Updated on 2022/04/12

写真a

 
NAKAMICHI Norihito
 
Organization
Graduate School of Bioagricultural Sciences Department of Applied Biosciences Professor
Graduate School
Graduate School of Bioagricultural Sciences
Undergraduate School
School of Agricultural Sciences Department of Applied Biosciences
Title
Professor
Contact information
メールアドレス

Degree 1

  1. Doctor (Agriculture) ( 2005.3   Nagoya University ) 

Research Interests 5

  1. バイオインフォマティクス

  2. ゲノム情報

  3. 概日リズム

  4. 概日時計

  5. 植物

Research Areas 2

  1. Life Science / Applied biochemistry

  2. Life Science / Plant molecular biology and physiology  / 植物、概日リズム

Current Research Project and SDGs 9

  1. 新奇時計関連因子の生化学的な解析

  2. 概日時計の周期安定性を内包する分子の解析

  3. 植物における時計関連タンパク質の生化学的解析

  4. バイオマス生産性の向上を指向した概日時計のシステム生物学

  5. 不規則な温度環境変化に対する時計システムのリジリエンス機構

  6. 改変型キナーゼとプロテオミクスで解く植物リン酸化ネットワーク

  7. 時計に依存する発生制御ネットワークアトラス

  8. 環境の時刻変動への適応を可能にする植物の時計転写ネットワークの包括的な解析

  9. 花成ホルモン誘導化合物の解析

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Research History 6

  1. Nagoya University   Graduate School of Bioagricultural Sciences   Professor

    2021.4

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

    2013.5 - 2021.3

  3. Assistant Professor, Institute for Advanced Research

    2011.4 - 2013.4

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    Country:Japan

  4. Nagoya University   Graduate School of Bioagricultural Sciences Department of Biological Mechanisms and Functions Division of Molecular and Cellular Biology   Special duty Assistant Professor

    2011.4 - 2013.4

  5. Special Postdoctoral Researcher, RIKEN

    2008.4 - 2011.3

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    Country:Japan

  6. Postdoctoral Fellowship, Japan Society for the Promotion of Science

    2005.4 - 2008.3

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    Country:Japan

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Education 2

  1. Nagoya University   Graduate School, Division of Agricultural Science

    - 2005.3

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    Country: Japan

  2. Nagoya University   Faculty of Agriculture

    1996.4 - 2000.3

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    Country: Japan

Professional Memberships 3

  1. The Japanese Society of Plant Physiologists

  2. 日本時間生物学会

  3. 日本農芸化学会

Awards 6

  1. 長瀬研究振興賞

    2020.4   長瀬科学技術振興財団   植物の成熟化を司る発生タイマーの解明へむけたケミカルバイオロジー

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    Award type:Award from publisher, newspaper, foundation, etc.  Country:Japan

  2. 文部科学大臣表彰若手科学者賞

    2017.4   文部科学省   植物の概日リズムに関する転写制御ネットワークの研究

    中道範人

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    Country:Japan

  3. 日本時間生物学会奨励賞

    2015.11   日本時間生物学会  

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    Award type:International academic award (Japan or overseas)  Country:Japan

  4. 日本植物生理学会奨励賞

    2014.3   日本植物生理学会  

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    Country:Japan

  5. Travel grant for 1st World Congress of Chronobiology

    2003.8   World Congress of Chronobiology  

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    Country:Japan

  6. Award for Excellence to Authors Publishing to Bioscience, Biotechnology, and Biochemistry

    2003.3  

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    Country:Japan

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Papers 58

  1. Plant clock modifications for adapting flowering time to local environments

    Maeda Akari E., Nakamichi Norihito

    PLANT PHYSIOLOGY     2022.3

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    Language:Japanese  

    DOI: 10.1093/plphys/kiac107

    Web of Science

  2. Phosphorylation of RNA Polymerase II by CDKC;2 Maintains the Arabidopsis Circadian Clock Period

    Uehara Takahiro N., Nonoyama Takashi, Taki Kyomi, Kuwata Keiko, Sato Ayato, Fujimoto Kazuhiro J., Hirota Tsuyoshi, Matsuo Hiromi, Maeda Akari E., Ono Azusa, Takahara Tomoaki T., Tsutsui Hiroki, Suzuki Takamasa, Yanai Takeshi, Kay Steve A., Itami Kenichiro, Kinoshita Toshinori, Yamaguchi Junichiro, Nakamichi Norihito

    PLANT AND CELL PHYSIOLOGY     2022.1

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    Language:Japanese  

    DOI: 10.1093/pcp/pcac011

    Web of Science

  3. The Transcriptional Network in the Arabidopsis Circadian Clock System Invited Reviewed International journal

    Nakamichi Norihito

    GENES   Vol. 11 ( 11 )   2020.11

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

    DOI: 10.3390/genes11111284

    Web of Science

  4. 3,4-Dibromo-7-Azaindole Modulates Arabidopsis Circadian Clock by Inhibiting Casein Kinase 1 Activity Reviewed International journal

    Ono Azusa, Sato Ayato, Fujimoto Kazuhiro J., Matsuo Hiromi, Yanai Takeshi, Kinoshita Toshinori, Nakamichi Norihito

    PLANT AND CELL PHYSIOLOGY   Vol. 60 ( 11 ) page: 2360 - 2368   2019.11

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

    DOI: 10.1093/pcp/pcz183

    Web of Science

  5. Structure-function study of a novel inhibitor of the casein kinase 1 family in Arabidopsis thaliana Reviewed International journal

    Saito Ami N., Matsuo Hiromi, Kuwata Keiko, Ono Azusa, Kinoshita Toshinori, Yamaguchi Junichiro, Nakamichi Norihito

    PLANT DIRECT   Vol. 3 ( 9 )   2019.9

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

    DOI: 10.1002/pld3.172

    Web of Science

  6. Casein kinase 1 family regulates PRR5 and TOC1 in the Arabidopsis circadian clock Reviewed International coauthorship International journal

    Uehara Takahiro N., Mizutani Yoshiyuki, Kuwata Keiko, Hirota Tsuyoshi, Sato Ayato, Mizoi Junya, Takao Saori, Matsuo Hiromi, Suzuki Takamasa, Ito Shogo, Saito Ami N., Nishiwaki-Ohkawa Taeko, Yamaguchi-Shinozaki Kazuko, Yoshimura Takashi, Kay Steve A., Itami Kenichiro, Kinoshita Toshinori, Yamaguchi Junichiro, Nakamichi Norihito

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 116 ( 23 ) page: 11528 - 11536   2019.6

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

    DOI: 10.1073/pnas.1903357116

    Web of Science

  7. Evolutionary Insight into the Clock-Associated PRR5 Transcriptional Network of Flowering Plants Reviewed International journal

    Toda Yosuke, Kudo Toru, Kinoshita Toshinori, Nakamichi Norihito

    SCIENTIFIC REPORTS   Vol. 9   2019.2

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

    DOI: 10.1038/s41598-019-39720-2

    Web of Science

  8. Direct repression of evening genes by CIRCADIAN CLOCK-ASSOCIATED 1 in the Arabidopsis circadian clock Reviewed

    Mari Kamioka, Saori Takao, Takamasa Suzuki, Kyomi Taki, Tetsuya Higashiyama, Toshinori Kinoshita, Norihito Nakamichi

    The Plant Cell   Vol. 28   page: 696-711   2016.3

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  9. Transcriptional repressor PRR5 directly regulates clock-output pathways Reviewed

    Norihito Nakamichi, Takatoshi Kiba, Mari Kamioka, Takamasa Suzuki, Takafumi Yamashino, Tetsuya Higashiyama, Hitoshi Sakakibara, Takeshi Mizuno

    Proceedings of the National Academy of Sciences   Vol. 109 ( 42 ) page: 17123-17128   2012.10

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  10. PSEUDO-RESPONSE REGULATORS 9, 7, and 5 are transcriptional repressors in the Arabidopsis circadian clock Reviewed

    Norihito Nakamichi, Takatoshi Kiba, Rossana Henriques, Takeshi Mizuno, Nam-Hai Chua, Hitoshi Sakakibara

    The Plant Cell   Vol. 22   page: 594-605   2010

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    An interlocking transcriptional-translational feedback loop of clock-associated genes is thought to be the central oscillator of the circadian clock in plants. TIMING OF CAB EXPRESSION1 (also called PSEUDO-RESPONSE REGULATOR1 [PRR1]) and two MYB transcription factors, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), play pivotal roles in the loop. Genetic studies have suggested that PRR9, PRR7, and PRR5 also act within or close to the loop; however, their molecular functions remain unknown. Here, we demonstrate that PRR9, PRR7, and PRR5 act as transcriptional repressors of CCA1 and LHY. PRR9, PRR7, and PRR5 each suppress CCA1 and LHY promoter activities and confer transcriptional repressor activity to a heterologous DNA binding protein in a transient reporter assay. Using a glucocorticoid-induced PRR5-GR (glucorticoid receptor) construct, we found that PRR5 directly downregulates CCA1 and LHY expression. Furthermore, PRR9, PRR7, and PRR5 associate with the CCA1 and LHY promoters in vivo, coincident with the timing of decreased CCA1 and LHY expression. These results suggest that the repressor activities of PRR9, PRR7, and PRR5 on the CCA1 and LHY promoter regions constitute the molecular mechanism that accounts for the role of these proteins in the feedback loop of the circadian clock.

  11. The singularity response reveals entrainment properties of the plant circadian clock Reviewed International journal

    Masuda Kosaku, Tokuda Isao T., Nakamichi Norihito, Fukuda Hirokazu

    NATURE COMMUNICATIONS   Vol. 12 ( 1 )   2021.2

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

    DOI: 10.1038/s41467-021-21167-7

    Web of Science

  12. Flowering time control in rice by introducing Arabidopsis clock-associated PSEUDO-RESPONSE REGULATOR 5 Reviewed International journal

    Nakamichi Norihito, Kudo Toru, Makita Nobue, Kiba Takatoshi, Kinoshita Toshinori, Sakakibara Hitoshi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   Vol. 84 ( 5 ) page: 970 - 979   2020.5

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

    DOI: 10.1080/09168451.2020.1719822

    Web of Science

  13. Regulation of stomatal opening and histone modification by photoperiod in Arabidopsis thaliana Reviewed International journal

    Aoki Saya, Toh Shigeo, Nakamichi Norihito, Hayashi Yuki, Wang Yin, Suzuki Takamasa, Tsuji Hiroyuki, Kinoshita Toshinori

    SCIENTIFIC REPORTS   Vol. 9   2019.7

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

    DOI: 10.1038/s41598-019-46440-0

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  14. Improvement of Arabidopsis Biomass and Cold, Drought and Salinity Stress Tolerance by Modified Circadian Clock-Associated PSEUDO-RESPONSE REGULATORs Reviewed International coauthorship

    Norihito Nakamichi, Saori Takao, Toru Kudo, Takatoshi Kiba, Yin Wang, Toshinori Kinoshita, Hitoshi Sakakibara

    Plant Cell Physiol.   Vol. 57   page: 1085-1097   2016.5

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  15. Transcriptional repression by MYB3R proteins regulates plant organ growth. Reviewed

    Kobayashi K, Suzuki T, Iwata E, Nakamichi N, Suzuki T, Chen P, Ohtani M, Ishida T, Hosoya H, Müller S, Leviczky T, Pettkó-Szandtner A, Darula Z, Iwamoto A, Nomoto M, Tada Y, Higashiyama T, Demura T, Doonan JH, Hauser MT, Sugimoto K, Umeda M, Magyar Z, Bögre L, Ito M.

    EMBO J   Vol. 34 ( 15 ) page: 1992-2007   2015.8

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  16. Adaptation to the local environment by modifications of the photoperiod response in crops. Reviewed

    Nakamichi N.

