Researcher Database

Junji Yamaguchi

Researcher Profile and Settings


  • President/Vice-Presidents

Job Title

  • Executive/Vice-President


J-Global ID

Research Interests

  • carbon dioxide   proteomics   plant   代謝   ubiquitin ligase   plant immunity   adaptation to environment   biomass   protein degradation   

Research Areas

  • Life sciences / Applied molecular and cellular biology
  • Life sciences / Plants: molecular biology and physiology

Academic & Professional Experience

  • 2020/10 - Today Hokkaido University Executive Director, Vice President
  • 2009/04 - Today Hokkaido University, Research Faculty of Science Division of Biological Sciences Professor
  • 2014/04 - 2020/09 Hokkaido University Vice President
  • 2014/04 - 2017/03 Hokkaido University Graduate School of Life Science Dean
  • 2010/09 - 2013/03 Hokkaido University Institute for the Advancement of Higher Education
  • 2003/04 - 2009/05 科学技術振興調整費 研究領域主管(プログラムオフィサー)
  • 2006/04 - 2009/03 Hokkaido University Faculty of Advanced Life Science
  • 2001/04 - 2006/03 Hokkaido University, Research Faculty of Science Professor
  • 1995/02 - 2001/03 名古屋大学生物分子応答研究センター 助教授
  • 1987/10 - 1995/01 Nagoya University School of Agricultural Sciences
  • 1986/10 - 1987/09 日本学術振興会 海外特別研究員(米国ロックフェラー大学留学)
  • 1985/10 - 1986/09 日本学術振興会 特別研究員
  • 1982/04 - 1985/03 名古屋大学大学院農学研究科 博士課程後期修了

