伴細胞から茎の伸長を導くさらなる作用が発生すると考えられている。実際に、er変異体やepfl4 epfl6二重変異体では背が低くなる。また一方で、epfl6単独変異体は、通常培養条件下では何の異常も見られないが、低温下では花器官に異常が生じ、受粉が達成できないという特徴を持つことも見えてきた。すなわち、EPFL6は低温時の受粉達成に必須の役割を果たすと考えられる。EPFL4とEPFL6によく似た因子としてEPFL5が存在するが、epfl4 epfl5 epfl6三重変異体を作成し観察したところ、この三重変異体は通常温度下でもepfl6変異体は低温下で見せるのと同じ花器官の異常を呈し受粉しなかった。このことから、通常温度下においてはEPFL4、EPFL5、EPFL6が機能冗長的に受粉達成に関わると考えられる。以上のような特徴を持つEPFL4、EPFL5、EPFL6の関わるシグナル経路が作動させる仕組みの解明を目指して、EPFL6シグナルをOFFからONに切り換えた際の遺伝子発現の変動をRNA-seqを実施し、それら変動遺伝子群の中でも特に篩部で働く可能性を持つ因子を約28個抽出して解析を行った結果、茎伸長に明確な異常を持つ因子は未だ見出してはいないが、変異体が受粉に異常を持つような因子は見つかった。さらにこの因子は篩部に特異的に発現していることも判明した。この因子は過去にその機能の報告が一切なされていない新規因子であり、今後の機能解明が期待される。
    令和元年度が最終年度であるため、記入しない。
    令和元年度が最終年度であるため、記入しない。

  8. 局所的なホルモン応答操作技術の創成による環境適応型植物成長システムの構成的理解

    Grant number:17KT0017  2017.7 - 2021.3

    打田 直行

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    Authorship:Principal investigator 

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

    芽生えた場所の環境に適応して生育する植物は、体の各所での局所的応答を個体全体の生長へ反映させるシステムを持つ。しかし、その局所応答を全身統御へつなげる構成的システムの理解は不足している。本研究では、多様な局所応答の起点の1つである低分子ホルモンのオーキシンに着目し、局所的オーキシン応答を操作する技術を創成して、局所的シグナル発信が植物個体の統御につながる様子を解析し理解する。そのために、人工化合物とそれのみを受容する改変型オーキシン受容体(変異体)から成る人工ペアを創成し、オーキシン応答の自在操作を行えるようになった。そこで、この改変受容体を様々な部位特異的プロモーターで発現させ人工化合物を局所投与することでオーキシン応答を自在操作することを目指し、この改変受容体を様々な細胞種に特異的なプロモーターで発現させた植物を作成した。これらの植物に対し、上述の人工化合物を添加し、その結果として植物体に生じた変化を解析したところ、過去にオーキシンとの関連が報告されたことのない生理応答が誘導される状況が複数のケースで見られた。従来のオーキシン添加実験では、オーキシン添加によって身体中の実に多様な細胞が一度に応答を起こすために、単独の細胞腫におけるオーキシン応答だけを抽出できなかったために見落とされてきた制御が多かったと想定できるが、本ツールは狙った細胞腫でのオーキシン応答だけを特異的に活性化できるため、これまでは見えていなかった新規の生理応答が見えてきたものと考えている。ただし、未報告のオーキシン反応を行う興味深い細胞群を発見したとはいえ、今回用いたプロモーターで分類できる以上にさらに細かい複数の細胞種に分類でき得る可能性も判明した。すなわち、これらのさらに細かい細胞種ごとにオーキシン応答を活性化できる植物を作成し解析を行う必要性も生じた。
    今年度は、創成した人工ペアを用いてオーキシン応答を局所的に操作するために、様々な細胞種特異的なプロモーターで改変受容体を発現させた各々の植物に対する人工化合物の添加実験を実施したところ、オーキシンとの関連が過去に見られたことのない生理応答が影響を受ける例を複数のケースで見つけることができた。これをきっかけにすることで、今後は独創的な研究が展開できることが期待される。以上のことから、現在までの進捗状況としては、「おおむね順調に進展している」と判断した。
    今年度に見えてきた、オーキシンが関わる過去に報告のない複数の生理応答に関しては、それぞれの現象をさらに詳細に解析する。また、今年度に用いたプロモーターの特異性以上にさらに細かい細胞種に分類して解析すべきことが見えてきたケースに関しては、その目的に資する新たなプロモーターで改変受容体を発現する植物体を作成し、その植物体を人工化合物で刺激した際に引き起こされる現象を観察することで、着目する生理応答を引き起こす鍵となる細胞種をさらに絞り込む。

