Updated on 2024/04/16

写真a

 
YOSHINARI Akira
 
Organization
Institute for Advanced Research Designated assistant professor
Institute of Transformative Bio-Molecules Designated assistant professor
Title
Designated assistant professor
Contact information
メールアドレス
External link

Degree 1

  1. Ph.D ( 2016.12   Hokkaido University ) 

Research Interests 9

  1. Cell Polarity

  2. Transporter

  3. Cell Biology

  4. Plant Nutrition

  5. Arabidopsis thaliana

  6. Endocytosis

  7. Receptor

  8. Transporter

  9. Plant Physiology

Research Areas 4

  1. Life Science / Plant nutrition and soil science  / Boron transporter

  2. Life Science / Plant molecular biology and physiology  / Cell polarity, Transporter, Receptor, Membrane proteins

  3. Life Science / Developmental biology  / 放射軸細胞極性

  4. Life Science / Plant molecular biology and physiology

Current Research Project and SDGs 2

  1. 受容体様キナーゼの極性スイッチの分子機構と生理学的意義の解明

  2. ケミカルジェネティクスによる植物の細胞極性形成機構の解明

Research History 7

  1. 国立研究開発法人科学技術振興機構(JST)さきがけ(兼任)

    2022.10 - 2026.3

  2. Nagoya University

    2022.4

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  3. JSPS   Fellowship PD

    2020.4 - 2022.3

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  4. Nagoya University   Institute of Transformative Bio-Molecules   Post-doctoral Fellow

    2017.9 - 2020.3

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  5. Osaka Prefecture University

    2017.1 - 2017.8

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  6. Osaka Prefecture University

    2016.4 - 2017.1

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  7. Japan Society for Promotion of Science   Fellowship DC1

    2013.4 - 2016.3

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Education 3

  1. Hokkaido University   Graduate School of Agriculture

    2013.4 - 2016.3

  2. Hokkaido University   Graduate School of Life Science

    2011.4 - 2013.3

  3. Tohoku University   Department of Agriculture   Applied Biochemistry

    2007.4 - 2011.3

Professional Memberships 5

  1. 日本土壌肥料学会

  2. 日本植物生理学会

  3. 日本植物生理学会

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  4. 日本植物学会

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  5. JAPANESE SOCIETY OF SOIL SCIENCE AND PLANT NUTRITION

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Awards 9

  1. 第42回(2024年度)日本土壌肥料学会奨励賞

    2024.9   日本土壌肥料学会  

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  2. 日本土壌肥料学会 2021 年度北海道大会 若手口頭発表優秀賞

    2021.9   日本土壌肥料学会   化学遺伝学による栄養素輸送体の極性局在機構の解明

    吉成 晃, 佐藤綾人, Wolf Frommer, 中村匡良

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  3. 日本植物生理学会 PCP論文賞

    2018.3   日本植物生理学会   DRP1-dependent endocytosis is essential for polar localization and boron-induced degradation of the borate transporter BOR1 in Arabidopsis thaliana.

  4. ITbM Award

    2018.2   トランスフォーマティブ生命分子研究所  

  5. ITbM Award

    2018.2  

    吉成 晃

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  6. 優秀発表賞

    2016.7   第11回トランスポーター研究会  

  7. 最優秀ポスター発表賞

    2015.9   第1回植物の栄養研究会  

  8. Best Poster Presentation Award

    2013.8   International Plant Nutrition Colloquium (IPNC) 2013  

  9. 修士論文発表会 最優秀発表者賞

    2013.3   北海道大学大学院 生命科学院  

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Papers 19

  1. Near‐infrared imaging of phytochrome‐derived autofluorescence in plant nuclei Reviewed

    Akira Yoshinari, Reika Isoda, Noriyoshi Yagi, Yoshikatsu Sato, Jelmer J. Lindeboom, David W. Ehrhardt, Wolf B. Frommer, Masayoshi Nakamura

    The Plant Journal     2024.3

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    Authorship:Lead author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    SUMMARY

    Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live‐cell imaging. In Arabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near‐infrared (NIR) range of the spectrum and devised a non‐invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non‐model plant species.

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  2. Rapid Vacuolar Sorting of the Borate Transporter BOR1 Requires the Adaptor Protein Complex AP-4 in Arabidopsis

    Akira Yoshinari, Yutaro Shimizu, Takuya Hosokawa, Akihiko Nakano, Tomohiro Uemura, Junpei Takano

        2023.12

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    Authorship:Lead author   Publisher:Cold Spring Harbor Laboratory  