Association Memberships

  • 植物細胞分子生物学会   日本植物化学調節学会   American Society of Plant Biologists   日本植物生理学会   

Research Activities

Published Papers

  • Enkhsukh Lkhamkhuu, Kazunori Zikihara, Hitomi Katsura, Satoru Tokutomi, Takafumi Hosokawa, Yoshihisa Usami, Mitsuyoshi Ichihashi, Junji Yamaguchi, Kenji Monde
    Plant Biotechnology 37 (1) 57 - 67 1342-4580 2020/03/25
  • Xingwen Li, Miho Sanagi, Yu Lu, Yuko Nomura, Sara Christina Stolze, Shigetaka Yasuda, Yusuke Saijo, Waltraud X Schulze, Regina Feil, Mark Stitt, John E Lunn, Hirofumi Nakagami, Takeo Sato, Junji Yamaguchi
    Frontiers in plant science 11 377 - 377 2020 [Refereed][Not invited]
    Nutrient availability, in particular the availability of sugar [carbon (C)] and nitrogen (N), is important for the regulation of plant metabolism and development. In addition to independent utilization of C and N nutrients, plants sense and respond to the balance of C and N nutrients (C/N-nutrient) available to them. High C/low N-nutrient stress has been shown to arrest early post-germinative growth while promoting progression to senescence in Arabidopsis. Although several signaling components of the C/N-nutrient response have been identified, the inclusive molecular basis of plant C/N-nutrient response remains unclear. This proteome analysis evaluated phosphorylation dynamics in response to high C/low N-nutrient stress. Phosphoproteomics under conditions of C/N-nutrient stress showed a global change in the phosphorylation status of proteins, including plasma membrane H+-ATPase, carbon and nitrogen metabolic enzymes and signaling proteins such as protein kinases and transcription factors. Further analyses suggested that SNF1-related protein kinase 1 (SnRK1) is involved in primary C/N-nutrient signal mediation via the transcriptional regulation of C/N-regulatory kinases. We also identified a leucine-rich repeat receptor-like kinase with extracellular malectin-like domain, named as LMK1, which was shown to possess cell death induction activity in plant leaves. These results provide important insight into the C/N-nutrient signaling pathways connecting nutrition stress to various cellular and physiological processes in plants.
  • Arae T, Morita K, Imahori R, Suzuki Y, Yasuda S, Sato T, Yamaguchi J, Chiba Y
    Plant & cell physiology 60 (9) 2015 - 2025 0032-0781 2019/09 [Refereed][Not invited]
  • Yongming Luo, Shoki Aoyama, Yoichiro Fukao, Yukako Chiba, Takeo Sato, Junji Yamaguchi
    Plant Biotechnology 36 (2) 107 - 112 1342-4580 2019/06/25
  • Miho Sanagi, Yu Lu, Shoki Aoyama, Yoshie Morita, Nobutaka Mitsuda, Miho Ikeda, Masaru Ohme-Takagi, Takeo Sato, Junji Yamaguchi
    Plant Biotechnology Japanese Society for Plant Cell and Molecular Biology 35 (2) 167  1342-4580 2018/06 [Refereed][Not invited]
  • Thais Huarancca Reyes, Andrea Scartazza, Antonio Pompeiano, Andrea Ciurli, Yu Lu, Lorenzo Guglielminetti, Junji Yamaguchi
    Plant and Cell Physiology 59 (6) 1248 - 1254 1471-9053 2018/06/01 [Refereed][Not invited]
    Environmental cues modulate the balance of carbon (C) and nitrogen (N) which are essential elements for plant metabolism and growth. In Arabidopsis, photochemical efficiency of PSII, phosphorylation status and localization of many enzymes, and the level of total soluble sugars were affected by an unbalanced C/N ratio. Since differences in C/N affect these parameters, here we checked whether different sources of N have different effects when a high C/N ratio is imposed. NO3- and NH4+ were separately provided in C/N medium. We investigated the effects on photochemical efficiency of PSII, the level of total soluble sugars and nitrate reductase activity under stressful C/N conditions compared with control conditions. We found that treated plants accumulated more total soluble sugars when compared with control. Photochemical efficiency of PSII did not show significant differences between the two sources of nitrogen after 24 h. The actual nitrate reductase activity was the result of a combination of activity, activation state and protein level. This activity constantly decreased starting from time zero in control conditions in contrast, the actual nitrate reductase activity showed a peak at 2 h after treatment with NO3-, and at 30 min with NH4+. This, according to the level of total soluble sugars, can be explained by the existence of a cross-talk between the sugars in excess and low nitrate in the medium that blocks the activity of nitrate reductase in stressful sugar conditions until the plant is adapted to the stress.
  • Shoki Aoyama, Saki Terada, Miho Sanagi, Yoko Hasegawa, Yu Lu, Yoshie Morita, Yukako Chiba, Takeo Sato, Junji Yamaguchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 491 (1) 33 - 39 0006-291X 2017/09 [Refereed][Not invited]
    Ubiquitin ligases play important roles in regulating various cellular processes by modulating the protein function of specific ubiquitination targets. The Arabidopsis Toxicos en Levadura (ATL) family is a group of plant-specific RING-type ubiquitin ligases that localize to membranes via their N-terminal transmembrane-like domains. To date, 91 ATL isoforms have been identified in the Arabidopsis genome, with several ATLs reported to be involved in regulating plant responses to environmental stresses. However, the functions of most ATLs remain unknown. This study, involving transcriptome database analysis, identifies ATL15 as a sugar responsive ATL gene in Arabidopsis. ATL15 expression was rapidly down-regulated in the presence of sugar. The ATL15 protein showed ubiquitin ligase activity in vitro and localized to plasma membrane and endomembrane compartments. Further genetic analyses demonstrated that the atI15 knockout mutants are insensitive to high glucose concentrations, whereas ATL15 overexpression depresses plant growth. In addition, endogenous glucose and starch amounts were reciprocally affected in the at115 knockout mutants and the ATL15 overexpressors. These results suggest that ATL15 protein plays a significant role as a membrane-localized ubiquitin ligase that regulates sugar responsive plant growth in Arabidopsis. (C) 2017 Elsevier Inc. All rights reserved.
  • Toshihiro Arae, Shiori Isai, Akira Sakai, Katsuhiko Mineta, Masami Yokota Hirai, Yuya Suzuki, Shigehiko Kanaya, Junji Yamaguchi, Satoshi Naito, Yukako Chiba
    PLANT AND CELL PHYSIOLOGY 58 (6) 1090 - 1102 0032-0781 2017/06 [Refereed][Not invited]
    Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat-binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.
  • Shigetaka Yasuda, Shoki Aoyama, Yoko Hasegawa, Takeo Sato, Junji Yamaguchi
    MOLECULAR PLANT 10 (4) 605 - 618 1674-2052 2017/04 [Refereed][Not invited]
    In response to the ratio of available carbon (C) and nitrogen (N) nutrients, plants regulate their metabolism, growth, and development, a process called the C/N-nutrient response. However, the molecular basis of C/N-nutrient signaling remains largely unclear. In this study, we identified three CALCINEURIN B-LIKE (CBL)-INTERACTING PROTEIN KINASES (CIPKs), CIPK7, CIPK12, and CIPK14, as key regulators of the C/N-nutrient response during the post-germination growth in Arabidopsis. Single-knockout mutants of CIPK7, CIPK12, and CIPK14 showed hypersensitivity to high C/low N conditions, which was enhanced in their triple-knockout mutant, indicating that they play a negative role and at least partly function redundantly in the C/N-nutrient response. Moreover, these CIPKs were found to regulate the function of ATL31, a ubiquitin ligase involved in the C/N-nutrient response via the phosphorylation-dependent ubiquitination and proteasomal degradation of 14-3-3 proteins. CIPK7, CIPK12, and CIPK14 physically interacted with ATL31, and CIPK14, acting with CBL8, directly phosphorylated ATL31 in a Ca2+-dependent manner. Further analyses showed that these CIPKs are required for ATL31 phosphorylation and stabilization, which mediates the degradation of 14-3-3 proteins in response to C/N-nutrient conditions. These findings provide new insights into C/N-nutrient signaling mediated by protein phosphorylation.
  • Takeo Sato, Shugo Maekawa, Mineko Konishi, Nozomi Yoshioka, Yuki Sasaki, Haruna Maeda, Tetsuya Ishida, Yuki Kato, Junji Yamaguchi, Shuichi Yanagisawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 483 (1) 380 - 386 0006-291X 2017/01 [Refereed][Not invited]
    Nitrate modulates growth and development, functioning as a nutrient signal in plants. Although many changes in physiological processes in response to nitrate have been well characterized as nitrate responses, the molecular mechanisms underlying the nitrate response are not yet fully understood. Here, we show that NLP transcription factors, which are key regulators of the nitrate response, directly activate the nitrate-inducible expression of BT1 and BT2 encoding putative scaffold proteins with a plant-specific domain structure in Arabidopsis. Interestingly, the 35S promoter-driven expression of BT2 partially rescued growth inhibition caused by reductions in NLP activity in Arabidopsis. Furthermore, simultaneous disruption of BT1 and BT2 affected nitrate-dependent lateral root development. These results suggest that direct activation of BT1 and BT2 by NLP transcriptional activators is a key component of the molecular mechanism underlying the nitrate response in Arabidopsis. (C) 2016 Elsevier Inc. All rights reserved.
  • Thais Huarancca Reyes, Andrea Scartazza, Yu Lu, Junji Yamaguchi, Lorenzo Guglielminetti
    PLANT PHYSIOLOGY AND BIOCHEMISTRY 105 195 - 202 0981-9428 2016/08 [Refereed][Not invited]
    Carbon (C) and nitrogen (N) nutrient sources are essential elements for metabolism, and their availability must be tightly coordinated for the optimal growth and development in plants. Plants are able to sense and respond to different C/N conditions via specific partitioning of C and N sources and the regulation of a complex cellular metabolic activity. We studied how the interaction between C and N signaling could affect carbohydrate metabolism, soluble sugar levels, photochemical efficiency of photosystem II (PSII) and the ability to drive the excess energy in Arabidopsis seedlings under moderated and disrupted C/N nutrient conditions. Invertase and sucrose synthase activities were markedly affected by C/N-nutrient status depending on the phosphorylation status, suggesting that these enzymes may necessarily be modulated by their direct phosphorylation or phosphorylation of proteins that form complex with them in response to C/N stress. In addition, the enzymatic activity of these enzymes was also correlated with the amount of sugars, which not only act as substrate but also as signaling compounds. Analysis of chlorophyll fluorescence in plants under disrupted C/N condition suggested a reduction of electron transport rate at PSII level associated with a higher capacity for non-radiative energy dissipation in comparison with plants under moderated C/N condition. In conclusion, the tight coordination between C and N not only affects the carbohydrates metabolism and their concentration within plant tissues, but also the partitioning of the excitation energy at PSII level between radiative (electron transport) and non radiative (heat) dissipation pathways. (C) 2016 Elsevier Masson SAS. All rights reserved.
  • Yu Lu, Shigetaka Yasuda, Xingwen Li, Yoichiro Fukao, Takayuki Tohge, Alisdair R. Fernie, Chiaki Matsukura, Hiroshi Ezura, Takeo Sato, Junji Yamaguchi
    JOURNAL OF PROTEOMICS 143 254 - 264 1874-3919 2016/06 [Refereed][Not invited]
    The 14-3-3 proteins participate in many aspects of plant physiology by interacting with phosphorylated proteins and thereby regulating target protein functions. In Arabidopsis plant, the ubiquitin ligase ATL31 controls 14-3-3 stability via both direct interaction and ubiquitination, and this consequently regulates post-germinative growth in response to carbon and nitrogen nutrient availability. Since 14-3-3 proteins regulate the activities of many key enzymes related to nutrient metabolism, one would anticipate that they should play an essential role not only in vegetative but also in reproductive tissue. Because fruit yield largely depends on carbon and nitrogen availability and their utilization, the function of 14-3-3 proteins was analyzed in tomato fruit tissue. Here, we isolated and characterized an ubiquitin ligase SIATL31 (Solyc03g112340) from tomato and demonstrated that SIATL31 has ubiquitin ligase activity as well as interaction with tomato 14-3-3 proteins, suggesting the possibility that the SIATL31 functions as an ubiquitin ligase for 14-3-3 similarly to its Arabidopsis ortholog. Furthermore, we performed proteomic analysis of 14-3-3 interacting proteins and identified 106 proteins as putative 14-3-3 targets including key enzymes for carbon metabolism and photosynthesis. This 14-3-3 interactome result and available transcriptome profile suggest a considerable yet complex role of 14-3-3 proteins in tomato fruit tissue.Biological significance: Considerable cumulative evidence exists which implies that 14-3-3 proteins are involved in the regulation of plant primary metabolism. Here we provide the first report of 14-3-3 interactome analysis and identify putative 14-3-3 targets in tomato fruit tissue, which may be highly important given the documented metabolic shifts, which occur during fruit development and ripening. These data open future research avenues by which to understand the regulation of the role of post-translational regulation in tomato fruit development. (C) 2016 Elsevier B.V. All rights reserved.
  • Yu Lu, Junji Yamaguchi, Takeo Sato
    PLANT SIGNALING & BEHAVIOR 10 (12) e1048940  1559-2316 2015/12 [Refereed][Not invited]
    Due to their immobility, plants have developed sophisticated mechanisms to robustly monitor and appropriately respond to dynamic changes in nutrient availability. Carbon (C) and nitrogen (N) are especially important in regulating plant metabolism and development, thereby affecting crop productivity. In addition to their independent utilization, the ratio of C to N metabolites in the cell, referred to as the C/N balance, is important for the regulation of plant growth, although molecular mechanisms mediating C/N signaling remain unclear. Recently ABI1, a protein phosphatase type 2C (PP2C), was shown to be a regulator of C/N response in Arabidopsis plants. ABI1 functions as a negative regulator of abscisic acid (ABA) signal transduction. ABA is versatile phytohormone that regulates multiple aspects of plant growth and adaptation to environmental stress. This review highlights the regulation of the C/N response mediated by a non-canonical ABA signaling pathway that is independent of ABA biosynthesis, as well as recent findings on the direct crosstalk between multiple cellular signals and the ABA signaling cascade.
  • Yu Lu, Yuki Sasaki, Xingwen Li, Izumi C. Mori, Takakazu Matsuura, Takashi Hirayama, Takeo Sato, Junji Yamaguchi
    Journal of Experimental Botany 66 (15) 4851  1460-2431 2015/08/01 [Refereed][Not invited]
  • Yu Lu, Yuki Sasaki, Xingwen Li, Izumi C. Mori, Takakazu Matsuura, Takashi Hirayama, Takeo Sato, Junji Yamaguchi
    JOURNAL OF EXPERIMENTAL BOTANY 66 (15) 4851 - 4851 0022-0957 2015/08 [Refereed][Not invited]
  • Yuya Suzuki, Toshihiro Arae, Pamela J. Green, Junji Yamaguchi, Yukako Chiba
    PLANT AND CELL PHYSIOLOGY 56 (5) 863 - 874 0032-0781 2015/05 [Refereed][Not invited]
    Removing the poly(A) tail is the first and rate-limiting step of mRNA degradation and apparently an effective step not only for modulating mRNA stability but also for translation of many eukaryotic transcripts. Carbon catabolite repressor 4 (CCR4) has been identified as a major cytoplasmic deadenylase in Saccharomyces cerevisiae. The Arabidopsis thaliana homologs of the yeast CCR4, AtCCR4a and AtCCR4b, were identified by sequence-based analysis; however, their role and physiological significance in plants remain to be elucidated. In this study, we revealed that AtCCR4a and AtCCR4b are localized to cytoplasmic mRNA processing bodies, which are specific granules consisting of many enzymes involved in mRNA turnover. Double mutants of AtCCR4a and AtCCR4b exhibited tolerance to sucrose application but not to glucose. The levels of sucrose in the seedlings of the atccr4a/4b double mutants were reduced, whereas no difference was observed in glucose levels. Further, amylose levels were slightly but significantly increased in the atccr4a/4b double mutants. Consistent with this observation, we found that the transcript encoding granule-bound starch synthase 1 (GBSS1), which is responsible for amylose synthesis, is accumulated to a higher level in the atccr4a/4b double mutant plants than in the control plants. Moreover, we revealed that GBSS1 has a longer poly(A) tail in the double mutant than in the control plant, suggesting that AtCCR4a and AtCCR4b can influence the poly(A) length of transcripts related to starch metabolism. Our results collectively suggested that AtCCR4a and AtCCR4b are involved in sucrose and starch metabolism in A. thaliana.
  • Yu Lu, Yuki Sasaki, Xingwen Li, Izumi C. Mori, Takakazu Matsuura, Takashi Hirayama, Takeo Sato, Junji Yamaguchi
    JOURNAL OF EXPERIMENTAL BOTANY 66 (9) 2763 - 2771 0022-0957 2015/05 [Refereed][Not invited]
    ABI1 was identified as the corresponding gene of the C/N-nutrient response mutant cni2-D. This study provides a new insight into the cross-talk between C/N and ABA signalling under the control of ABI1.Plants are able to sense and mediate the balance between carbon (C) and nitrogen (N) nutrient availability to optimize metabolism and growth, described as the C/N response. To clarify the C/N signalling mechanism, C/N-insensitive plants were obtained from an Arabidopsis FOX hunting population, which over-expresses full-length cDNAs for individuals. The resulting cni2-D (carbon/nitrogen insensitive 2-dominant) plant was found to overcome the post-germination growth checkpoint and to expand green cotyledons in disrupted high C/low N stress conditions. The CNI2 gene encodes ABI1, a phosphatase type 2C protein, which negatively regulates abscisic acid (ABA) signal transduction. Over-expressors of ABI1 were found to be insensitive to disrupted C/N stress, whereas the loss-of function mutant abi1-2 was hypersensitive, suggesting that ABI1 plays an essential role in the plant C/N response. By contrast, the C/N-dependent growth phenotype observed in wild-type plants was not associated with endogenous ABA content. Accordingly, the ABA-insensitive mutant abi1-1, which could not bind to the ABA-ABA receptor complex, was not insensitive and restored normal sensitivity to high C/low N stress. The canonical ABA signalling mutants abi4 and abi5 were also sensitive to disrupted C/N stress. Further gene expression analysis demonstrated that several genes in the SnRK2s and SnRK1s pathways are transcriptionally affected by high C/low N stress in wild-type plants regardless of the lack of increased endogenous ABA contents, whereas the expression of these genes were significantly suppressed in ABI1 over-expressors. Taken together, these results suggest direct cross-talk between C/N and non-canonical ABA signalling pathways, regulated by ABI1, in plants.
  • Thais Huarancca Reyes, Shugo Maekawa, Takeo Sato, Junji Yamaguchi
    PLANT BIOTECHNOLOGY 32 (1) 11 - + 1342-4580 2015/03 [Refereed][Not invited]