    Plant Cell Physiol.   Vol. 56 ( 4 ) page: 594-604   2015.4

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  17. A flowering integrator, SOC1, affects stomatal opening in Arabidopsis thaliana Reviewed

    Kimura Y, Aoki S, Ando E, Kitatsuji A, Watanabe A, Ohnishi M, Takahashi K, Inoue S, Nakamichi N, Tamada Y, Kinoshita T

    Plant Cell Physiol.   Vol. 56 ( 4 ) page: 640-649   2015.4

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    DOI: 10.1093/pcp/pcu214

  18. The LNK1 night light-inducible and clock-regulated gene is induced also in response to warm-night through the circadian clock nighttime repressor in Arabidopsis thaliana

    Takeshi Mizuno, Aya Takeuchi, Yuichi Nomoto, Norihito Nakamichi, Takafumi Yamashino

    Plant signaling & behavior   Vol. 9 ( 3 ) page: e28505   2014.1

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  19. Clock-Controlled and FLOWERING LOCUS T (FT)-Dependent Photoperiodic Pathway in Lotus japonicus II: Characterization of a MicroRNA Implicated in the Control of Flowering Time. Reviewed

    Takafumi Yamashino, Saori Yamawaki, Emi Hagui, Kai Ishida, Hanayo Ueoka-Nakanishi, Norihito Nakamichi, Takeshi Mizuno

    Bioscience, biotechnology, and biochemistry   Vol. 77 ( 6 ) page: 1179-1185   2013.6

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  20. Circadian clock and PIF4-mediated external coincidence mechanism coordinately integrates both of the cues from seasonal changes in photoperiod and temperature to regulate plant growth in Arabidopsis thaliana

    Yuji Nomoto, Saori Kubozono, Miki Miyachi, Takafumi Yamashino, Norihito Nakamichi, Takeshi Mizuno

    Plant signaling & behavior   Vol. 8 ( 2 ) page: e22863   2013.2

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  21. Clock-controlled and FLOWERING LOCUS T (FT)-dependent photoperiodic pathway in Lotus japonicus I: verification of the flowering-associated function of an FT homolog. Reviewed

    Takafumi Yamashino, Saori Yamawaki, Emi Hagui, Hanayo Ueoka-Nakanishi, Norihito Nakamichi, Shogo Ito, Takeshi Mizuno

    Bioscience, biotechnology, and biochemistry   Vol. 77 ( 4 ) page: 747-753   2012.12

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  22. Molecular mechanisms of circadian rhythm in Lotus japonicus and Arabidopsis thaliana are sufficiently compatible to regulate heterologous core clock genes robustly. Reviewed

    Hanayo Ueoka-Nakanishi, Takafumi Yamashino, Kai Ishida, Mari Kamioka, Norihito Nakamichi, Takeshi Mizuno

    Bioscience, biotechnology, and biochemistry   Vol. 76 ( 12 ) page: 2332-2334   2012.12

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  23. Circadian clock-and PIF4-controlled plant growth: a coincidence mechanism directly integrates a hormone signaling network into the photoperiodic control of plant architectures in Arabidopsis thaliana Reviewed

    Yuichi Nomoto, Saori Kubozono, Takafumi Yamashino, Norihito Nakamichi, Takeshi Mizuno

    Plant and Cell Physiology   Vol. 53 ( 11 ) page: 1950-1964   2012.11

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  24. A circadian clock-and PIF4-mediated double coincidence mechanism is implicated in the thermosensitive photoperiodic control of plant architectures in Arabidopsis thaliana Reviewed

    Yuichi Nomoto, Saori Kubozono, Miki Miyachi, Takafumi Yamashino, Norihito Nakamichi, Takeshi Mizuno

    Plant and Cell Physiology   Vol. 53 ( 11 ) page: 1965-1973   2012.11

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  25. Traveling waves of circadian gene expression in lettuce

    Kazuya UKAI, Koji INAI, Norihito NAKAMICHI, Hiroki ASHIDA, Akiho YOKOTA, Yusuf HENDRAWAN, Haruhiko MURASE, Hirokazu FUKUDA

    Environment control in biology   Vol. 50 ( 3 ) page: 237-246   2012.9

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  26. Molecular mechanisms underlying the Arabidopsis circadian clock Reviewed

      Vol. 52 ( 10 ) page: 1709-1718   2011.10

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  27. Phytochrome-interacting factor 4 and 5 (PIF4 and PIF5) activate the homeobox ATHB2 and auxin-inducible IAA29 genes in the coincidence mechanism underlying photoperiodic control of plant growth of Arabidopsis thaliana Reviewed

    Atsushi Kunihiro, Takafumi Yamashino, Norihito Nakamichi, Yusuke Niwa, Hanayo Nakanishi, Takeshi Mizuno

    Plant and Cell Physiology   Vol. 52 ( 8 ) page: 1315-1329   2011.8

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  28. LIGHT-REGULATED WD1 and PSEUDO-RESPONSE REGULATOR9 Form a Positive Feedback Regulatory Loop in the Arabidopsis Circadian Clock Reviewed

    Ying Wang, Jing-Fen Wu, Norihito Nakamichi, Hitoshi Sakakibara, Hong-Gil Nam, Shu-Hsing Wu

    The Plant Cell   Vol. 23   page: 486-498   2011

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    In Arabidopsis thaliana, central circadian clock genes constitute several feedback loops. These interlocking loops generate an ~24-h oscillation that enables plants to anticipate the daily diurnal environment. The identification of additional clock proteins can help dissect the complex nature of the circadian clock. Previously, LIGHT-REGULATED WD1 (LWD1) and LWD2 were identified as two clock proteins regulating circadian period length and photoperiodic flowering. Here, we systematically studied the function of LWD1/2 in the Arabidopsis circadian clock. Analysis of the lwd1 lwd2 double mutant revealed that LWD1/2 plays dual functions in the light input pathway and the regulation of the central oscillator. Promoter:luciferase fusion studies showed that activities of LWD1/2 promoters are rhythmic and depend on functional PSEUDO-RESPONSE REGULATOR9 (PRR9) and PRR7. LWD1/2 is also needed for the expression of PRR9, PRR7, and PRR5. LWD1 is preferentially localized within the nucleus and associates with promoters of PRR9, PRR5, and TOC1 in vivo. Our results support the existence of a positive feedback loop within the Arabidopsis circadian clock. Further mechanistic studies of this positive feedback loop and its regulatory effects on the other clock components will further elucidate the complex nature of the Arabidopsis circadian clock.

  29. Transcript profiling of an Arabidopsis PSEUDO RESPONSE REGULATOR arrhythmic triple mutant reveals a role for the circadian clock in cold stress response Reviewed

    Norihito Nakamichi, Miyako Kusano, Atsushi Fukushima, Masanori Kita, Shogo Ito, Takafumi Yamashino, Kazuki Saito, Hitoshi Sakakibara, Takeshi Mizuno

    Plant Cell Physiol   Vol. 50   page: 447-462   2009

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  30. A genetic study of the Arabidopsis circadian clock with reference to the TIMING OF CAB EXPRESSION 1 (TOC1) gene Reviewed

    Shogo Ito, Hideaki Kawamura, Yosuke Niwa, Norihito Nakamichi, Takafumi Yamashino, Takeshi Mizuno

    Plant Cell Physiol   Vol. 50   page: 290-303   2009

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    In Arabidopsis thaliana, a consistent multiloop clock model has been widely adopted in many recent publications. This tentative model consists of three interactive feedback loops, namely the core CCA1/LHY-TOC1/X loop, the morning CCA1/LHY-PRR9/PRR7 loop and the evening Y-TOC1 loop, in which the undefined Y gene might be GI. The model in its current form provides us with a basis on which to address a number of fundamental issues for a better understanding of the molecular mechanism by which the central oscillator generates circadian rhythms. We have been conducting a series of genetic studies through the establishment of a set of combinatorial mutants. We have already characterized a prr9 prr7 double loss-of-function mutant that has lost the morning loop, and a cca1 lhy toc1 triple mutant that lacks the core loop. Extension of this line of study required characterization of a gi toc1 double loss-of-function mutant, which is expected to have no evening loop, and a prr9 prr7 toc1 triple mutant, lacking both the morning and evening loops. Genetic analysis of both these lines is reported here. From the results, we have clarified the genetic linkages between GI and TOC1 and those between PRR9/PRR7 and TOC1 with reference to the circadian clock-associated phenotypes, including: (i) length of hypocotyls during early photomor-phogenesis; (ii) photoperiodic control of flowering time; and (iii) expression profiles of CCA1 and LHY under free-running conditions. These results indicate that GI is not sufficient to fulfill the Y role, but plays more complicated clock-associated roles and, interestingly, that no epistatic interaction between PRR9/PRR7 and TOC1 was observed. Furthermore, these clock-defective mutants could still generate robust, free-running rhythms at the level of transcription. Therefore, we speculate that an as yet undefined oscillator (or loop) continues to generate rhythms within the plants lacking GI/TOC1 or PRR9/PRR7/TOC

  31. Linkage between circadian clock and tricarboxylic acid cycle in Arabidopsis

    Norihito Nakamichi, Atsushi Fukushima, Miyako Kusano, Hitoshi Sakakibara, Takeshi Mizuno, Kazuki Saito

    Plant Signal Behav   Vol. 4   page: 660-662   2009

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    The transcriptional/translational feedback loop is thought to play a central role in the circadian clock in Arabidopsis. The loop includes close paralogs of MYB transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1, PSEUDO RESPONSE REGULATOR 1[PRR1]), PRR9, PRR7 and PRR5. The prr9 prr7 prr5 triple mutants (d975) and overexpression line of CCA1 (CCA1-ox) are arrhythmic under continuous light conditions followed by light and dark cycles. We recently demonstrated a tight link between the circadian clock and the tricarboxylic acid (TCA) cycle, from the metabolome analysis of d975. The levels of metabolites belonging to TCA cycle, such as 2-oxoglutarate, succinate, fumarate, citrate and malate increased in d975, whereas those of arginine and ornithine decreased. In this addendum, we further demonstrate that metabolism belonging to TCA cycle in CCA1-ox was partly similar to that of d975. This profiling also supported the linkage between circadian clock and metabolism associated to TCA cycle.

  32. Impact of clock-associated Arabidopsis pseudo-response regulators in metabolic coordination Reviewed

    Atsushi Fukushima, Miyako Kusano, Norihito Nakamichi, Makoto Kobayashi, Naomi Hayashi, Hitoshi Sakakibara, Takeshi Mizuno, Kazuki Saito

    Proc Natl Acad Sci U S A   Vol. 106   page: 7251-7256   2009

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    In higher plants, the circadian clock controls a wide range of cellular processes such as photosynthesis and stress responses. Understanding metabolic changes in arrhythmic plants and determining output-related function of clock genes would help in elucidating circadian-clock mechanisms underlying plant growth and development. In this work, we investigated physiological relevance of PSEUDO-RESPONSE REGULATORS (PRR 9, 7, and 5) in Arabidopsis thaliana by transcriptomic and metabolomic analyses. Metabolite profiling using gas chromatography-time-of-flight mass spectrometry demonstrated well-differentiated metabolite phenotypes of seven mutants, including two arrhythmic plants with similar morphology, a PRR 9, 7, and 5 triple mutant and a CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1)-overexpressor line. Despite different light and time conditions, the triple mutant exhibited a dramatic increase in intermediates in the tricarboxylic acid cycle. This suggests that proteins PRR 9, 7, and 5 are involved in maintaining mitochondrial homeostasis. Integrated analysis of transcriptomics and metabolomics revealed that PRR 9, 7, and 5 negatively regulate the biosynthetic pathways of chlorophyll, carotenoid and abscisic acid, and alpha-tocopherol, highlighting them as additional outputs of pseudo-response regulators. These findings indicated that mitochondrial functions are coupled with the circadian system in plants.