    Abstract

    Plants maintain nutrient homeostasis by controlling the activities and abundance of nutrient transporters. InArabidopsis thaliana, the borate (B) transporter BOR1 plays a role in the efficient translocation of B under low-B conditions. BOR1 undergoes polyubiquitination in the presence of sufficient B and is then transported to the vacuole via multivesicular bodies (MVBs) to prevent B accumulation in tissues at a toxic level. A previous study indicated that BOR1 physically interacts with μ subunits of adaptor protein complexes AP-3 and AP-4, both involved in vacuolar sorting pathways. In this study, we investigated the roles of AP-3 and AP-4 subunits in BOR1 trafficking in Arabidopsis. The lack of AP-3 subunits did not affect either vacuolar sorting or polar localization of BOR1-GFP, whereas the absence of AP-4 subunits resulted in a delay in high-B-induced vacuolar sorting without affecting polar localization. Super-resolution microscopy revealed a rapid sorting of BOR1-GFP into AP-4-positive spots in thetrans-Golgi network (TGN) upon high-B supply. These results indicate that AP-4 is involved in sequestration of ubiquitinated BOR1 into a TGN-specific subdomain “vacuolar-trafficking zone,” and is required for efficient sorting to MVB and vacuole. Our findings elucidate the rapid vacuolar sorting process facilitated by AP-4 in plant nutrient transporters.

    Subject areas

    (7) Membrane and transport

    DOI: 10.1101/2023.12.06.570355

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  3. Advances in Synthetic Fluorescent Probe Labeling for Live Cell Imaging in Plants.

    Noriyoshi Yagi, Akira Yoshinari, Ryu J Iwatate, Reika Isoda, Wolf B Frommer, Masayoshi Nakamura

    Plant & cell physiology   Vol. 62 ( 8 ) page: 1259 - 1268   2021.7

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    Fluorescent probes are a powerful tool for visualizing cellular and subcellular structures, their dynamics, and cellular molecules in living cells, and enables us to monitor cellular processes in a spatio-temporal manner within complex and crowded systems. In addition to popular fluorescent proteins, a wide variety of small-molecule dyes have been synthesized through close association with the interdisciplinary field of chemistry and biology, ranging from those suitable for labeling cellular compartments such as organelles to those for labeling intracellular biochemical and biophysical processes and signaling. In recent years, self-labeling technologies including the SNAP-tag system have allowed us to attach these dyes to cellular domains or specific proteins, and are beginning to be employed in plant studies. In this mini review, we will discuss the current range of synthetic fluorescent probes that have been exploited for live cell imaging and the recent advances in the application that enable genetical tagging of synthetic probes in plant research.

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  4. GNOM‐dependent endocytosis maintains polar localization of the borate exporter BOR1 in Arabidopsis Reviewed

    Akira Yoshinari, Yosuke Toda, Junpei Takano

    Biology of the Cell   Vol. 113 ( 5 ) page: 264 - 269   2021.1

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

    DOI: 10.1111/boc.202000106

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  5. Using Genetically Encoded Fluorescent Biosensors for Quantitative In Vivo Imaging. Reviewed International journal

    Akira Yoshinari, Jacob Moe-Lange, Thomas J Kleist, Heather N Cartwright, David A Quint, David W Ehrhardt, Wolf B Frommer, Masayoshi Nakamura

    Methods in molecular biology (Clifton, N.J.)   Vol. 2200   page: 303 - 322   2021

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

    Fluorescent biosensors are powerful tools for tracking analytes or cellular processes in live organisms and allowing visualization of the spatial and temporal dynamics of cellular regulators. Fluorescent protein (FP)-based biosensors are extensively employed due to their high selectivity and low invasiveness. A variety of FP-based biosensors have been engineered and applied in plant research to visualize dynamic changes in pH, redox state, concentration of molecules (ions, sugars, peptides, ATP, reactive oxygen species, and phytohormones), and activity of transporters. In this chapter, we briefly summarize reported uses of FP-based biosensors in planta and show simple methods to monitor the dynamics of intracellular Ca2+ in Arabidopsis thaliana using a ratiometric genetically encoded Ca2+ indicator, MatryoshCaMP6s.

    DOI: 10.1007/978-1-0716-0880-7_14

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  6. Transport-coupled ubiquitination of the borate transporter BOR1 for its boron-dependent degradation Reviewed

    Akira Yoshinari, Takuya Hosokawa, Marcel Pascal Beier, Keishi Oshima, Yuka Ogino, Chiaki Hori, Taichi E Takasuka, Yoichiro Fukao, Toru Fujiwara, Junpei Takano

    The Plant Cell   Vol. 33 ( 2 ) page: 420 - 438   2020.12

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

    <title>Abstract</title>
    Plants take up and translocate nutrients through transporters. In Arabidopsis thaliana, the borate exporter BOR1 acts as a key transporter under boron (B) limitation in the soil. Upon sufficient-B supply, BOR1 undergoes ubiquitination and is transported to the vacuole for degradation, to avoid overaccumulation of B. However, the mechanisms underlying B-sensing and ubiquitination of BOR1 are unknown. In this study, we confirmed the lysine-590 residue in the C-terminal cytosolic region of BOR1 as the direct ubiquitination site and showed that BOR1 undergoes K63-linked polyubiquitination. A forward genetic screen identified that amino acid residues located in vicinity of the substrate-binding pocket of BOR1 are essential for the vacuolar sorting. BOR1 variants that lack B-transport activity showed a significant reduction of polyubiquitination and subsequent vacuolar sorting. Coexpression of wild-type (WT) and a transport-defective variant of BOR1 in the same cells showed degradation of the WT but not the variant upon sufficient-B supply. These findings suggest that polyubiquitination of BOR1 relies on its conformational transition during the transport cycle. We propose a model in which BOR1, as a B transceptor, directly senses the B concentration and promotes its own polyubiquitination and vacuolar sorting for quick and precise maintenance of B homeostasis.

