研究者情報

マスター

アカウント（マスター）

• 氏名

  山口　淳二(ヤマグチ　ジユンジ), ヤマグチ　ジユンジ

所属（マスター）

• 総長、理事・副学長

所属（マスター）

• 総長、理事・副学長

独自項目

syllabus

• 2021, 大学院共通授業科目（一般科目）：複合領域, Inter-Graduate School Classes(General Subject):Inter-Disciplinary Sciences, 修士課程, 大学院共通科目, インターンシップ、海外研修、就業体験、キャリア・デザイン，企業，グローバル
• 2021, 大学院共通授業科目（一般科目）：複合領域, Inter-Graduate School Classes(General Subject):Inter-Disciplinary Sciences, 修士課程, 大学院共通科目, インターンシップ，海外研修，就業体験，キャリア・デザイン，企業，グローバル
• 2021, インターンシップA, Internship A, 学士課程, 全学教育, インターンシップ、海外研修、就業体験、キャリア・デザイン，企業，グローバル
• 2021, インターンシップB, Internship B, 学士課程, 全学教育, インターンシップ、海外研修、就業体験、キャリア・デザイン，企業，グローバル

PositionHistory

• アドミッションセンター長, 2020年10月1日, 2022年3月31日
• アドミッションセンター長, 2022年4月1日, 2024年3月31日
• 大学院理学研究院附属ゲノムダイナミクス研究センター長, 2019年4月1日, 2020年9月30日
• ダイバーシティ・インクルージョン推進本部長, 2022年4月1日, 2024年3月31日
• 教育改革室長, 2020年10月1日, 2022年3月31日
• 教育改革室長, 2022年4月1日, 2024年3月31日
• 教育改革室室員, 2013年4月1日, 2014年3月31日
• 教育改革室室員, 2017年4月1日, 2019年3月31日
• 教育改革室室員, 2019年4月1日, 2020年9月30日
• 教育研究評議会評議員, 2014年4月1日, 2016年3月31日
• 教育研究評議会評議員, 2016年4月1日, 2017年3月31日
• 経営戦略室室長, 2021年4月1日, 2022年3月31日
• 経営戦略室室長, 2022年4月1日, 2024年3月31日
• 高等教育推進機構長, 2020年10月1日, 2022年3月31日
• 高等教育推進機構長, 2022年4月1日, 2024年3月31日
• 高等教育推進機構副機構長, 2019年4月1日, 2020年9月30日
• 人材育成本部長, 2020年10月1日, 2022年3月31日
• 大学院生命科学院長, 2014年4月1日, 2016年3月31日
• 大学院生命科学院長, 2016年4月1日, 2017年3月31日
• 大学力強化推進本部副本部長, 2020年10月1日, 2024年3月31日
• 副学長, 2014年4月1日, 2015年3月31日
• 副学長, 2015年4月1日, 2017年3月31日
• 副学長, 2017年4月1日, 2019年3月31日
• 副学長, 2019年4月1日, 2020年9月30日
• 役員補佐, 2013年4月1日, 2014年3月31日

researchmap

プロフィール情報

学位

• 農学博士（名古屋大学）

プロフィール情報

• 姓

  山口, ヤマグチ

• 名

  淳二, ジュンジ

• ID各種

  200901009980317817

業績リスト

研究キーワード

• 二酸化炭素   プロテオミクス   植物   代謝   ユビキチンリガーゼ   免疫   環境適応   バイオマス   タンパク質分解   

研究分野

• ライフサイエンス / 応用分子細胞生物学
• ライフサイエンス / 植物分子、生理科学

経歴

• 2020年10月 - 現在 北海道大学 理事・副学長
• 2022年04月 - 2024年03月 北海道大学 大学院理学研究院 特任教授
• 2009年04月 - 2022年03月 北海道大学大学院理学研究院 生物科学部門 教授
• 2014年04月 - 2020年09月 北海道大学 副学長
• 2014年04月 - 2017年03月 北海道大学 生命科学院 学院長
• 2010年09月 - 2013年03月 北海道大学高等教育推進機構 副機構長・総合教育部長
• 2003年04月 - 2009年05月 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年04月 - 2009年03月 北海道大学大学院先端生命科学研究院 教授
• 2001年04月 - 2006年03月 北海道大学大学院理学研究科生物科学専攻（生物学） 理学(系)研究科(研究院) 教授
• 1995年02月 - 2001年03月 名古屋大学生物分子応答研究センター 助教授
• 1987年10月 - 1995年01月 名古屋大学農学部 助手
• 1986年10月 - 1987年09月 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年10月 - 1986年09月 日本学術振興会 特別研究員
• 1982年04月 - 1985年03月 名古屋大学大学院農学研究科 博士課程後期修了

委員歴

• 2013年10月 - 2016年09月   日本植物化学調節学会   評議員
• 2010年01月 - 2015年12月   植物細胞分子生物学会   評議員   植物細胞分子生物学会
• 2009年01月 - 2014年12月   植物細胞分子生物学会   Plant Biotechnology 編集委員   植物細胞分子生物学会
• 2007年06月 - 2013年06月   北海道食の安全・安心委員会   特別委員
• 2006年01月 - 2009年12月   日本植物生理学会   評議員
• 2004年01月 - 2009年06月   科学技術振興調整費   研究領域主管
• 2004年04月 - 2006年03月   日本農芸化学会   評議員
• 2004年 - 2005年   日本育種学会   幹事   日本育種学会
• 2003年03月 - 2005年01月   文部科学省   科学技術調査委員
• 2002年07月 - 2004年06月   J. Applied Glycosci.   編集委員
• 2002年 - 2004年   日本応用糖質学会   J. Applied Glycoscience編集委員   日本応用糖質学会
• 2000年01月 - 2003年12月   日本植物生理学会   評議員
• 2000年 - 2003年   日本植物生理学会   Plant and Cell Physiology誌編集委員
• 2001年04月 - 2002年03月   理化学研究所植物科学研究センター   研究評価委員
• 2000年 - 2001年   Trends in Glycosci. Glycotech. Glycotopic   執筆委員

受賞

• 2023年09月 日本植物バイオロジー学会 日本植物バイオロジー学会論文賞
   3-Phynyllactic acid is converted to phenylacetic acid andinduces auxin-responsive root growth in Arabidopsis plants. 