  9. ペプチドホルモン経路と低分子ホルモン経路の干渉を介した発生・成長システムの解明

    Grant number:17H03695  2017.4 - 2021.3

    打田 直行

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    Authorship:Principal investigator 

    Grant amount:\17420000 ( Direct Cost: \13400000 、 Indirect Cost:\4020000 )

    高等植物の体は様々な細胞群が協調して機能することで成り立つが、それら細胞間のコミュニケーションを秩序立てて処理する仕組みや、その仕組みが発生・成長の制御において担う役割に関して、過去に解明された例は未だ限定的である。そのような中、研究代表者は、共通の受容体ファミリーが複数の現象で共通して働くものの、現象ごとに異なるリガンドを受容し、さらにその受容体経路の下流では現象ごとに異なる低分子ホルモン経路の働きが調節される、という興味深い受容体システムに関する研究を展開してきた。本研究ではこの研究経緯を発展させ、リガンドの選択性を現象ごとに変化させる分子機序、異なる低分子ホルモン経路の働きが現象ごとに調節されるメカニズム、に焦点を当てた解析を行い、これらの仕組みで中心的な役割を担う新規制御因子群の同定までを目指す。
    本年度は、本研究で解析対象とするERファミリー受容体群の関わる発生現象のうち、リガンドが同定されていないケースでの未同定リガンドの同定作業のうち、昨年度までに葉脈パターン形成でのリガンド候補として発達中の葉脈で生まれるリンド候補を見出していたので、その解析に用いる植物の作成などを進めた。まず既存の変異体が存在していなかったので、CRISPR/Cas9により機能失変異体の作成を進め、複数の機能欠失アレルをホモの状態で確立した。また、エシトラジオール誘導型の過剰発現体も確立も完了した。他にも、扱っている各種のリガンドに関して、機能冗長性を考慮した機能欠失多重変異体の作製も進めた。これらのラインは今後解析を進める。さらに、着目している各現象ごとにERファミリー経路の下流で調節される低分子ホルモン経路の探索を進めた結果、花器官の成長に関わるペプチドホルモンの下流で低分子植物ホルモンのジベレシン経路が調節されている可能性を見出した。
    解析に必要となる機能欠失体や過剰発現体のライン整備が順調に進んでいることにくわえて、受容体シグナルの下流で低分子ホルモンの作用が変化している可能性もつかんだ。以上のことから、現在までの進捗状況としては、「おおむね順調に進展している」と判断した。
    着目している因子群に関してまずは確立が進んできた過剰発現体と機能欠失体の解析を進める。機能欠失体に分かりやすい異常が生じていない場合は、機能冗長性により機能が補完されている可能性を考慮し、ファミリー因子群との多重機能欠損体を作製する。花器官の成長に関わるペプチドホルモンの下流で低分子植物ホルモンのジベレシン経路が調節されている可能性を見出したのと同様に、他の着目現象においても今回の受容体下流でどのような低分子ホルモン経路が作動しているのかの解析を引き続き進める。また、本受容体下流でこれら低分子ホルモン経路がどのような機序で活性調節をされているのかの解析を進める予定である。

  10. 篩部から発信される茎成長シグナルの解析

    Grant number:16H01462  2016.4 - 2018.3

    新学術領域研究(研究領域提案型)

    打田 直行

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    Authorship:Principal investigator 

    Grant amount:\10660000 ( Direct Cost: \8200000 、 Indirect Cost:\2460000 )