  33. Involvement of Arabidopsis clock-associated pseudo-response regulators in diurnal oscillations of gene expression in the presence of environmental time cues Reviewed

    Takafumi Yamashino, Shogo Ito, Yosuke Niwa, Atsushi Kunihiro, Norihito Nakamichi, Takeshi Mizuno

    Plant Cell Physiol   Vol. 49   page: 1839-1850   2008

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    In plants, the circadian clock is implicated in the biological system that generates diurnal oscillations in cellular and physiological activities. The circadian clock must be synchronized (or entrained) to local time by environmental time cues, such as light/dark and/or hot/cold cycles. In Arabidopsis thaliana, although a number of clock-associated components have been uncovered over the last decade, the clock-associated elements that are involved in entrainment to environmental time cues are largely unknown. In this regard, we have been characterizing one core group of clock components that together control the pace of the central oscillator, including PSEUDO-RESPONSE REGULATOR9 (PRR9), PRR7, PRR5 and TIMING OF CAB2 EXPRESSION 1 (TOC1; or PRR1). The primary aim of this study is to clarify whether these PRR members are implicated in entrainment of the circadian clock to environmental time cues. For this purpose, the diurnal oscillation profiles of clock-controlled genes in the presence of environmental time cues were determined in a set of prr mutants, including a prr9 prr7 prr5 toc1 quadruple mutant. As an extreme phenotype, the prr9-10 prr7-11 prr5-11 toc1-2 quadruple mutant showed an arrhythmia phenotype even under light/dark and hot/cold cycles. In contrast, a cca1-1 lhy-11 toc1-2 triple mutant maintained robust oscillations in the presence of these environmental time cues, although their phases were markedly affected. Based on these results, we propose that the clock components PRR9, PRR7 and PRR5 together might represent elements necessary for the circadian clock to entrain properly to local time in response to light/dark and hot/cold cycles in natural habitats.

  34. Insight into missing genetic links between two evening-expressed pseudo-response regulator genes TOC1 and PRR5 in the circadian clock-controlled circuitry in Arabidopsis thaliana Reviewed

    Shogo Ito, Yosuke Niwa, Norihito Nakamichi, Hideaki Kawamura, Takafumi Yamashino, Takeshi Mizuno

    Plant Cell Physiol   Vol. 49   page: 201-213   2008

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    In Arabidopsis thaliana, many circadian clock-associated genes have been identified. Among them, the evening-expressed TOC1 (TIMING OF CAB EXPRESSION 1) gene plays a role by forming a transcriptional feedback core loop together with the morning-expressed CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) gene and its homologous LHY (LATE ELONGATED HYPOCOTYL) gene. TOC1 encodes a member of the PSEUDO-RESPONSE REGULATOR (PRR) family, including PRR9, PRR7, PRR5, PRR3,and PRR1/TOC1. The PRR genes other than TOC1 (or PRR1) also appear to be crucial for certain circadian-associated events. To clarify missing genetic linkages amongst these PRR genes, here we constructed a toc1 prr5 double knockdown mutant. In free-running circadian rhythms, the resulting toc1-2 prr5-11 mutant plants showed an extremely short period and reduced amplitude phenotype, which was more severe than that of the toc1-2 single mutant plant, suggesting a non-linear genetic interaction between TOC1 and PRR5. Surprisingly, the hallmark early flowering phenotype of toc1-2 in the short-day conditions had been converted to a markedly late flowering phenotype in the long-day conditions, when combined with the prr5-11 allele, which itself showed a subtle flowering phenotype. This unexpected genetic result (i.e. phenotypic sign conversion) suggested that the TOC1 and PRR5 genes are coordinately implicated in a non-linear and closed genetic circuitry. In the toc1-2 prr5-11 double mutant, the diurnal expression profile of CDF1 (CYCLING DOF FACTOR 1) was markedly de-repressed in the evening in the long-day conditions. These and other results of this study led us to propose the novel view that TOC1 might play bipartite roles in the control of flowering time within a closed circuitry; the one is a GI (GIGANTEA)-dependent negative role through CCA1/LHY, and the other is a CDF1-dependent positive role through cooperating closely with PRR5.

  35. The function of the clock-associated transcriptional regulator CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) in Arabidopsis thaliana Reviewed

    Masakazu Kawamura, Shogo Ito, Nakamichi Nakamichi, Takafumi Yamashino, Takeshi Mizuno

    Biosci Biotechnol Biochem   Vol. 72   page: 1307-1316   2008

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    In the model higher plant Arabidopsis thaliana, the CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) gene plays important circadian clock-associated roles. The CCA1 protein is a member of a small subfamily of single MYB-related transcription factors. This family consists of several homologous CCA1-like transcription factors, including the closest homolog LHY (LATE ELONGATED HYPOCOTYL). To gain insight into the molecular function of CCA1 and its homologs, here we took a unique genetic approach that was recently developed for Arabidopsis thaliana. Through this strategy, referred to as CRES-T (Chimeric REpressor Silencing Technology), a transgenic plant was constructed to produce a dominant negative transcriptional repressor (designated CCA1-SRDX). By employing the resulting transgenic lines, together with previously established cca1 lhy double mutant and CCA1-ox (over-expressing) plants, their circadian clock-associated phenotypes were examined and compared with each other. The observed clock-associated phenotypes of the CCA1-SRDX plants were very similar to those of CCA1-ox, but not to those of cca1 lhy, suggesting that CCA1 acts predominantly as a transcriptional repressor in nature. However, the developmental morphology (or architecture) of adult CCA1-SRDX plants were quite different from that of CCA1-ox, suggesting that CCA1 might also be implicated, directly or indirectly, in an as yet unknown circadian-associated output pathway at a late developmental stage.

  36. The common function of a novel subfamily of B-Box zinc finger proteins with reference to circadian-associated events in Arabidopsis thaliana Reviewed

    Takeshi Kumagai, Shogo Ito, Norihito Nakamichi, Yosuke Niwa, Masaya Murakami, Takafumi Yamashino, Takeshi Mizuno

    Biosci Biotechnol Biochem   Vol. 72   page: 1539-1549   2008

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    Over 1,600 genes encoding putative transcription factors have been identified in the Arabidopsis genome sequence, however, their physiological functions are not yet fully understood. In this study, a small subfamily of double B-box zinc finger (DBB, DOUBLE B-BOX) genes, encoding eight putative transcription factors, were characterized with reference to the circadian rhythm and the early photomorphogenic regulation of hypocotyl elongation in response to light signals. Among these, it was found that the transcriptions of five DBB genes were under the control of circadian rhythm. To gain insight into the physiological roles of these putative transcription factors, forward and reverse genetic studies were carried out. The results suggested that they are commonly implicated in light signal transduction during early photomorphogenesis, however, their functions are not totally redundant, as judged by the fact that their circadian-expression profiles (or phases) were distinctive from each other, and by the fact that some DBBs (named DBB1a, DBB1b, STO, and STH) were apparently implicated in light signal transduction in a negative manner, whereas another (named DBB3) was implicated in a positive manner with regard to light-induced inhibition of elongation of hypocotyls. We also found that homologous B-box zinc finger genes are widely conserved in higher plants (e.g., Oryza sativa). Taking this altogether, it is probable that in addition to previously characterized bZIP-type transcription factors (e.g., HY5 and HYH) and bHLH-type transcription factors (e.g., PIF4 and PIF5/PIL6), a set of B-box zinc finger transcription factors should also be taken into consideration for a better understanding of the complex molecular mechanisms underlying the early photomorphogenic development of Arabidopsis thaliana.

  37. Characterization of genetic links between two clock-associated genes, GI and PRR5 in the current clock model of Arabidopsis thaliana Reviewed

    Hideaki Kawamura, Shogo Ito, Takafumi Yamashino, Yosuke Niwa, Norihito Nakamichi, Takeshi Mizuno

    Biosci Biotechnol Biochem   Vol. 72   page: 2770-2774   2008

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    The GI (GIGANTEA) and PRR5 (PSEUDO-RESPONSE REGULATOR 5) genes are crucially implicated in the Arabidopsis circadian clock. We characterized a gi prr5 double loss-of-function mutant for the first time with reference to circadian clock-associated phenotypes. The results of this study revealed the genetic linkages between GI and PRR5 in the control of free running circadian oscillation of gene expression, early photomorphogenesis and flowering time. A mathematical clock model consisting of three interactive transcriptional feedback loops has recently been proposed. The model includes the hypothetical evening Y-TOC1 feedback loop, in which "Y" is suspected to be GI and/or PRR5. This issue was also addressed in this study; perhaps GI and PRR5 are not sufficient to fulfill the Y role.

  38. Synchronization of Plant Circadian Oscillators with a Phase Delay Effect of Vein Network Reviewed

    Hirokazu Fukuda, Norihito Nakamichi, Mihoe Hisatsune, Haruhiko Murase, Takeshi Mizuno

    Physical Review Letters   Vol. 99   page: 098102   2007

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    Synchronization phenomena in coupled circadian oscillators of plant leaves were investigated experimentally using bioluminescence technology for a clock gene. Analyzing the phase of circadian oscillation, the phase-wave propagations and the phase delay caused by the vein network were observed. We describe these phase dynamics using a two-layer model with coupled Stuart-Landau equations. Global synchronization of circadian oscillators in the leaf is also investigated.

  39. Characterization of Circadian-associated pseudo-response regulators: I. Comparative studies on a series of transgenic lines misexpressing five distinctive PRR Genes in Arabidopsis thaliana Reviewed

    Akinori Matsushika, Masaya Murakami, Shogo Ito, Norihito Nakamichi, Takafumi Yamashino, Takeshi Mizuno

    Biosci Biotechnol Biochem   Vol. 71   page: 527-534   2007

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    Every member of a small family of Pseudo-Response Regulator (PRR) genes, including Timing of Cab Expression 1 (TOC1 [or PRR1]), are believed to play roles close to the circadian clock in the model higher plant Arabidopsis thaliana. In this study we established a transgenic line that misexpresses (or overexpresses) the PRR7 gene. As compared with wild-type plants, the resulting PRR7-misexpressing plants (designated PRR7-ox) showed characteristic phenotypes as to hallmarked circadian-associated biological events: (i) early flowering in a manner independent of photoperiodicity, (ii) hypersensitive response to red light during early photomorphogenesis, and (iii) altered free-running rhythms with long period of clock-associated genes. Finally, a series of all transgenic lines (PRR1-ox, PRR3-ox, PRR5-ox, PRR7-ox, and PRR9-ox) were characterized comparatively with regard to their clock-associated roles. The results suggested that the five homologous PRR factors play coordinate roles, distinctively from one another, and closely to the circadian clock in higher plants.

  40. Mutants of circadian-associated PRR genes display a novel and visible phenotype as to light responses during de-etiolation of Arabidopsis thaliana seedlings Reviewed

    Takahiko Kato, Masaya Murakami, Yuko Nakamura, Shogo Ito, Norihito Nakamichi, Takafumi Yamashino, Takeshi Mizuno

    Biosci Biotechnol Biochem   Vol. 71   page: 834-839   2007

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    In Arabidopsis thaliana, it is currently accepted that certain mutants with lesions in clock-associated genes commonly display hallmarked phenotypes with regard to three characteristic biological events: (i) altered rhythmic expression of circadian-controlled genes, (ii) changes in flowering time, and (iii) altered sensitivity to red light in elongation of hypocotyls. During the course of examination of the clock-associated mutants of PSEUDO-RESPONSE REGULATORS, PRRs, including TOC1 (PRR1), we found that they commonly show another visible phenotype of anomalous greening responses upon the onset to light exposure of etiolated seedlings. These findings are indicative of a novel link between circadian rhythms and chloroplast development.