    ATL31, an Arabidopsis RING-type ubiquitin ligase, plays a critical role in plant carbon/nitrogen (C/N)-nutrient responses during post-germinative growth and in defense responses to pathogen attack. ATL31 expression under these stress conditions suggested the presence of transcriptional regulators mediated by these stress signals. We recently reported that the expression pattern of WRKY33, a transcription factor involved in plant defense responses, is highly correlated with that of ATL31. In this study, we investigated the detailed relationship between the ATL31 gene and WRKY33. Using transient reporter analysis, we found that WRKY33 could significantly activate ATL31 transcription in plant cells. Transcript analysis of stable transgenic Arabidopsis plants overexpressing WRKY33 confirmed that the expression of ATL31 in response to the PAMPs flg22 and chitin was enhanced compared with wild-type plants, while expression was repressed in wrky33 mutants. Further detailed transient reporter analysis revealed that transactivation by WRKY33 is required and mediated through a specific W-box cis-acting element in the promoter region of the ATL31 gene. In contrast, WRKY33 did not regulate ATL31 expression during the C/N response. Taken together, these results demonstrate that WRKY33 acts as a transcription factor of ATL31 and positively regulates its expression during activation of plant defense responses.
  • Shugo Maekawa, Atsushi Takabayashi, Thais Huarancca Reyes, Hiroko Yamamoto, Ayumi Tanaka, Takeo Sato, Junji Yamaguchi
    PLOS ONE 10 (2) e0117662  1932-6203 2015/02 [Refereed][Not invited]
    Arabidopsis ubiquitin ligases ATL31 and homologue ATL6 control the carbon/nitrogen nutrient and pathogen responses. A mutant with the loss-of-function of both atl31 and atl6 developed light intensity-dependent pale-green true leaves, whereas the single knockoutmutants did not. Plastid ultrastructure and Blue Native-PAGE analyses revealed that pale-green leaves contain abnormal plastid structure with highly reduced levels of thylakoid proteins. In contrast, the pale-green leaves of the atl31/atl6 mutant showed normal Fv/Fm. In the pale-green leaves of the atl31/atl6, the expression of HEMA1, which encodes the key enzyme for 5-aminolevulinic acid synthesis, the rate-limiting step in chlorophyll biosynthesis, was markedly down-regulated. The expression of key transcription factor GLK1, which directly promotes HEMA1 transcription, was also significantly decreased in atl31/atl6 mutant. Finally, application of 5-aminolevulinic acid to the atl31/atl6 mutants resulted in recovery to a green phenotype. Taken together, these findings indicate that the 5-aminolevulinic acid biosynthesis step was inhibited through the down-regulation of chlorophyll biosynthesis-related genes in the pale-green leaves of atl31/atl6 mutant.
  • Kaori Sako, Yuki Yanagawa, Tomoyuki Kanai, Takeo Sato, Motoaki Seki, Masayuki Fujiwara, Yoichiro Fukao, Junji Yamaguchi
    JOURNAL OF PROTEOME RESEARCH 13 (7) 3223 - 3230 1535-3893 2014/07 [Refereed][Not invited]
    The 26S proteasome is an ATP-dependent proteinase complex that is responsible for regulated proteolysis of polyubiquitinated proteins in eukaryotic cells. Here, we report novel 26S proteasome interacting proteins in Arabidopsis as revealed by LC-MS/MS analysis. We performed a two-step screening process that involved affinity purification of the 26S proteasome using Arabidopsis plants expressing a FLAG-tagged RPT2a subunit and partial purification of the 26S proteasome from cultured cells by glycerol density gradient centrifugation (GDG). Two plastid proteins, LTA2 and PDH E1 alpha, which were commonly identified by both affinity purification and GDG, interacted with the 26S proteasome both in vitro and in vivo, and the transit peptides of LTA2 and PDH E1 alpha were necessary for the interaction. Furthermore, the degradation of both LTA2 and PDH E1 alpha was inhibited by MG132, a proteasome inhibitor. Similar to those two proteins, 26S proteasome subunits RPT2a/b and RPT5a interacted with the transit peptides of three other chloroplast proteins, which are known to be substrates of the ubiquitin-26S proteasome system. These results suggest that a direct interaction between the 26S proteasome and a transit peptide is important for the degradation of unimported plastid protein precursors to maintain cellular homeostasis.
  • Shigetaka Yasuda, Takeo Sato, Shugo Maekawa, Shoki Aoyama, Yoichiro Fukao, Junji Yamaguchi
    JOURNAL OF BIOLOGICAL CHEMISTRY 289 (22) 15179 - 15193 0021-9258 2014/05 [Refereed][Not invited]
    Background: Ubiquitin ligase ATL31 and the target, 14-3-3 proteins, function in plant nutrient response. Results: ATL31 binds to 14-3-3 proteins via phosphorylation of specific residues. These residues are essential for the function of ATL31. Conclusion: ATL31 targets 14-3-3 proteins for degradation in a phosphorylation-dependent manner to regulate nutrient response. Significance: Phosphorylation of ubiquitin ligase ATL31 controls plant nutrient response. Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr(209), Ser(247), Ser(270), and Ser(303) as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr(209) and Ser(247) on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr(209) peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response.
  • Shoki Aoyama, Yu Lu, Junji Yamaguchi, Takeo Sato
    Plant Signaling and Behavior 9 (5) e28839  1559-2324 2014/04/16 [Refereed][Not invited]
    Elevated atmospheric CO2 concentration is a serious global environmental problem. Elevated CO2 affects plant growth by changing primary metabolism, closely related to carbon (C) and nitrogen (N) availability. Under sufficient N conditions, plant growth is dramatically promoted by elevated CO2. When N availability is limited, however, elevated CO2 disrupts the balance between cellular C and N (C/N). Disruption of the C/N balance is regarded as an important factor in plant growth defects. Here we highlight the regulation of senescence in higher plants by atmospheric CO2 and N, and the physiological function of C/N-related ubiquitin ligase ATL31 under condition of elevated CO2. We also provide an overview of the ubiquitin ligases and related enzymes involved in regulating senescence in plants. © 2014 Landes Bioscience.
  • Shugo Maekawa, Noriko Inada, Shigetaka Yasuda, Yoichiro Fukao, Masayuki Fujiwara, Takeo Sato, Junji Yamaguchi
    PLANT PHYSIOLOGY 164 (2) 879 - 887 0032-0889 2014/02 [Refereed][Not invited]
    The carbon/nitrogen (C/N) balance of plants is not only required for growth and development but also plays an important role in basal immunity. However, the mechanisms that link C/N regulation and basal immunity are poorly understood. We previously demonstrated that the Arabidopsis (Arabidopsis thaliana) Arabidopsis Toxicos en Levadura31 (ATL31) ubiquitin ligase, a regulator of the C/N response, positively regulates the defense response against bacterial pathogens. In this study, we identified the plasma membrane-localized soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor SYNTAXIN OF PLANTS121 (SYP121) as a novel ATL31 interactor. The syp121-1 loss-of-function mutant showed similar hypersensitivity to C/N stress conditions as the atl31 atl6 double mutant. SYP121 is essential for resistance to penetration by powdery mildew fungus and positively regulates the formation of cell wall appositions (papillae) at fungal entry sites. Microscopic analysis demonstrated that ATL31 was specifically localized around papillae. In addition, ATL31 overexpressors showed accelerated papilla formation, enhancing their resistance to penetration by powdery mildew fungus. Together, these data indicate that ATL31 plays an important role in connecting the C/N response with basal immunity by promoting papilla formation through its association with SYP121.
  • Shoki Aoyama, Thais Huarancca Reyes, Lorenzo Guglielminetti, Yu Lu, Yoshie Morita, Takeo Sato, Junji Yamaguchi
    PLANT AND CELL PHYSIOLOGY 55 (2) 293 - 305 0032-0781 2014/02 [Refereed][Not invited]
    Carbon (C) and nitrogen (N) are essential elements for metabolism, and their availability, called the C/N balance, must be tightly coordinated for optimal growth in plants. Previously, we have identified the ubiquitin ligase CNI1/ATL31 as a novel C/N regulator by screening plants grown on C/N stress medium containing excess sugar and limited N. To elucidate further the effect of C/N balance on plant growth and to determine the physiological function of ATL31, we performed C/N response analysis using an atmospheric CO2 manipulation system. Under conditions of elevated CO2 and sufficient N, plant biomass and total sugar and starch dramatically increased. In contrast, elevated CO2 with limited N did not increase plant biomass but promoted leaf chlorosis, with anthocyanin accumulation and increased senescence-associated gene expression. Similar results were obtained with plants grown in medium containing excess sugar and limited N, suggesting that disruption of the C/N balance affects senescence progression. In ATL31-overexpressing plants, promotion of senescence under disrupted CO2/N conditions was repressed, whereas in the loss-of-function mutant it was enhanced. The ATL31 gene was transcriptionally up-regulated under N deficiency and in senescent leaves, and ATL31 expression was highly correlated with WRKY53 expression, a key regulator of senescence. Furthermore, transient protoplast analysis implicated the direct activation of ATL31 expression by WRKY53, which was in accordance with the results of WRKY53 overexpression experiments. Together, these results demonstrate the importance of C/N balance in leaf senescence and the involvement of ubiquitin ligase ATL31 in the process of senescence in Arabidopsis.
  • Yosuke Maruyama, Natsuko Yamoto, Yuya Suzuki, Yukako Chiba, Ken-ichi Yamazaki, Takeo Sato, Junji Yamaguchi
    PLANT SCIENCE 213 79 - 87 0168-9452 2013/12 [Refereed][Not invited]
    Complex plant defenses that include the hypersensitive response (HR) are mediated by plant hormones, such as salicylic acid (SA), jasmonic acid (JA) and ethylene. We previously isolated the Arabidopsis DEAR1 (DREB AND EAR MOTIF PROTEIN 1) regulator and showed that its overexpression DEAR1 (DEAR1ox) resulted in a dwarf phenotype and lesion-like cell death, accompanied by elevated expression of PR (PATHOGENESIS-RELATED) genes. Here, we show that transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) has enhanced resistance to the necrotrophic fungus Botrytis cinerea (B. cinerea). This result indicates that DEAR1 represses negative regulators of plant defense responses, including transcriptional repressors belonging to the ERF (ETHYLEN RESPONSE FACTOR) family. Knockout mutants of ERF9 (erf9), which were down-regulated in DEAR1ox plants, showed transcriptional promotion of PDF1.2 (PATHOGEN-INDUCIBLE PLANT DEFENSIN) genes, which serve as positive markers for the ethylene/jasmonic acid (JA) signaling pathway and provide enhanced resistance to B. cinerea. Biochemical assays demonstrated that the ERF9 in capable of binding to the GCC box, a cis-element contained in the promoters of the PDF1.2 gene that possesses trans-repression activity. Moreover, infection with B. cinerea resulted in the promotion of the PDF1.2 expression, coinciding with suppression of the ERF9 gene under the control of the DEAR1 gene. These results indicate that the transcriptional repressor ERF9 participates in plant defense mechanisms against necrotic fungi mediated by the DEAR1-dependent ethylene/JA signaling pathway. (C) 2013 Elsevier Ireland Ltd. All rights reserved.
  • Hui H. Sun, Yoichiro Fukao, Sakiko Ishida, Hiroko Yamamoto, Shugo Maekawa, Masayuki Fujiwara, Takeo Sato, Junji Yamaguchi
    JOURNAL OF PROTEOME RESEARCH 12 (11) 5084 - 5095 1535-3893 2013/11 [Refereed][Not invited]
    The proteasome is a large multisubunit complex that plays a crucial role in the removal of damaged or selective ubiquitinated proteins, thereby allowing quality control of cellular proteins and restricted regulation of diverse cellular signaling in eukaryotic cells. Proteasome-dependent protein degradation is involved in almost all aspects of plant growth and responses to environmental stresses including pathogen resistance. Although the molecular mechanism for specifying targets by ubiquitin ligases is well understood, the detailed characterization of the plant proteasome complex remains unclear. One of the most important features of the plant proteasome is that most subunits are encoded by duplicate genes, suggesting the highly heterogeneous composition of this proteasome. Here, we performed affinity purification and a combination of 2-dimensional electrophoresis and mass spectrometry, which identified the detailed composition of paralogous and modified proteins. Moreover, these proteomics approaches revealed that specific subunit composition and proteasome peptidase activity were affected by pathogen-derived MAMPs, flg22 treatment. Interestingly, flg22 treatment did not alter mRNA expression levels of the peptidase genes PBA, PBB1/2, PBE1/2, and total proteasome levels remained unchanged by flg22 as well. These results demonstrate the finely tuned mechanism that regulates proteasome function via putative post-translational modifications in response to environmental stress in plants.
  • Yukako Chiba, Katsuhiko Mineta, Masami Y. Hirai, Yuya Suzuki, Shigehiko Kanaya, Hiro Takahashi, Hitoshi Onouchi, Junji Yamaguchi, Satoshi Naito
    Plant and Cell Physiology 54 (2) 181 - 194 1471-9053 2013 [Refereed][Not invited]
    Control of mRNA half-life is a powerful strategy to adjust individual mRNA levels to various stress conditions, because the mRNA degradation rate controls not only the steady-state mRNA level but also the transition speed of mRNA levels. Here, we analyzed mRNA half-life changes in response to cold stress in Arabidopsis cells using genome-wide analysis, in which mRNA half-life measurements and transcriptome analysis were combined. Half-lives of average transcripts were determined to be elongated under cold conditions. Taking this general shift into account, we identified more than a thousand transcripts that were classified as relatively stabilized or relatively destabilized. The relatively stabilized class was predominantly observed in functional categories that included various regulators involved in transcriptional, post-transcriptional and post-translational processes. On the other hand, the relatively destabilized class was enriched in categories related to stress and hormonal response proteins, supporting the idea that rapid decay of mRNA is advantageous for swift responses to stress. In addition, pentatricopeptide repeat, cyclin-like F-box and Myb transcription factor protein families were significantly over-represented in the relatively destabilized class. The global analysis presented here demonstrates not only the importance of mRNA turnover control in the cold stress response but also several structural characteristics that might be important in the control of mRNA stability. © 2011 The Author 2012. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved.
  • Shugo Maekawa, Takeo Sato, Yutaka Asada, Shigetaka Yasuda, Midori Yoshida, Yukako Chiba, Junji Yamaguchi
    PLANT MOLECULAR BIOLOGY 79 (3) 217 - 227 0167-4412 2012/06 [Refereed][Not invited]
    In higher plants, the metabolism of carbon (C) and nitrogen nutrients (N) is mutually regulated and referred to as the C and N balance (C/N). Plants are thus able to optimize their growth depending on their cellular C/N status. Arabidopsis and encode a RING-type ubiquitin ligases which play a critical role in the C/N status response (Sato et al. in Plant J 60:852-864, 2009). Since many ATL members are involved in the plant defense response, the present study evaluated whether the C/N response regulators ATL31 and ATL6 are involved in defense responses. Our results confirmed that and expression is up-regulated with the microbe-associated molecular patterns elicitors flg22 and chitin as well as with infections with pv. DC3000 ( DC3000). Moreover, transgenic plants overexpressing and displayed increased resistance to DC3000. In accordance with these data, loss of ATL31 and ATL6 function in an double knockout mutant resulted in reduced resistance to DC3000. In addition, the molecular cross-talk between C/N and the defense response was investigated by mining public databases. The analysis identified the transcription factors MYB51 and WRKY33, which are involved in the defense response, and their transcripts levels correlate closely with and . Further study demonstrated that the expression of , and defense marker genes including and were regulated by C/N conditions. Taken together, these results indicate that ATL31 and ATL6 function as key components of both C/N regulation and the defense response in Arabidopsis.
  • Kaori Sako, Yuko Maki, Tomoyuki Kanai, Eriko Kato, Shugo Maekawa, Shigetaka Yasuda, Takeo Sato, Masaaki K. Watahiki, Junji Yamaguchi
    PLOS ONE 7 (5) e37086  1932-6203 2012/05 [Refereed][Not invited]
    The ubiquitin/proteasome pathway plays a crucial role in many biological processes. Here we report a novel role for the Arabidopsis 19S proteasome subunit RPT2a in regulating gene activity at the transcriptional level via DNA methylation. Knockout mutation of the RPT2a gene did not alter global protein levels; however, the transcriptional activities of reporter transgenes were severely reduced compared to those in the wild type. This transcriptional gene silencing (TGS) was observed for transgenes under control of either the constitutive CaMV 35S promoter or the cold-inducible RD29A promoter. Bisulfite sequencing analysis revealed that both the transgene and endogenous RD29A promoter regions were hypermethylated at CG and non-CG contexts in the rpt2a mutant. Moreover, the TGS of transgenes driven by the CaMV 35S promoters was released by treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine, but not by application of the inhibitor of histone deacetylase Trichostatin A. Genetic crosses with the DNA methyltransferase met1 single or drm1drm2cmt3 triple mutants also resulted in a release of CaMV 35S transgene TGS in the rpt2a mutant background. Increased methylation was also found at transposon sequences, suggesting that the 19S proteasome containing AtRPT2a negatively regulates TGS at transgenes and at specific endogenous genes through DNA methylation.
  • Proteomics approach to study metabolic regulation mediated by ubiquitin-proteasome system in Arabidopsis
    Yasuda S, Maekawa S, Sato T, Yamaguchi J
    Frontiers of Agriculture Proteome Research 39 - 44 2012 [Refereed][Not invited]
  • Sun Huihui, Sako Kaori, Suzuki Yuya, Maekawa Shugo, Yasuda Shigetaka, Chiba Yukako, Sato Takeo, Yamaguchi Junji
    PLANT BIOTECHNOLOGY 29 (3) 279 - 284 1342-4580 2012 [Refereed][Not invited]
  • Takeo Sato, Shugo Maekawa, Shigetaka Yasuda, Yukie Domeki, Kuni Sueyoshi, Masayuki Fujiwara, Yoichiro Fukao, Derek B. Goto, Junji Yamaguchi
    PLANT JOURNAL 68 (1) 137 - 146 0960-7412 2011/10 [Refereed][Not invited]
    The balance between carbon (C) and nitrogen (N) availability is an important determinant for various phases of plant growth; however, the detailed mechanisms regulating the C/N response are not well understood. We previously described two related ubiquitin ligases, ATL31 and ATL6, that function in the C/N response in Arabidopsis thaliana. Here, we used FLAG tag affinity purification and MS analysis to identify proteins targeted by ATL31, and thus likely to be involved in regulating the phase transition checkpoint based on C/N status. This analysis revealed that 14-3-3 proteins were associated with ATL31, and one of these, 14-3-3 chi, was selected for detailed characterization. The interaction between ATL31 and 14-3-3 chi was confirmed by yeast two-hybrid and co-immunoprecipitation analyses. In vitro assays showed that ubiquitination of 14-3-3 chi is catalyzed by ATL31. Degradation of 14-3-3 chi in vivo was shown to be correlated with ATL31 activity, and to occur in a proteasome-dependent manner. Furthermore, 14-3-3 protein accumulation was induced by a shift to high-C/N stress conditions in Arabidopsis seedlings, and this regulated response required both ATL31 and ATL6. It was also shown that over-expression of 14-3-3 chi leads to hypersensitivity of Arabidopsis seedlings to C/N stress conditions. These results indicate that ATL31 targets and ubiquitinates 14-3-3 proteins for degradation via the ubiquitin-proteasome system during the response to cellular C/N status.
