Development of a regulatory system of membrane potential in plant using microbial rhodopsin

• Project/Area Number: 17K19230
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Research Field: Agricultural chemistry and related fields
• Research Institution: The University of Tokyo (2019); Nagoya Institute of Technology (2017-2018)
• Principal Investigator: Konno Masae 東京大学, 物性研究所, 特任研究員 (60459732)
• Project Period (FY): 2017-06-30 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥6,370,000 (Direct Cost: ¥4,900,000、Indirect Cost: ¥1,470,000); Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2018: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2017: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
• Keywords: オプトジェネティクス / 膜電位 / 微生物型ロドプシン / 植物細胞 / イオン輸送 / 光遺伝学 / 膜電位制御 / 植物培養細胞 / 遺伝子導入 / 植物 / 生体分子 / 代謝

Outline of Final Research Achievements

We aimed to construct a membrane potential regulatory system in plant cells by using light-dependent ion transport of the microbial rhodopsin. Six microbial rhodopsin were transiently expressed as fusion proteins with the fluorescent protein Venus in cultured plant cells. We observed the plasma membrane localization of the expressed protein in the outward-facing sodium pump KR2, the cation channel GtCCR4, and the anion channel GtACR1. Light-dependent hyperpolarization of the membrane potential was observed in cells expressing KR2 and GtCCR4 upon exposure to 530 nm light. We further characterized the molecular properties of the cation channel GtCCR4 and the inward-facing proton pump SzR as novel microbial rhodopsins.

Academic Significance and Societal Importance of the Research Achievements

本研究でKR2およびGtCCR4発現細胞における光依存的な膜電位の過分極が観測されたことは、微生物型ロドプシンが植物細胞の膜電位を操作するためのオプトジェネティクスツールとして機能することを示した初めての例である。また、新奇微生物型ロドプシンとしてカチオンチャネルGtCCR4及び内向きプロトンポンプSzRの機能解析を行い、新たな膜電位操作ツールとしての可能性を提示した。特に、GtCCR4はこれまで利用されてきたChR2とは性質が異なるツールとしての可能性が期待されている。