    高等植物の体は様々な細胞群が協調して振舞うことで成り立つが、その全身的な協調を達成するための「長距離シグナル」の実体や作用メカニズムに関する知見は極めて少ない。特に、長距離シグナルはそもそもは局所的な細胞応答をきっかけに生まれ、その部位から周囲に向け発信されると想定されるが、その最初の段階である「シグナルの発生の仕組み」に関する知見は圧倒的に不足している。本研究では、茎全体の協調的な伸長の制御で茎の内皮細胞から分泌される機能冗長的なペプチドEPFL4とEPFL6が、それらの受容体であるERECTA(ER)によって篩部伴細胞で受容されると、篩部伴細胞から茎全体の協調した伸長を導くさらなるシグナルが発生する、という事象において、この篩部伴細胞で生まれて茎全体に作用するシグナルの実体、ならびに、このシグナル作用が及ぶ範囲や作用先が受ける影響、の解明を目指している。
    本年度は、昨年度までに完成させたEPFL6シグナルをOFFからONに切り替えることができる発現誘導系と最適な発現誘導条件を用いて、EPFL6刺激をONに切り換えた際の遺伝子発現の変動を網羅的に解析するためのRNA-seqを実施した。それら遺伝子群に関して、篩部伴細胞での発現の有無、分泌シグナルの有無、ホルモン関連遺伝子かどうか、などで分類し、本事象での重要因子候補としての順位付けを行った。興味深いことに、この中には、篩部から周辺領域に遠隔作用を発揮できる可能性を持つ因子が複数含まれている。今後は、各因子の個別解析を進めていく。
    29年度が最終年度であるため、記入しない。
    29年度が最終年度であるため、記入しない。

  11. Signal transduction and specificity of peptide ligand-receptor pairs specifying plant growth

    Grant number:26291057  2014.4 - 2017.3

    TORII KEIKO

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    Authorship:Collaborating Investigator(s) (not designated on Grant-in-Aid) 

    This proposal aims at understanding the mechanism of signal discrimination and further unraveling the function of orphan EPFL peptides during Arabidopsis development. Using the promoter swapping approach, we learned that the sub-family of EPF/EPFL peptides have shared and specific activities. We further revealed that EPFL2-ERECTA-family ligand-receptor pair is required for leaf margin morphogenesis. Furthermore, we demonstrated that a negative feedback circuitry between auxin and EPFL2 signaling promotes leaf teeth growth by maintaining the auxin maxima throughout the dynamic process of leaf expansion. During secondary growth, ERECTA-family ensure the timing of wood tissue expansion, a process mediated by GA. Our work reveals that EPF/EPFL-ERECTA-family ligand-receptor pairs control specific developmental programs through interaction with phytohormones, and that restricted expression of peptide ligands in part contribute to signal specificity.

  12. 植物免疫とF1壊死の多様性構築の基礎となるR遺伝子への新規変異導入現象の解析

    Grant number:26113707  2014.4 - 2016.3

    新学術領域研究(研究領域提案型)

    打田 直行

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    Authorship:Principal investigator 

    Grant amount:\10790000 ( Direct Cost: \8300000 、 Indirect Cost:\2490000 )