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  7. Sensors for quantification, localization and analysis of dynamics of plant hormones. Reviewed International journal

    Reika Isoda, Akira Yoshinari, Yuuma Ishikawa, Mayuri Sadoine, Rüdiger Simon, Wolf B Frommer, Masayoshi Nakamura

    The Plant journal : for cell and molecular biology   Vol. 105 ( 2 ) page: 542 - 557   2020.11

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

    Plant hormones play important roles in plant growth and development, physiology and in acclimation to environmental changes. The hormone signaling networks are highly complex and interconnected. It is thus important to not only know where the hormones are produced, how they are transported and how and where they are perceived, but also to quantitatively monitor their distribution quantitatively, ideally in a non-invasive manner. Here we summarize the diverse set of tools available for quantifying and visualizing hormone distribution and dynamics. We provide an overview over the tools that are currently available - including transcriptional reporters, and degradation sensors, luciferase and fluorescent sensors - and compare the tools and their suitability for different purposes.

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  8. Covalent Self-Labeling of Tagged Proteins with Chemical Fluorescent Dyes in BY-2 Cells and Arabidopsis Seedlings. Reviewed International journal

    Ryu J Iwatate, Akira Yoshinari, Noriyoshi Yagi, Marek Grzybowski, Hiroaki Ogasawara, Mako Kamiya, Toru Komatsu, Masayasu Taki, Shigehiro Yamaguchi, Wolf B Frommer, Masayoshi Nakamura

    The Plant cell   Vol. 32 ( 10 ) page: 3081 - 3094   2020.10

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    Synthetic chemical fluorescent dyes promise to be useful for many applications in biology. Covalent, targeted labeling, such as with a SNAP-tag, uses synthetic dyes to label specific proteins in vivo for studying processes such as endocytosis or for imaging via super-resolution microscopy. Despite its potential, such chemical tagging has not been used effectively in plants. A major drawback has been the limited knowledge regarding cell wall and membrane permeability of the available synthetic dyes. Of 31 synthetic dyes tested here, 23 were taken up into BY-2 cells, while eight were not. This creates sets of dyes that can serve to measure endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine, and silicon-rhodamine 647, were used to SNAP-tag α-tubulin. Successful tagging was verified by live cell imaging and visualization of microtubule arrays in interphase and during mitosis in Arabidopsis (Arabidopsis thaliana) seedlings. Fluorescence activation-coupled protein labeling with DRBG-488 was used to observe PIN-FORMED2 (PIN2) endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the actively forming cell plate during mitosis. Together, the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety of cellular and biological processes.

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  9. Polar Localization of the Borate Exporter BOR1 Requires AP2-Dependent Endocytosis. Reviewed International journal

    Akira Yoshinari, Takuya Hosokawa, Taro Amano, Marcel Pascal Beier, Tadashi Kunieda, Tomoo Shimada, Ikuko Hara-Nishimura, Satoshi Naito, Junpei Takano

    Plant physiology   Vol. 179 ( 4 ) page: 1569 - 1580   2019.4

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    Boron (B) is an essential element in plants but is toxic when it accumulates to high levels. In root cells of Arabidopsis (Arabidopsis thaliana), the borate exporter BOR1 is polarly localized in the plasma membrane toward the stele side for directional transport of B. Upon high-B supply, BOR1 is rapidly internalized and degraded in the vacuole. The polar localization and B-induced vacuolar sorting of BOR1 are mediated by endocytosis from the plasma membrane. To dissect the endocytic pathways mediating the polar localization and vacuolar sorting, we investigated the contribution of the clathrin adaptor protein, ADAPTOR PROTEIN2 (AP2) complex, to BOR1 trafficking. In the mutants lacking µ- or σ-subunits of the AP2 complex, the polar localization and constitutive endocytosis of BOR1 under low-B conditions were dramatically disturbed. A coimmunoprecipitation assay showed association of the AP2 complex with BOR1, while it was independent of YxxΦ sorting motifs, which are in a cytosolic loop of BOR1. A yeast two-hybrid assay supported the interaction of the AP2 complex µ-subunit with the C-terminal tail but not with the YxxΦ motifs in the cytosolic loop of BOR1. Intriguingly, lack of the AP2 subunit did not affect the B-induced rapid internalization/vacuolar sorting of BOR1. Consistent with defects in the polar localization, the AP2 complex mutants showed hypersensitivity to B deficiency. Our results indicate that AP2-dependent endocytosis maintains the polar localization of BOR1 to support plant growth under low-B conditions, whereas the B-induced vacuolar sorting of BOR1 is mediated through an AP2-independent endocytic pathway.