  41. Arabidopsis Clock-Associated Pseudo-Response Regulators PRR9, PRR7 and PRR5 Coordinately and Positively Regulate Flowering Time through the Canonical CONSTANS-Dependent Photoperiodic Pathway Reviewed

    Norihito Nakamichi, Masanori Kita, Kanae Niinuma, Shogo Ito, Takafumi Yamashino, Tsuyoshi Mizuguchi, Takeshi Mizuno

    Plant Cell Physiol   Vol. 48   page: 822-832   2007

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    Photoperiodism allows organisms to measure daylength, or external photoperiod, and to anticipate coming seasons. Daylength measurement requires the integration of light signal and temporal information by the circadian clock. In the long-day plant Arabidopsis thaliana, CONSTANS (CO) plays a crucial role in integrating the circadian rhythm and environmental light signals into the photoperiodic flowering pathway. Nevertheless, the molecular mechanism by which the circadian clock modulates the cyclic expression profile of CO is poorly understood. Here, we first showed that the clock-associated genes PSEUDO-RESPONSE REGULATOR (PRR) PRR9, PRR7 and PRR5 are involved in activation of CO expression during the daytime. Then, extensive genetic studies using CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) double mutants (cca1/lhy) and prr7/prr5 were conducted. The results suggested that PRR genes act coordinately in a manner parallel with and antagonistic to CCA/LHY, upstream of the canonical CO-FLOWERING LOCUS T (FT) photoperiodic flowering pathway. Finally, we provided evidence to propose a model, in which CCA1/LHY repress CO through GIGANTEA (GI), while PRR9, PRR7 and PRR5 activate CO predominantly by repressing CYCLING DOF FACTOR1 (CDF1) encoding a DNA-binding transcriptional repressor.

  42. Genetic Linkages between Circadian Clock-Associated Components and Phytochrome-Dependent Red Light-Signal Transduction in Arabidopsis thaliana Reviewed

    Shogo Ito, Norihito Nakamichi, Yuko Nakamura, Yosuke Niwa, Takahuko Kato, Masaya Murakami, Masanori Kita, Tsuyoshi Mizoguchi, Kanae Niinuma, Takafumi Yamashino, Takeshi Mizuno

    Plant Cell Physiol   Vol. 48   page: 971-983   2007

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    The current best candidates for Arabidopsis thaliana clock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and its homolog LHY (LATE ELONGATED HYPOCOTYL). In addition, five members of a small family, PSEUDO-RESPONSE REGULATORS (including PRR1, PRR3, PRR5, PRR7 and PRR9), are believed to be another type of clock component. The originally described member of PRRs is TOC1 (or PRR1) (TIMING OF CAB EXPRESSION 1). Interestingly, seedlings of A. thaliana carrying a certain lesion (i.e. loss-of-function or misexpression) of a given clock-associated gene commonly display a characteristic phenotype of light response during early photomorphogenesis. For instance, cca1 lhy double mutant seedlings show a shorter hypocotyl length than the wild type under a given fluence rate of red light (i.e. hypersensitivity to red light). In contrast, both toc1 single and prr7 prr5 double mutant seedlings with longer hypocotyls are hyposensitive under the same conditions. These phenotypes are indicative of linkage between the circadian clock and red light signal transduction mechanisms. Here this issue was addressed by conducting combinatorial genetic and epistasis analyses with a large number of mutants and transgenic lines carrying lesions in clock-associated genes, including a cca1 lhy toc1 triple mutant and a cca1 lhy prr7 prr5 quadruple mutant. Taking these results together, we propose a genetic model for clock-associated red light signaling, in which CCA1 and LHY function upstream of TOC1 (PRR1) in a negative manner, in turn, TOC1 (PRR1) serves as a positive regulator. PRR7 and PRR5 also act as positive regulators, but independently from TOC1 (PRR1). It is further suggested that these signaling pathways are coordinately integrated into the phytochrome-mediated red light signal transduction pathway, in which PIF3 (PHYTOCHROME-INTERACTING FACTOR 3) functions as a negative regulator immediately downstream of phyB.

  43. Genetic linkages of the circadian clock-associated genes, TOC1, CCA1 and LHY, in the photoperiodic control of flowering time in Arabidopsis thaliana Reviewed

    Yosuke Niwa, Shogo Ito, Norihito Nakamichi, Tsuyoshi Mizoguchi, Kanae Niinuma, Takafumi Yamashino, Takeshi Mizuno

    Plant Cell Physiol   Vol. 48   page: 925-937   2007

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    In Arabidopsis thaliana, the flowering time is regulated through the circadian clock that measures day-length and modulates the photoperiodic CO-FT output pathway in accordance with the external coincidence model. Nevertheless, the genetic linkages between the major clock-associated TOC1, CCA1 and LHY genes and the canonical CO-FT flowering pathway are less clear. By employing a set of mutants including an extremely early flowering toc1 cca1 lhy triple mutant, here we showed that CCA1 and LHY act redundantly as negative regulators of the photoperiodic flowering pathway. The partly redundant CCA1/LHY functions are largely, but not absolutely, dependent on the upstream TOC1 gene that serves as an activator. The results of examination with reference to the expression profiles of CO and FT in the mutants indicated that this clock circuitry is indeed linked to the CO-FT output pathway, if not exclusively. For this linkage, the phase control of certain flowering-associated genes, GI, CDF1 and FKF1, appears to be crucial. Furthermore, the genetic linkage between TOC1 and CCA1/LHY is compatible with the negative and positive feedback loop, which is currently believed to be a core of the circadian clock. The results of this study suggested that the circadian clock might open an exit for a photoperiodic output pathway during the daytime. In the context of the current clock model, these results will be discussed in connection with the previous finding that the same clock might open an exit for the early photomorphogenic output pathway during the night-time.

  44. Rhythmic and light-inducible appearance of clock-associated pseudo-response regulator protein PRR9 through programmed degradation in the dark in Arabidopsis thaliana Reviewed

    Shogo Ito, Norihito Nakamichi, Takatoshi Kiba, Takafumi Yamashino, Takeshi Mizuno

    Plant Cell Physiol   Vol. 48   page: 1644-1651   2007

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    In Arabidopsis thaliana, it is currently believed that the members of a small family of PSEUDO-RESPONSE REGULATOR (PRR) proteins, including TOC1 (TIMING OF CAB EXPRESSION 1), coordinately play roles close to the circadian clock. Among these PRR members, the PRR9 gene is unique in that not only does its transcription oscillate diurnally, but it is also rapidly induced by light in a manner dependent on phytochromes. These events at the level of transcription must be crucial for the clock-associated functions of PRR9. Nonetheless, little is known about the expression of the PRR9 protein product itself in plant cells. Here, we show that PRR9 polypeptides themselves oscillate diurnally, and that they accumulate rapidly in response to light. Our work further suggests that the presence of PRR9 polypeptides is controlled through proteasome-mediated programmed degradation in the dark.

  45. Psuedo-Pesponse Regulators(PRRs) or True Oscillator Components(TOCs) Reviewed

    Takeshi Mizuno, Norihito Nakamichi

    Plant Cell Physiol   Vol. 46   page: 677-685   2005

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    In Arabidopsis thaliana, AUTHENTIC RESPONSE REGULATORS (ARRs) act as downstream components of the His-to-Asp phosphorelay (two-component) signaling pathway that is propagated primarily by the cytokinin receptor kinases, AUTHENTIC HIS-KINASES (AHK2, AHK3 and AHK4/CRE1). Thus, this bacterial type of signaling system is essential for responses to a class of hormones in plants. Interestingly, this higher plant has also evolved its own atypical (or unique) variants of two-component signal transducers, PSEUDO-RESPONSE REGULATORS (PRRs). Several lines of recent results suggest that the functions of PRRs are closely relevant to the plant clock (oscillator) that is central to circadian rhythms, the underlying mechanisms of which have long been the subject of debate. Through an overview of recent results, the main issue addressed here is whether or not the pseudo-response regulators (PRRs) are true oscillator components (TOCs).

  46. Three Psudo-Resoponse Regulators, PRR9, PRR7, and PRR5 Together Play an Essential Role Close to the Circadian Clock of Alabidopsis thaliana Reviewed

    Norihito Nakamichi, Masanori Kita, Shogo Ito, Takafumi Yamashino, Takeshi Mizuno

    Plant Cell Physiol   Vol. 46   page: 686-698   2005

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    In Arabidopsis thaliana, a number of clock-associated protein components have been identified. Among them, CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1)/LHY (LATE ELONGATED HYPOCOTYL) and TOC1 (TIMING OF CAB EXPRESSION 1) are believed to be the essential components of the central oscillator. CCA1 and LHY are homologous and partially redundant Myb-related DNA-binding proteins, whereas TOC1 is a member of a small family of proteins, designated as PSEUDO-RESPONSE REGULATOR. It is also believed that these two different types of clock components form an autoregulatory positive/negative feedback loop at the levels of transcription/translation that generates intrinsic rhythms. Nonetheless, it was not yet certain whether or not other PRR family members (PRR9, PRR7, PRR5 and PRR3) are implicated in clock function per se. Employing a set of prr9, prr7 and prr5 mutant alleles, here we established all possible single, double and triple prr mutants. They were examined extensively by comparing them with each other with regard to their phenotypes of circadian rhythms, photoperiodicity-dependent control of flowering time and photomorphogenic responses to red light during de-etiolation. Notably, the prr9 prr7 prr5 triple lesions in plants resulted in severe phenotypes: (i) arrhythmia in the continuous light conditions, and an anomalous phasing of diurnal oscillation of certain circadian-controlled genes even in the entrained light/dark cycle conditions; (ii) late flowering that was no longer sensitive to the photoperiodicity; and (iii) hyposensitivity (or blind) to red light in the photomorphogenic responses. The phenotypes of the single and double mutants were also characterized extensively, showing that they exhibited circadian-associated phenotypes characteristic for each. These results are discussed from the viewpoint that PRR9/PRR7/PRR5 together act as period-controlling factors, and they play overlapping and distinctive roles close to (or within) the central oscillator in which the relative, PRR1/TOC1, plays an essential role.

  47. The Arabidopsis Pseudo-Response Regulators, PRR5 and PRR7, Coordinately Play Essential Roles for Circadian Clock Reviewed

    Norihito Nakamichi, Masanori Kita, Shogo Ito, Eriko Sato, Takafumi Yamashino, Takeshi Mizuno

    Plant Cell Physiol   Vol. 46   page: 609-619   2005

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    In Arabidopsis thaliana, a number of clock-associated protein factors have been identified. Among them, TOC1 (TIMING OF CAB EXPRESSION 1) is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATOR, including PRR1/TOC1, PRR3, PRR5, PRR7 and PRR9. It has not been certain whether or not other PRR family members are also implicated in clock function per se. To clarify this problem, here we constructed a double mutant line, which is assumed to have severe lesions in both the PRR5 and PRR7 genes. Resulting homozygous prr5-11 prr7-11 young seedlings showed a marked phenotype of hyposensitivity to red light during de-etiolation. In addition, they displayed a phenotype of extremely late flowering under long-day photoperiod conditions, but not short-day conditions. The rhythms at the level of transcription of certain clock-controlled genes were severely perturbed in the double mutant plants when they were released into continuous light (LL) and darkness (DD). The observed phenotype was best interpreted as 'arrhythmic in both LL and DD' and/or 'very short period with markedly reduced amplitude'. Even under the light entrainment (LD) conditions, the mutant plants showed anomalous diurnal oscillation profiles with altered amplitude and/or phase with regard to certain clock-controlled genes, including the clock component CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) gene. Such events were observed even under temperature entrainment conditions, suggesting that the prr5-11 prr7-11 lesions cannot simply be attributed to a defect in the light signal input pathway. These pleiotropic circadian-associated phenotypes of the double mutant were very remarkable, as compared with those observed previously for each single mutant. Taking these results together, we propose for the first time that PRR5 and PRR7 coordinately (or synergistically) play essential clock-associated roles close to the central oscillator.