  受賞者: Yuko Maki, Hiroshi Soejima, Tamizi Sugiyama, Masaaki K. Watahiki, Takeo Sato, Junji Yamaguchi
• 2018年08月 日本植物細胞分子生物学会 学術賞
   栄養応答を中心とした植物環境適応機構の解明 

  受賞者: 山口 淳二

論文

• Histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 proteins affect plant growth under nitrogen deficient conditions in Arabidopsis thaliana

  Linnan Jie, Miho Sanagi, Yongming Luo, Haruna Maeda, Yoichiro Fukao, Yukako Chiba, Shuichi Yanagisawa, Junji Yamaguchi, Junpei Takagi, Takeo Sato

  PLANT BIOTECHNOLOGY 40 1 93 - 98 2023年03月 

  Nitrogen (N) availability is one of the most important factors regulating plant metabolism and growth as it affects global gene expression profiles. Dynamic changes in chromatin structure, including histone modifications and nucleosome assembly/disassembly, have been extensively shown to regulate gene expression under various environmental stresses in plants. However, the involvement of chromatin related changes in plant nutrient responses has been demonstrated only in a few studies to date. In this study, we investigated the function of histone chaperone NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) proteins under N deficient conditions in Arabidopsis. In the nap1;1 nap1;2 nap1;3 triple mutant (m123-1), the expression of N-responsive marker genes and growth of lateral roots were decreased under N deficient conditions. In addition, the m123-1 plants showed a delay in N deficiency-induced leaf senescence. Taken together, these results suggest that NAP1s affect plant growth under N deficient conditions in Arabidopsis.
• Deubiquitinating enzymes UBP12 and UBP13 regulate carbon/nitrogen-nutrient stress responses by interacting with the membrane-localized ubiquitin ligase ATL31 in Arabidopsis

  Yongming Luo, Shigetaka Yasuda, Junpei Takagi, Yoko Hasegawa, Yukako Chiba, Junji Yamaguchi, Takeo Sato

  Biochemical and Biophysical Research Communications 2022年10月
• Conjugates of 3-phenyllactic acid and tryptophan enhance root-promoting activity without adverse effects in Vigna angularis.

  Yuko Maki, Hiroshi Soejima, Tamizi Sugiyama, Takeo Sato, Junji Yamaguchi, Masaaki K Watahiki

  Plant biotechnology (Tokyo, Japan) 39 2 173 - 177 2022年06月25日 

  3-Phenyllactic acid (PLA) is a common secondary product of Lactobacillus sp. and promotes adventitious-root formation in Azuki beans (Vigna angularis). Root promotion activity of PLA is synergistically enhanced by tryptophan (Trp). In this study, stereoisomers of PLA and Trp amide conjugates and their alkyl esters were synthesized to investigate the structure-activity relationships on root-promotion activity. The rooting activity of D-PLA-L-Trp conjugate shows more than 40 times higher than that of the mixture of D-PLA and L-Trp. Modification of PLA-Trp with ethyl ester showed the highest activity at 3,400 times of a mixture of D-PLA and L-Trp. However, L-or D-PLA-D-Trp conjugate and the isopropyl ester of PLA-Trp conjugates, both lost the root promotion activity and implicated that a requirement for steric structure for PLA related root promotion mechanism. Unlike auxin substances, which are commonly used as rooting agents that displayed high activity in low concentrations, PLA-Trp ethyl ester exhibited far less phytotoxicity at high concentration of 1 mM, despite its high rooting activity. Innovation of PLA-Trp ethyl ester may be expected for agricultural aspects with low environmental impact.
• 3-Phenyllactic acid is converted to phenylacetic acid and induces auxin-responsive root growth in Arabidopsis plants.

  Yuko Maki, Hiroshi Soejima, Tamizi Sugiyama, Masaaki K Watahiki, Takeo Sato, Junji Yamaguchi

  Plant biotechnology (Tokyo, Japan) 39 2 111 - 117 2022年06月25日 

  Many microorganisms have been reported to produce compounds that promote plant growth and are thought to be involved in the establishment and maintenance of symbiotic relationships. 3-Phenyllactic acid (PLA) produced by lactic acid bacteria was previously shown to promote root growth in adzuki cuttings. However, the mode of action of PLA as a root-promoting substance had not been clarified. The present study therefore investigated the relationship between PLA and auxin. PLA was found to inhibit primary root elongation and to increase lateral root density in wild-type Arabidopsis, but not in an auxin signaling mutant. In addition, PLA induced IAA19 promoter fused β-glucuronidase gene expression, suggesting that PLA exhibits auxin-like activity. The inability of PLA to promote degradation of Auxin/Indole-3-Acetic Acid protein in a yeast heterologous reconstitution system indicated that PLA may not a ligand of auxin receptor. Using of a synthetic PLA labeled with stable isotope showed that exogenously applied PLA was converted to phenylacetic acid (PAA), an endogenous auxin, in both adzuki and Arabidopsis. Taken together, these results suggest that exogenous PLA promotes auxin signaling by conversion to PAA, thereby regulating root growth in plants.
• Deubiquitinating enzymes UBP12 and UBP13 stabilize the brassinosteroid receptor BRI1.

  Yongming Luo, Junpei Takagi, Lucas Alves Neubus Claus, Chao Zhang, Shigetaka Yasuda, Yoko Hasegawa, Junji Yamaguchi, Libo Shan, Eugenia Russinova, Takeo Sato

  EMBO reports 23 4 e53354  2022年04月05日 

  Protein ubiquitination is a dynamic and reversible post-translational modification that controls diverse cellular processes in eukaryotes. Ubiquitin-dependent internalization, recycling, and degradation are important mechanisms that regulate the activity and the abundance of plasma membrane (PM)-localized proteins. In plants, although several ubiquitin ligases are implicated in these processes, no deubiquitinating enzymes (DUBs), have been identified that directly remove ubiquitin from membrane proteins and limit their vacuolar degradation. Here, we discover two DUB proteins, UBP12 and UBP13, that directly target the PM-localized brassinosteroid (BR) receptor BR INSENSITIVE1 (BRI1) in Arabidopsis. BRI1 protein abundance is decreased in the ubp12i/ubp13 double mutant that displayed severe growth defects and reduced sensitivity to BRs. UBP13 directly interacts with and effectively removes K63-linked polyubiquitin chains from BRI1, thereby negatively modulating its vacuolar targeting and degradation. Our study reveals that UBP12 and UBP13 play crucial roles in governing BRI1 abundance and BR signaling activity to regulate plant growth.
• The TGN/EE SNARE protein SYP61 and the ubiquitin ligase ATL31 cooperatively regulate plant responses to carbon/nitrogen conditions in Arabidopsis.