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  10. Nodulin Intrinsic Protein 7;1 Is a Tapetal Boric Acid Channel Involved in Pollen Cell Wall Formation.

    Routray P, Li T, Yamasaki A, Yoshinari A, Takano J, Choi WG, Sams CE, Roberts DM

    Plant physiology   Vol. 178 ( 3 ) page: 1269 - 1283   2018.11

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    Authorship:Lead author, Last author, Corresponding author   Language:English   Publisher:Plant Physiology  

    Boron is an essential plant micronutrient that plays a structural role in the rhamnogalacturonan II component of the pectic cell wall. To prevent boron deficiency under limiting conditions, its uptake, distribution, and homeostasis are mediated by boric acid transporters and channel proteins. Among the membrane channels that facilitate boric acid uptake are the type II nodulin intrinsic protein (NIP) subfamily of aquaporin-like proteins. Arabidopsis (Arabidopsis thaliana) possesses three NIP II genes (NIP5;1, NIP6;1, and NIP7;1) that show distinct tissue expression profiles (predominantly expressed in roots, stem nodes, and developing flowers, respectively). Orthologs of each are represented in all dicots. Here, we show that purified and reconstituted NIP7;1 is a boric acid facilitator. By using native promoter-reporter fusions, we show that NIP7;1 is expressed predominantly in anthers of young flowers in a narrow developmental window, floral stages 9 and 10, with protein accumulation solely within tapetum cells, where it is localized to the plasma membrane. Under limiting boric acid conditions, loss-of-function T-DNA mutants (nip7;1-1 and nip7;1-2) show reduced fertility, including shorter siliques and an increase in aborted seeds, compared with the wild type. Under these conditions, nip7;1 mutant pollen grains show morphological defects, increased aggregation, defective exine cell wall formation, reduced germination frequency, and decreased viability. During stages 9 and 10, the tapetum is essential for supplying materials to the pollen microspore cell wall. We propose that NIP7;1 serves as a gated boric acid channel in developing anthers that aids in the uptake of this critical micronutrient by tapetal cells.

    DOI: 10.1104/pp.18.00604

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  11. TOL proteins mediate vacuolar sorting of the borate transporter BOR1 in Arabidopsis thaliana. Reviewed

    Yoshinari A, Korbei B, Takano J

    Soil Sci Plant Nutr   Vol. 64 ( 5 ) page: 598 - 605   2018

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Soil Science and Plant Nutrition  

    Boron (B) is an essential micronutrient for plants; however, it shows cytotoxicity at high concentrations. A borate transporter BOR1 is required for efficient transport of B toward the root stele in Arabidopsis thaliana. BOR1 shows polar localization in the plasma membrane of various root cells toward the stele-side under B limitation. To avoid over-accumulation of B, BOR1 in the plasma membrane is rapidly internalized and transported into the vacuole for proteolysis after high-B supply in an ubiquitination-dependent manner. Although BOR1 has been predicted to be transported into multi-vesicular bodies/late endosomes (MVB/LEs) via the endosomal sorting complex required for transport (ESCRT) machinery, experimental evidence was absent so far. In this study, we investigated the intracellular localization of BOR1 by visualizing endomembrane compartments, and tested the involvement of ESCRT-0-like proteins TOM1-LIKEs (TOLs) in the vacuolar sorting of BOR1. Under low-B conditions, a large portion of cytoplasmic BOR1 was localized in the trans-Golgi networks/early endosomes (TGN/EEs) labeled with VHA-a1 subunit. Pharmacological interference of endosomal recycling using brefeldin A-induced colocalization of BOR1 with RabA5D, which labels recycling vesicles associated with the TGN. On the other hand, under high-B conditions, BOR1 was localized in the inside of TOL5-positive MVB/LEs. To examine the roles of TOL proteins in intracellular trafficking of BOR1, we analyzed BOR1-GFP localization in the TOL quintuple mutant (tolQ; tol2-1tol3-1tol5-1tol6-1tol9-1) after high-B supply. In the tolQ mutant, vacuolar sorting of BOR1 was delayed, while the polar localization of BOR1 was not disturbed. Taken together, BOR1 is constantly transported to the TGN/EE by endocytosis and recycled to the plasma membrane likely via RabA5D-positive endomembrane compartments under low-B conditions. On the other hand, BOR1 is transported to the vacuole via TOL5-positive MVB/LEs under high-B conditions. TOL proteins are required for sorting of ubiquitinated BOR1 into MVB/LE for vacuolar degradation.