Research Products

• [Journal Article] Schizorhodopsins: A novel family of rhodopsins from Asgard archaea that function as light-driven inward H+ pumps (2020)
  • Author(s): Keiichi Inoue, Satoshi P. Tsunoda, Manish Singh, Sahoko Tomida, Shoko Hososhima, Masae Konno, Ryoko Nakamura, Hiroki Watanabe, Paul-Adrian Bulzu, Horia L. Banciu, Adrian Andrei, Takayuki Uchihashi, Rohit Ghai, Oded Beja, Hideki Kandori
  • Journal Title: Science Advances Volume: 6 Issue: 15
  • DOI: 10.1126/sciadv.aaz2441
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Crystal structure of heliorhodopsin (2019)
  • Author(s): W. Shihoya, K. Inoue, M. Singh, M. Konno, S. Hososhima, K. Yamashita, K. Ikeda, A. Higuchi, T. Izume, Okazaki S, Hashimoto M, Mizutori R, Tomida S, Yamauchi Y, Abe-Yoshizumi R, K. Katayama, SP. Tsunoda, M. Shibata, Y. Furutani, A. Pushkarev, O. Béjà, T. Uchihashi, H. Kandori, O. Nureki
  • Journal Title: Nature Volume: 574 Issue: 7776 Pages: 132-136
  • DOI: 10.1038/s41586-019-1604-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Engineered Functional Recovery of Microbial Rhodopsin without Retinal-Binding Lysine. (2019)
  • Author(s): Yumeka Yamauchi, Masae Konno, Daichi Yamada, Kei Yura, Keiichi Inoue, Oded Beja, Hideki Kandori
  • Journal Title: Photochem. Photobiol. Volume: 印刷中 Issue: 5 Pages: 1116-1121
  • DOI: 10.1111/php.13114
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A distinct abundant group of microbial rhodopsins discovered using functional metagenomics. (2018)
  • Author(s): A.Pushkarev, K. Inoue, S. Larom, J. Flores-Uribe, M. Singh, M. Konno, S. Tomida, S. Ito, R. Nakamura, S. P. Tsunoda, A. Philosof, I. Sharon, N. Yutin, E. V. Koonin, H. Kandori, O. Béjà
  • Journal Title: Nature Volume: 558 Issue: 7711 Pages: 595-599
  • DOI: 10.1038/s41586-018-0225-9
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy (2018)
  • Author(s): Shibata Mikihiro、Inoue Keiichi、Ikeda Kento、Konno Masae、Singh Manish、Kataoka Chihiro、Abe-Yoshizumi Rei、Kandori Hideki、Uchihashi Takayuki
  • Journal Title: Scientific Reports Volume: 8 Issue: 1 Pages: 8262-8262
  • DOI: 10.1038/s41598-018-26606-y
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Molecular properties of a DTD channelrhodopsin from <i>Guillardia theta</i> (2017)
  • Author(s): Yamauchi Yumeka、Konno Masae、Ito Shota、Tsunoda Satoshi P.、Inoue Keiichi、Kandori Hideki
  • Journal Title: Biophysics and Physicobiology Volume: 14 Issue: 0 Pages: 57-66
  • DOI: 10.2142/biophysico.14.0_57
  • NAID: 130007040873
  • ISSN: 2189-4779
  • Peer Reviewed / Open Access
• [Presentation] Structural basis of proton transport in Schizorhodopsin (2019)
  • Author(s): A. Higuchi, W. Shihoya, K. Inoue, M. Konno, H. Kandori, O. Nureki
  • Organizer: The 57th Annual Meeting of the Biophysical Society of Japan
  • Int'l Joint Research
• [Presentation] Engineering microbial rhodopsin without retinal-binding lysine to gain photosensitive function (2019)
  • Author(s): Y. Yamauchi, M. Konno, D. Yamada, K. Yura, K. Inoue, O. Beja, H.Kandori
  • Organizer: The 57th Annual Meeting of the Biophysical Society of Japan
  • Int'l Joint Research
• [Presentation] Molecular characterization of heliorhodopsin from marine giant virus light-dependently infecting to Emiliania huxleyi. (2019)
  • Author(s): R. Mizutori, M. Konno, K. Inoue, O. Beja, H. Kandori
  • Organizer: The 57th Annual Meeting of the Biophysical Society of Japan
  • Int'l Joint Research
• [Presentation] Characterization of the inward proton transport pathway in Schizorhodopsin (2019)
  • Author(s): M. Konno, K. Inoue, R. Ghai, O. Beja, H. Kandori
  • Organizer: The 57th Annual Meeting of the Biophysical Society of Japan
  • Int'l Joint Research
• [Presentation] Structure and biophysical characterization of the heliorhodopsin (2019)
  • Author(s): W. Shihoya、K. Inoue、M. Singh、M. Konno、S. Hososhima、K. Yamashita、K. Ikeda、A. Higuchi、T. Izume、S. Okazaki、M. Hashimoto、R. Mizutori、S. Tomida、Y. Yamauchi、R. Abe-Yoshizumi、K. Katayama、S.P. Tsunoda、M. Shibata、Y. Furutani、A. Pushkarev、O. Beja、T. Uchihashi、H. Kandori、O. Nureki