  Yoko Hasegawa, Thais Huarancca Reyes, Tomohiro Uemura, Anirban Baral, Akari Fujimaki, Yongming Luo, Yoshie Morita, Yasushi Saeki, Shugo Maekawa, Shigetaka Yasuda, Koki Mukuta, Yoichiro Fukao, Keiji Tanaka, Akihiko Nakano, Junpei Takagi, Rishikesh P Bhalerao, Junji Yamaguchi, Takeo Sato

  The Plant cell 34 4 1354 - 1374 2022年03月29日 

  Ubiquitination is a post-translational modification involving the reversible attachment of the small protein ubiquitin to a target protein. Ubiquitination is involved in numerous cellular processes, including the membrane trafficking of cargo proteins. However, the ubiquitination of the trafficking machinery components and their involvement in environmental responses are not well understood. Here, we report that the Arabidopsis thaliana trans-Golgi network/early endosome localized SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein SYP61 interacts with the transmembrane ubiquitin ligase ATL31, a key regulator of resistance to disrupted carbon (C)/nitrogen/(N)-nutrient conditions. SYP61 is a key component of membrane trafficking in Arabidopsis. The subcellular localization of ATL31 was disrupted in knockdown mutants of SYP61, and the insensitivity of ATL31-overexpressing plants to high C/low N-stress was repressed in these mutants, suggesting that SYP61 and ATL31 cooperatively function in plant responses to nutrient stress. SYP61 is ubiquitinated in plants, and its ubiquitination level is upregulated under low C/high N-nutrient conditions. These findings provide important insights into the ubiquitin signaling and membrane trafficking machinery in plants.
• Low nitrogen conditions accelerate flowering by modulating the phosphorylation state of FLOWERING BHLH 4 in Arabidopsis.

  Miho Sanagi, Shoki Aoyama, Akio Kubo, Yu Lu, Yasutake Sato, Shogo Ito, Mitsutomo Abe, Nobutaka Mitsuda, Masaru Ohme-Takagi, Takatoshi Kiba, Hirofumi Nakagami, Filip Rolland, Junji Yamaguchi, Takato Imaizumi, Takeo Sato

  Proceedings of the National Academy of Sciences of the United States of America 118 19 2021年05月11日 

  Nitrogen (N) is an essential nutrient that affects multiple plant developmental processes, including flowering. As flowering requires resources to develop sink tissues for reproduction, nutrient availability is tightly linked to this process. Low N levels accelerate floral transition; however, the molecular mechanisms underlying this response are not well understood. Here, we identify the FLOWERING BHLH 4 (FBH4) transcription factor as a key regulator of N-responsive flowering in Arabidopsis Low N-induced early flowering is compromised in fbh quadruple mutants. We found that FBH4 is a highly phosphorylated protein and that FBH4 phosphorylation levels decrease under low N conditions. In addition, decreased phosphorylation promotes FBH4 nuclear localization and transcriptional activation of the direct target CONSTANS (CO) and downstream florigen FLOWERING LOCUS T (FT) genes. Moreover, we demonstrate that the evolutionarily conserved cellular fuel sensor SNF1-RELATED KINASE 1 (SnRK1), whose kinase activity is down-regulated under low N conditions, directly phosphorylates FBH4. SnRK1 negatively regulates CO and FT transcript levels under high N conditions. Together, these results reveal a mechanism by which N levels may fine-tune FBH4 nuclear localization by adjusting the phosphorylation state to modulate flowering time. In addition to its role in flowering regulation, we also showed that FBH4 was involved in low N-induced up-regulation of nutrient recycling and remobilization-related gene expression. Thus, our findings provide insight into N-responsive growth phase transitions and optimization of plant fitness under nutrient-limited conditions.
• 3-Phenyllactic acid, a root-promoting substance isolated from Bokashi fertilizer, exhibits synergistic effects with tryptophan.

  Yuko Maki, Hiroshi Soejima, Toru Kitamura, Tamizi Sugiyama, Takeo Sato, Masaaki K Watahiki, Junji Yamaguchi

  Plant biotechnology (Tokyo, Japan) 38 1 9 - 16 2021年03月25日 

  Bokashi fertilizer, an organic fertilizer made of plant residue, has been used in Japan not only to fertilize plants but to regulate their growth. Lactic acid bacteria have been found to play an important role in the fermentation process of Bokashi, but the relationship between these bacteria and plant growth activity has not been clarified. Using the adzuki rooting assay, this study identified 3-phenyllactic acid (PLA) produced by lactic acid bacteria as a root promoting compound in Bokashi. PLA showed synergistic effect with tryptophan, but no stem elongation activity. Lactic acid bacteria produced equal quantities of the L- and D-forms of PLA, which have similar root promoting activity. PLA did not significantly affect the amount of endogenous indole-3-acetic acid (IAA), although the chemical structure of PLA is highly similar to that of L-2-aminooxy-3-phenypropionic acid (L-AOPP), which inhibits IAA biosynthesis. These results indicate that the root promoting activity of PLA is not simply due to its increase in the amount of active auxin.
• Effect of circularly polarized light on germination, hypocotyl elongation and biomass production of arabidopsis and lettuce: Involvement of phytochrome B.

  Enkhsukh Lkhamkhuu, Kazunori Zikihara, Hitomi Katsura, Satoru Tokutomi, Takafumi Hosokawa, Yoshihisa Usami, Mitsuyoshi Ichihashi, Junji Yamaguchi, Kenji Monde

  Plant biotechnology (Tokyo, Japan) 37 1 57 - 67 2020年03月25日 

  Circular dichroism (CD), defined as the differential absorption of left- and right-handed circularly polarized light (CPL), is a useful spectroscopic technique for structural studies of biological systems composed of chiral molecules. The present study evaluated the effects of CPL on germination, hypocotyl elongation and biomass production of Arabidopsis and lettuce. Higher germination rates were observed when Arabidopsis and lettuce seedlings were irradiated with red right-handed CPL (R-CPL) than with red left-handed CPL (L-CPL). Hypocotyl elongation was effectively inhibited when Arabidopsis and lettuce seedlings were irradiated with red R-CPL than with red L-CPL. This difference was not observed when a phytochrome B (phyB) deficient mutant of Arabidopsis was irradiated, suggesting that inhibition of elongation by red R-CPL was mediated by phyB. White R-CPL induced greater biomass production by adult Arabidopsis plants, as determined by their fresh shoot weight, than white L-CPL. To determine the molecular basis of these CPL effects, CD spectra and the effect of CPL on the photoreaction of a sensory module of Arabidopsis phyB were measured. The red light-absorbing form of phyB showed a negative CD in the red light-absorbing region, consistent with the results of germination, inhibition of hypocotyl elongation and biomass production. L-CPL and R-CPL, however, did not differ in their ability to induce the interconversion of the red light-absorbing and far-red light-absorbing forms of phyB. These findings suggest that these CPL effects involve phyB, along with other photoreceptors and the photosynthetic process.
• Proteome Analysis of 14-3-3 Targets in Tomato Fruit Tissues.