    DOI: 10.1080/00380768.2018.1504322

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  12. Insights into the Mechanisms Underlying Boron Homeostasis in Plants Reviewed

    Akira Yoshinari, Junpei Takano

    FRONTIERS IN PLANT SCIENCE   Vol. 8   page: 1951   2017.11

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:FRONTIERS MEDIA SA  

    Boron is an essential element for plants but is toxic in excess. Therefore, plants must adapt to both limiting and excess boron conditions for normal growth. Boron transport in plants is primarily based on three transport mechanisms across the plasma membrane: passive diffusion of boric acid, facilitated diffusion of boric acid via channels, and export of borate anion via transporters. Under boron -limiting conditions, boric acid channels and borate exporters function in the uptake and translocation of boron to support growth of various plant species. In Arabidopsis thaliana, NIP5;1 and BOR1 are located in the plasma membrane and polarized toward soil and stele, respectively, in various root cells, for efficient transport of boron from the soil to the stele. Importantly, sufficient levels of boron induce downregulation of NIP5;1 and BOR1 through mRNA degradation and proteolysis through endocytosis, respectively. In addition, borate exporters, such as Arabidopsis BOR4 and barley Bot1, function in boron exclusion from tissues and cells under conditions of excess boron. Thus, plants actively regulate intracellular localization and abundance of transport proteins to maintain boron homeostasis. In this review, the physiological roles and regulatory mechanisms of intracellular localization and abundance of boron transport proteins are discussed.

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  13. Polar Localization of the NIP5;1 Boric Acid Channel Is Maintained by Endocytosis and Facilitates Boron Transport in Arabidopsis Roots Reviewed

    Sheliang Wang, Akira Yoshinari, Tomoo Shimada, Ikuko Hara-Nishimura, Namiki Mitani-Ueno, Jian Feng Ma, Satoshi Naito, Junpei Takano

    PLANT CELL   Vol. 29 ( 4 ) page: 824 - 842   2017.4

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

    Boron uptake in Arabidopsis thaliana is mediated by nodulin 26-like intrinsic protein 5;1 (NIP5;1), a boric acid channel that is located preferentially on the soil side of the plasma membrane in root cells. However, the mechanism underlying this polar localization is poorly understood. Here, we show that the polar localization of NIP5;1 in epidermal and endodermal root cells is mediated by the phosphorylation of Thr residues in the conserved TPG (ThrProGly) repeat in the N-terminal region of NIP5;1. Although substitutions of Ala for three Thr residues in the TPG repeat did not affect lateral diffusion in the plasma membrane, these substitutions inhibited endocytosis and strongly compromised the polar localization of GFP-NIP5;1. Consistent with this, the polar localization was compromised in m subunit mutants of the clathrin adaptor AP2. The Thr-to-Ala substitutions did not affect the boron transport activity of GFP-NIP5;1 in Xenopus laevis oocytes but did inhibit the ability to complement boron translocation to shoots and rescue growth defects in nip5;1-1 mutant plants under boron-limited conditions. These results demonstrate that the polar localization of NIP5;1 is maintained by clathrin-mediated endocytosis, is dependent on phosphorylation in the TPG repeat, and is necessary for the efficient transport of boron in roots.

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  14. Plant Aquaporin Trafficking

    Takano Junpei, Yoshinari Akira, Luu Doan-Trung

    PLANT AQUAPORINS: FROM TRANSPORT TO SIGNALING     page: 47 - 81   2017

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    Authorship:Lead author, Last author, Corresponding author   Language:Japanese  

    DOI: 10.1007/978-3-319-49395-4_3

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  15. Near-infrared imaging of phytochrome-derived autofluorescence in plant nuclei

    Akira Yoshinari, Reika Isoda, Noriyoshi Yagi, Yoshikatsu Sato, Jelmer J. Lindeboom, David W. Ehrhardt, Wolf B. Frommer, Masayoshi Nakamura

        2023.9

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    Publisher:Cold Spring Harbor Laboratory  

    Abstract

    Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live-cell imaging. InArabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near-infrared (NIR) range of the spectrum and devised a non-invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non-model plant species.

    DOI: 10.1101/2023.09.18.558255

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  16. SWEET13 transport of sucrose, but not gibberellin, restores male fertility in <i>Arabidopsis sweet13;14</i>.

    Isoda R, Palmai Z, Yoshinari A, Chen LQ, Tama F, Frommer WB, Nakamura M

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 119 ( 42 ) page: e2207558119   2022.10

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    Authorship:Lead author, Last author, Corresponding author   Language:English  

    DOI: 10.1073/pnas.2207558119

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  17. The manifold actions of signaling peptides on subcellular dynamics of a receptor specify stomatal cell fate. Reviewed International journal

    Xingyun Qi, Akira Yoshinari, Pengfei Bai, Michal Maes, Scott M Zeng, Keiko U Torii

    eLife   Vol. 9   page: 1 - 29   2020.8

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    Receptor endocytosis is important for signal activation, transduction, and deactivation. However, how a receptor interprets conflicting signals to adjust cellular output is not clearly understood. Using genetic, cell biological, and pharmacological approaches, we report here that ERECTA-LIKE1 (ERL1), the major receptor restricting plant stomatal differentiation, undergoes dynamic subcellular behaviors in response to different EPIDERMAL PATTERNING FACTOR (EPF) peptides. Activation of ERL1 by EPF1 induces rapid ERL1 internalization via multivesicular bodies/late endosomes to vacuolar degradation, whereas ERL1 constitutively internalizes in the absence of EPF1. The co-receptor, TOO MANY MOUTHS is essential for ERL1 internalization induced by EPF1 but not by EPFL6. The peptide antagonist, Stomagen, triggers retention of ERL1 in the endoplasmic reticulum, likely coupled with reduced endocytosis. In contrast, the dominant-negative ERL1 remained dysfunctional in ligand-induced subcellular trafficking. Our study elucidates that multiple related yet unique peptides specify cell fate by deploying the differential subcellular dynamics of a single receptor.