  • Organizer: The 57th Annual Meeting of the Biophysical Society of Japan
  • Int'l Joint Research
• [Presentation] Molecular properties of new type microbial rhodopsins (2019)
  • Author(s): M.Konno
  • Organizer: International Symposium on “Optobiotechnology”
  • Int'l Joint Research / Invited
• [Presentation] 微生物型ロドプシンを用いた植物細胞の膜電位操作系の開発 (2019)
  • Author(s): 今野雅恵, 神取秀樹
  • Organizer: 第60回日本植物生理学会年会
• [Presentation] The effect of protein expression host on the ion-transport analysis of microbial rhodopsins (2018)
  • Author(s): M. Konno, K. Inoue, H. Kandori
  • Organizer: 18th International Conference on Retinal Proteins
  • Int'l Joint Research
• [Presentation] Gene expression analysis of rhodopsin-like proteins from marine algae Guillardia theta. (2018)
  • Author(s): Y. Yamauchi, M. Konno, K. Inoue, H. Kandori
  • Organizer: 18th International Conference on Retinal Proteins
  • Int'l Joint Research
• [Presentation] Gene expression analysis of 44 microbial rhodopsin-like proteins from marine algae Guillardia theta. (2018)
  • Author(s): Y. Yamauchi, M. Konno, K. Inoue, H. Kandori
  • Organizer: .” The 56rd Annual Meeting of the Biophysical Society of Japan
  • Int'l Joint Research
• [Presentation] Microbial rhodopsins leading to development of optogenetic tool. (2018)
  • Author(s): M. Konno
  • Organizer: Symposium “Dive into Brain Abyss by Optogenetics.” The 56rd Annual Meeting of the Biophysical Society of Japan
  • Int'l Joint Research / Invited
• [Presentation] Molecular characterization of heliorhodopsin from Emiliania huxleyi virus (2018)
  • Author(s): R. Mizutori, M. Konno, K. Inoue, O, B&eacute;j&agrave;, H. Kandori.
  • Organizer: The 56rd Annual Meeting of the Biophysical Society of Japan
  • Int'l Joint Research
• [Presentation] Identification and functional analysis of two lycopene cyclase genes (crtL-b and crtL-e) in Guillrdia theta (2018)
  • Author(s): M. Konno, H. Kandori, S. Takaichi
  • Organizer: ISPR International Conference on Microbial Photosynthesis
  • Int'l Joint Research
• [Presentation] 海洋性クリプト藻由来の微生物型ロドプシン様遺伝子の発現解析 (2018)
  • Author(s): 山内夢叶、今野雅恵、井上圭一、神取秀樹
  • Organizer: 第20回日本光生物学協会年会
• [Presentation] 海洋ブルームに影響を与える巨大ウイルスに由来する新奇ロドプシンの特性研究 (2018)
  • Author(s): 水鳥律、今野雅恵、井上圭一、Oded B&eacute;j&agrave;、神取秀樹
  • Organizer: 第20回日本光生物学協会年会
• [Presentation] 海洋性真核藻類がもつ光駆動カチオンチャネルGtCCR4の光反応解析 (2018)
  • Author(s): 山内夢叶，今野雅恵，伊藤奨太，井上圭一，神取秀樹
  • Organizer: 第59回日本植物生理学会年会
• [Presentation] Molecular properties of the light-gated cation channel from cryptophyta Guillardia theta (2017)
  • Author(s): Y. Yamauchi, M. Konno, S. Ito, Y. Kato, S. Tsunoda, K. Inoue, H. Kandori
  • Organizer: 253rd American Chemical Society National Meeting & Exposition
  • Int'l Joint Research
• [Presentation] Molecular properties of a DTD cation channelrhodopsin from marine algae (2017)
  • Author(s): Y. Yamauchi, M. Konno, S. Ito, S. Tsunoda, K. Inoue, H. Kandori
  • Organizer: International Symposium on Biophysics of Rhodopsins
  • Int'l Joint Research
• [Presentation] 海洋性クリプト藻Guillardia thetaが持つ微生物型ロドプシン様遺伝子の発現解析 (2017)
  • Author(s): 今野雅恵、井上圭一、神取秀樹
  • Organizer: 日本植物学会第81回大会
• [Presentation] Spectroscopic analysis of a light-gated cation channel GtCCR4 from marine algae (2017)
  • Author(s): Y. Yamauchi, M. Konno, S. Ito, S. Tsunoda, K. Inoue, H. Kandori
  • Organizer: The 55th Annual Meeting of the Biophysical Society of Japan
  • Int'l Joint Research
• [Presentation] 光駆動カチオンチャネルGtCCR4を対象とする光反応過程の解析 (2017)
  • Author(s): 山内夢叶、今野雅恵、伊藤奨太、角田聡、井上圭一、神取秀樹
  • Organizer: 第7回CSJ化学フェスタ2017
• [Presentation] Gene Expression Analysis of Microbial Rhodopsin-Like Genes in Marine Cryptomonad (2017)
  • Author(s): M. Konno, Y. Yamauchi, K. Inoue, H. Kandori
  • Organizer: Taiwan-Japan Plant Biology 2017
  • Int'l Joint Research
• [Presentation] 海洋性真核藻類がもつ光駆動カチオンャネルGtCCR4の光反応中間体の解析 (2017)
  • Author(s): 山内夢叶、今野雅恵、伊藤奨太、井上圭一、神取秀樹
  • Organizer: 日本生体エネルギー研究会第43回討論会