  Yongming Luo, Yu Lu, Junji Yamaguchi, Takeo Sato

  Methods in molecular biology (Clifton, N.J.) 2139 289 - 296 2020年 

  Tomato is a major crop plant and an important constituent of the human diet. Exclusive features such as bearing fleshy fruits and undergoing a phase transition from partially photosynthetic to fully heterotrophic metabolism make tomato fruit a model system for fruit development studies. Although the tomato genome has been completely sequenced, functional proteomics studies are still at their starting stage. Proteomics technologies, especially the combination of multiple approaches, provide a very powerful tool to accurately identify functional proteins and investigate certain sets of proteins in more detail. The direct binding of plant 14-3-3 proteins to their multiple target proteins modulates the functions of the latter, suggesting that these 14-3-3 proteins are directly involved in various physiological pathways. This chapter outline methods for the identification of 14-3-3 protein complexes in tomato fruit tissues. These methods include detailed protocols for protein extraction, coimmunoprecipitation, SDS-PAGE, SYPRO Ruby staining, in-gel trypsin digestion, and LC-MS/MS analysis for 14-3-3 interactomics.
• Protein Phosphorylation Dynamics Under Carbon/Nitrogen-Nutrient Stress and Identification of a Cell Death-Related Receptor-Like Kinase in Arabidopsis.

  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年 [査読有り][通常論文]
   

  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.
• Identification of Arabidopsis CCR4-NOT Complexes with Pumilio RNA-Binding Proteins, APUM5 and APUM2.

  Toshihiro Arae, Kotone Morita, Riko Imahori, Yuya Suzuki, Shigetaka Yasuda, Takeo Sato, Junji Yamaguchi, Yukako Chiba

  Plant & cell physiology 60 9 2015 - 2025 2019年09月01日 [査読有り][通常論文]
   

  CCR4/CAF1 are widely conserved deadenylases in eukaryotes. They form a large complex that includes NOT1 as a scaffold protein and various NOT proteins that are core components of multiple levels of gene expression control. The CCR4-NOT complex also contains several RNA-binding proteins as accessory proteins, which are required for target recognition by CCR4/CAF1 deadenylases. AtCCR4a/b, orthologs of human CCR4 in Arabidopsis, have various physiological effects. AtCCR4 isoforms are likely to have specific target mRNAs related to each physiological effect; however, AtCCR4 does not have RNA-binding capability. Therefore, identifying factors that interact with AtCCR4a/b is indispensable to understand its function as a regulator of gene expression, as well as the target mRNA recognition mechanism. Here, we identified putative components of the AtCCR4-NOT complex using co-immunoprecipitation in combination with mass spectrometry using FLAG-tagged AtCCR4b and subsequent verification with a yeast two-hybrid assay. Interestingly, four of 11 AtCAF1 isoforms interacted with both AtCCR4b and AtNOT1, whereas two isoforms interacted only with AtNOT1 in yeast two-hybrid assays. These results imply that Arabidopsis has multiple CCR4-NOT complexes with various combinations of deadenylases. We also revealed that the RNA-binding protein Arabidopsis Pumilio 5 and 2 interacted with AtCCR4a/b in the cytoplasm with a few foci.
• Involvement of the membrane-localized ubiquitin ligase ATL8 in sugar starvation response in Arabidopsis.

  Yongming Luo, Shoki Aoyama, Yoichiro Fukao, Yukako Chiba, Takeo Sato, Junji Yamaguchi

  Plant biotechnology (Tokyo, Japan) 36 2 107 - 112 2019年06月25日 

  As major components of the ubiquitin system, ubiquitin ligases mediate the transfer of ubiquitin to specific target substrates, thereby playing important roles in regulating a wide range of cellular processes. The Arabidopsis Tóxicos en Levadura (ATL) family is a group of plant-specific RING-type ubiquitin ligases with N-terminal transmembrane-like domains. To date, 91 ATL isoforms have been identified in the Arabidopsis genome, with some reported to regulate plant responses to environmental stresses. However, the functions of most ATLs remain unclear. This study showed that ATL8 is a sugar starvation response gene and that ATL8 expression was significantly increased by sugar starvation conditions but repressed by exogenous sugar supply. The ATL8 protein was found to possess ubiquitin ligase activity in vitro and to localize to membrane-bound compartments in plant cells. In addition, Starch Synthase 4 was identified as a putative interactor with ATL8, suggesting that ATL8 may be involved in modulating starch accumulation in response to sugar availability. These findings suggest that ATL8 functions as a membrane-localized ubiquitin ligase likely to be involved in the adaptation of Arabidopsis plants to sugar starvation stress.
• Sugar-responsive transcription factor bZIP3 affects leaf shape in Arabidopsis plants.

  Miho Sanagi, Yu Lu, Shoki Aoyama, Yoshie Morita, Nobutaka Mitsuda, Miho Ikeda, Masaru Ohme-Takagi, Takeo Sato, Junji Yamaguchi

  Plant biotechnology (Tokyo, Japan) 35 2 167 - 170 2018年06月25日 [査読有り][通常論文]
   

  Sugars are essential for plant metabolism, growth and development. Plants must therefore manage their growth and developmental processes in response to sugar availability. Sugar signaling pathways constitute a complicated molecular network and are associated with global transcriptional regulation. However, the molecular mechanisms underlying sugar signaling remain largely unclear. This study reports that the protein basic-region leucine zipper 3 (bZIP3) is a novel sugar-responsive transcription factor in Arabidopsis plants. The expression of bZIP3 was rapidly repressed by sugar. Genetic analysis indicated that bZIP3 expression was modulated by the SNF1-RELATED KINASE 1 (SnRK1) pathway. Moreover, transgenic plants overexpressing bZIP3 and dominant repressor form bZIP3-SRDX showed aberrant shaped cotyledons with hyponastic bending. These findings suggest that bZIP3 plays a role in plant responses to sugars and is also associated with leaf development.
• Nitrate Reductase Modulation in Response to Changes in C/N Balance and Nitrogen Source in Arabidopsis.