    DOI: 10.7554/eLife.58097

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  18. Analysis of Endocytosis and Intracellular Trafficking of Boric Acid/Borate Transport Proteins in Arabidopsis. Reviewed International journal

    Akira Yoshinari, Junpei Takano

    Methods in molecular biology (Clifton, N.J.)   Vol. 2177   page: 1 - 13   2020

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

    Plants take up inorganic nutrients from the soil by transport proteins located in the plasma membrane of root cells. Boron (B) is an essential element for plant growth; it taken up and translocated by boric acid channels such as NIP5;1 and borate exporters such as BOR1 in Arabidopsis. NIP5;1 and BOR1 are localized to the plasma membrane of various root cells in polar manners toward soil- and stele-side, respectively, for efficient transport of B. In response to elevated B concentration, BOR1 undergoes vacuolar sorting for degradation to avoid accumulation of B to a toxic level in tissues. The polar localization and vacuolar sorting of the transport proteins are regulated through differential mechanisms of endocytosis and intracellular trafficking. In this chapter, we describe methods for quantitative live-cell imaging of GFP-NIP5;1 and BOR1-GFP as markers for the polar and vacuolar trafficking.

    DOI: 10.1007/978-1-0716-0767-1_1

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  19. Lack of ACTPK1, an STY kinase, enhances ammonium uptake and use, and promotes growth of rice seedlings under sufficient external ammonium Reviewed

    Marcel P. Beier, Mitsuhiro Obara, Akiko Taniai, Yuki Sawa, Jin Ishizawa, Haruki Yoshida, Narumi Tomita, Tsuyoshi Yamanaka, Yawara Ishizuka, Syuko Kudo, Akira Yoshinari, Shiho Takeuchi, Soichi Kojima, Tomoyuki Yamaya, Toshihiko Hayakawa

    Plant Journal   Vol. 93 ( 6 ) page: 992 - 1006   2018.3

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

    Ammonium influx into plant roots via the high-affinity transport system (HATS) is down-modulated under elevated external ammonium, preventing ammonium toxicity. In ammonium-fed Arabidopsis, ammonium transporter 1 (AMT1) trimers responsible for HATS activity are allosterically inactivated in a dose-dependent manner via phosphorylation of the conserved threonine at the carboxyl-tail by the calcineurin B-like protein 1-calcineurin B-like protein-interacting protein kinase 23 complex and other yet unidentified protein kinases. Using transcriptome and reverse genetics in ammonium-preferring rice, we revealed the role of the serine/threonine/tyrosine protein kinase gene OsACTPK1 in down-modulation of HATS under sufficient ammonium. In wild-type roots, ACTPK1 mRNA and protein accumulated dose-dependently under sufficient ammonium. To determine the function of ACTPK1, two independent mutants lacking ACTPK1 were produced by retrotransposon Tos17 insertion. Compared with segregants lacking insertions, the two mutants showed decreased root growth and increased shoot growth under 1 mm ammonium due to enhanced ammonium acquisition, via aberrantly high HATS activity, and use. Furthermore, introduction of OsACTPK1 cDNA fused to the synthetic green fluorescence protein under its own promoter complemented growth and the HATS influx, and suggested plasma membrane localization. Root cellular expression of OsACTPK1 also overlapped with that of ammonium-induced OsAMT1
    1 and OsAMT1
    2. Meanwhile, threonine-phosphorylated AMT1 levels were substantially decreased in roots of ACTPK1-deficient mutants grown under sufficient ammonium. Bimolecular fluorescence complementation assay further confirmed interaction between ACTPK1 and AMT1
    2 at the cell plasma membrane. Overall, these findings suggest that ACTPK1 directly phosphorylates and inactivates AMT1
    2 in rice seedling roots under sufficient ammonium.

    DOI: 10.1111/tpj.13824

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Books 1

  1. Plant Aquaporin Trafficking

    Takano J, Yoshinari A, Luu DT( Role: Joint author)

    2017 

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MISC 2

  1. The adaptor protein complex AP-4 plays a role in vacuolar targeting of a borate transporter BOR1.

    HOSOKAWA Takuya, YOSHINARI Akira, YOSHINARI Akira, KUNIEDA Tadashi, KUNIEDA Tadashi, SHIMADA Tomoo, HARA-NISHIMURA Ikuko, TAKANO Junpei

    日本植物生理学会年会(Web)   Vol. 60th   2019

  2. The role of adaptor protein complex 4 (AP-4) in vacuolar targeting of a borate transporter AtBOR1

    HOSOKAWA Takuya, YOSHINARI Akira, KUNIEDA Tadashi, KUNIEDA Tadashi, SHIMADA Tomoo, HARA-NISHIMURA Ikuko, TAKANO Junpei