  48. Molecular dissection of the promoter of the light-induced and circadian-controlled APRR9 gene encoding a clock associated component of Arabidopsis thaliana Reviewed

    Shogo Ito, Norihito Nakamichi, Akinori Matsushika, Toru Fujimori, Takafumi Yamashino, Takeshi Mizuno

    Biosci.Biotechnol.Biochem   Vol. 69   page: 382-390   2005

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    In the model higher plant Arabidopsis thaliana, a number of circadian clock-associated protein components have recently been identified. Among them, a small family of ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9) is interesting because the most probable clock component TIMING OF CAB EXPRESSION 1 (TOC1) belongs to this family. Several lines of evidence have already been accumulated to support the view that not only APRR1/TOC1 but also other APRR family members are crucial for a better understanding of the molecular link between circadian rhythm and light-signal transduction. Among the APRR1/TOC1 family members, the circadian-controlled APRR9 gene is unique in that its expression is rapidly induced by light at the level of transcription. In this study we dissected the regulatory cis-elements of the light-induced and/or circadian-controlled APRR9 promoter by employing not only a mutant plant carrying a T-DNA insertion in the APRR9 promoter, but also a series of APRR9-promoter::LUC (luciferase) reporters that were introduced into an Arabidopsis cultured cell line (T87 cells). Taking the results of these approaches together, we provide several lines of evidence that the APRR9 promoter contains at least two distinctive and separable regulatory cis-elements: an "L element" responsible for the light-induced expression, followed by an "R element" necessary for the fundamental rhythmic expression of APRR9. Furthermore, APRR1/TOC1 was implicated in the L-element-mediated light response of APRR9, directly or indirectly.

  49. Rapid response of Arabidopsis T87 cultured cells to cytokinin through His-to-Asp phosphorelay signal transduction Reviewed

    Hisami Yamada, Nozomu Koizumi, Norihito Nakamichi, Takatoshi Kiba, Takafumi Yamashino, Takeshi Mizuno

    Biosci Biotechnol Biochem   Vol. 68   page: 1966-1976   2004

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    According to the current consistent model for the higher plant Arabidopsis thaliana, the scheme for an immediate early response to the plant hormone cytokinin can be formulated as Arabidopsis histidine kinase (AHK) cytokinin receptor-mediated His --> Asp phosphorelay signal transduction. Nonetheless, clarification of the comprehensive picture of cytokinin-mediated signal transduction in this higher plant is at a very early stage. As a new approach to this end, we studied whether or not a certain Arabidopsis cell line (named T87) would be versatile for such work on cytokinin signal transduction. We show that T87 cells had the ability to respond to cytokinin, displaying the immediate early induction of type-A Arabidopsis response regulator (ARR) family genes (e.g., ARR6) at the transcriptional level. This event was further confirmed by employing the stable transgenic lines of T87 cells with a set of ARR::LUC reporter transgenes. We also show that T87 cells had the ability to respond to auxin when the expression of a set of AUX/IAA genes (e.g., IAA5) was examined. As postulated for intact plants, in T87 cells too, the induction of IAA5 by auxin was selectively inhibited in the presence of a proteasome inhibitor, while the induction of ARR6 by cytokinin was not significantly affected under the same conditions. Through transient expression assays with T87 protoplasts, it is shown that the intracellular localization profiles of the phosphorelay intermediate Arabidopsis histidine-containing phosphotransfer factor (AHPs; e.g., AHP1 and AHP4) were markedly affected in response to cytokinin, but those of type-A ARRs were not (e.g., ARR15 and ARR16). Taken together, we conclude that, in T87 cells, the AHK-dependent His --> Asp phosphorelay circuitry appears to be propagated in response to cytokinin, as in the case of plants, as far as the immediate early responses were concerned. This cultured cell system might therefore provide us with an alternative means to further characterize the mechanisms underlying cytokinin (and also auxin) responses at the molecular level.

  50. Characterization of Plant Circadian Rhythms by Employing Arabidopsis Cultured Cells with Bioluminescence Reporters Reviewed

    Norihito Nakamichi, Shogo Ito, Tokitaka Oyama, Takafumi Yamashino, Takao Kondo, Takeshi Mizuno

    Plant Cell Physiol   Vol. 45   page: 57-67   2004

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    Recent intensive studies have begun to shed light on the molecular mechanisms underlying the plant circadian clock in Arabidopsis thaliana. During the course of these previous studies, the most powerful technique, elegantly adopted, was a real-time bioluminescence monitoring system of circadian rhythms in intact plants carrying a luciferase (LUC) fusion transgene. We previously demonstrated that Arabidopsis cultured cells also retain an ability to generate circadian rhythms, at least partly. To further improve the cultured cell system for studies on circadian rhythms, here we adopted a bioluminescence monitoring system by establishing the cell lines carrying appropriate reporter genes, namely, CCA1::LUC and APRR1::LUC, with which CCA1 (CIRCADIAN CLOCK-ASSOCIATED1) and APRR1 (or TOC1) (ARABIDOPSIS PSEUDO-RESPONSE REGULATORS1 or TIMING OF CAB EXPRESSION1) are believed to be the components of the central oscillator. We report the results that consistently supported the view that the established cell lines, equipped with such bioluminescence reporters, might provide us with an advantageous means to characterize the plant circadian clock.

  51. Comparative Genetic Studies on the APRR5 and APRR7 Genes Belonging to the APRR1/TOC1 Quintet Implicated in Circadian Rhythm, Control of Flowering, and Early Photomorphogenesis Reviewed

    Yoko Yamamoto, Eriko Sato, Tomo Shimizu, Norihito Nakamichi, Shusei Sato, Tomohiko Kato, Satoshi Tabata, Akira Nagatani, Takafumi Yamashino, Takeshi Mizuno

    Plant Cell Physiol   Vol. 44   page: 1119-1130   2003

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    In Arabidopsis thaliana, a number of circadian-associated factors have been identified. Among those, TOC1 (TIMING OF CAB EXPRESSION 1) is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as Arabidopsis PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9). Nonetheless, it is not very clear whether or not the APRR family members other than APRR1/TOC1 are also implicated in the mechanisms underlying the circadian rhythm. To address this issue further, here we characterized a set of T-DNA insertion mutants, each of which is assumed to have a severe lesion in each one of the quintet genes (i.e. APRR5 and APRR7). For each of these mutants (aprr5-11 and aprr7-11) we demonstrate that a given mutation singly, if not directly, affects the circadian-associated biological events simultaneously: (i) flowering time in the long-day photoperiod conditions, (ii) red light sensitivity of seedlings during the early photomorphogenesis, and (iii) the period of free-running rhythms of certain clock-controlled genes including CCA1 and APRR1/TOC1 in constant white light. These results suggest that, although the quintet members other than APRR1/TOC1 may not be directly integrated into the framework of the central oscillator, they are crucial for a better understanding of the molecular mechanisms underlying the Arabidopsis circadian clock.

  52. Characterization of the Prr1 response regulator with special reference to sexual development in Schizosaccharomyces pombe Reviewed

    Norihito Nakamichi, Hisami Yanada, Hirofumi Aiba, Keisuke Aoyama, Ryosuke Ohmiya, Takeshi Mizuno

    Biosci Biotechnol Biochem   Vol. 67   page: 547-555   2003

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    The histidine (His)-to-Aspartate (Asp) phosphorelay is a paradigm of intracellular signaling systems through protein phosphorylation in both prokaryotes and eukaryotes. The fission yeast Schizosaccharomyces pombe has three histidine kinases (Phk1/Mak2, Phk2/Mak3, and Phk3/Mak1), together with two response regulators (Mcs4 and Prr1). The results of recent extensive studies suggested that these His-to-Asp phosphorelay components are involved in oxidative stress responses through the transcriptional regulation of several scavenger genes for toxic free radicals. It was also suggested that they were somehow implicated in control of both the mitotic and meiotic cell proliferations. Among these S. pombe His-to-Asp phosphorelay components, however, the function of Prr1 is less clear. We here characterized a mutant, named prr1-D418N, specifying an altered Prr1 protein that presumably acts as a gain-of-function (or constitutive-active) mutant, with special reference to sexual development. The mutant cells showed a striking phenotype in that they underwent mating even in a nitrogen-sufficient medium, under which conditions the wild-type cells hardly did so. Furthermore, the mutant cells underwent mating very rapidly in a nitrogen-deficient medium. Under anaerobic (or micro-aerobic) growth conditions, the wild-type cells were not capable of undergoing sexual development even in a nitrogen-deficient medium. The prr1-D418N cells underwent mating efficiently under such anaerobic growth conditions. Taken these together, it was suggested that the function of Prr1 is closely linked to the well-characterized signaling pathways for induction of the sexual development, in a way that this response regulator regulates a critical step of the initiation of meiosis through activating the transcription of ste11+, mam2+, and mei2+, in S. pombe.

  53. Cell autonomous circadian waves of the APRR1/TOC1 quintet in an established cell line of Arabidopsis thaliana Reviewed

    Norihito Nakamichi, Akinori Matsushika, Takafumi Yamashino, Takeshi Mizuno

    Plant Cell Physiol   Vol. 44   page: 360-365   2003

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    A small family of genes, named Arabidopsis Pseudo Response Regulator (APRR), are intriguing with special reference to circadian rhythms in plants, based on the fact that one of the members (APRR1) is identical to TOC1 (Timing of CAB Expression 1) that is believed to encode a clock component. In Arabidopsis plants, each transcript of the APRR1/TOC1 quintet genes starts accumulating after dawn rhythmically and one after another at intervals in the order of APRR9 --> APRR7 --> APRR5 --> APRR3 --> APRR1/TOC1. To characterize such intriguing circadian-associated events, we employed an established Arabidopsis cell line (named T87). When T87 cells were grown in an appropriate light and dark cycle, cell autonomous diurnal oscillations of the APRR1/TOC1 quintet genes were observed at the level of transcription, as seen in intact plants. After transfer to the conditions without any environmental time cues, particularly in constant dark, we further showed that free-running circadian rhythms persisted in the cultured cells, not only for the APRR1/TOC1 quintet genes, but also other typical circadian-controlled genes including CCA1 (Circadian Clock Associated 1), LHY (Late Elongated Hypocotyl) and CCR2 (Cold Circadian Rhythm RNA Binding 2). To our knowledge, this is the first indication of cell autonomous circadian rhythms in cultured cells in Arabidopsis thaliana, which will provide us with an alternative and advantageous means to characterize the plant biological clock.

  54. The APRR3 component of the clock-associated APRR1/TOC1 quintet is phosphorylated by a novel protein kinase belonging to the WNK family, the gene for which is also transcribed rhythmically in Arabidopsis thaliana Reviewed

    Masaya Murakami-Kojima, Norihito Nakamichi, Takafumi Yamashino, Takeshi Mizuno

    Plant Cell Physiol   Vol. 43   page: 675-683   2002

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    In higher plants, clock-controlled circadian rhythms are a longstanding issue in physiology, and a newly emerging paradigm of molecular biology. In the model higher plant Arabidopsis thaliana, several genes have been proposed to encode potential clock-associated components, including a member (APRR1/TOC1) of the pseudo-response regulator family. We previously showed that transcripts of the APRR1/TOC1 family start accumulating after dawn rhythmically and sequentially at approximately 2 h intervals in the order of APRR9-->APRR7-->APRR5-->APRR3-->APRR1/ TOC1. This and other results led us to propose that this APRR1/TOC1 quintet might play coordinate roles in the mechanism underlying circadian rhythms in higher plants. To gain further insight as to such an idea, we here attempt to identify proteins that interact with one of the quintet members, APRR3. The identified component is a novel protein kinase, named WNK1, which is considerably similar to, but clearly distinct from, mitogen-activated protein kinases (MAPKs). It was found that APRR3 is a substrate of this novel protein kinase, the gene for which also shows a rhythmic transcription profile that is well coincident with the APRR3 rhythm. These findings give new insight into the mechanisms underlying the circadian rhythm in A. thaliana, providing a molecular link between the putative clock component, APRR3, and WNK1, a novel protein kinase that might be implicated as a signal transducer.