  Thais Huarancca Reyes, Andrea Scartazza, Antonio Pompeiano, Andrea Ciurli, Yu Lu, Lorenzo Guglielminetti, Junji Yamaguchi

  Plant & cell physiology 59 6 1248 - 1254 2018年06月01日 [査読有り][通常論文]
   

  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.
• Membrane-localized ubiquitin ligase ATL15 functions in sugar-responsive growth regulation in Arabidopsis

  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 2017年09月 [査読有り][通常論文]
   

  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.
• Co-ordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells

  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 2017年06月 [査読有り][通常論文]
   

  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.
• Arabidopsis CBL-Interacting Protein Kinases Regulate Carbon/Nitrogen-Nutrient Response by Phosphorylating Ubiquitin Ligase ATL31

  Shigetaka Yasuda, Shoki Aoyama, Yoko Hasegawa, Takeo Sato, Junji Yamaguchi

  MOLECULAR PLANT 10 4 605 - 618 2017年04月 [査読有り][通常論文]
   

  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.
• Direct transcriptional activation of BT genes by NLP transcription factors is a key component of the nitrate response in Arabidopsis

  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 2017年01月 [査読有り][通常論文]
   

  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.
• Effect of carbon/nitrogen ratio on carbohydrate metabolism and light energy dissipation mechanisms in Arabidopsis thaliana

  Thais Huarancca Reyes, Andrea Scartazza, Yu Lu, Junji Yamaguchi, Lorenzo Guglielminetti

  PLANT PHYSIOLOGY AND BIOCHEMISTRY 105 195 - 202 2016年08月 [査読有り][通常論文]
   

  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.
• Characterization of ubiquitin ligase SlATL31 and proteomic analysis of 14-3-3 targets in tomato fruit tissue (Solanum lycopersicum L.)

  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 2016年06月 [査読有り][通常論文]
   

  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.
• P4-1-15 硝酸シグナル応答型転写因子NLPはBT群の遺伝子発現制御を介して側根伸長を制御する（ポスター，4-1 植物の多量栄養素，2016年度佐賀大会）

  前川 修吾, 吉岡 希, 小西 美稲子, 石田 哲也, 加藤 祐樹, 佐々木 勇樹, 佐藤 長緒, 山口 淳二, 柳澤 修一

  日本土壌肥料学会講演要旨集 62 55 - 55 一般社団法人 日本土壌肥料学会 2016年
• Integration of C/N-nutrient and multiple environmental signals into the ABA signaling cascade

  Yu Lu, Junji Yamaguchi, Takeo Sato

  PLANT SIGNALING & BEHAVIOR 10 12 e1048940  2015年12月 [査読有り][通常論文]
   

  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.
• Erratum: ABI1 regulates carbon/nitrogen-nutrient signal transduction independent of ABA biosynthesis and canonical ABA signalling pathways in Arabidopsis (Journal of Experimental Botany (2015) 66:9 (2763-2771))

  Yu Lu, Yuki Sasaki, Xingwen Li, Izumi C. Mori, Takakazu Matsuura, Takashi Hirayama, Takeo Sato, Junji Yamaguchi

  Journal of Experimental Botany 66 15 4851  2015年08月01日 [査読有り][通常論文]
  
• ABI1 regulates carbon/nitrogen-nutrient signal transduction independent of ABA biosynthesis and canonical ABA signalling pathways in Arabidopsis (vol 66, pg 2763, 2015)

  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 2015年08月 [査読有り][通常論文]
  
• AtCCR4a and AtCCR4b are Involved in Determining the Poly(A) Length of Granule-bound starch synthase 1 Transcript and Modulating Sucrose and Starch Metabolism in Arabidopsis thaliana

  Yuya Suzuki, Toshihiro Arae, Pamela J. Green, Junji Yamaguchi, Yukako Chiba

  PLANT AND CELL PHYSIOLOGY 56 5 863 - 874 2015年05月 [査読有り][通常論文]
   

  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.
• ABI1 regulates carbon/nitrogen-nutrient signal transduction independent of ABA biosynthesis and canonical ABA signalling pathways in Arabidopsis

  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 2015年05月 [査読有り][通常論文]
   

  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.
• The Arabidopsis ubiquitin ligase ATL31 is transcriptionally controlled by WRKY33 transcription factor in response to pathogen attack

  Thais Huarancca Reyes, Shugo Maekawa, Takeo Sato, Junji Yamaguchi

  PLANT BIOTECHNOLOGY 32 1 11 - + 2015年03月 [査読有り][通常論文]
   

  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.
• Pale-Green Phenotype of atl31 atl6 Double Mutant Leaves Is Caused by Disruption of 5-Aminolevulinic Acid Biosynthesis in Arabidopsis thaliana

  Shugo Maekawa, Atsushi Takabayashi, Thais Huarancca Reyes, Hiroko Yamamoto, Ayumi Tanaka, Takeo Sato, Junji Yamaguchi

  PLOS ONE 10 2 e0117662  2015年02月 [査読有り][通常論文]
   

  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.
• 植物の自然免疫に関わる膜局在型ユビキチンリガーゼの標的タンパク質の探索

  安田 盛貴, 長谷川 陽子, 門田 康弘, 深尾 陽一朗, 佐藤 長緒, 山口 淳二

  日本プロテオーム学会大会要旨集 2015 232 - 232 日本プロテオーム学会（日本ヒトプロテオーム機構） 2015年
• Proteomic Analysis of the 26S Proteasome Reveals Its Direct Interaction with Transit Peptides of Plastid Protein Precursors for Their Degradation

  Kaori Sako, Yuki Yanagawa, Tomoyuki Kanai, Takeo Sato, Motoaki Seki, Masayuki Fujiwara, Yoichiro Fukao, Junji Yamaguchi

  JOURNAL OF PROTEOME RESEARCH 13 7 3223 - 3230 2014年07月 [査読有り][通常論文]
   

  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.
• Phosphorylation of Arabidopsis Ubiquitin Ligase ATL31 Is Critical for Plant Carbon/Nitrogen Nutrient Balance Response and Controls the Stability of 14-3-3 Proteins

  Shigetaka Yasuda, Takeo Sato, Shugo Maekawa, Shoki Aoyama, Yoichiro Fukao, Junji Yamaguchi

  JOURNAL OF BIOLOGICAL CHEMISTRY 289 22 15179 - 15193 2014年05月 [査読有り][通常論文]
   

  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.
• Regulation of senescence under elevated atmospheric CO₂ via ubiquitin modification.