  • Emiko Okubo-Kurihara, Takumi Higaki, Yukio Kurihara, Natsumaro Kutsuna, Junji Yamaguchi, Seiichiro Hasezawa
    JOURNAL OF PLANT RESEARCH 124 (3) 395 - 403 0918-9440 2011/05 [Not refereed][Not invited]
    Sucrose plays an important role in several cellular processes since it is a general source of metabolic energy, serves as a precursor for starch and cellulose synthesis, and is a metabolic starting point for carboxylate- and amino acid synthesis. While plant vacuole is the main cellular storage pool, where sucrose accumulates to high concentrations, only a small number of vacuolar sugar transporters have been identified and characterized to date. We initially identified a vacuolar sucrose transporter (NtSUT4) from tobacco BY-2 cells and established transgenic tobacco BY-2 cell lines that overexpress NtSUT4-GFP (BY-SUTG cells). Using a model system for synchronous cell elongation in miniprotoplasts (evacuolated cells) prepared from tobacco BY-2 cells, we found that NtSUT4-GFP overexpression inhibited cell growth towards the cell major axis. Moreover, under the same conditions, we found that the cell walls were well stained by calcofluor in BY-SUTG cells than in wild type BY-2 cells. These results suggest that NtSUT4 is involved in cell shape via sucrose homeostasis in plant cells.
  • Takuya Sakamoto, Takehiro Kamiya, Kaori Sako, Junji Yamaguchi, Mutsumi Yamagami, Toru Fujiwara
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 (3) 561 - 567 0916-8451 2011/03 [Refereed][Not invited]
    RPTs (regulatory particle triple-A-ATPase) are components of 26S proteasome. We found novel roles of RPT2a and RPT5a in Zn deficiency-tolerance. Arabidopsis thaliana mutants carrying T-DNA in RPT2a and RPT5a were more sensitive to Zn deficiency than the wild-type. In the;pi mutants, the shoot Zn contents were similar to those Or the wild-type. Transcripts of Zn deficiency-inducible genes were highly accumulated in the rpt mutants, suggesting that the rpt mutants suffer from various Zn deficiency symptoms, although the Zn levels are not reduced. Lipid peroxidation levels, known to be increased under Zn deficiency, were higher in the rpt mutants than in the wild-type. Poly-ubiquitinated proteins were accumulated upon exposure to Zn deficiency, especially in the rpt mutants. Overall, this study indicates that RPT2a and RPT5a are involved in Zn deficiency-tolerance, possibly through alleviation of oxidative stresses and/or processing of poly-ubiquitinated proteins.
  • Yutaka Asada, Masako Yamamoto, Tomokazu Tsutsui, Junji Yamaguchi
    PLANT BIOTECHNOLOGY 28 (1) 9 - 15 1342-4580 2011 [Not refereed][Not invited]
    The Arabidopsis nsl2 (necrotic spotted lesion 2) mutant, which has been originally reported as the cad1 (constitutively activated cell death 1), shows a phenotype that mimics a hypersensitive response (HR)-like cell death. The NSL2 protein is suggested to negatively control the salicylic acid (SA)-mediated pathway of HR-like cell death in plant immunity. The induction of systemic acquired resistance (SAR) results in the induction of pathogenesis-related (PR) genes in systemic organs triggered by the local HR. In this report, we establish a NSL2 knockdown system in transgenic Arabidopsis based on constitutive or dexamethasone (DEX)-induced RNAi. The former showed a nsl2-like phenotype. In DEX-induced RNAi, localized knockdown resulted in the induction of PR1 gene expression and the restriction of bacterial growth in both DEX-treated and systemic leaves. These results indicate that NSL2 negatively controls SAR via HR.
  • Plant and Signaling Behavior 6 1465 - 1468 2011 [Not refereed][Not invited]
  • Takeo Sato, Shugo Maekawa, Shigetaka Yasuda, Junji Yamaguchi
    Plant Signaling and Behavior 6 (10) 1465 - 1468 1559-2324 2011 [Refereed][Not invited]
    The ubiquitin-proteasome system (UPS) is a unique protein degradation mechanism conserved in the eukaryotic cell. In addition to the control of protein quality, UPS regulates diverse cellular signal transduction via the fine-tuning of target protein degradation. Protein ubiquitylation and subsequent degradation by the 26S proteasome are involved in almost all aspects of plant growth and development and response to biotic and abiotic stresses. Recent studies reveal that the UPS plays an essential role in adaptation to carbon and nitrogen availability in plants. Here we highlight ubiquitin ligase ATL31 and the homolog ATL6 target 14-3-3 proteins for ubiquitylation to be degraded, which control signaling for carbon and nitrogen metabolisms and C/N balance response. We also give an overview of the UPS function involved in carbon and nitrogen metabolisms. © 2011 Landes Bioscience.
  • Kaori Sako, Yuko Maki, Kumiko K. Imai, Takashi Aoyama, Derek B. Goto, Junji Yamaguchi
    JOURNAL OF PLANT RESEARCH 123 (5) 701 - 706 0918-9440 2010/09 [Not refereed][Not invited]
    The ubiquitin/26S proteasome pathway plays a central role in the degradation of short-lived regulatory proteins to control many cellular events. The Arabidopsis knockout mutant rpt2a, which contains a defect in the AtRPT2a subunit of the 26S proteasome regulatory particle, showed enlarged leaves caused by increased cell size that correlated with increased ploidy caused by extended endoreduplication. To clarify the role of RPT2a in endoreduplication control, trichome development was genetically examined in further detail. RHL1 and GL3 encode proteins that have a role in the positive regulation of endocycle progression in trichomes. The rhl1 mutants are stalled at 8C and have trichomes with only a single branch. The rpt2a mutation did not alter the rhl1 mutant phenotype, and trichomes of double rpt2a rhl1 mutants resembled that of single rhl1 mutants. On the other hand, the rpt2a mutation suppressed the gl3 phenotype (stalled at 16C, two trichome branches), and trichomes of the double rpt2a gl3 mutant resembled those of the wild type (WT) plants. Together, these data suggest that RPT2a functions to negatively regulate endocycle progression following completion of the third endoreduplication step mediated by RHL1 (8C-16C).
  • Kaori Sako, Junji Yamaguchi
    Plant Signaling and Behavior 5 (9) 1119 - 1120 1559-2316 2010/09 [Refereed][Not invited]
    The ubiquitin/26S proteasome pathway plays a central role in the degradation of short-lived regulatory proteins to control many cellular events. The Arabidopsis genome contains two genes, AtRPT2a and AtRPT2b, which encode paralog molecules of the RPT2 subunit of 19S proteasome. We demonstrated that mutation of the AtRPT2a gene causes a specific phenotype of enlarged leaves due to increased cell size in correlation with expanded endoreduplication. This phenotype was also observed in the knockout mutant of AtRPT5a, which encodes one of the paralogs of the RPT5 subunit. Taken together, this suggests that a cell size-specific proteasome consisting of AtRPT2a and AtRPT5a is involved in controlling cell size during leaf development. © 2010 Landes Bioscience.
  • 佐久間 太, 植原 健人, 近藤 則夫, 山口 淳二
    Nematological Research 40 (1) 15 - 19 0919-6765 2010 [Not refereed][Not invited]
  • Sato T, Maekawa S, Yasuda S, Sonoda Y, Katoh E, Ichikawa T, Nakazawa M, Seki M, Shinozaki K, Matsui M, Goto DB, Ikeda A, Yamaguchi J
    The Plant journal : for cell and molecular biology 60 (5) 852 - 864 0960-7412 2009/12 [Refereed][Not invited]
  • Tomokazu Tsutsui, Wataru Kato, Yutaka Asada, Kaori Sako, Takeo Sato, Yutaka Sonoda, Satoshi Kidokoro, Kazuko Yamaguchi-Shinozaki, Masanori Tamaoki, Keita Arakawa, Takanari Ichikawa, Miki Nakazawa, Motoaki Seki, Kazuo Shinozaki, Minami Matsui, Akira Ikeda, Junji Yamaguchi
    JOURNAL OF PLANT RESEARCH 122 (6) 633 - 643 0918-9440 2009/11 [Not refereed][Not invited]
    Plants have evolved intricate mechanisms to respond and adapt to a wide variety of biotic and abiotic stresses in their environment. The Arabidopsis DEAR1 (DREB and EAR motif protein 1; At3g50260) gene encodes a protein containing significant homology to the DREB1/CBF (dehydration-responsive element binding protein 1/C-repeat binding factor) domain and the EAR (ethylene response factor-associated amphiphilic repression) motif. We show here that DEAR1 mRNA accumulates in response to both pathogen infection and cold treatment. Transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) showed a dwarf phenotype and lesion-like cell death, together with constitutive expression of PR genes and accumulation of salicylic acid. DEAR1ox also showed more limited P. syringae pathogen growth compared to wild-type, consistent with an activated defense phenotype. In addition, transient expression experiments revealed that the DEAR1 protein represses DRE/CRT (dehydration-responsive element/C-repeat)-dependent transcription, which is regulated by low temperature. Furthermore, the induction of DREB1/CBF family genes by cold treatment was suppressed in DEAR1ox, leading to a reduction in freezing tolerance. These results suggest that DEAR1 has an upstream regulatory role in mediating crosstalk between signaling pathways for biotic and abiotic stress responses.
  • Yutaka Sonoda, Kaori Sako, Yuko Maki, Naoko Yamazaki, Hiroko Yamamoto, Akira Ikeda, Junji Yamaguchi
    PLANT JOURNAL 60 (1) 68 - 78 0960-7412 2009/10 [Refereed][Not invited]
    The ubiquitin/26S proteasome pathway plays a central role in the degradation of short-lived regulatory proteins, to control many cellular events. To further understand this pathway, we focused on the RPT2 subunit of the 26S proteasome regulatory particle. The Arabidopsis genome contains two genes, AtRPT2a and AtRPT2b, which encode paralog molecules of the RPT2 subunit, with a difference of only three amino acids in the protein sequences. Both genes showed similar mRNA accumulation patterns. However, the rpt2a mutant showed a specific phenotype of enlarged leaves caused by increased cell size, in correlation with increased ploidy. Detailed analyses revealed that cell expansion is increased in the rpt2a mutant by extended endoreduplication early in leaf development. The transcription of genes encoding cell cycle-related components, for DNA replication licensing and the G2/M phase, was also promoted in the rpt2a mutant, suggesting that extended endoreduplication was caused by increased DNA replication, and disrupted regulation of the G2/M checkpoint, at the proliferation stage of leaf development.
  • Takuto Tojo, Kenichi Tsuda, Takeshi Yoshizumi, Akira Ikeda, Junji Yamaguchi, Minami Matsui, Ken-ichi Yamazaki
    PLANT AND CELL PHYSIOLOGY 50 (2) 254 - 264 0032-0781 2009/02 [Not refereed][Not invited]
    Multiprotein bridging factor 1 (MBF1) is known as a transcriptional co-activator that enhances transcription of its target genes by bridging between transcription factors and TATA-box-binding protein in eukaryotes. Arabidopsis thaliana has three MBF1 genes: AtMBF1aAtMBF1c. However, details of the functions of AtMBF1 remain unclear. For this study, transgenic Arabidopsis overexpressing AtMBF1 fused to an active transcriptional repression domain (SRDX) was constructed. The chimeric protein putatively functions as a transcriptional co-repressor and as a suppressor of functions of endogenous AtMBF1in transgenic plants. Transgenic Arabidopsis overexpressing AtMBF1SRDX (AtMBF1-SRDX(OE)) showed an extremely small leaf phenotype under a continuous white light condition. Its leaf cellsespecially those around vascular tissues, where strong expression of endogenous AtMBF1s is observedwere much smaller than those from the wild type (WT). In addition, a lower cell number was observed in leaves from AtMBF1-SRDX(OE) plants. Time course analysis of cell size revealed that cell expansion of leaves of AtMBF1-SRDX(OE) plants was dramatically suppressed during the late leaf developmental stage (cell expansion stage), when endogenous AtMBF1b is strongly expressed in the WT. The results show that ploidy levels of leaves from AtMBF1-SRDX(OE) plants were dramatically lower than those from the WT; moreover, expression levels of several negative regulators of endoreduplication were more elevated in AtMBF1s-SRDX(OE) plants than those in the WT. These observations suggest that AtMBF1SRDX interacts with regulators of endoreduplication. Therefore, AtMBF1s are considered to affect not only leaf cell expansion but also regulation of the ploidy level in leaf cells during the leaf expansion stage.
  • Tomokazu Tsutsui, Yutaka Asada, Masanori Tamaoki, Akira Ikeda, Junji Yamaguchi
    PLANT SCIENCE 175 (4) 604 - 611 0168-9452 2008/10 [Not refereed][Not invited]
    The Arabidopsis cad1 (constitutively activated cell death 1) mutant shows a phenotype of hypersensitive response (HR)-like cell death and increased levels of endogenous salicylic acid (SA), indicating that the CAD1 protein negatively controls the SA-mediated pathway in plant immunity. To clarify the relationship between the CAD1 protein and SA-mediated defense pathways, a molecular genetic analysis was carried out using mutants related to SA biosynthesis. A genetic cross between the cad1 mutant and the pad4 and sid2 mutants only partially suppressed the cad1 phenotypes. These double mutants still showed acquired resistance to Pst DC3000 infection similar to the cad1 single mutant. These results indicate that the CAD1 negatively controls not only the SA-dependent defense pathway but also the SA-independent pathway. To clarify the CAD1-mediated SA-independent pathway, a revertant was isolated from an M2 population of the sid2 cad1 mutant. The revertant can1t mutant did not show cell death phenotype and the plant and leaf size recovered to those of the wild-type. The can1t mutant also showed pathogen susceptibility comparable to wild type, indicating that the can It mutant looses the pathogen-resistance that the cad I mutant acquires. There results suggested that CAN1 is a positive regulator of the novel plant immunity under the control of CAD1. (c) 2008 Elsevier Ireland Ltd. All rights reserved.
  • Shan-Guo Yao, Yutaka Sonoda, Tomokazu Tsutsui, Hidemitsu Nakamura, Hiroaki Ichikawa, Akira Ikeda, Junji Yamaguchi
    BREEDING SCIENCE 58 (3) 201 - 207 1344-7610 2008/09 [Not refereed][Not invited]
    We previously reported two rice ammonium transporter (OsAMT) genes, OsAMT1;2 and OsAMT1;3, which show root-specific expression at the vegetative stage. To further understand the functional differences between the two genes, we used the promoter::gusA reporter gene to investigate their detailed expression patterns. We show that in transgenic rice plants carrying individual 4-kb promoter::gusA fusion genes, the expression of the two genes was confined to roots during the entire plant life cycle, suggesting the housekeeping roles of the two transporters in rice. Detailed observation of the above transgenic lines further shows that OsAMT1;3 was preferentially expressed in the apices of seminal and lateral roots under nitrogen-deficient conditions, and was repressed when supplied with ammonium. By contrast, OsAMT1;2 expression was induced most intensely in the root elongation zone under nitrogen-sufficient conditions. These observations suggest that the two genes have distinct roles in nitrogen utilization: OsAMT1;3 appears to function as a nitrogen sensor and OsAMT1;2 as an assimilator. In contrast to 4-kb promoters, gusA-reporter expression was not detected in the roots under both nitrogen-deficient and -sufficient conditions when using individual 2-kb promoter sequences proximal to transcription initiation sites for the two genes, indicating that distal 2-kb promoter sequences are essential for the root-specific expression and conditional responsiveness to nitrogen of OsAMT1;2 and OsAMT1;3 genes.
  • Shin-ichiro Mitsunaga, Midori Kobayashi, Satoe Fukui, Kayoko Fukuoka, Osamu Kawakami, Junji Yamaguchi, Masahiro Ohshima, Toshiaki Mitsui
    PLANT PHYSIOLOGY AND BIOCHEMISTRY 45 (12) 922 - 925 0981-9428 2007/12 [Not refereed][Not invited]
    The hydrolytic enzyme v.-amylase (EC is produced mainly in aleurone cells of germinating cereals, and the phytohormone gibberellin (GA) is essential for its induction. However, in rice (Oryza sativa L.), sulfuric acid (H2SO4) induces alpha-amylase production in aleurone tissue even in the absence of GA. Here, the pre-treatment of rice aleurone cells with H2SO4 and incubation in water induced a-amylase activity, as if the cells had been incubated in GA solution. (C) 2007 Elsevier Masson SAS. All rights reserved.
  • Yutaka Sonoda, Shan-Guo Yao, Kaori Sako, Takeo Sato, Wataru Kato, Masa-aki Ohto, Takanari Ichikawa, Minami Matsui, Junji Yamaguchi, Akira Ikeda
    PLANT JOURNAL 50 (4) 586 - 596 0960-7412 2007/05 [Refereed][Not invited]
    Post-embryonic plant growth is dependent on a functional shoot apical meristem (SAM) that provides cells for continuous development of new aerial organs. However, how the SAM is dynamically maintained during vegetative development remains largely unclear. We report here the characterization of a new SAM maintenance mutant, sha1-1 (shoot apical meristem arrest 1-1), that shows a primary SAM-deficient phenotype at the adult stage. The SHA1 gene encodes a novel RING finger protein, and is expressed most intensely in the shoot apex. We show that, in the sha1-1 mutant, the primary SAM develops normally during the juvenile vegetative stage, but cell layer structure becomes disorganized after entering the adult vegetative stage, resulting in a dysfunctional SAM that cannot initiate floral primordia. The sha1-1 SAM terminates completely at the stage when the wild-type begins to bolt, producing adult plants with a primary inflorescence-deficient phenotype. These observations indicate that SHA1, a putative E3 ligase, is required for post-embryonic SAM maintenance by controlling proper cellular organization.
  • Tolllokazu Tsutsui, Chlzuko Morita-Yamamuro, Yutaka Asada, Ellchl Minami, Naoto Shibuya, Aklra Ikeda, Junji Yamaguchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 70 (9) 2042 - 2048 0916-8451 2006/09 [Not refereed][Not invited]
    The Arabidopsis mutant cad1 (constitutively activated cell death 1) shows a phenotype that mimics hypersensitive response (HR)-like cell death. The CAD1 gene, which encodes a protein containing a domain with significant homology to the MACPF (membrane attach complex and perforin) domain of complement components and perforin, is likely to control plant immunity negatively and has a W-box cis-element in its promoter region. We found that expression of the CAD1 gene and other W-box containing genes, such as NPR1 and PR2, was promoted by salicylic acid (SA) and benzothiadiazole (BTH) as a SA agonisf. The CAD1 gene was also stimulated by a purified chitin oligosaccharide elicitor (degree of polymerization = 8). This latter control was not under SA, because CAD1 expression was not suppressed in 35SnahG transgenic plants, which are unable to accumulate SA. These expression profiles were confirmed by promoter analysis using pCAD1::GUS transgenic plants. The CAD1 expression promoted by BTH and the chitin elicitor was not suppressed in the npr1 mutant, which is insensitive to SA signaling. These results indicate that the CAD1 gene is regulated by two distinct pathways involving SA and a chitin elicitor: viz., SA signaling mediated through an NPR1-independent pathway, and chitin elicitor signaling, through an SA-independent pathway. Three CAD1 homologs that have multiple W-box elements in their promoters were also found to be under the control of SA.