  55. His-to-Asp Phosphorelay Circuitry for Regulation of Sexual Development in Schizosaccharomyces pombe Reviewed

    Norihito Nakamichi, Hisami Yamada, Ryusuke Ohmiya, Keisuke Aoyama, Hirofumi Aiba, Takeshi Mizuno

    Biosci.Biotechnol.Biochem   Vol. 66   page: 2663-2672   2002

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    The fission yeast Schizosaccharomyces pombe has three histidine kinases (Phk1/Mak2, Phk2/Mak3, and Phk3/Mak1), and two response regulators (Mcs4 and Prr1). The results of recent extensive studies on the S. pombe His-to-Asp phosphorelay circuitry suggested that it is involved in oxidative stress responses through the transcriptional regulation of several scavenger genes for toxic free radicals. The functions of these histidine kinases have not yet been fully characterized. Here we characterize a homothallic (h90) mutant lacking the genes for all the histidine kinases, with special reference to sexual development. Homothallic phk1/2/3delta cells underwent mating precociously in a nitrogen-deficient medium. Surprisingly, the mutant cells underwent mating even in a nitrogen-sufficient medium, under which conditions wild-type cells did so rarely if at all. Under anaerobic (or microaerobic) growth conditions, wild-type cells did not undergo sexual development even in a nitrogen-deficient medium, but the homothallic phk1/2/3delta cells mated efficiently. Oxidative reagents such as H2O2 induced sexual development in wild-type cells grown anaerobically. On the basis of these results, we propose the novel view that the S. pombe His-to-Asp phosphorelay, initiated by the Phk histidine kinases, is crucial for regulation of sexual development. This Phk-mediated signaling pathway is linked to the well-documented canonical pathway for induction of the sexual development, in that both converge at the initiation of meiosis through activation of ste11+, mam2+, and mei2+ transcription.

  56. Compilation and characterization of a novel WNK family of protein kinases in Arabiodpsis thaliana with reference to circadian rhythms Reviewed

    Norihito Nakamichi, Masaya Murakami-Kojima, Eriko Sato, Yasuko Kishi, Takafumi Yamashino, Takeshi Mizuno

    Biosci Biotechnol Biochem   Vol. 66   page: 2429-2436   2002

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    The complete genome sequence of Arabidopsis thaliana revealed that this higher plant has a tremendous number of protein kinases. We recently isolated a novel type of protein kinase, named AtWNK1, which shows an in vitro ability to phosphorylate the APRR3 member of the APRR1/TOC1 quintet that has been implicated in a mechanism underlying circadian rhythms in Arabidopsis. We here address two issues, one general and one specific, as to this novel protein kinase. We first asked the general question of how many WNK family members are present in this higher plant, then whether or not other members are also relevant to circadian rhythms. The results of our analyses showed that Arabidopsis has at least 9 members of the WNK1 family of protein kinases (designated here as WNK1 to WNK9), the structural design of which is clearly distinct from those of other known protein kinases, such as receptor-like kinases and mitogen-activated protein kinases. They were examined with special reference to the circadian-related APRR1/TOC1 quintet. It was found that not only the transcription of the WNK1 gene, but also those of three other members (WNK2, WNK4, and WNK6) are under the control of circadian rhythms. These results suggested that certain members of the WNK family of protein kinases might play roles in a mechanism that generates circadian rhythms in Arabidopsis.

  57. Aberrant Expression of the Arabidopsis Circadian-Regulated APRR5 Gene Belonging to the APRR1/TOC1 Quintet Result in Early Flowering and Hypersensitive to Light in Early Photomorphogenesis Reviewed

    Eriko Sato, Norihito Nakamichi, Takafumi Yamashino, Takeshi Mizuno

    Plant Cell Physiol   Vol. 43   page: 1374-1385   2002

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    In Arabidopsis thaliana, the transcripts of the APRR1/TOC1 family genes each start accumulating after dawn rhythmically and one after another at intervals in the order of APRR9-->APRR7-->APRR5-->APRR3-->APRR1/TOC1 under continuous light. Except for the well-characterized APRR1/TOC1, however, no evidence has been provided that other APRR1/TOC1 family genes are indeed implicated in the mechanisms underlying circadian rhythms. We here attempted to provide such evidence by characterizing transgenic plants that constitutively express the APRR5 gene. The resulting APRR5-overexpressing (APRR5-ox) plants showed intriguing properties with regard to not only circadian rhythms, but also control of flowering time and light response. First, the aberrant expression of APRR5 in such transgenic plants resulted in a characteristic phenotype with regard to transcriptional events, in which free-running rhythms were considerably altered for certain circadian-regulated genes, including CCA1, LHY, APRR1/TOC1, other APRR1/TOC1 members, GI and CAB2, although each rhythm was clearly sustained even after plants were transferred to continuous light. With regard to biological events, APRR5-ox plants flowered much earlier than wild-type plants, more or less, in a manner independent of photoperiodicity (or under short-day conditions). Furthermore, APRR5-ox plants showed an SRL (short-hypocotyls under red light) phenotype that is indicative of hypersensitiveness to red light in early photomorphogenesis. Both APRR1-ox and APRR9-ox plants also showed the same phenotype. Therefore, APRR5 (together with APRR1/TOC1 and APRR9) must be taken into consideration for a better understanding of the molecular links between circadian rhythms, control of flowering time through the photoperiodic long-day pathway, and also light signaling-controlled plant development.

  58. Identification and characterization of a novel gene, hos2+, the function of which is necessary for growth under high osmotic stress in fission yeast Reviewed

    Norihito Nakamichi, Eiji Yamamoto, Hisami Yamada, Hirofumi Aiba, Takeshi Mizuno

    Biosci Biotechnol Biochem   Vol. 64   page: 2493-2496   2000

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    hos2 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth. An S. pombe strain carrying the hos2-M10 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously. In this study, the hos2+ gene was identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases. The hos2-M10 mutation resulted in Gln-62 to TAG-termination codon. A Hos2-defective (hos2delta) strain, which we then constructed, showed the phenotype of high osmolarity sensitivity, as in the case of the original hos2-M10 mutant. For this hos2delta mutant, three multicopy suppressor genes were isolated and one of which was identified as the pgk1+ gene, encoding a phosphoglycerate kinase.

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Books 4

  1. 有用品種から紐解く植物の概日時計メカニズム Reviewed

    中道範人( Role: Sole author)

    化学と生物  2020.12 

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    Language:Japanese Book type:Textbook, survey, introduction

  2. 植物の概日リズム研究に関して Reviewed

    中道範人( Role: Sole author)

    日本時間生物学会誌「時間生物学」  2016 

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    Language:Japanese Book type:Textbook, survey, introduction

  3. Biological clock in plants

    Norihito Nakamichi, Takeshi Mizuno( Role: Joint author)

    Kagaku-to-Seibutsu  2006 

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    Language:Japanese

  4. His-Asp phosphorelay signal transduction involved in spore-formation in fission yeast

    Norihito Nakamichi, Hisami Yamada, Keisuke Aoyama, Ryusuke Ohmiya, Hirohumi Aiba, Takeshi Mizuno( Role: Joint author)

    Nihon-nougeikagakukai-kaishi  2003 

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    Language:Japanese

Presentations 35

  1. Small-molecule modulators of the plant clock Invited International conference

    Norihito Nakamichi

    The 5th Asian Forum on Chronobiology  2022.7.16 

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    Event date: 2022.7

    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Kaifeng-Online   Country:China  

  2. 時計長周期化化合物と標的タンパクの結合モデル

    中道範人

    第63回日本植物生理学会年会  2022.3.23 

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    Event date: 2022.3

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:つくば・オンライン   Country:Japan  

  3. Small molecules targeting Casein Kinase 1 family modulate Arabidopsis circadian clock Invited International conference

    Norihito Nakamichi

    Frontiers in plant environmental response research  2019.11.18 

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    Event date: 2019.11

    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Nagoya   Country:Japan  

  4. 植物の時間を操る技術 Invited

    中道範人

    平成30年度アグリビジネス創出フェアin東海  2019.1.29 

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    Event date: 2019.1

    Language:Japanese   Presentation type:Oral presentation (keynote)  

    Venue:名古屋   Country:Japan  

  5. Molecular mechanism underpinning the plant circadian clock Invited International conference

    Norihito Nakamichi

    The Universities of Edinburgh and Nagoya Joint PhD Degree Program meeting  2018.11.9  The University of Edinburgh

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    Event date: 2018.11

    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Edinburgh   Country:United Kingdom  

  6. 農芸化学的な植物体内時計の解析 Invited

    中道範人

    農芸化学Frontiersシンポシウム  2018.3.18 

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    Event date: 2018.3

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

  7. Molecular study of circadian clock with small molecules Invited International conference

    Norihito Nakamichi

    Taiwan-Japan Plant Biology 2017  2017.11.4 

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    Event date: 2017.11

    Language:English   Presentation type:Oral presentation (invited, special)  

  8. A small molecule modulating circadian clocks both in plant and animal

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    Event date: 2017.10

    Language:English   Presentation type:Poster presentation  

    Country:Japan  

  9. 植物の時間を調節するトランスフォーマティブ生命分子 Invited

    中道範人

    生物リズム若手研究者の集い  2017.8.6 

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    Event date: 2017.8

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

  10. シロイヌナズナの概日リズム周期を調節する低分子化合物群

    中道範人 他

    日本植物生理学会年会 

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    Event date: 2017.3

    Language:Japanese   Presentation type:Poster presentation  

    Venue:鹿児島   Country:Japan  

  11. 時計周期を調整する合成化合物 Invited

    中道範人

    第23回日本時間生物学会年会 

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    Event date: 2016.11

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:名古屋   Country:Japan  

  12. バイオマス生産性の向上を指向した概日時計のシステム生物学 Invited

    中道範人

    植物の環境適応戦略をひもとく-JST植物科学のいま- 

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    Event date: 2016.10

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:東京   Country:Japan  

  13. Synthetic molecules changing plant circadian clock Invited International conference

    The 64th NIBB Conference -Evolution of Seasonal Timers- 

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    Event date: 2016.4

    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Okazaki   Country:Japan  

  14. 植物の概日時計機能に関わるタンパク質PRRの研究 Invited

    中道範人

    第22回日本時間生物学会年会 

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    Event date: 2015.11

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:東京   Country:Japan  

  15. Synthetic molecules controlling plant circadian rhythms Invited International conference

    Norihito Nakamichi

    2nd International Symposium of Transformative Bio-molecules 

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    Event date: 2014.5

    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Nagoya   Country:Japan  

  16. 概日リズムに関わる転写制御ネットワークの発見

    中道範人

    第55回日本植物生理学会年会 

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    Event date: 2014.3

    Language:Japanese   Presentation type:Symposium, workshop panel (nominated)  

    Country:Japan  

  17. 概日時間情報はPRRタンパク質を介して出力系現象に伝達する

    中道範人、木羽隆敏、神岡真理、鈴木孝征、山篠貴史、東山哲也、榊原均、水野猛

    第54回日本植物生理学会年会 

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    Event date: 2013.3

    Language:Japanese   Presentation type:Oral presentation (general)  