    植物のRタンパク質群は病原因子由来の物質を認識して免疫反応を引き起こすが、各々のRタンパク質は特異的な因子しか認識せず、ゲノムに有限個のR遺伝子しか存在しないので、新たな病原因子には新たなR遺伝子が生まれない限り対応できない。しかし、高等植物の遺伝子の変化は進化的なタイムスパンでしか捉えられない現象であることから、R遺伝子の多様化メカニズムに実験科学的に迫った知見はほぼ皆無である。本研究では、R遺伝子の多様化現象に実験科学的に迫るユニークな系を発見したことを受け、上記の現象について実験科学的な知見を得ることを目的とした。
    これまでに、uni-1D変異体では、R遺伝子の一つであるUNI遺伝子で高頻度に変異が起こることを見出し、同様の変異現象をゲノムワイドに解析することを目指してきた。しかし、昨年までに進めた手法では、将来的に種となって次世代に遺伝する変異しか捉えられない、という欠点が存在した。そこで今年度は、体内の体細胞の各々に別個に導入される変異を、変異の内容(機能獲得、機能欠失、アミノ酸の変化を伴わない変異)を問わずにモニターできる新方法論の開発を行った。野生型の植物体全体から採取したゲノムを用いて、ゲノム内のUNI遺伝子を含む複数のR遺伝子群といくつかのハウスキーピング遺伝子群を特異的に濃縮したのちに、次世代シークエンサーを用いて約3万のDepthでシークエンスを行ったところ、UNI遺伝子に加えてUNI遺伝子と配列の類似したUNI-LIKE遺伝子群ではハウスキーピング遺伝子群よりも変異頻度が上昇していた。またその際の変異箇所は5’領域とコーディング領域に集中していた。これらの結果は、UNI遺伝子ファミリーは野生型背景でそもそも早く変化しており、この現象を駆動する仕組みの存在が示唆される。
    27年度が最終年度であるため、記入しない。
    27年度が最終年度であるため、記入しない。

  13. 細胞壁空間を交錯する類似情報分子群を秩序立てて区別・認識するメカニズムの解析

    Grant number:25114511  2013.4 - 2015.3

    新学術領域研究(研究領域提案型)

    打田 直行

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    Authorship:Principal investigator 

    Grant amount:\10400000 ( Direct Cost: \8000000 、 Indirect Cost:\2400000 )

    高等植物の細胞間コミュニケーションでは、「細胞外に分泌されるリガンドが受容体によって受容される」制御が重要だが、数多くのシグナル情報を秩序立てて処理する仕組みの解明は遅れている。本研究では、様々な形態制御に関わる受容体ファミリー、ERECTA(ER)ファミリーに着目する。ERファミリーのリガンドはこれまでに5つが同定され、全てEPFL分泌ペプチドファミリーに属す。このファミリーにはさらに6つの機能未知因子が含まれ、ERファミリーに作用しうる。そこでEPFL・ERファミリーの各メンバーが未知の制御でもリガンド・受容体ペアとして働く可能性を追求する。また、各々のリガンドは別の現象のために分泌され混在し、どのリガンドがどの現象を制御すべきか受容体には区別できない事態に見えるが、受容体はそれぞれのリガンド刺激に応じた現象の制御を区別して行う。そこで、この仕組みに関わる新たな知見の獲得も目指している。
    今年度はまず、葉の形態制御に関しては、EPFLファミリーのメンバーの一つがERファミリーとともに新しいリガンド・受容体ペアとして働くことを遺伝学的・生化学的に明らかとした。各々の発現部位の解析と部位特異的プロモーターによる発現実験から、このEPFLファミリーメンバーは鋸歯の周辺部で、ERファミリーは鋸歯の先端で機能することが重要であることが分かった。これは、過去に想定されていない細胞間における新しい細胞間コミュニケーションを提唱することにつながる。また、鋸歯の成長においては鋸歯先端部でのオーキシン反応の活性化が重要であることが以前より知られているが、このEPFLファミリーメンバーとERファミリーはこのオーキシン反応性の制御に関わっていた。
    26年度が最終年度であるため、記入しない。
    26年度が最終年度であるため、記入しない。

  14. Analysis of novel regulatory mechanisms for plant stem growth

    Grant number:24570050  2012.4 - 2015.3

    UCHIDA Naoyuki

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    Authorship:Principal investigator 

    Grant amount:\5590000 ( Direct Cost: \4300000 、 Indirect Cost:\1290000 )

    In this project, we aimed to identify novel genes that regulate stem growth of plants. To that purpose, we used er mutant and epfl4 epfl6 mutnat. By comparing wild type, er and epfl4 epfl6 plants, it was revealed that cell proliferation decreases around the tip of stems in mutants compared with wild type. Then by performing comprehensive gene expression analysis, we identified a novel receptor gene whose expression level is down-regulated in mutants. Because there is a similar gene to the identified gene, we made the double mutant for these two genes. The double mutant showed defects in stem growth, indicating that these receptor genes play a important role in the regulation of stem growth. Thus, we achieved the goal of this project, that is, identification of novel genes that regulate stem growth.