  • L. Guglielminetti, A. Morita, J. Yamaguchi, E. Loreti, P. Perata, A. Alpi
    JOURNAL OF PLANT RESEARCH 119 (4) 351 - 356 0918-9440 2006/07 [Not refereed][Not invited]
    Fructokinases (EC may play an important role in carbohydrate metabolism of Oryza sativa L. (rice) seedlings under anoxia. We present here the molecular and biochemical characterizations of two rice fructokinases, namely OsFK1 and OsFK2. The results show that, at both a transcriptional and a transductional level, OsFK1 is preferentially expressed under aerobic conditions, whereas OsFK2 is induced under anoxia. Substrate inhibition was demonstrated for OsFK1, while OsFK2 appears to be largely unaffected by fructose concentrations up to 10 mM. Sugar modulation of anoxia-induced proteins has been proposed, but our results on rice calli treated with or without glucose (10, 30 or 90 mM) for different time indicate that neither OsFK1 nor OsFK2 are sugar-regulated. We propose that OsFK2 plays a major role in fructose phosphorylation under anoxic conditions.
  • H Saika, M Nakazono, A Ikeda, J Yamaguchi, S Masaki, M Kanekatsu, K Nemoto
    PLANT SCIENCE 169 (1) 239 - 244 0168-9452 2005/07 [Not refereed][Not invited]
    Although germinating rice seeds typically show high beta-amylase activity, some japonica rice cultivars have almost no beta-amylase activity in the seedlings. This study was conducted to clarify the genetic basis for this suppression of beta-amylases in germinating rice seeds. Quantitative trait locus (QTL) analysis using 'Nipponbare' (a japonica cultivar with very low beta-amylase activity) x 'Kasalath' (an indica cultivar with normal beta-amylase activity) backcross inbred lines revealed a major QTL (R-2 = 0.64) at the marker interval C451-R1357 on chromosome 7, where the 'Nipponbare' allele dramatically decreased beta-amylase activity. According to databases, this region is syntenic with the barley Bmy2 beta-amylase locus, and carries two rice beta-amylase genes (BAC83770 and BAC83773) in tight linkage. An RT-PCR experiment showed that 'Nipponbare' has suppressed BAC83773 expression (less than 1% that of 'Kasalath') but normal BAC83770 expression, indicating that BAC83773 is a candidate for the QTL. Insertion of a Mutator-like transposon in the BAC83773 promoter region was considered responsible for this suppression in 'Nipponbare' BAC83773 expression. (c) 2005 Elsevier Ireland Ltd. All rights reserved.
  • C Morita-Yamamuro, T Tsutsui, M Sato, H Yoshioka, M Tamaoki, D Ogawa, H Matsuura, T Yoshihara, A Ikeda, Uyeda, I, J Yamaguchi
    PLANT AND CELL PHYSIOLOGY 46 (6) 902 - 912 0032-0781 2005/06 [Refereed][Not invited]
    To clarify the processes involved in plant immunity, we have isolated and characterized a single recessive Arabidopsis mutant, cad1 (constitutively activated cell death 1), which shows a phenotype that mimics the lesions seen in the hypersensitive response (HR). This mutant shows spontaneously activated expression of pathogenesis-related (PR) genes, and leading to a 32-fold increase in salicylic acid (SA). Inoculation of cad1 mutant plants with Pseudomonas syringae pv tomato DC3000 shows that the cad1 mutation results in the restriction of bacterial growth. Cloning of CAD1 reveals that this gene encodes a protein containing a domain with significant homology to the MACPF (membrane attack complex and perforin) domain of complement components and perforin proteins that are involved in innate immunity in animals. Furthermore, cell death is suppressed in transgenic cad1 plants expressing nahG, which encodes an SA-degrading enzyme. We therefore conclude that the CAD1 protein negatively controls the SA-mediated pathway of programmed cell death in plant immunity.
  • MORITA‐YAMAMURO Chizuko, MORITA‐YAMAMURO Chizuko, TSUTSUI Tomokazu, SATO Masanao, YOSHIOKA Hirofumi, TAMAOKI Masanori, OGAWA Daisuke, MATSUURA Hideyuki, YOSHIHARA Teruhiko, IKEDA Akira, UYEDA Ichiro, YAMAGUCHI Junji, YAMAGUCHI Junji
    Plant Cell Physiology 46 (6) 902 - 912 0032-0781 2005 [Not refereed][Not invited]
  • 光永伸一郎, 川上修, 山口淳二, 三ツ井敏明
    Journal of Applied Glycoscience 52 (4) 399 - 402 1344-7882 2005 [Not refereed][Not invited]
  • C Morita-Yamamuro, T Tsutsui, A Tanaka, J Yamaguchi
    PLANT AND CELL PHYSIOLOGY 45 (6) 781 - 788 0032-0781 2004/06 [Not refereed][Not invited]
    To clarify the mechanism of sugar-response of higher plants, the ghs1 (glucose hypersensitive) mutant of Arabidopsis was isolated and characterized. The ghs1 mutant had an increased sensitivity to glucose, showing a dramatic inhibition of chlorophyll synthesis and developmental arrest of leaves when grown on medium containing more than 5% glucose; the wild type required exposure to 7% glucose to show the same response. The ghs1 mutant is a single recessive loss-of-function mutation caused by a T-DNA insertion in the GHS1 gene (At3g27160), which encodes the plastid 30S ribosomal protein S21. The mutant showed: (1) reduction in the translation product but not the transcript for plastid-encoded rbcL, (2) reduction in photosynthetic activity monitored with pulse-amplitude modulated fluorometry, (3) impaired chloroplast development, as observed by electron microscopy. These results indicate that the deficiency of such chloroplast functions as photosynthetic activity observed in the ghs1 mutant is caused by impaired plastid protein synthesis associated with loss of ribosomal S21 protein. Relationships between the GHS1 gene and sugar-response are discussed.
  • Chizuko Morita-Yamamuro, Paolo Vernieri, Junji Yamaguchi
    Plant Biotechnology 21 (1) 9 - 14 1342-4580 2004 [Not refereed][Not invited]
    Antagonistic interaction between endogenous sugar status and abscisic acid (ABA) signaling was examined in Arabidopsis. Endogenous ABA content in suspension-cultured cells and seedlings grown in the dark increased following reduction in endogenous sugar content. Expression of Arabidopsis 9-cis epoxycarotenoid dioxygenase (AtNCED) genes, which encode key enzymes for ABA biosynthesis and ABSCISIC ACID INSENSITIVE 3 (ABI 3) which controls ABA sensitivity was enhanced by sugar starvation. GUS activity analysis in transgenic Arabidopsis carrying GUS controlled by the ABI3 promoter showed that ABI3 was expressed, when sugar was depleted and thereby leaf development was terminated, in the shoot apical meristem. We conclude that sugar depletion promotes ABA signaling to terminate leaf development in Arabidopsis seedlings. Antagonistic effects of sugar on ABA signaling in the process of vegetative quiescence processs are discussed.
  • Y Sonoda, A Ikeda, S Saiki, T Yamaya, J Yamaguchi
    PLANT AND CELL PHYSIOLOGY 44 (12) 1396 - 1402 0032-0781 2003/12 [Not refereed][Not invited]
    The three members of the rice OsAMT1 gene family of ammonium transporters show distinct expression patterns; constitutive and ammonium-promoted expression in shoots and roots for OsAMT1;1; root-specific and ammonium-inducible expression for OsAMT1;2; root-specific and nitrogen-repressible expression for OsAMT1;3 [Sonoda et al. (2003), Plant Cell Physiol. 44: 726]. To clarify the feedback mechanisms, and to identify regulatory factors of the OsAMT1 genes, the accumulation of the three mRNAs and its dependence on endogenous nitrogen compounds (as quantified by capillary electrophoresis) was studied. Ammonium application to roots following a period of nitrogen starvation induced accumulation of OsAMT1;1 and OsAMT1;2 mRNA, but a decrease of OsAMT1;3 mRNA levels. The expression patterns of the three genes showed good correlation (positive in OsAMT1;1 and OsAMT1;2, negative in OsAMT1;3) with the root tissue contents of glutamine but not of ammonium. The ammonium effects on OsAMT1 expression were prevented by methionine sulfoximine, an inhibitor of glutamine synthetase. Moreover, glutamine had the same effect on transcriptional regulation of OsAMT1 genes as ammonium, indicating that glutamine rather than ammonium controls the expression of ammonium transporter genes in rice. These results imply that rice possesses unique mechanisms of adaptation to variable nitrogen sources in the soil.
  • Y Sonoda, A Ikeda, S Saiki, N von Wiren, T Yamaya, J Yamaguchi
    PLANT AND CELL PHYSIOLOGY 44 (7) 726 - 734 0032-0781 2003/07 [Not refereed][Not invited]
    To study the regulation of ammonium uptake into rice roots, three ammonium transporter genes (OsAMT];1, 1;2 and 1;3; Oryza sativa ammonium transporter) were isolated and examined. OsAMT1s belong to AMT1 family, containing 11 putative transmembrane-spanning domains. Southern blot analysis and screening of the rice genome database confirmed that with OsAMT1;1-1;3 the complete AMT1 family of rice had been isolated. Heterologous expression of OsAMT1s in the yeast Saccharomyces cerevisiae mutant 31019b showed that all three OsAMT1s exhibit ammonium transport activity. Northern blot analysis showed a distinct expression pattern for the three genes; more constitutive expression in shoots and roots for OsAMT1;1, root-specific and ammonium-inducible expression for OsAMT1;2, and root-specific and nitrogen-derepressible expression for OsAMT1;3. In situ mRNA detection revealed that OsAMT1; 2 is expressed in the central cylinder and cell surface of root tips. This gene expression analysis revealed a distinct nitrogen-dependent regulation for AMTs in rice, differing from that in tomato or Arabidopsis.
  • E Loreti, J Yamaguchi, A Alpi, P Perata
    PLANT AND SOIL 253 (1) 137 - 143 0032-079X 2003/06 [Not refereed][Not invited]
    Production of alpha-amylase during the germination of rice grains is thought to play an important role for tolerance to anoxia of these cereal grains. Under aerobic conditions alpha-amylases production is enhanced in response to gibberellins produced by the embryos, but the role of these hormones is less clear under anoxia. In this paper we analysed alpha-amylase gene expression in a rice mutant (Tan-ginbozu) severely impaired in gibberellin biosynthesis. Expression of alpha-amylase genes others than the gibberellin-induced Amy1A gene is observed. The expression of the Amy3D gene, which does dot require gibberellins to be induced, is high under anoxia in the Tan-ginbozu mutant suggesting that germination under anoxia can proceed thanks to the activity of the alpha-amylase isoform encoded by the Amy3D gene. Amy3D gene expression is repressed in the presence of high levels of soluble carbohydrates, indicating that the anaerobic expression of this gene can be triggered by a lower carbohydrate content of rice grains kept under anoxia. Germination under anoxia of Tan-ginbozu grains can proceed even in absence of exogenously-added gibberellic acid. Overall, results indicate that gibberellins are not required for the anaerobic germination of rice grains.
  • B Ngampanya, A Sobolewska, T Takeda, K Toyofuku, J Narangajavana, A Ikeda, J Yamaguchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 67 (3) 556 - 562 0916-8451 2003/03 [Not refereed][Not invited]
    cDNA of a monosaccharide transporter in rice, OsMST5 ((O) under bar ryza (s) under bar ativa (m) under bar onosaccharide (t) under bar ransporter 5) was cloned and its sugar transport activity was characterized by heterologous expression analysis. The amino acid sequence and topology were similar to the sequences and topology of other plant monosaccharide transporters. Yeast cells co-expressed with OsMST5 cDNA transported some monosaccharide substrates. The transport rate increased when ethanol as an electron donor was added, so the transporter was an energy-dependent active one. Most of the OsMST5 was expressed in panicles before pollination, indicating that it is associated with pollen development in rice.
  • S Otsuki, A Ikeda, T Sunako, S Muto, J Yazaki, K Nakamura, F Fujii, K Shimbo, Y Otsuka, K Yamamoto, K Sakata, T Sasaki, N Kishimoto, S Kikuchi, J Yamaguchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 67 (2) 347 - 353 0916-8451 2003/02 [Not refereed][Not invited]
    A cDNA encoding a predicted 15-kDa protein was earlier isolated from sugar-induced genes in rice embryos (Oryza sativa L.) by cDNA microarray analysis. Here we report that this cDNA encodes a novel Ca2+-binding protein, named OsSUR1 (for Oryza sativa sugar-up-regulated-1). The recombinant OsSUR1 protein expressed in Escherichia coli had Ca-45(2+)-binding activity. Northern analysis showed that the OsSUR1 gene was expressed mainly in the internodes of mature plants and in embryos at an early stage of germination. Expression of the OsSUR1 gene was induced by sugars that could serve as substrates of hexokinase, but expression was not repressed by Ca2+ signaling inhibitors, calmodulin antagonists and inhibitors of protein kinase or protein phosphatase. These results suggested that OsSUR1 gene expression was stimulated by a hexokinase-dependent pathway not mediated by Ca2+.
  • A Suenaga, K Moriya, Y Sonoda, A Ikeda, N von Wiren, T Hayakawa, J Yamaguchi, T Yamaya
    PLANT AND CELL PHYSIOLOGY 44 (2) 206 - 211 0032-0781 2003/02 [Not refereed][Not invited]
    To characterize ammonium transport pathways in rice, two cDNAs with high homology to MEP/AMT2-type ammonium transporters, OsAMT2;1 and OsAMT3;1, were isolated. Expression of OsAMT2;1 in an ammonium-uptake-defective yeast mutant showed that this gene encodes functional ammonium transporters. OsAMT2;1 was constitutively expressed in both roots and shoots irrespective of the supply of inorganic nitrogen to the medium, whereas OsAMT3;1 expression was relatively weak. A database search with the amino acid sequence of OsAMT2;1 showed that there are 10 putative OsAMT genes in rice, i.e. three each for OsAMT1, OsAMT2 and OsAMT3, respectively, and one for OsAMT4.
  • E Loreti, J Yamaguchi, A Alpi, P Perata
    ANNALS OF BOTANY 91 (2) 143 - 148 0305-7364 2003/01 [Not refereed][Not invited]
    Tolerance to low oxygen availability is likely to be due to the interaction of several factors. Sugar availability is one of the elements required to support anaerobic metabolism. In cereal grains the availability of soluble sugars is limited, while starch is stored in large amounts. Degradation of starch under anoxia is therefore needed to avoid sugar starvation leading to rapid cell death. The striking difference in the ability to produce alpha-amylase when comparing the anoxia-tolerant rice (Oryza sativa L.) grains with grains of other cereals is not easily explained. Rice is able to respond to gibberellins under anoxia, but the response is too slow to explain the rapid production of alpha-amylase enzyme. In the present work we demonstrated that alpha-amylase production during the first 2 d after imbibition is mostly due to the activity of the Ramy3D gene, encoding for the G and H isoforms of alpha-amylase. The induction of Ramy3D transcription is likely to result from a low sugar content in the grains incubated under anoxia. The ability of rice embryos to sense sugars under anoxia is reported. (C) 2003 Annals of Botany Company.
  • Journal of Applied Glycoscience 50 (2) 237 - 240 1344-7882 2003 [Not refereed][Not invited]
  • A Ikeda, Y Sonoda, P Vernieri, P Perata, H Hirochika, J Yamaguchi
    PLANT AND CELL PHYSIOLOGY 43 (9) 974 - 979 0032-0781 2002/09 [Not refereed][Not invited]
    The slender rice (slr1-1) mutant, carrying a lethal and recessive single mutation, has a constitutive gibberellin (GA)-response phenotype and behaves as if it were saturated with GAs [Ikeda et al. (2001) Plant Cell 13, 999]. The SLR1 gene, with sequence homology to members of the plant-specific GRAS gene family, is a mediator of the GA signal transduction process. In the slender rice, GA-inducible a-amylase was produced from the aleurone layer without applying GA. GA-independent alpha-amylase production in the mutant was inhibited by applying abscisic acid (ABA). Shoot elongation in the mutant was also suppressed by ABA, indicating that the slender rice responds normally to ABA. Interestingly, shoot ABA content was 10-fold higher in the mutant than in the wild type, while there was no difference in root ABA content. Expression of the Rab16A gene, which is known to be ABA inducible, was about 10-fold higher in shoots of the mutant than in those of the wild type. These results indicate that constitutive activation of the GA signal transduction pathway by the slr1-1 mutation promotes the endogenous ABA level.
  • A Ikeda, M Ueguchi-Tanaka, Y Sonoda, H Kitano, M Koshioka, Y Futsuhara, M Matsuoka, J Yamaguchi
    PLANT CELL 13 (5) 999 - 1010 1040-4651 2001/05 [Not refereed][Not invited]
    The rice slender mutant (slr1-1) is caused by a single recessive mutation and results in a constitutive gibberellin (GA) response phenotype. The mutant elongates as if saturated with GAs, In this mutant, (1) elongation was unaffected by an inhibitor of GA biosynthesis, (2) GA-inducible a-amylase was produced by the aleurone layers without gibberellic acid application, and (3) endogenous GA content was lower than in the wild-type plant. These results indicate that the product of the SLR1 gene is an intermediate of the GA signal transduction pathway. SLR1 maps to OsGAI in rice and has significant homology with height-regulating genes, such as RHT-1Da in wheat, D8 in maize, and GAI and RGA in Arabidopsis, The GAI gene family is likely to encode transcriptional factors belonging to the GRAS gene superfamily. DNA sequence analysis revealed that the slr1-1 mutation is a single basepair deletion of the nuclear localization signal domain, resulting in a frameshift mutation that abolishes protein production. Furthermore, introduction of a 6-kb genomic DNA fragment containing the wild-type SLR1 gene into the slr1-1 mutant restored GA sensitivity to normal. These results indicate that the slr1-1 mutant is caused by a loss-of-function mutation of the SLR1 gene, which is an ortholog of GAI, RGA, RHT, and D8, We also succeeded in producing GA-insensitive dwarf rice by transforming wild-type rice with a modified SLR1 gene construct that has a 17-amino acid deletion affecting the DELLA region. Thus, we demonstrate opposite GA response phenotypes depending on the type of mutations in SLR1.
  • T Takeda, K Toyofuku, C Matsukura, J Yamaguchi
    JOURNAL OF PLANT PHYSIOLOGY 158 (4) 465 - 470 0176-1617 2001/04 [Not refereed][Not invited]
    Developing seeds offer a fine experimental system for study on sugar transport mechanism(s) in sink tissue. The sugar transporters identified as being specific to the seed development may play a crucial role for flowering and grain development. The rice sucrose transporter OsSUT1 and the monosaccharide transporters OsMST1-3 have been previously characterized. To investigate sugar transport processes during flowering and in developing grains of rice, we newly isolated two genomic clones OsSUT2 (Oryza Sativa sucrose transporter 2) and OsMST5 (Oryza sativa monosaccharide transporter 5) and their corresponding cDNAs. OsSUT2 and OsMST5 are encoded by open reading frames of 1485 and 1557 bp encoding 495 and 519 amino acids, respectively. The putative amino acid sequence of OsSUT2 showed 63.0 and 62.4% identity to that of OsSUT1 and barley transporter HvSUT1, respectively. Northern blot analysis revealed that OsSUT2 and OsMST5 mRNA accumulates in panicles before pollination. in situ hybridization analysis showed that OsSUT2 transcript is specific to the developing pollen. These data presented suggest that both OsSUT2 and OsMST5 play a role during the development at the early stage of the seed development. On the other hand OsSUT1 was expressed at the early stage of the grain development, suggesting a different physiological role compared to OsSUT2.