    Country:Japan  

  18. 概日時計に関連したPSEUDO-RESPONSE REGULATOR 5遺伝子の発現を制御する新規転写因子

    神岡真理、光田展隆、山溝千尋、大宮あけみ、山篠貴史、高木優、水野猛、中道範人

    第54回日本植物生理学会年会 

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    Event date: 2013.3

    Language:Japanese   Presentation type:Oral presentation (general)  

    Country:Japan  

  19. ChIP-seq法による植物時計の出力系の遺伝子ネットワーク構造の解析

    中道範人、木羽隆敏、神岡真理、鈴木孝征、山篠貴史、東山哲也、榊原均、水野猛

    第19回日本時間生物学会学術大会 

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    Event date: 2012.9

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Country:Japan  

  20. 機能的ゲノミクスによる高等植物の新規時計関連因子の探索

    神岡真理、光田展隆、山篠貴史、高木優、水野猛、中道範人

    第19回日本時間生物学会学術大会 

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    Event date: 2012.9

    Language:Japanese   Presentation type:Poster presentation  

    Country:Japan  

  21. TRANSCRIPTIONAL REPRESSOR PSEUDO-RESPONSE REGULATOR 5 DIRECTLY REGULATES CLOCK OUTPUT PATHWAYS International conference

    Norihito Nakamichi, Takatoshi Kiba, Mari Kamioka, Takamasa Suzuki, Takafumi Yamashino, Tetsuya Higashiyama, Hitoshi Sakakibara, Takeshi Mizuno

    The 23rd International Conference on Arabidopsis Research 

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    Event date: 2012.7

    Language:English   Presentation type:Poster presentation  

    Country:Austria  

  22. Repressors for the morning time in Arabidopsis circadian system

    Norihito Nakamichi, Takatoshi Kiba, Hitoshi Sakakibara, Takeshi Mizuno

    第53回日本植物生理学会年会 

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    Event date: 2012.3

    Language:English   Presentation type:Symposium, workshop panel (nominated)  

    Country:Japan  

  23. 植物の概日リズム現象

    中道範人

    第14回植物オルガネラワークショップ 

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    Event date: 2012.3

    Language:Japanese   Presentation type:Symposium, workshop panel (nominated)  

    Country:Japan  

  24. 高等植物の生物時計:分子機構からみるグリーンテクノロジーへの貢献の可能性

    中道範人

    第18回日本時間生物学会学術大会 

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    Event date: 2011.11

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Country:Japan  

  25. 植物の概日リズムに関わる転写ネットワーク構造

    中道範人 木羽隆敏 水野猛 榊原均

    第18回日本時間生物学会学術大会 

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    Event date: 2011.11

    Language:Japanese   Presentation type:Poster presentation  

    Country:Japan  

  26. Molecular mechanism for regulation of various circadian rhythms

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    Event date: 2011.9

    Language:Japanese   Presentation type:Symposium, workshop panel (nominated)  

    Country:Japan  

  27. Arabidopsis PSEUDO-RESPONSE REGULATOR9, 7, and 5 are Repressors in the Circadian Clock International conference

    Norihito Nakamichi, Takatoshi Kiba, Rossana Henriques, Takeshi Mizuno, Nam-Hai Chua, Hitoshi Sakakibara

    21st International Conference on Arabidopsis Research 

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    Event date: 2010.6

    Language:English   Presentation type:Poster presentation  

    Country:Japan  

  28. PSEUDO-RESPONSE REGULATORS 9 (PRR9), PRR7 and PRR5 are Transcriptional Repressors in the Arabidopsis Circadian Clock International conference

    Norihito Nakamichi, Takatoshi Kiba, Rossana Henriques, Takeshi Mizuno, Nam-Hai Chua, Hitoshi Sakakibara

    2010 SRBR Meeting 

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    Event date: 2010.5

    Language:English   Presentation type:Oral presentation (general)  

    Country:Japan  

  29. シロイヌナズナの疑似レスポンスレギュレーターは生物時計で機能する転写抑制因子である

    中道範人, 木羽隆敏, Rossana Henriques, 水野猛, Nam-Hai Chua, 榊原均

    第51回日本植物生理学会年会 

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    Event date: 2010.3

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:熊本   Country:Japan  

  30. PSEUDO-RESPONSE REGULATOR 9, 7 and 5 are Repressors of CCA1 and LHY Transcription in Arabidopsis Circadian Clock International conference

    Norihito Nakamichi, Takatoshi Kiba, Rossana Henriques, Takeshi Mizuno, Nam-Hai Chua, Hitoshi Sakakibara

    Plant Biology 2009 

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    Event date: 2009.7

    Language:English   Presentation type:Oral presentation (general)  

    Country:Japan  

  31. 時計変異体のマイクロアレイ解析

    中道範人, 草野都, 福島敦史, 北雅規, 伊藤照悟, 山篠貴史, 斉藤和季, 榊原均, 水野猛

    第50回日本植物生理学会年会 

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    Event date: 2009.3

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:名古屋   Country:Japan  

  32. シロイヌナズナ培養細胞系をもちいたPRRタンパクの解析

    中道範人, 伊藤照悟, 山篠貴史, 小山時隆, 近藤孝男, 水野猛

    第49回日本植物生理学会年会 

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    Event date: 2008.3

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:札幌   Country:Japan  

  33. Arabidopsis Pseudo Response Regulators PRR9, PRR7 and PRR5 are Involved in the Circadian Phase-Transition of the Transcriptome International conference

    Norihito Nakamichi, Miyako Kusano, Atushi Fukushima, Masanori Kita, Shogo Ito, Takafumi Yamashino, Kazuki Saito, Hitoshi Sakakibara, Takeshi Mizuno

    18th International Conference on Arabidopsis Research 

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    Event date: 2007.6

    Language:English   Presentation type:Poster presentation  

    Country:Japan  

  34. 高等植物の分子時計

    中道範人, 山篠貴史, 近藤孝男, 水野猛

    第13回日本時間生物学会年会 

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    Event date: 2006.12

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:東京   Country:Japan  

  35. Circadian Rhythms in Plant International conference

    Norihito Nakamichi, Takeshi Mizuno

    International Symposium on Plant Metabolism 

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    Event date: 2005.8

    Language:English   Presentation type:Oral presentation (keynote)  

    Country:Japan  

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Research Project for Joint Research, Competitive Funding, etc. 7

  1. タンパク質複合体ダイナミクスから探る植物時計のペースメーカーの実体

    2021.9

    武田科学振興財団 生命科学研究助成 

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    Authorship:Principal investigator 

    Grant amount:\10000000

  2. 植物の成熟化を司る発生タイマーの解明へむけたケミカルバイオロジー

    2020.4 - 2021.3

    長瀬科学技術振興財団 研究助成 

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    Authorship:Principal investigator 

  3. 重複性遺伝子がおりなす複雑系生命現象「植物体内時計」の解明

    2018.4 - 2019.3

    豊田理研スカラー 

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    Authorship:Principal investigator 

    Grant amount:\1000000

  4. ケミカルバイオロジーで解く植物時計の多様性

    2016.10 - 2018.3

    豊秋奨学会 研究費助成 

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    Authorship:Principal investigator 

  5. 新規時計調整化合物を用いた植物の乾燥耐性付与技術の開発

    2016.4 - 2017.3

    鳥取大学乾燥地研究センター共同研究 若手奨励研究 

    中道範人

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    Authorship:Principal investigator  Grant type:Competitive

  6. 植物の概日時計の統合的な解析

    2015.4 - 2016.3

    内藤記念財団 特定研究助成 

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    Authorship:Principal investigator 

  7. バイオマス生産性の向上を指向した概日時計のシステム生物学

    Grant number:JPMJPR11B9  2011.12 - 2015.3

    さきがけ「二酸化炭素資源化を目指した植物の物質生産力強化と生産物活用のための基盤技術の創出」領域 

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\52000000 ( Direct Cost: \40000000 、 Indirect Cost:\12000000 )

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KAKENHI (Grants-in-Aid for Scientific Research) 9

  1. 不規則な温度環境変化に対する時計システムのリジリエンス機構

    Grant number:21H05656  2021.9 - 2023.3

    科学研究費助成事業  学術変革領域研究(A)

    中道 範人

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    Authorship:Principal investigator 

    Grant amount:\9360000 ( Direct Cost: \7200000 、 Indirect Cost:\2160000 )

  2. 改変型キナーゼとプロテオミクスで解く植物リン酸化ネットワーク

    Grant number:20K21272  2020.7 - 2022.3

    科学研究費助成事業  挑戦的研究(萌芽)

    中道 範人

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\6500000 ( Direct Cost: \5000000 、 Indirect Cost:\1500000 )

    我々が開発したユニークな植物のタンパク質リン酸化酵素(カゼインキナーゼ1 :CK1) の阻害剤や, 化合物との結合状態に基づいて人工設計するCK1を利用したプロテオミクス解析によって, 遺伝的重複性が障壁になって不明な点の多いシロイヌナズナのCK1ファミリーの関わる生理現象を明らかにする. 本研究の成果は, 遺伝的重複性が問題となって解析が遅れている植物の多くのキナーゼの機能解明へ向けたプロテオミクス研究のモデル系となる.
    我々が開発したユニークな植物のタンパク質リン酸化酵素(カゼインキナーゼ1 :CK1) の阻害剤や, 化合物との結合状態に基づいて人工設計するCK1を利用したプロテオミクス解析によって, 遺伝的重複性が障壁になって不明な点の多いシロイヌナズナのCK1ファミリーの関わる生理現象を明らかにする. 本研究の成果は, 遺伝的重複性が問題となって解析が遅れている植物の多くのキナーゼの機能解明へ向けたプロテオミクス研究のモデル系となる.
    本年度は, すでにリコビナントタンパク質として合成されていたシロイヌナズナのCKL4をCK1ファミリーの代表酵素として解析した. 合成ATPアナログを用いたプロテオミクス解析へ向けて, 合成ATPアナログをCKL4が加水分解し, 基質に対してガンマ位のチオリン酸を転移できるかを解析した. チオリン酸化は, p-ニトロベンジルメチレートと抗チオフォスフェートエステル抗体を用いたウエスタンブロッティングで検出した. ATPのガンマ位がチオリン酸化したアナログのうち, アデニン骨格を持つものはCKL4タンパクによってガンマ位が加水分解され, 基質がチオリン酸化されたことが確認できた. 弱いながらも, アデニン骨格に分子修飾されたATPガンマ位チオリン酸アナログ2種類も, CKL4によって基質へガンマ位のチオリン酸が転移されていた. これらの反応はCKL阻害剤であるAMI-331によって完全に阻害されることも判明した.
    次にアデニン骨格が分子修飾されたATPガンマ位チオリン酸アナログ2種類を効率的に使うことができることが期待されたCKL4変異体タンパクを3種類合成し, この可能性を解析した. 結果として, これら3種のCKL4変異体タンパクは, いずれのATPアナログの加水分解能力が落ちていたことが分かった.
    本年度は, 本研究計画全体の中で最も困難だと考えられていた実験を進めた. その結果, 実験系の確立には成功した. 一方で, CKL4のATP結合部位の構造を基にしたCKL4変異体を3種類作成したが, いずれの変異体タンパクもATPアナログの加水分解能力は落ちていた. これは変異によって, ATPアナログの加水分解能力が落ちていたというよりも, ATPそのものの加水分解能力も落ちたことを示唆する結果も得ている. したがって, これらの変異は適切ではなかったことが考えられる. しかしながら, 実験系が確立できたことから, この系を使ってATPアナログを効率的に加水分解できるCKL4タンパク変異体を探索することも可能である.
    また次年度の準備としてCKL遺伝子群の多重変異体の作成に着手している. 複数の遺伝子座を一度の形質転換でゲノム編集することを狙った実験系の立ち上げにも成功し, 複数の多重変異体を得ている. またCKL4の過剰発現の作出を試みたが, 汎用されているgateway binary vectorを使った方法では発現量がとても低くなることが判明した. そこで, より強いプロモーターをもつbinary vectorにCKL4をクローニングし, 形質転換体を作成したところ, ウエスタンブロッティングで検出できるほどのタンパク量を発現する形質転換体の作出に成功した. またCKL阻害剤AMI331の植物への処理方法を検討することで, 従来よりも強い効果を得る条件を見出した. この発見は当初予想されなかったものである.
    引き続き, チオリン酸化反応を指標にして, アデニン骨格に分子修飾されたATPガンマ位チオリン酸アナログ2種類を十分に加水分解できるCKL4の作出を試みる. 仮にこれらアナログを十分に加水分解できるCKL4が作出できれば, これを植物体で発現させ, チオリン酸化されたタンパクを抗体で濃縮し, これらをMS解析に持ち込むことで, CKL4の生体内での基質の同定が可能となる. また, これまでに作出したckl多重変異体やCKL4過剰発現体のリン酸化プロテオミクスを行うことで, 基質の同定が可能である. これら2つの方法を並列させることで, CKLにリン酸化される基質の同定を行いたい.
    またリン酸化プロテオミクスやATPアナログの解析が不調であっても, CKLの基質の同定へ向けて, 近接タンパク修飾法を用いてCKLの相互作用タンパク質を同定する. このための形質転換体の作出も順調に進んでいるため, この形質転換体を使った実験も早く取り組む.
    さらにckl変異体やCKL過剰発現体の表現型観察や網羅的遺伝子発現解析から, CKLが制御する生理現象を見出すことができると考えている. そしてその生理現象や遺伝子発現に関わる鍵タンパク質がCKLによるリン酸化を受けている可能性が高い. 網羅的なプロテオミクスと並行して, 仮説検証型のアプローチも進めることで, 確実に本研究の成果につなげていくことができると考えている.