  Aoyama S, Lu Y, Yamaguchi J, Sato T

  Plant signaling & behavior 9 5 e28839  2014年04月16日 [査読有り][通常論文]
   

  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.
• The Carbon/Nitrogen Regulator ARABIDOPSIS TOXICOS EN LEVADURA31 Controls Papilla Formation in Response to Powdery Mildew Fungi Penetration by Interacting with SYNTAXIN OF PLANTS121 in Arabidopsis

  Shugo Maekawa, Noriko Inada, Shigetaka Yasuda, Yoichiro Fukao, Masayuki Fujiwara, Takeo Sato, Junji Yamaguchi

  PLANT PHYSIOLOGY 164 2 879 - 887 2014年02月 [査読有り][通常論文]
   

  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.
• Ubiquitin Ligase ATL31 Functions in Leaf Senescence in Response to the Balance Between Atmospheric CO2 and Nitrogen Availability in Arabidopsis

  Shoki Aoyama, Thais Huarancca Reyes, Lorenzo Guglielminetti, Yu Lu, Yoshie Morita, Takeo Sato, Junji Yamaguchi

  PLANT AND CELL PHYSIOLOGY 55 2 293 - 305 2014年02月 [査読有り][通常論文]
   

  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.
• The Arabidopsis transcriptional repressor ERF9 participates in resistance against necrotrophic fungi

  Yosuke Maruyama, Natsuko Yamoto, Yuya Suzuki, Yukako Chiba, Ken-ichi Yamazaki, Takeo Sato, Junji Yamaguchi

  PLANT SCIENCE 213 79 - 87 2013年12月 [査読有り][通常論文]
   

  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.
• Proteomics Analysis Reveals a Highly Heterogeneous Proteasome Composition and the Post-translational Regulation of Peptidase Activity under Pathogen Signaling in Plants

  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 2013年11月 [査読有り][通常論文]
   

  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.
• Changes in mRNA stability associated with cold stress in arabidopsis cells

  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 2013年 [査読有り][通常論文]
   

  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.
• The Arabidopsis ubiquitin ligases ATL31 and ATL6 control the defense response as well as the carbon/nitrogen response

  Shugo Maekawa, Takeo Sato, Yutaka Asada, Shigetaka Yasuda, Midori Yoshida, Yukako Chiba, Junji Yamaguchi

  PLANT MOLECULAR BIOLOGY 79 3 217 - 227 2012年06月 [査読有り][通常論文]
   

  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.
• Arabidopsis RPT2a, 19S Proteasome Subunit, Regulates Gene Silencing via DNA Methylation

  Kaori Sako, Yuko Maki, Tomoyuki Kanai, Eriko Kato, Shugo Maekawa, Shigetaka Yasuda, Takeo Sato, Masaaki K. Watahiki, Junji Yamaguchi

  PLOS ONE 7 5 e37086  2012年05月 [査読有り][通常論文]
   

  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年 [査読有り][通常論文]
  
• Sugar-inducible RPT2a, a subunit of 26S proteasome, participates in sugar response in Arabidopsis

  Sun Huihui, Sako Kaori, Suzuki Yuya, Maekawa Shugo, Yasuda Shigetaka, Chiba Yukako, Sato Takeo, Yamaguchi Junji

  PLANT BIOTECHNOLOGY 29 3 279 - 284 日本植物細胞分子生物学会 2012年 [査読有り][通常論文]
   

  The ubiquitin/26S proteasome system (UPS) plays a central role in the degradation of short-lived regulatory proteins that control many cellular events. In this study, the Arabidopsis knockout mutant rpt2a, which contains a defect in the AtRPT2a subunit of the 26S proteasome regulatory particle, showed hypersensitivity to sugars as well as enlarged leaves. When the role of RPT2a in sugar response was examined in further detail it was found that putatively only the AtRPT2a gene of 19S proteasome was markedly transcriptionally promoted by sugar application. Notably, poly-ubiquitinated proteins degraded by the UPS accumulated significantly in rpt2a mutant under 6% sucrose conditions compared to wild type. In addition, the AtRPT2a gene in gin2, a glucose insensitive mutant with a defective glucose-sensing hexokinase, was not upregulated by sugar application, indicating that AtRPT2a is involved in hexokinase-dependent sugar response. Taken together, the above findings indicate that AtRPT2a plays an essential role in the maintenance of proteasome-dependent proteolysis activity in response to sugars.
• Identification of 14-3-3 proteins as a target of ATL31 ubiquitin ligase, a regulator of the C/N response in Arabidopsis

  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 2011年10月 [査読有り][通常論文]
   

  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.
• Sucrose transporter NtSUT4 from tobacco BY-2 involved in plant cell shape during miniprotoplast culture

  Emiko Okubo-Kurihara, Takumi Higaki, Yukio Kurihara, Natsumaro Kutsuna, Junji Yamaguchi, Seiichiro Hasezawa

  JOURNAL OF PLANT RESEARCH 124 3 395 - 403 2011年05月 [査読無し][通常論文]
   

  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.
• Arabidopsis thaliana 26S Proteasome Subunits RPT2a and RPT5a Are Crucial for Zinc Deficiency-Tolerance

  Takuya Sakamoto, Takehiro Kamiya, Kaori Sako, Junji Yamaguchi, Mutsumi Yamagami, Toru Fujiwara

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 3 561 - 567 2011年03月 [査読有り][通常論文]
   

  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.
• The Arabidopsis NSL2 negatively controls systemic acquired resistance via hypersensitive response

  Yutaka Asada, Masako Yamamoto, Tomokazu Tsutsui, Junji Yamaguchi

  PLANT BIOTECHNOLOGY 28 1 9 - 15 2011年 [査読無し][通常論文]
   

  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.
• Carbon and nitrogen metabolism regulated by ubiquitin-proteasome system.

  Plant and Signaling Behavior 6 1465 - 1468 2011年 [査読無し][通常論文]
  
• Carbon and nitrogen metabolism regulated by the ubiquitin-proteasome system

  Takeo Sato, Shugo Maekawa, Shigetaka Yasuda, Junji Yamaguchi

  Plant Signaling and Behavior 6 10 1465 - 1468 2011年 [査読有り][通常論文]
   

  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.
• プロテオミクスを用いた植物ユビキチン－プロテアソームシステムの解明―基幹代謝を例として―

  山口 淳二, 佐藤 長緒

  日本プロテオーム学会大会要旨集 2011 64 - 64 日本プロテオーム学会（日本ヒトプロテオーム機構） 2011年
• Control of endoreduplication of trichome by RPT2a, a subunit of the 19S proteasome in Arabidopsis

  Kaori Sako, Yuko Maki, Kumiko K. Imai, Takashi Aoyama, Derek B. Goto, Junji Yamaguchi

  JOURNAL OF PLANT RESEARCH 123 5 701 - 706 2010年09月 [査読無し][通常論文]
   

  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).
• How does the plant proteasome control leaf size?

  Kaori Sako, Junji Yamaguchi

  Plant Signaling and Behavior 5 9 1119 - 1120 2010年09月 [査読有り][通常論文]
   

  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.
• In planta observation of live fluorescent plant endoparasitic nematodes during early stages of infection.