  • S Mitsunaga, O Kawakami, T Numata, J Yamaguchi, K Fukui, T Mitsui
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 65 (3) 662 - 665 0916-8451 2001/03 [Not refereed][Not invited]
    A polymorphism in rice amylases at an early stage of seed germination is analyzed by zymogram, In nonglutinous cultivars of rite, alpha -amylase isozymes are mainly confirmed in germinating seeds. However, in glutinous cultivars, beta -amylase isozymes, which are not confirmed in nonglutinous cultivars, make up the major part of the total amylase activity and the expression of alpha -amylases are repressed.
  • C Matsukura, T Saitoh, T Hirose, R Ohsugi, P Perata, J Yamaguchi
    PLANT PHYSIOLOGY 124 (1) 85 - 93 0032-0889 2000/09 [Refereed][Not invited]
    We investigated sugar uptake and transport in rice (Oryza sativa) embryo during grain germination. Endogenous sugar levels, accumulation of starch granules, and gene expression of a rice sucrose transporter (OsSUT1) were examined using rice embryos germinated with or without exogenous sugar supply. Starch granules remarkably accumulated in the cells around vascular bundles as a consequence of the sugar taken up by the embryos, indicating that the taken-up sugars are transiently converted into starch. In situ detection for OsSUT1 mRNA indicated its localization in the phloem companion cells. Furthermore, northern-blot and in situ hybridization analyses showed that OsSUT1 expression is not detectable in embryos subjected to sugar starvation conditions, whereas its expression is enhanced by an increased endogenous sugar level. Overall results indicate that the expression of companion cell-specific sucrose transporter, OsSUT1 is regulated by the endogenous sugar status as well as light exposure.
  • K Toyofuku, M Kasahara, J Yamaguchi
    PLANT AND CELL PHYSIOLOGY 41 (8) 940 - 947 0032-0781 2000/08 [Refereed][Not invited]
    This study deals with the cloning and characterization of monosaccharide transporter cDNAs in rice. OsMST1-3 (Oryza sativa monosaccharide transporters 1-3) have two sets of putative six transmembrane domains separated by a central long hydrophilic region. Heterologous expression of OsMST3 in the yeast Saccharomyces cerevisiae indicated that OsMST3 has transport activity for some monosaccharides in an energy-dependent H+ co-transport manner, Northern blot and in situ hybridization analyses showed that OsMST3 mRNA is detectable in leaf blades, leaf sheaths, calli and roots, especially the xylem as well as in sclerenchyma cells in the root. These results suggested that OsMST3 is involved in the accumulation of monosaccharides required for cell wall synthesis at the stage of cell thickening.
  • Motoko Awazuhara, Atsuko Nakagawa, Junji Yamaguchi, Toru Fujiwara, Hiroaki Hayashi, Keiko Hatae, Mitsuo Chino, Atsuko Shimada
    Journal of Agricultural and Food Chemistry 48 (2) 245 - 252 0021-8561 2000/02 [Refereed][Not invited]
    The thermal dependency and stability of enzymes producing reducing sugar (RS) were examined in bran, the exterior 13% part (outer endosperm), and the remaining inner endosperm of rice grains. RS-producing enzymes in the inner endosperm showed a higher optimum temperature than those in other parts of the rice grain. Diethylaminoethyl-Sephacel chromatography of crude extracts revealed two peaks of RS-producing activity with different optimum temperatures (60 and 37 °C) in all three parts. α-Glucosidase (EC and α-amylase (EC isoform G were thought to be major components of the RS-producing activities with high and low optimum temperatures, respectively. The peak with a high optimum temperature was a more abundant component in the inner endosperm, compared with other parts of the rice grain. Thus, different parts of rice were found to have distinct enzyme sets having different thermal dependency and to be involved in starch degradation to various sugars.
  • K Toyofuku, E Loreti, P Vernieri, A Alpi, P Perata, J Yamaguchi
    PLANT MOLECULAR BIOLOGY 42 (3) 451 - 460 0167-4412 2000/02 [Refereed][Not invited]
    Glucose effects on the expression of the abscisic acid-inducible Rab16A gene were examined in rice and barley embryos. Glucose feeding to rice embryos negatively affects the endogenous abscisic acid content and represses the promoter activity of the Rab16A gene. Glucose repression of the Rab16A gene takes place both at a transcriptional and a post-transcriptional level. Modulation of the abscisic acid content in rice embryos triggered by glucose did not directly influence the expression of the rice alpha-amylase gene RAmy3D, which is known to be under glucose control. The possible interaction between the glucose and abscisic acid signaling pathway is discussed.
  • J. Huang, K. Toyofuku, J. Yamaguchi, S. Akita
    Plant Production Science 3 (1) 32 - 37 1343-943X 2000 [Refereed][Not invited]
    A significant difference in the seed germinability was observed between the two rice cultivars, 'Nipponbare' and 'Suweon 287', under anoxia (i.e., during germination in submerged soil at 18°C), although little difference was seen under aerobic (in air) or hypoxic (in water) conditions. The number of α-amylase isoforms synthesized in germinating seeds was inversely proportional to the O2 concentrations at the early germination stage. The formation of isoform B was promoted by oxygen supply, while isoform H was undetectable if the seeds were unable to germinate. The activity of isoform H was highly correlated with the coleoptile length in the submerged soil at 18°C, indicating that isoform H is a critical factor for seed germination under anoxia. The expression of the rice α-amylase RAmy1A gene was repressed when the seeds germinated under hypoxia or anoxia. The interactions between oxygen stress, gibberellin, and carbon metabolites on the expression of α-amylase in rice are discussed.
  • L Guglielminetti, P Perata, A Morita, E Loreti, J Yamaguchi, A Alpi
    PHYTOCHEMISTRY 53 (2) 195 - 200 0031-9422 2000/01 [Refereed][Not invited]
    Hexose kinases in rice embryos have been characterized. Six isoforms were detected: i.e. three glucokinases (GK1-3), two hexokinases (HK1 and HK2) and one fructokinase (FK1). Out of these, CK3, HK1 and HK2 were inhibited by mannoheptulose and glucosamine, known inhibitors of hexokinase activity. These inhibitors are also known to be modulators of sugar sensing processes. The results suggest that GK3, HK1 and HK2 may play a role in sensing the cellular sugar status in the rice embryo. (C) 2000 Elsevier Science Ltd. All rights reserved.
  • Glucose repression of a-amylase in barley embryos is independent of GAMYB transcription.
    Loreti;E, C., Matsukura, F., Gubler, A., Alpi, J., Yamaguchi, P., Perata
    Plant Molecular Biology 44 85-90  2000/01 [Refereed][Not invited]
  • C Matsukura, M Kawai, K Toyofuku, RA Barrero, H Uchimiya, J Yamaguchi
    ANNALS OF BOTANY 85 (1) 19 - 27 0305-7364 2000/01 [Refereed][Not invited]
    In monocotyledons, the leaf vascular network consists of a hierarchical sequence of vertical vascular bundles and numerous transverse veins that interconnect adjacent vertical veins. In the leaf sheath of these species, especially grasses, lysigenous gas cavities (gas spaces) are developed into intervascular spaces and provide a gas conducting system to non-aerial Darts under flooded conditions. The spatial relationship between gas space formation and transverse vein differentiation was investigated using the leaf sheath of rice (Oryza sativa L.). Histochemical observation showed that patterns of differentiation of the transverse vein are distinct from those of vertical vascular bundles. On the other hand, gas spaces are formed through the processes of cell death (collapse). Both events are initiated at a specific cell position in the middle layers of the leaf sheath, from which the vascular system of the leaf is derived; this indicates that differentiation of transverse veins is associated with gas space formation. The cell-to-cell movement of fluorescein isothiocyanate-conjugated dextran injected into middle layer cells coincided with the area where cell collapse occurred, indicating a close relationship between the middle and adaxial cell layers, but not abaxial cell layers. A uniform cell number between each transverse vein in the leaf sheath suggested the involvement of spatial regulation in transverse vein formation regardless of clonal history at the later stage of leaf vein canalization. (C) 2000 Annals of Botany Company.
  • Differences in sugar regulation between two isoforms of alpha-amylase in suspension-cultured cells of rice.
    N. Geshi, T. Mitsui, T. Akazawa, J. Yamaguchi
    J. Appl. Glycosci. 46 (2) 111 - 119 1999/12 [Refereed][Not invited]
  • Wakako Takeuchi, Hironori Masui, Junji Yamaguchi
    Bioscience, Biotechnology and Biochemistry 63 (3) 510 - 514 1347-6947 1999/01/01 [Refereed][Not invited]
    The effects of reducing agents on solubilization and activation of the debranching enzyme (pullulanase) were examined using rice flour. The activity of the debranching enzyme was observed in a buffer solution (pH 7.5) in which rice flour was incubated together with thiol-reducing reagents, (dithiothreitol, 2-mercaptoethanol etc.), but there was only low activity in the absence of reducing agents. Immunochemical measurement and the specific activity of the enzyme showed that the activation caused by the reductant was due to solubilization of the enzyme protein besides the enzyme activation. © 1999, Taylor & Francis Group, LLC. All rights reserved.
  • Jirong Huang, Junji Yamaguchi, Shigemi Akita
    Plant Production Science 2 (1) 12 - 13 1343-943X 1999 [Refereed][Not invited]
  • Sugar repression of a-amylase genes in rice embryos.
    Yamaguchi;J, K., Toyofuku, A., Morita, A., Ikeda, C., Matsukura, P., Perata
    8th International Symposium on Pre-harvest Sprouting in Cereals 136-145  1999/01 [Not refereed][Not invited]
  • J Yamaguchi, S Itoh, T Saitoh, A Ikeda, T Tashiro, Y Nagato
    THEORETICAL AND APPLIED GENETICS 98 (1) 32 - 38 0040-5752 1999/01 [Refereed][Not invited]
    beta-Amylase deficiency in various cultivars of rice was examined at the molecular level. Using an antibody against beta-amylase purified from germinating seeds of rice, we were able to demonstrate the expression and organization of the beta-amylase gene in normal and deficient cultivars. Although beta-amylase is a starch-hydrolyzing enzyme, as is alpha-amylase, the beta-amylase protein/gene is expressed differently from the alpha-amylase protein/gene; i.e. (1) beta-amylase is synthesized only in aleurone cells, (2) the enzyme production in the embryo-less half-seeds is not under hormonal control, We identified some cultivars of rice that are deficient for beta-amylase activity. We present new evidence that synthesis is blocked at the level of mRNA synthesis in the deficient cultivars. The usefulness of beta-amylase as a crop trait is also discussed.
  • J Yamaguchi
    BREEDING SCIENCE 48 (4) 365 - 370 1344-7610 1998/12 [Refereed][Not invited]
    As a first step to clarify the alpha-amylase production in embryos, preparation of mass-isolated rice embryos was established. Morphological and biochemical characterization of the mass- isolated embryos revealed that the embryos are viable and suitable for characterization of alpha-amylase production. Activity staining for alpha-amylase revealed that the production of RAmy1A-encoded protein (isoform A) is under hormonal control, but that of other isoforms including RAmy3D-encoded protein (isoforms G and H) are not. All isoforms were repressed by glucose. The physiological role of alpha-amylase produced in the embryos was discussed.
  • S Mitsunaga, K Fukui, H Ohyama, J Yamaguchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 62 (9) 1812 - 1814 0916-8451 1998/09 [Refereed][Not invited]
    Binding of nuclear proteins to the promoter region was studied by a nonradioactive gel-retardation assay. The procedure uses biotinylated oligonucleotides in combination with streptavidin and biotin-conjugated alkaline phosphatase. This method offers sensitivity comparable to radioactive detection, and the advantage of the high stability of probes. Moreover the hazards of usage associated with radiation are avoided.
  • C Matsukura, S Itoh, K Nemoto, E Tanimoto, J Yamaguchi
    PLANTA 205 (2) 145 - 152 0032-0935 1998/06 [Refereed][Not invited]
    The mechanism of gibberellin (GA)-induced leaf sheath growth was examined using a dwarf mutant of rice (Oryza sativa L. cv. Tan-ginbozu) treated in advance with an inhibitor of GA biosynthesis. Gibberellic acid (GA(3)) enhanced the growth of the second leaf sheath, but auxins did not. Measurement of the mitotic index and cell size revealed that cell elongation rather than cell division is promoted by GA(3). Gibberellic acid increased the extensibility of cell walls in the elongation zone of the leaf sheath. It also increased the total amount of osmotic solutes including sugars in the leaf sheath, but did not increase the osmotic concentration of the cell sap, due to an accompanying increase in cell volume by water absorption. In the later stage of GA(3)-induced growth, starch granules completely disappeared from leaf sheath cells, whereas dense granules remained in control plants. These findings indicate that GA enhances cell elongation by increasing wall extensibility, osmotic concentration being kept unchanged by starch degradation.
  • K Toyofuku, T Umemura, J Yamaguchi
    FEBS LETTERS 428 (3) 275 - 280 0014-5793 1998/05 [Refereed][Not invited]
    There is increasing evidence shelving that cereal alpha-amylase gene expression is controlled not only by the classical hormonal regulation, but also by feed-back sugar repression. We demonstrated by in situ hybridization that the sugar repression of rice alpha-amylase gene RAmy3D takes place in scutellar epithelium cells of callus-forming rice embryos. We also used a transient expression system to study the cia-acting elements involved in the sugar repression of the RAmy3D promoter activity. Site-directed mutagenesis of the 50-bp nucleotide sequence from -172 to -123 revealed that consensus sequences of G motif (TACGTA) and TATCCA TIC motif(GATA motif as its antisense sequence) are responsible for sugar repression. The promoter sequences required for sugar repression are reported and discussed. (C) 1998 federation of European Biochemical Societies.
  • T Umemura, P Perata, Y Futsuhara, J Yamaguchi
    PLANTA 204 (4) 420 - 428 0032-0935 1998/04 [Refereed][Not invited]
    We used a transient expression system to study the mechanism by which carbohydrates repress a lice (Oryza sativa L.) alpha-amylase (EC gene. Exogenously fed metabolizable carbohydrates are able to elicit repression of the alpha-amylase gene RAmy3D in the rice embryo, and our results indicate that repression is also triggered efficiently by endogenous carbohydrates. Glucose analogs that are taken up by plant cells but not phosphorylated by hexokinase are unable to repress the alpha-amylase gene studied, while 2-deoxyglucose, which is phosphorylable but not further metabolized, downregulates RAmy3D promoter activity, indicating a role for hexokinase in the sugar-sensing mechanism triggering repression of the RAmy3D gene. We tested two different hexokinase inhibitors, mannoheptulose and glucosamine, but only the latter was able to relieve RAmy3D promoter activity from repression by endogenous carbohydrates. This correlates with the higher ability of glucosamine to inhibit the activity of rice hexokinases in vitro. The glucosamine-mediated relief of RAmy3D promoter activity from repression by endogenous carbohydrates does not correlate with a reduced rate of carbohydrate utilization.
  • N Sugimoto, G Takeda, Y Nagato, J Yamaguchi
    PLANT AND CELL PHYSIOLOGY 39 (3) 323 - 333 0032-0781 1998/03 [Refereed][Not invited]
    We have analyzed the in situ hybridization of alpha-amylase genes in rice and barley seeds. There are some similarities in the expression pattern between the rice and barley alpha-amylase genes, RAmy1A and Amy1. Both are gibberellin-inducible, the expression of these genes initiates in the scutellar epithelium and continues in the aleurone layer, and the gene expression in the aleurone layer initiates from a region close to the embryo. However, we also identified some differences in the pattern of gene expression between these species. (i) expression of the rice a-amylase gene is initiated in the terminal regions of scutellar epithelium, but in the middle-basal region in barley, (ii) the progress of the gene expression in the rice aleurone layer is faster than that in barley. Additionally, analyses using embryo mutants of rice indicate that the scutellum has a responsibility for RAmy1A gene expression in aleurone layer cells.
  • A Morita, T Umemura, M Kuroyanagi, Y Futsuhara, P Perata, J Yamaguchi
    FEBS LETTERS 423 (1) 81 - 85 0014-5793 1998/02 [Refereed][Not invited]
    The gibberellin-inducible rice alpha-amylase gene, RAmy1A, was demonstrated to be sugar repressed in rice embryos and functional dissection of the promoter of RAmy1A in relation of its sugar-modulated expression was performed. Gibberellin-response cis-elements of GARE (TAACAAA) and pyrimidine box (CCTTTT) were partially involved in the sugar repression. (C) 1998 Federation of European Biochemical Societies.
  • J. Yamaguchi
    Breeding Science 48 (4) 365 - 370 0536-3683 1998 [Refereed][Not invited]
    As a first step to clarify the α-amylase production in embryos, preparation of mass-isolated rice embryos was established. Morphological and biochemical characterization of the mass-isolated embryos revealed that the embryos are viable and suitable for characterization of α - amylase production. Activity staining for α- amylase revealed that the production of RAmy1A-encoded protein (isoform A) is under hormonal control, but that of other isoforms including RAmy3D-encoded protein (isoforms G and H) are not. All isoforms were repressed by glucose. The physiological role of α-amylase produced in the embryos was discussed.
  • E Loreti, L Guglielminetti, J Yamaguchi, S Gonzali, A Alpi, P Perata
    JOURNAL OF PLANT PHYSIOLOGY 152 (1) 44 - 50 0176-1617 1998/01 [Refereed][Not invited]
    In barley seeds beta-amylase is synthesized during the development of the grain, where it accumulates bound to starch granules. As germination proceeds, beta-amylase is released through proteolytic processing leading to a free form of the enzyme having a 5 kD lower molecular weight. Under anoxia, barley seeds are unable to germinate and both alpha-amylase synthesis and beta-amylase processing are inhibited. We report here data on the effects of anoxia on the induction of the protease responsible for the release of beta-amylase in barley seeds. The results show that barley seeds kept under anoxia fail to produce the endoprotease involved in the processing of beta-amylase. This is likely the consequence of the lack of response of-barley seeds to GA(3) under anoxic conditions.
  • Starch degradation during gibberellin-induced leaf sheath growth in rice.
    Matsukura;C, J., Yamaguchi, 松倉, 千昭
    Rice Genetics Newsletter 15 114-117  1998/01 [Refereed][Not invited]
  • Sugar repression of a gibberellin-dependent signaling pathway in barley embryos.
    Perata;P, C., Matsukura, P., Vernieri, J., Yamaguchi