    日本植物生理学会年会(Web)   Vol. 59th   2018

Presentations 1

  1. Elucidation of molecular mechanism underlying inward/outward lateral cell polarity in plants Invited

    2024.1.23 

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Research Project for Joint Research, Competitive Funding, etc. 1

  1. 植物の細胞極性を制御する分子基盤の解明

    2022.10 - 2026.3

    さきがけ「植物分子の機能と制御」領域 

KAKENHI (Grants-in-Aid for Scientific Research) 7

  1. Clarification of cell polarization mechanism in plants

    Grant number:1199518  2022.10 - 2026.3

    JST  PRESTO 

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  2. Mechanisms for boron sensing by borate transceptors

    Grant number:26712007  2014.4 - 2018.3

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

    TAKANO Junpei, YOSHINARI Akira, WANG Sheliang

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    Plants sense mineral concentrations in soils or tissues and regulate membrane transport proteins to response to environmental changes. Boron is an essential element but is toxic when accumulated at high levels. Arabidopsis BOR1 is an borate exporter at the plasma membrane and responsible for boron transport toward root stele. BOR1 is transferred from the plasma membrane to the vacuole for degradation upon high-B supply to avoid boron toxicity. Our results in this study suggested that BOR1 functions as a borate/boric acid transceptor (transporter and receptor) regulating own accumulation in the plasma membrane to maintain boron homeostasis.

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  3. 受容体様キナーゼの極性スイッチの分子機構と生理学的意義の解明

    Grant number:22K15139  2022.4 - 2027.3

    科学研究費助成事業  若手研究

    吉成 晃

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    Authorship:Principal investigator 

    Grant amount:\4550000 ( Direct Cost: \3500000 、 Indirect Cost:\1050000 )

    植物細胞では、様々な受容体や輸送体タンパク質が特定の細胞膜ドメインに局在することで、形態形成やシグナリング、方向性をもった物質輸送が行われている。本研究では、シロイヌナズナのロイシンリッチリピート受容体様キナーゼ (LRR-RLK)のうち、極性局在性の受容体様キナーゼを多く包含する「VIIサブファミリー」に着目し、申請者が発見したDUAL POLAR KINASE (DPK) の「極性スイッチ」機構の解明を軸として、極性スイッチの普遍的分子機構とその生理学的意義を明らかにするとともに、植物進化の過程でどのように極性スイッチが生まれ多様化したのかを解明する。

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  4. ケミカルジェネティクスによる植物の細胞極性形成機構の解明

    Grant number:20J01129  2020.4 - 2023.3

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

    吉成 晃

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    植物細胞において、「内側/外側の細胞極性」を決定する根本的な要素は全く分かっていない。本研究では、合成小分子および環状ペプチドライブラリーを用いたケミカルジェネティクスおよび分泌性ペプチド遺伝子の異所発現系によって「内側/外側の細胞極性」を決定するモルフォゲンと下流の遺伝子群を一挙に同定する。
    細胞極性の形成は多細胞生物の発生、生育、そして環境応答に必須であり、植物においても様々な極性軸に基づく細胞極性が存在することが知られている。しかしながら、細胞極性を制御する分子機構については不明な点が多い。この理由として、細胞極性の形成が胚発生において重要なプロセスであること、遺伝子重複による変異体獲得の困難さ等が挙げられる。本研究では、ケミカルジェネティクスによって、変異体スクリーニングでは獲得できなかった細胞極性を司る遺伝子群を同定し、植物において細胞極性を形成し維持するメカニズムを明らかにするものである。
    現在までに、約7,300種類(目標の18.3%)の小分子化合物のスクリーニングを終えており、BOR1やNIP5;1の極性局在パターンを変調するヒット化合物として2種類の分子を同定している。これらの分子については、標的タンパク質を同定するための実験を行っている。
    また、細胞層特異的な極性局在パターンを示すユニークな受容体様キナーゼを新たに発見し、この受容体様キナーゼの極性局在を制御する分子機構についての解析も始めた。この受容体様キナーゼの一部のアミノ酸配列を削った変異体を複数種類作製し、これらの細胞内局在を解析した結果、キナーゼドメイン近傍のドメインが内皮細胞層特異的な中心柱に向かった極性局在パターンに重要であることがわかった。質量分析によってこのドメイン内の翻訳後修飾を網羅的に解析したところ、リジン残基のユビキチン化とセリン残基のリン酸化が検出された。現在、これらの翻訳後修飾が、細胞層特異的な極性パターンのスイッチとして機能していると仮説を立てて検証を行っている。
    BOR1-GFPを細胞極性マーカーとして、細胞極性を変調する化合物をスクリーニングしている。現在までに約7,300種類の小分子化合物のスクリーニングを終えており、BOR1-GFPの極性パターンを変調する化合物を2種類獲得している。また、細胞極性マーカーとなりうる機能未知の受容体様キナーゼを同定した。この受容体様キナーゼは、細胞層特異的な極性局在パターンを示すことから、植物細胞における膜タンパク質の「極性スイッチ」機構のモデルとしても用いることができる。現在までに、この受容体様キナーゼの極性パターンに重要なドメインを同定し、このドメイン内の翻訳後修飾を、質量分析によって同定している。
    今後、現在進めている化合物スクリーニングを継続するととともに、ヒット化合物の標的タンパク質を同定する。また、新たに発見した受容体様キナーゼの極性パターンを制御する仕組みと生理的機能を明らかにするため、アミノ酸置換変異の導入、相互作用因子の同定、変異体の獲得を行う。また、受容体様キナーゼの部分配列(ドメイン)が極性パターンに関わっていることから、このドメインがタンパク質の動態にどのような影響を与えるのかを、コンピュータシミュレーションによって明らかにする。