  佐久間 太, 植原 健人, 近藤 則夫, 山口 淳二

  Nematological Research 40 1 15 - 19 日本線虫学会 2010年 [査読無し][通常論文]
   

  内部寄生性植物線虫の感染初期の研究は重要であるが、寄主植物根内の線虫を生きたまま観察できないことが障害となっていた。そこで、二酢酸フルオレセイン（FDA）の0.001％溶液を用い、生きたネコブセンチュウとネグサレセンチュウを蛍光標識して、非破壊的な蛍光観察を行った。その結果、FDAで処理したネコブセンチュウは生物活性を保っており、寄主のトマト根内で感染場所が検出できることが示された。実験系として用いたシロイヌナズナでは、寄主植物根の中に侵入した線虫を落射蛍光顕微鏡下で直接観察することができ、FDA由来の蛍光が処理後少なくとも4日間検出された。FDAで処理したネグサレセンチュウも容易に寄主植物に感染したが、ネグサレセンチユウは検出可能な自己蛍光を示したため、非破壊的な検出のためのFDA処理は必要ないことが分かった。この報告は、線虫感染の初期段階において、高度な時空間分析に基づく非破壊的解析が可能であることを実証した。
• CNI1/ATL31, a RING-type ubiquitin ligase that functions in the carbon/nitrogen response for growth phase transition in Arabidopsis seedlings.

  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 2009年12月 [査読有り][通常論文]
   

  Plants are able to sense and respond to changes in the balance between carbon (C) and nitrogen (N) metabolite availability, known as the C/N response. During the transition to photoautotrophic growth following germination, growth of seedlings is arrested if a high external C/N ratio is detected. To clarify the mechanisms for C/N sensing and signaling during this transition period, we screened a large collection of FOX transgenic plants, overexpressing full-length cDNAs, for individuals able to continue post-germinative growth under severe C/N stress. One line, cni1-D (carbon/nitrogen insensitive 1-dominant), was shown to have a suppressed sensitivity to C/N conditions at both the physiological and molecular level. The CNI1 cDNA encoded a predicted RING-type ubiquitin ligase previously annotated as ATL31. Overexpression of ATL31 was confirmed to be responsible for the cni1-D phenotype, and a knock-out of this gene resulted in hypersensitivity to C/N conditions during post-germinative growth. The ATL31 protein was confirmed to contain ubiquitin ligase activity using an in vitro assay system. Moreover, removal of this ubiquitin ligase activity from the overexpressed protein resulted in the loss of the mutant phenotype. Taken together, these data demonstrated that CNI1/ATL31 activity is required for the plant C/N response during seedling growth transition.
• DEAR1, a transcriptional repressor of DREB protein that mediates plant defense and freezing stress responses in Arabidopsis

  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 2009年11月 [査読無し][通常論文]
   

  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.
• Regulation of leaf organ size by the Arabidopsis RPT2a 19S proteasome subunit

  Yutaka Sonoda, Kaori Sako, Yuko Maki, Naoko Yamazaki, Hiroko Yamamoto, Akira Ikeda, Junji Yamaguchi

  PLANT JOURNAL 60 1 68 - 78 2009年10月 [査読有り][通常論文]
   

  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.
• Arabidopsis MBF1s Control Leaf Cell Cycle and its Expansion

  Takuto Tojo, Kenichi Tsuda, Takeshi Yoshizumi, Akira Ikeda, Junji Yamaguchi, Minami Matsui, Ken-ichi Yamazaki

  PLANT AND CELL PHYSIOLOGY 50 2 254 - 264 2009年02月 [査読無し][通常論文]
   

  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.
• Arabidopsis CAD1 negatively controls plant immunity mediated by both salicylic acid-dependent and -independent signaling pathways

  Tomokazu Tsutsui, Yutaka Asada, Masanori Tamaoki, Akira Ikeda, Junji Yamaguchi

  PLANT SCIENCE 175 4 604 - 611 2008年10月 [査読無し][通常論文]
   

  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.
• Promoter analysis of OsAMT1;2 and OsAMT1;3 implies their distinct roles in nitrogen utilization in rice

  Shan-Guo Yao, Yutaka Sonoda, Tomokazu Tsutsui, Hidemitsu Nakamura, Hiroaki Ichikawa, Akira Ikeda, Junji Yamaguchi

  BREEDING SCIENCE 58 3 201 - 207 2008年09月 [査読無し][通常論文]
   

  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.
• alpha-amylase production is induced by sulfuric acid in rice aleurone cells

  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 2007年12月 [査読無し][通常論文]
   

  The hydrolytic enzyme v.-amylase (EC 3.2.1.1) 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.
• SHA1, a novel RING finger protein, functions in shoot apical meristem maintenance in Arabidopsis

  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 2007年05月 [査読有り][通常論文]
   

  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.
• Isolation and characterization of sugar insensitive mutant in Arabidopsis

  Takeo Sato, Wataru Kato, Yutaka Sonoda, Takanari Ichikawa, Miki Nakazawa, Miki Fujita, Motoaki Seki, Kazuo Shinozaki, Minami Matsui, Akira Ikeda, Junji Yamaguchi

  PLANT AND CELL PHYSIOLOGY 48 S132 - S132 2007年 [査読有り][通常論文]
  
• Salicylic acid and a chitin elicitor both control expression of the CAD1 gene involved in the plant immunity of Arabidopsis

  Tolllokazu Tsutsui, Chlzuko Morita-Yamamuro, Yutaka Asada, Ellchl Minami, Naoto Shibuya, Aklra Ikeda, Junji Yamaguchi

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 70 9 2042 - 2048 2006年09月 [査読無し][通常論文]
   

  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.
• Differential expression of two fructokinases in Oryza sativa seedlings grown under aerobic and anaerobic conditions

  L. Guglielminetti, A. Morita, J. Yamaguchi, E. Loreti, P. Perata, A. Alpi

  JOURNAL OF PLANT RESEARCH 119 4 351 - 356 2006年07月 [査読無し][通常論文]
   

  Fructokinases (EC 2.7.1.4) 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.
• A transposon-induced spontaneous mutation results in low beta-amylase content in rice

  H Saika, M Nakazono, A Ikeda, J Yamaguchi, S Masaki, M Kanekatsu, K Nemoto

  PLANT SCIENCE 169 1 239 - 244 2005年07月 [査読無し][通常論文]
   

  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.
• The Arabidopsis gene CAD1 controls programmed cell death in the plant immune system and encodes a protein containing a MACPF domain

  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 2005年06月 [査読有り][通常論文]
   

  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.
• The Arabidopsis gene CAD1 controls programmed cell death in the plant immune system and encodes a protein containing a MACPF domain.