    Plant Cell 9 2197-2208  1997/01 [Refereed][Not invited]
  • The first leaf growth promoted by gibberellins in dwarf mutant of rice.
    Matsukura;C, J., Yamaguchi, 松倉, 千昭
    Rice Genetics Newsletter 14 153-155  1997/01 [Refereed][Not invited]
  • T Mitsui, J Yamaguchi, T Akazawa
    PLANT PHYSIOLOGY 110 (4) 1395 - 1404 0032-0889 1996/04 [Refereed][Not invited]
    We have identified, purified, and characterized 10 alpha-amylase isoforms from suspension-cultured rice (Oryza sativa L.) cells having different isoelectric point values. They had distinguishable optimum temperatures for enzymatic activity and molecular sizes. The results of immunoblotting indicated that polyclonal anti-A + B antibodies bound well to isoforms A, B, Y, and Z but weakly or not at all to E, F, G, H, I, and J. However, the anti-A + B antibodies inhibited the enzyme activities of only isoforms A and B. Polyclonal anti-H antibodies strongly bound to isoforms F, G, H, I, and J, whereas polyclonal anti-E antibodies preferentially recognized isoform E. A monoclonal antibody against isoform H (H-G49) inhibited the activities of isoforms E, G, H, I, and J, whereas it did not inhibit those of isoforms A, B, Y, and Z. Judging from their physicochemical and serological properties, we classified the rice cu-amylase isoforms into two major classes, class I (A, B, Y,and Z) and class II (E, F, G, H, I, and J), and into four subgroups, group 1 (A and B), group 2 (Y and Z), group 3 (E), and group 4 (F, G, H, I, and J). Partial amino acid sequences for isoforms A, E, G, and H were also determined. In addition, the recombinant alpha-amylases expressed by plasmid pEno/103 containing the rice alpha-amylase gene RAmy1A in yeast were identified as both isoforms A and B. These analyses indicated that isoforms A and B were encoded by the gene RAmy1A, isoforms G and H were encoded by the gene RAmy3D, and isoform E was encoded by RAmy3E. The results strongly suggest that some isoforms within subgroups are formed by posttranslational modifications.
  • Masayuki Nakase, Takehisa Yamada, Takahiro Kira, Junji Yamaguchi, Naohito Aoki, Ryo Nakamura, Tsukasa Matsuda, Takahiro Adachi
    Plant Molecular Biology 32 (4) 621 - 630 0167-4412 1996 [Refereed][Not invited]
    Expression of rice seed storage-protein genes is dramatically regulated over a short period of seed maturation. To characterize the expression mechanism of the rice seed storage protein genes, their expression of major storage protein genes (16 kDa albumin, 13 kDa prolamin and type II glutelin) were compared by RNA blot analysis. Their coordinate expression suggests that the transcriptional regulatory machinery is shared among the glutelin, prolamin and albumin-genes. We isolated two novel genomic genes for prolamins (PG5a and PG5b) and obtained the promoter region of the glutelin gene by PCR. The 5′-flanking regions of these three rice seed storage protein genes were found to contain some similar conserved sequences. Nuclear extract partially purified from maturing rice seeds was used for the gel shift assay of the 5′ region of the RA gene. We identified two DNA sequences of RA gene which were recognized by independent DNA-binding proteins. The complexes of these DNA sequences and DNA-binding proteins were inhibited by the fragments containing the 5′ regions of the prolamin and glutelin genes, suggesting that these three genes share transcription factors. © 1996 Kluwer Academic Publishers.
  • Toshinori Abe, Masumi Kudo, Yasuhiro Oka, Junji Yamaguchi, Takeo Sasahara
    Journal of Plant Physiology 149 (5) 592 - 598 0176-1617 1996 [Refereed][Not invited]
    Changes in α-amylase activity in rice calli during organogenesis were investigated in 5 rice varieties that exhibit different abilities for plant regeneration. During organ differentiation in rice callus tissues, samples were taken at 5-day intervals up to 35 days and α-amylase (EC. activities were measured. The activity in regenerative calli began to increase 15 to 20 days after transfer to the regeneration medium and were elevated 7-11 fold during the culture period, while the calli transferred to callus maintenance medium did not increase and maintained a stable state. The increase in α-amylase activity in regenerative calli was more rapid in the calli that showed higher regenerative abilities (Sasanishiki, Tadukan and Tetep) than in the calli that showed lower regenerative abilities (Fujisaka 5 and Nipponbare). Correlation coefficients between frequencies of organ differentiation and α-amylase activity in the calli 35 days after transfer to the regeneration medium was highly significant. Expression of a rice α- amylase gene (RAmy1A) in callus tissues during organ differentiation was examined by Northern blot and Western blot analyses. Messenger RNA from the RAmy1A gene, which is a main transcript in germinating rice seeds, was highly expressed in the regenerating calli 15 days after transfer. The translated product from the mRNA of the RAmy1A gene and α-amylase isozyme band I (1A and 1B) were highly expressed 20 days after transfer, especially in the regeneration medium.
  • Lorenzo Guglielminetti, Junji Yamaguchi, Pierdomenico Perata, Amedeo Alpi
    Plant Physiology 109 (3) 1069 - 1076 0032-0889 1995 [Refereed][Not invited]
    An adequate carbohydrate supply contributes to the survival of seeds under conditions of limited oxygen availability. The amount of soluble, readily fermentable carbohydrates in dry cereal seeds is usually very limited, with starch representing the main storage compound. Starch breakdown during the germination of cereal seeds is the result of the action of hydrolytic enzymes and only through the concerted action of α-amylase (EC, β-amylase (EC, debranching enzyme (EC, and α-glucosidase (EC can starch be hydrolyzed completely. We present here data concerning the complete set of starch-degrading enzymes in three cereals, rice (Oryza sativa L.), which is tolerant to anaerobiosis, and wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), which are unable to germinate under anoxia. Among the cereal seeds tested under anoxia, only rice is able to degrade nonboiled, soluble starch, reflecting the ability to degrade the starch granules in vivo. This is explained by the presence of the complete set of enzymes needed to degrade starch completely either as the result of de novo synthesis (α-amylase, β-amylase) or activation of preexisting, inactive forms of the enzyme (debranching enzyme, α-glucosidase). These enzymes are either absent or inactive in wheat and barley seeds kept under anaerobic conditions.
    PLANT PHYSIOLOGY 107 (1) 25 - 31 0032-0889 1995/01 [Refereed][Not invited]
    Transgenic seeds of rice (Oryza sativa L.) were used to investigate temporal, spatial, and hormonal regulation of a rice Lu-amylase gene, RAmy1A. Two overlapping segments of the RAmy1A promoter were fused to the coding region of the bacterial reporter gene, gusA. The resulting promoter-gusA fusions, pE4/GUS (-232 to +31) and pH4/GUS (-748 to +31), were used separately to transform rice protoplasts. beta-Glucuronidase (GUS) activity was detected in germinated transgenic seeds, although the two constructs showed no significant difference in timing or location of GUS expression. Both constructs first expressed GUS in the scutellar epithelium and then in the aleurone layer. Aleurone expression of GUS activity was strongly induced when embryoless half-seeds were treated with gibberellic acid. GUS expression in the aleurone layer was also suppressed by abscisic acid. These results indicate that the 5' regulatory region from -232 to +31 is sufficient for temporal, spatial, and hormonal regulation of RAmy1A gene expression.
    NUCLEIC ACIDS RESEARCH 22 (11) 1948 - 1953 0305-1048 1994/06 [Refereed][Not invited]
    The expression of a rice gene for alpha-amylase, RAmy3D, in suspension-cultured cells is induced at the transcriptional level by the deprivation of sugars. Binding of a nuclear protein from suspension-cultured rice cells to the promoter region of the RAmy3D gene was studied by gel-retardation and DNase I footprinting assays. Gel-retardation assays indicated that a 358-bp fragment of the promoter region interacted specifically with a protein factor from suspension-cultured cells. DNase I footprinting analysis allowed us to define three protein-binding regions. Each of these protein-binding sequences contained the GCCG G/C CG motif, which is specifically present in the promoter region of the sugar-regulated gene, RAmy3D, for rice alpha-amylase and not in that of the gibberellin-regulated RAmy1A gene. Subsequent cross-competition experiments using gel-retardation assay and synthetic oligonucleotides showed that the GCCG G/C CG motifs directly mediated the binding of a nuclear protein. These observations are discussed in relation to expression of the gene for alpha-amylase in suspension-cultured cells.
    THEORETICAL AND APPLIED GENETICS 87 (6) 705 - 712 0040-5752 1994/01 [Refereed][Not invited]
    In rice, many dwarf mutants have been isolated and characterized. We have investigated the relationship between dwarfism and the gibberellin (GA)-mediated control of physiological processes. Twenty-three rice cultivars and mutants (9 normal, 3 semi-dwarf, 11 dwarf) were analyzed in terms of two GA-mediated processes, namely, elongation of shoots and production of alpha-amylase activity in the endosperm. As a result, we identified four different groups (groups N,T,D and E). Two-dimensional plotting of the extent of induction of alpha-amylase in the endosperm versus the extent of enhancement of shoot elongation upon treatment with exogenous gibberellic acid (GA(3)) provided a useful method for the rapid allocation of large numbers of dwarf mutants of rice to the various groups. Members of group N (normal type), which included all normal cultivars and semi-dwarf mutants, showed a slight increase in elongation of shoots and a remarkable increase in production of alpha-amylase with the application of GA(3) during germination. All of the dwarf mutants were classified as being members of the other three groups. Members of group T (Tan-ginbozu type), including three dwarf mutants, were highly responsive to exogenous GA(3) in terms of elongation of shoots and production of alpha-amylase, with associated lower levels of endogenous GA. In contrast, members of the other three groups, including group N, had normal levels of endogenous GAs. Members of group D (Daikoku type) were only slightly responsive to exogenous GA(3), an indication that they are GA-insensitive mutants. Members of group E (Ebisu type) had responses to GA(3) similar to those of group N, not only in terms of elongation of shoots but also in terms of alpha-amylase production, an indication that they are dwarf mutants that can be considered as neither GA-deficient nor GA-insensitive mutants. We also examined a GA-insensitive mutant selected from among 19 near-isogenic dwarflines of 'Shiokari', and we concluded that the d-1 gene is associated with the phenotype of GA-insensitive dwarf mutants.
    PLANTA 191 (3) 402 - 408 0032-0935 1993/08 [Refereed][Not invited]
    The effect of anoxia on the induction of alpha-amylase in seeds of rice (Oryza sativa L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), oat (Avena sativa L.) and rye (Secale cereale L.) was studied. The results showed that only rice is able to synthesize alpha-amylase under anoxia, while other cereal seeds fail to produce the enzyme and do not germinate. The inhibitory effect of anoxia on alpha-amylase induction is concluded to be due to the inability of cereal seeds to respond to gibberellic acid under conditions of total oxygen deprivation. Rice appears to be an exception to this rule among the cereal seeds tested. We found that alpha-amylase mRNA fails to accumulate in embryoless barley half-seeds treated with gibberellic acid, indicating that the action of anoxia may be at the transcriptional level.
    PLANT AND CELL PHYSIOLOGY 34 (2) 243 - 249 0032-0781 1993/03 [Refereed][Not invited]
    A system for induction of synthesis of alpha-amylase during germination by gibberellin using intact seeds, similar to that involving embryo-less half-seeds, was established using a combination of an inhibitor of the biosynthesis of gibberellin, uniconazole, and the rice dwarf mutant, Waito-C (dy mutant). Waito-C is a dwarf mutant in which the biosynthetic pathway to gibberellins is genetically blocked. In the presence of uniconazole, the synthesis of alpha-amylase by Waito-C during germination was repressed. The repression caused by uniconazole could be overcome by a subsequent treatment with GA3. The results of activity staining and immunoblotting showed that this repression of the synthesis of alpha-amylase occurred at the protein level.
    PLANTA 188 (4) 611 - 618 0032-0935 1992/11 [Refereed][Not invited]
    The capabilities of rice (Oryza sativa L.) and wheat (Triticum aestivum L.) seeds (caryopses) to degrade starchy reserves present in the endosperm tissue were compared under anaerobic conditions. The results showed that rice, a species highly tolerant to anoxia, can readily break down starch under anaerobiosis concomitant with germination, while wheat does not germinate and fails to degrade starch present in the endosperm. This clearly distinct behavior is likely the consequence of the successful inducible formation of alpha-amylase (EC in rice under anoxia, whereas the enzyme is not produced in wheat seeds. We found that rice seeds possess a set of enzymes allowing starch and its degradative products to be utilized under anoxic conditions. Wheat seeds were shown to germinate even under anoxia if fed glucose or sucrose exogenously. The overall results indicate that induction of alpha-amylase appears to be one of the factors permiting rice seeds to germinate in totally anaerobic environments.
    PLANT PHYSIOLOGY 100 (2) 1069 - 1071 0032-0889 1992/10 [Refereed][Not invited]
    FEBS LETTERS 309 (3) 283 - 287 0014-5793 1992/09 [Refereed][Not invited]
    Results presented in a previous report from this laboratory indicated the presence, in crude extracts from sycamore (Acer pseudoplatanus) and spinach (Spinacea oleracea), of a sucrose synthase (EC showing high affinity for ADP as the glucose acceptor in the sucrose-cleaving reaction. In the present paper we report that the modified enzymatic method previously used to measure sucrose synthase activities leads to the detection of artifactual ADP-dependent sucrose synthase, which in fact arises from the combined action of invertase (EC and nucleoside diphosphate kinase (EC activities. We also present data on the partial purification of nucleoside diphosphate kinase from sycamore cells.
  • Junji Yamaguchi, Poh-Yam Lim, Takashi Akazawa, Kazunii Aratani
    Cell Structure and Function 17 (2) 87 - 92 1347-3700 1992 [Refereed][Not invited]
    A method has been developed to isolate pure preparations of nuclei in high yield from commercially available viable rice embryos (germ), employing extraction with buffer solution containing glycerol (without detergent) and polyamine, followed by centrifugation on a 30% Percoll cushion. The intactness of the isolated nuclei was confirmed by light microscopy as well as electron microscopy. The protein profiles of both whole nuclei and nuclear extracts obtained by SDS-PAGE, organellar marker enzyme activities, DNA and RNA analyses, and in vitro RNA synthesis, all indicate that the highly purified nuclei are isolated from rice embryos. © 1992, Japan Society for Cell Biology. All rights reserved.
  • Christian Chevalier, Junji Yamaguchi, Peter McCourt
    Plant Physiology 99 (4) 1726 - 1728 0032-0889 1992 [Refereed][Not invited]
  • Shoji Hatano, Junji Yamaguchi, Atsushi Hirai
    Plant Science 83 (1) 55 - 64 0168-9452 1992 [Refereed][Not invited]
    We have isolated high molecular weight DNA of over 5.7 Mb in length from isolated rice germ nuclei. The high-molecular-weight DNA was digested with a number of restriction endonucleases. An ethidium-stained gel after pulsed-field electrophoresis revealed that few of these endonucleases have the ability to cleave rice chromosomal DNA to large fragments of over 200 kb in length, perhaps because of the lower level of methylation of nucleotides in the rice genome than in other genomes. Some restriction endonucleases that do not cut rDNA repeats produced fragments with arrays of rDNA repeats of more than 1.1 Mb, as estimated by Southern analysis. Therefore, it appears that the rice rDNA cluster is constructed with a 'physical' succession of more than 130 copies of 8-kb repeating units of rDNA. Genes for α-amylase were also detected by the Southern method. It indicates that our procedure is suitable for analysis of low-copy-number genes. © 1992.
    FEBS LETTERS 291 (2) 233 - 237 0014-5793 1991/10 [Refereed][Not invited]
    The standardized enzyme coupling method for assaying sucrose synthase activities in the direction of sucrose cleavage was reexamined using enzyme preparations from cultured cells of sycamore (Acer pseudoplatanus L.) and spinach leaves (Spinacea oleracea). Both ATP and Tris, commonly utilized in assay systems to measure sucrose synthase, were found to inhibit non-competitively the ADPG-synthesizing activities of the enzyme. Upon substituting ATP by either GTP or UTP, and Tris by HEPES, we found that the sucrose synthase is capable of producing ADPG effectively, recognizing ADP as the principal substrate (K(m) = 5.3-mu-M (sycamore) and 16.8-mu-M (spinach)). The V(max) value for the synthesis of ADPG clearly surpasses the V(max) observed for the synthesis of UDPG by the enzyme. It was found that UDP is not inhibitory on the synthesis of ADPG by SS, which behaves allosterically with respect to the concentration level of sucrose.
    FEBS LETTERS 213 (2) 329 - 332 0014-5793 1987/03 [Refereed][Not invited]
  • Junji Yamaguchi, Mikio Nishimura, Takashi Akazawa
    Plant and Cell Physiology 28 219 - 226 0032-0781 1987/03 [Refereed][Not invited]
    The catalase molecule in germinating pumpkin cotyledons is synthesized as a precursor (59-kDa) form, whose relative molecular mass is larger than the mature enzyme (55-kDa). Although both types of molecules are localized in the microbodies, the 59-kDa species has been shown to be present predominantly in the leaf peroxisomes isolated from green cotyledons, while the 55-kDa species is predominantly in the glyoxysomes from etiolated cotyledons [Yamaguchi et al. (1984) Proc. Natl. Acad. Sci. USA, 81: 4809]. We examined the distribution of the 59- and 55-kDa catalase molecules in dark- and light-grown tissues of pumpkin seedlings as well as in other plant species, using the immunoblotting technique. The ratios of the 59- and 55-kDa catalase species differed in the pumpkin tissues examined. Light interferes with the conversion of the 59-kDa precursor to the 55-kDa form, especially in the cotyledons. The effect of light was less pronounced in the roots and hypocotyls, indicating that the light regulation of the conversion is tissue-specific. Dark- and light-grown cotyledons from cucumber and watermelon seedlings showed a similar light regulation, suggesting that cucurbitaceous plants possess similar light-regulatory mechanism. From the analysis of catalase protein from various plant tissues, a limited correlation between molecular forms of catalase and different microbody populations was observed. © 1987. The Japanese Society of Plant Physiologists (JSPP).
    EUROPEAN JOURNAL OF BIOCHEMISTRY 159 (2) 315 - 322 0014-2956 1986/09 [Refereed][Not invited]
    PLANT PHYSIOLOGY 81 (1) 313 - 316 0032-0889 1986/05 [Not refereed][Not invited]