  3. Developmental regulatory network atlas under the clock control

    Grant number:20H05411  2020.4 - 2022.3

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

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\8450000 ( Direct Cost: \6500000 、 Indirect Cost:\1950000 )

  4. 環境の時刻変動への適応を可能にする植物の時計転写ネットワークの包括的な解析

    Grant number:18H02136  2018.4 - 2022.3

    科学研究費助成事業  基盤研究(B)

    中道 範人

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\17810000 ( Direct Cost: \13700000 、 Indirect Cost:\4110000 )

    独自のスクリーニング系で発見していた超長周期化化合物(PAC3)の作用機序の解明を目指した。まず、PAC3の構造活性研究を行い、活性を損なわない形でアガロースビーズに結合させた。この化合物ビーズに結合するタンパク質をシロイヌナズナの細胞抽出液から探索した。その際、化合物ビーズと標的タンパク質の結合を阻害するために、PAC3を含む実験サンプルも用意した。化合物ビーズと結合するが、その結合がPAC3によって阻害されるというタンパク質がPAC3の標的として考えられる。以上の仮定のもと、プロテオミクス解析の実施したところ、あるタンパク質キナーゼが標的候補として得られた。そこでこのタンパク質キナーゼをコードする遺伝子を破壊した株を複数アリル作成し、それらの株の概日リズム周期を発光レポーター検出系を用いて解析したところ、これらの株は概日リズムが長周期化することが分かった。しかし、これら変異株における長周期化の程度は、化合物処理による長周期化よりも弱かった。そこで候補遺伝子とよく似た配列の遺伝子が、冗長的に機能する可能性が考えられた。これらの両遺伝子の二重機能欠損株は致死となったが、二重ノックダウン株の作成には成功し、この株はリズムが超長周期化すること、その超長周期化は化合物PAC3による効果と似ていることを突き止めた。またこの遺伝子の翻訳産物であるキナーゼの活性を試験管内で解析する実験系を確立し、PAC3がこのキナーゼの阻害剤であることを明らかにした。さらにPAC3とキナーゼの結合モデルを、in silico解析と生化学実験によって得ることができた。また、PAC3を植物に処理することで、このキナーゼの基質のリン酸化レベルが低下することを見出した。このキナーゼはこれまで時計に関与していることが報告されていないものであった。このように独自の化合物の作用機序を探る研究によって、新たな時計関連キナーゼを見出すことができた。
    昨年度の成果に引き続き、別の新たな時計長周期化化合物(PAC3)の作用機序の解明を達成した。この化合物は、これまで時計に関わると報告のなかったタンパク質キナーゼのリン酸化阻害剤であったため、新たな時計関連タンパク質の発見へと繋がった。またPAC3を植物に処理することで、このキナーゼの基質のリン酸化状態を低減するさせることも見出した。このように、独自の化合物の作用機序を探る研究によって、新たな時計関連キナーゼを見出すことができたことは、植物時計のメカニズムの研究においてブレークスルーといえる。
    また、このキナーゼをコードする遺伝子は2つ存在するが、これらの完全な二重ノックアウトは致死であった。したがって、キナーゼは生育に必要な働きを持ち、このような必須遺伝子を変異体スクリーニングを起点とした研究で見出すことは困難である。この研究は、化合物スクリーニングを起点とした研究の新たな技術的な有利性「必須性を回避して関連遺伝子を見出す」ということを明示した例となった。
    さらに、新たな分子プローブ(PAC3アガロースビーズ)およびプロテオミクスを使ったアプローチにより、化合物の標的タンパク質を同定することを達成した。このことは、我々の研究方法(アフィニティープロテオミクス)がより広範囲な構造をもつ化合物へも展開できる一般的方法となることを示唆しており、今後植物科学分野において多様な生物活性分子の作用機序を探る研究への技術的指針となると自負している。
    PAC3の作用機序が明らかとなったことをうけ、PAC3の構造類似化合物(アナログ)の解析を進める。試験的な解析では、PAC3よりも強力な活性をもつ化合物を得ているが、この化合物は標的タンパク質(キナーゼ)に結合する様式は、従来の結合モデルからは推論できない。そこで、化合物の標的タンパク質への結合の分子モデルを得るとともに、この化合物の活性を様々な生物活性テストから評価していく。化合物とタンパク質の新たな結合モデルが得られることで、このタンパク質の活性と時計周期との関連性がさらに理解されるであろう。
    標的タンパク質の同定が未だ困難な2つの時計周期変調化合物については、化合物プローブを使ったアフィニティープロテオミクスと化合物処理後の遺伝子発現変化の俯瞰を組み合わせた方法の研究を進める。化合物処理で変化した遺伝子を同定したのちに、これら遺伝子の発現に関わる転写因子やその修飾酵素などが、化合物の作用経路で働くと考えられる。このような状況を念頭におきながら、遺伝子の発現に関わる転写因子の相互作用因子を同定する。この同定方法に関して、すでに新たな方法を考案しており、この方法を使うことで効率的に進めたい。また近接タンパク質ラベリング法(tarboID)もこの目的のために汎用される方法であるため、この方法も取り入れて研究を進める。
    以上のように時計周期を変調させる化合物の働きや作用経路の解明によって、植物の時計に関わる新たな生体分子やその機能について理解を深めていく。

  5. Analysis of small molecule that induces flowering

    Grant number:17K19229  2017.6 - 2021.3

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Research (Exploratory)

    Nakamichi Norihito

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    Authorship:Principal investigator  Grant type:Competitive

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

    We obtained a small molecule that induces the expression of the florigen gene FT from a large-scale chemical screening. In this study, we aimed to elucidate the mode of action of the molecule by gene expression profiling. As a result, it was clarified that the molecule does not affect the expression of genes related to the gibberellin pathway and the vernalization pathway, but affects the photoperiodic pathway genes. Especially, the expression of clock genes that control the photoperiod pathway were affected by the molecule. Furthermore, was found that the molecule regulates the flowering time by changing the stability of the clock proteins that regulate the clock genes.

  6. 環境刺激による気孔開度制御機構の解析

    Grant number:15H05956  2015.10 - 2020.3

    科学研究費補助金  新学術領域研究

    木下俊則

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    Authorship:Coinvestigator(s) 

  7. 新奇時計関連因子の生化学的な解析

    Grant number:26870267  2014.4 - 2016.3

    科学研究費補助金  若手研究(B)

    中道範人

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    Authorship:Principal investigator 

  8. 概日時計の周期安定性を内包する分子の解析

    Grant number:24770042  2012.4 - 2014.3

    科学研究費補助金  若手研究(B)

    中道範人

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    Authorship:Principal investigator  Grant type:Competitive

  9. 植物における時計関連タンパク質の生化学的解析

    Grant number:20870040  2008.4 - 2010.3

    科学研究費補助金 

    中道範人

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    Authorship:Principal investigator 

    Grant amount:\3302000 ( Direct Cost: \2540000 、 Indirect Cost:\762000 )

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Industrial property rights 3

  1. 花成時期調節剤、農薬組成物及び植物の花成時期の調節方法

    中道範人, 上原貴大, 山口潤一郎, 松尾宏美, 佐藤綾人, 伊丹健一郎, 木下俊則

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    Applicant:国立大学法人名古屋大学

    Application no:特願2017-151824  Date applied:2017.8

    Country of applicant:Domestic  

  2. 植物概日リズム調整剤

    中道範人, 山口潤一郎, 伊丹健一郎, 上原貴大, 大松亨介, 古川由季乃, 木下俊則, 大井貴史, 佐藤綾人

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    Applicant:名古屋大学

    Application no:特願2014-164097  Date applied:2014.8

    Country of applicant:Domestic  

  3. バイオマスが増大し、かつ環境ストレス耐性が向上した形質転換植物およびその作出方法

    榊原均、中道範人、槇田庸絵

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    Applicant:RIKEN, 独立行政法人理化学研究所

    Application no:PCT/JP2010/069229  Date applied:2010.10

    Announcement no:WO2011049243 A1 

    Country of applicant:Domestic  

 

Teaching Experience (On-campus) 16

  1. 分子生理学I

    2017

  2. 分子生物学演習I

    2017

  3. 基礎生理学I

    2017

  4. 生物学基礎I

    2017

  5. 分子生物学演習 I

    2016

  6. 生物学基礎 Ⅰ

    2016

  7. 基礎生理学 I

    2016

  8. 分子生理学 I

    2016

  9. 生物学基礎 Ⅰ

    2015

  10. 分子生理学 I

    2015

  11. 基礎生理学 I

    2015

  12. 生物学基礎Ⅰ

    2014

  13. 分子生理学 I

    2014

  14. 生物学基礎Ⅰ

    2013

  15. 分子生理学 I

    2013

  16. 分子細胞機構学 III

    2010

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Social Contribution 4

  1. 植物の生物時計「みなさん、時計変異体を食べていると知っていますか?」

    Role(s):Lecturer

    長野県飯山高校_SSH事業  2020.10

  2. 愛知教育大学附属高校 「1・2年生対象出前講義」

    2015.9

  3. スーパーサイエンスハイスクール 研究者ライブ

    2015.8

  4. 春日井東高等学校 研究室訪問

    2015.8

Media Coverage 7

  1. 先端人 Newspaper, magazine

    朝日新聞社  2019.6

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    Author:Other 

  2. 植物の生物時計を変化させる化合物 Newspaper, magazine

    中日新聞社  2019.5

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    Author:Other 

  3. 農作物の開花早める化合物 名大が開発 Newspaper, magazine

    日本経済新聞社  2017.7

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    Author:Other 

  4. 植物の体内時計を調節する遺伝子を発見 Internet

    2016.3

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    Author:Other 

  5. 植物の時間情報を制御 Newspaper, magazine

    科学新聞社  2012.10

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    Author:Other 

  6. 植物眠らせるタンパク質特定 Newspaper, magazine

    中日新聞社  2012.10

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    Author:Other 

  7. 遺伝子60種 概日リズムに関与 Newspaper, magazine

    日刊工業新聞社  2012.10

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