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  5. 膜電位の可視化による植物の膜電位シグナリング機構の解明

    Grant number:19K16164  2019.4 - 2022.3

    日本学術振興会  科学研究費助成事業 若手研究  若手研究

    吉成 晃

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    Authorship:Principal investigator 

    Grant amount:\4160000 ( Direct Cost: \3200000 、 Indirect Cost:\960000 )

    近年、植物の細胞間情報伝達メカニズムとして「膜電位の変化」が注目されている。特に傷害に伴う膜電位の変化は傷害を受けた部位から離れた部位まで伝達され、組織・器官間の高速・長距離シグナル伝達機構として機能することが示されている。また、膜輸送タンパク質を介した物質輸送には、プロトン勾配や膜電位が密接に関わっていることから、膜電位の大小や局在が細胞毎に多様化している可能性がある。本研究では、植物の膜電位を可視化する新規の蛍光プローブを作製し、定量的なイメージングによって植物の膜電位シグナリング機構を理解する。
    近年、植物の細胞間情報伝達メカニズムとして「膜電位の変化」が注目されている。特に傷害に伴う膜電位の変化は傷害を受けた部位から離れた部位まで伝達され、組織・器官間の高速・長距離シグナル伝達機構として機能することが示されている。また、膜輸送タンパク質を介した物質輸送には、プロトン勾配や膜電位が密接に関わっていることから、膜電位の大小や局在が細胞毎に多様化している可能性がある。
    本研究では、(1) 植物の膜電位変化を可視化する新規の蛍光タンパク質膜電位プローブ (Plant-GEVI) の開発、(2) Plant-GEVIを用いて、個体・細胞レベルでの膜電位イメージング解析、(3) Plant-GEVIを用いた根の膜電位マップの作製を通して、植物の膜電位制御と膜電位を介した長距離膜電位シグナリング機構を理解する。
    これまでに、FlicR1、Mermaid2といった既存のGEVIが、植物の細胞膜に局在することを確かめている。さらに、FlicR1のVoltage sensing domain (VSD)にアミノ酸置換変異を導入することで、反応可能な膜電位の値を、植物の膜電位域に近づけることに成功している。現在、変異型FlicR1およびMermaid2を植物の根や孔辺細胞に発現させ、これらの膜電位変化に対する応答を調べている。一方、ASAP3と呼ばれるGEVIを植物に導入したところ、ASAP3は小胞体にスタックしてしまうことがわかった。
    孔辺細胞におけるイメージングを行っているが、観察中に組織が動いてしまうという問題がある。医療用接着剤による組織のカバーグラスへの固定を検討している。また、ASAP3に小胞体からの分泌を促すと期待できるSTP1C末端領域や、ゴルジ体以降シグナルを導入したものの、植物細胞においてASAP3を細胞膜に局在させることはできなかった。FlicR1のcpmApple部位にGO-Matryoshkaを導入し、FlicR1をratiometric GEVIに改変する実験を行っており、膜電位に応答して蛍光量が変化するプロトタイプが複数得られているものの、未だに蛍光変化のレベルが小さく、実用化には程遠い。
    現在のところ、膜電位の反応域を改変した変異型FlicR1およびMermaid2が、最も期待できるGEVIである。今後は、これらを発現する形質転換体を用いた詳細なイメージング解析を中心に行う。また、FlicR1をratiometric GEVIに改変するために、るVSDとGO-Matryoshka部位の間のリンカーのスクリーニングを継続する。

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  6. Mechanical manipulation and in vitro reconstitution of microtubule severing

    Grant number:18KK0195  2018.10 - 2022.3

    Grants-in-Aid for Scientific Research  Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))

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  7. 微小管の力学的特性計測と再構成系による微小管切断装置の階層的理解

    2018 - 2021

    日本学術振興会  2018年度 国際共同研究加速基金(国際共同研究強化(B)) 

    中村 匡良

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    Grant type:Competitive

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Social Contribution 1

  1. 第101回 名大カフェ「根っこの科学 〜植物の栄養素獲得をめぐるアンダーグラウンドな世界〜」

    Role(s):Appearance

    名古屋大学 学術研究・産学官連携推進本部  2024.3

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