Books etc

  • Plant Proteomics: Methods in Molecular Biology, vol. 1072
    Sato T, Sako K, Yamaguchi J (ContributorChapter 45 Assay for proteasome-dependent protein degradation and ubiquitinated proteins)
    Humana Press 2013 (ISBN: 9781627036306) 655-663
  • Frontiers in Agriculture Proteome Research
    Yasuda S, Maekawa S, Sato T, Yamaguchi J (ContributorProteomics approach to study metabolic regulation mediated by ubiquitin-proteasome system in Arabidopsis)
    NARO Institute of Crop Science 2012 (ISBN: 9784904633991) 39-44
  • 植物の百科事典
    山口淳二 (Contributor炭素代謝)
    朝倉書店 2009 (ISBN: 9784254171372) 9
  • Botanical encyclopedia
    Asakura 2009
  • 種子の科学とバイオテクノロジー
    佐藤長緒, 山口淳二 (Contributor種子デンプンの分解)
    学会出版センター 2009 (ISBN: 9784762230592) 88-91
  • バイオとナノの融合Ⅰ
    園田 裕, 佐藤長緒, 山崎直子, 佐古香織, 池田 亮, 山口淳二 (Contributorプロテアソームを介した細胞制御-高等植物の細胞サイズ制御を中心として)
    北海道大学出版会 2007 239-255
  • 朝倉植物生理学講座第2巻代謝
    武田泰斗, 山口淳二 (Contributorデンプンの合成と分解)
    朝倉書店 2001 (ISBN: 4254176562) 94-102
  • 新農学大事典
    山口淳二, 松倉千昭 (Contributor発芽)
    養賢堂 2001
  • 植物ホルモンと細胞の形
    山口淳二, 松倉千昭 (Contributorα-アミラーゼの誘導)
    学会出版センター 1998 (ISBN: 4762218707) 43-52
  • 植物化学調節実験マニュアル
    山口 淳二 (Contributorアリューロン層細胞を用いたα-アミラーゼ誘導検定法)
    全国農村教育協会 1997 (ISBN: 4881370634) 2-7
  • 種子のバイオサイエンス
    山口 淳二 (Contributor種子デンプン(炭水化物)の合成と分解)
    学会出版センター 1995 (ISBN: 476228789X) 87-93
  • 酵素実験法(1)
    山口淳二, 赤沢堯 (Contributor植物酵素の精製)
    1993 (ISBN: 4567243609) 80-87
  • 遺伝子工学実験
    山口 淳二 (Contributor植物細胞における遺伝子クローンの発現)
    日本アイソトープ協会 1991 143-150
  • 新生化学実験講座 Vol 1
    山口淳二, 赤沢堯 (Joint work植物組織と抽出法)
    東京化学同人 1990 (ISBN: 4807910477) 20-27


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Awards & Honors

  • 2018/08 Japanese Society for Plant Cell and Molecular Biology Studies on environmental adaptation in response to nutrients in plants
     The JSPCMB Award for Distinguished Research 
    受賞者: YAMAGUCHI Junji

Research Grants & Projects

Educational Activities

Teaching Experience

  • 特別講義
    開講年度 : 2019
    課程区分 : 学士課程
    開講学部 : 全学

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