  15. 植物免疫とF1壊死の多様性構築の基礎となるR遺伝子への新規変異導入現象の解析

    Grant number:24113513  2012.4 - 2014.3

    新学術領域研究(研究領域提案型)

    打田 直行

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    Authorship:Principal investigator 

    Grant amount:\11700000 ( Direct Cost: \9000000 、 Indirect Cost:\2700000 )

    植物のRタンパク質群は病原因子由来の物質を認識して免疫反応を引き起こすが、各々のRタンパク質は特異的な因子しか認識せず、ゲノムに有限個のR遺伝子しか存在しないので、新たな病原因子には新たなR遺伝子が生まれない限り対応できない。しかし、R遺伝子の多様化メカニズムの知見は少ない。また、種内外の異系統間のF1個体で親の組み合わせにより起こる壊死現象にR遺伝子が関わるケースが存在し、R遺伝子の変化はF1交雑の適合・不適合の変化にも関わる。本研究では、R遺伝子の多様化現象に実験科学的に迫るユニークな系を発見したことを受け、上記の現象について実験科学的な知見を得ることを目指している。
    uni-1D変異体は、UNI遺伝子に変異が高頻度に誘発されるユニークな変異体である。これまではUNI遺伝子に焦点を絞った解析を行って来たが、今年度はこの事象に関してゲノム全体を対象にした解析を進めた。単一のuni-1D変異体を起源とする野生型とuni-1D変異体に関して、独立した系譜として5世代経た複数ラインを確立し、それらに生じる新生SNPを解析するためのゲノムデータは次世代シークエンサーを用いて解析した。その結果、まず野生型においては、過去の研究からの示唆に沿うように、ゲノムに生じる変異の大部分は紫外線(本実験条件では蛍光灯由来の微弱紫外線と想定される)によって生じるタイプの塩基置換が大半を占めていた。それに対して、uni-1D変異体では、紫外線によらないタイプの塩基置換の占める割合が極めて高く上昇していた。すなわち、uni-1D変異体では、通常時とは異なる変異導入の仕組みが発動しており、これがR遺伝子の早い変化の原動力である可能性が考えられ、この結果は今後の解析の重要な足がかりとなると期待できる。
    25年度が最終年度であるため、記入しない。
    25年度が最終年度であるため、記入しない。

  16. Molecular analysis of the relation between R-protein and morphogenesis in plant

    Grant number:22370019  2010 - 2012

    TASAKA Masao

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    Authorship:Coinvestigator(s) 

    The constitutive active R-protein, uni-1D, induced abnormal morphogenesis in Arabidopsis shoot. We have isolated suppressors of uni-1D. One of them encodes RPT2, which is a component of 26S proteasome and binds directly to UNI. Erecta, which is a receptor kinase in a plasma membrane, is also one of the suppressors. ER family genes expressed outside of SAM, however, affect the SAM activity. ER expressed in phloem affect the inflorescence stem elongation and the EPFL4 and 6, both of which expressed in endoderm cells, function as a ligand in this process.

  17. A novel model system to study the rapid diversification of R genes

    Grant number:22657015  2010 - 2011

    TASAKA Masao

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    Authorship:Coinvestigator(s) 

    Plants use disease resistance(R) genes, most of which encode nucleotide-binding leucine-rich repeat(NB-LRR) protein, to recognize pathogens. Each R protein recognizes the specific effector protein. To counter the rapid diversification of pathogen effector genes, it thought that R genes also evolve rapidly. This idea is supported that high degree of polymorphism is observed in R-genes. However, little is known about the mechanisms underlying the R-gene diversification. We analyzed Arabidopsis uni-1D mutant, harbors a semi-dominant and gain-of-function allele of UNI gene, an R gene that has a NB-LRR-related structure. The uni-1D has a constitutively active R protein to induce resistance responses without any pathogen infection. Furthermore, uni-1D heterozygous mutant(hereafter uni1D/+) shows rapidly consume stem cells in the shoot apical meristem of the inflorescence stem, resulting in formation of very short stem. Interestingly, under normal growth condition, we infrequently but repeatedly observed that uni-1D/+produced chimeric sectors display the morphology of wild-type-like long inflorescence stem. When we checked nucleotide sequences in this chimeric stem, we always found additional mutations, which presumably disrupted the uni-1D protein function. This reversion event occurs less than 0. 5% of individuals among the population. When we tried with EMS, an alkylating agent, we succeeded to increase the reversion frequency about 30%. Furthermore, when we treated uni-1D/+with zeocin, which causes DNA double-strand breaks, or hydroxyurea(HU), which induces defects of DNA repair and replication by depletion by depleting deoxynucleotide triphosphate pools, the reversion frequency significantly increased. These suggest that the uni-1D systems can easily and efficiently detect various types of nucleotide alterations in the uni-1D gene. Currently, we are analyzing molecular mechanisms underlying the rapid diversification of R genes using this system and our preliminary results imply the involvement of DNA repair machinery in this phenomenon.

  18. Analysis of long-distance signals for regulation of the shoot apical meristem of plants

    Grant number:22770038  2010 - 2011

    UCHIDA Naoyuki

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    Authorship:Principal investigator 

    Grant amount:\4290000 ( Direct Cost: \3300000 、 Indirect Cost:\990000 )

    The shoot apical meristem(SAM) creates all above-ground tissues of plant body. It is implied that the SAM is regulated by signals that is originated in tissues outside the SAM. However, little is known about such a non-cell-autonomous signals. In this research, I focused on a receptor-kinase called ERECTA, which has been implicated in regulation of the SAM but its ligand for the SAM regulation is unclear. I succeeded in the identification of new ligands for ERECTA and showed that the novel cell-cell communication via the ligand-receptor pair plays a significant role for the regulation of plant architecture.

  19. Analysis of the regulation of axillary meristem formation using uni-1D mutant

    Grant number:20770035  2008 - 2009

    UCHIDA Naoyuki

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    Authorship:Principal investigator 

    Grant amount:\4290000 ( Direct Cost: \3300000 、 Indirect Cost:\990000 )

    Through the analysis of uni-1D mutants harboring unique phenotypes in formation and maintenance of shoot apical meristems and axillary meristems, ERECTA receptor kinase was shown to play a role in the regulation of shoot apical meristems and ERECTA family members was shown to function in the the regulation of axillary meristems.

  20. Regulation mechanisms of shoot apical meristem

    Grant number:19060007  2007 - 2012

    TASAKA Masao

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    Authorship:Coinvestigator(s) 

    We have isolated the pid enhancer mutants, macchi-bou 2 an4. MAB4 is a member of NPH3 and function to control the endocytosis of PIN proteins, which is an Auxin efflux carrier. MAB 2 is a member of CDK8 sub-complex to function a transcription repressor worked with Mediator complex. This protein suppressed the ARF transcription factor activity by making big protein complex with AUN/IAA and TOPLESS. We also isolated the enhancer of uni-1D, and isolated the enhancer genes by a map base cloning and by using next generation sequencer. One of the enhancers is erecta (er), which expressed without a shoot apical meristem but affected the maintenance of stem cells in it with the two other family members to affect the cytokinin-signaling pathway. ER, which expressed in the phloem, is important for the inflorescence stem elongation and the small peptides EPFL4 and 6 expressed in endoderm cells function as a ligand in this process.

  21. Crosstalk between morphogenesis and immunity in Arabidopsis thaliana

    Grant number:18207003  2006 - 2009

    TASAKA Masao

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    Authorship:Coinvestigator(s) 

    Semi-dominant uni-1Dmutant of Arabidopsis thaliana showed interesting phenotypes, loss of shoot apical meristem activity, extra lateral shoot meristem formation and constitutive PR genes activation. UNI encodes one of the R-protein suggesting there is a crosstalk between shoot morphogenesis and immunity. We have isolate some extragenic suppressors including erecta. We also make clear that UNI protein binds with RPT2a protein, which is a member of 26S proteasome, and the rpt2a can also suppress uni-1D.

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