  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 2005年 [査読無し][通常論文]
  
• Measurement of the concentration of bioactive gibberellin in germinating rice seed using the α-amylase induction from aleurone cells.

  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 The Japanese Society of Applied Glycoscience 2005年 [査読無し][通常論文]
   

  穀類アリューロン細胞の活性化に伴う貯蔵デンプン分解の分子機構を理解するうえで, 発芽種子内に含まれる活性型ジベレリン (GA) の濃度を把握しておくことはきわめて重要である. そこで, アリューロン細胞におけるα-アミラーゼ合成誘導を指標に, イネの発芽種子に含まれる活性型GA濃度の簡易測定を試みた. さまざまな濃度のGAをアリューロン細胞に加えた場合に合成誘導されるα-アミラーゼの酵素活性を測定し, それらを指標に種子内の活性型GA濃度を推測した. また, 抗α-アミラーゼ抗体を用いた免疫ブロッティングにより, α-アミラーゼタンパク質の発現量をニトロセルロース膜上で検出し, それらを利用したGA濃度の測定も行った. その結果, 発芽種子内における活性型GA濃度を, 0.1から1nMの範囲に特定することができた. また, GA濃度が1nM以上に上昇したか否かを判断するための指標となりうる42kDaのα-アミラーゼアイソフォームの存在を, 同時に見出すことができた. このアイソフォームは, イネのアリューロン細胞に存在する高濃度GAシグナリングを解析するために有効であると考えられる.
• The Arabidopsis gene CAD1 controls programmed cell death in the plant immune system and encodes a protein containing a MACPF domain.

  C Yamamuro, T Tsutsui, Y Asada, H Yoshioka, M Tamaoki, D Ogawa, H Matsuura, T Yoshihara, A Ikeda, J Yamaguchi

  PLANT AND CELL PHYSIOLOGY 46 S120 - S120 2005年 [査読有り][通常論文]
  
• Knock-out of the plastid ribosomal protein S21 causes impaired photosynthesis and sugar-response during germination and seedling development in Arabidopsis thaliana

  C Morita-Yamamuro, T Tsutsui, A Tanaka, J Yamaguchi

  PLANT AND CELL PHYSIOLOGY 45 6 781 - 788 2004年06月 [査読無し][通常論文]
   

  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.
• Low sugar status promotes endogenous ABA level and ABA sensitivity in Arabidopsis

  Chizuko Morita-Yamamuro, Paolo Vernieri, Junji Yamaguchi

  Plant Biotechnology 21 1 9 - 14 2004年 [査読無し][通常論文]
   

  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.
• Feedback regulation of the ammonium transporter gene family AMT1 by glutamine in rice

  Y Sonoda, A Ikeda, S Saiki, T Yamaya, J Yamaguchi

  PLANT AND CELL PHYSIOLOGY 44 12 1396 - 1402 2003年12月 [査読無し][通常論文]
   

  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.
• Distinct expression and function of three ammonium transporter genes (OsAMT1;1-1;3) in rice

  Y Sonoda, A Ikeda, S Saiki, N von Wiren, T Yamaya, J Yamaguchi

  PLANT AND CELL PHYSIOLOGY 44 7 726 - 734 2003年07月 [査読無し][通常論文]
   

  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.
• Gibberellins are not required for rice germination under anoxia

  E Loreti, J Yamaguchi, A Alpi, P Perata

  PLANT AND SOIL 253 1 137 - 143 2003年06月 [査読無し][通常論文]
   

  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.
• Characterization of rice functional monosaccharide transporter, OsMST5

  B Ngampanya, A Sobolewska, T Takeda, K Toyofuku, J Narangajavana, A Ikeda, J Yamaguchi

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 67 3 556 - 562 2003年03月 [査読無し][通常論文]
   

  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.
• Novel gene encoding a Ca2+-binding protein and under hexokinase-dependent sugar regulation

  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 2003年02月 [査読無し][通常論文]
   

  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+.
• Constitutive expression of a novel-type ammonium transporter OsAMT2 in rice plants

  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 2003年02月 [査読無し][通常論文]
   

  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.
• Sugar modulation of alpha-amylase genes under anoxia

  E Loreti, J Yamaguchi, A Alpi, P Perata

  ANNALS OF BOTANY 91 2 143 - 148 2003年01月 [査読無し][通常論文]
   

  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.
• Sugar transporters involved in flowering and grain development of rice

  Ngampanya Budsaraporn, Takeda Taito, Narangajavana Jarunya, IKEDA Akira, YAMAGUCHI Junji

  Journal of Applied Glycoscience 50 2 237 - 240 The Japanese Society of Applied Glycoscience 2003年 [査読無し][通常論文]
   

  Sucrose and hexoses derived from sucrose breakdown are importance for early grain development in cereals. These assimilates were used to promote division and elongation, differentiation of the cells and deposition in storage organs. OsMST5 (Oryza sativa monosaccharide transporter 5), OsSUT1 (Oryza sativa sucrose transporter 1) and OsSUT2 (Oryza saliva sucrose transporter 2) have been cloned from rice. All mRNAs of these functional sugar transporters were detected during flowering and grain filling stage. The OsMST5 and OsSUT2 mRNAs were exclusively detectable before the pollinating stage by Northern analysis and the Os- SUT2 mRNA was localized in archespores and pollen mother cells by in situ mRNA detection, indicating that these gene products are involved in import of sucrose to promote pollen development at the early stage. Moreover, the OsSUT1 whiclh is known to be phloem companion cell-specific expression in source organs, was also expressed in the sink organs as well as modified aleurone cells of the developing endosperm. These results indicate that the OsSUT1 plays a role in re-uptake of sucrose from endosperm cavity to accumulate starch in grain filling stage.
• The slender rice mutant, with constitutively activated gibberellin signal transduction, has enhanced capacity for abscisic acid level

  A Ikeda, Y Sonoda, P Vernieri, P Perata, H Hirochika, J Yamaguchi

  PLANT AND CELL PHYSIOLOGY 43 9 974 - 979 2002年09月 [査読無し][通常論文]
   

  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.
• イネアンモニウムトランスポーターOsAMT1;3の機能解析(第37回大会研究発表抄録)

  齋木 里文, 園田 裕, 池田 亮, 山谷 知行, 山口 淳二

  植物の生長調節 37 2 221 - 221 一般社団法人植物化学調節学会 2002年
• slender rice, a constitutive gibberellin response mutant, is caused by a null mutation of the SLR1 gene, an ortholog of the height-regulating gene GAI/RGA/RHT/D8

  A Ikeda, M Ueguchi-Tanaka, Y Sonoda, H Kitano, M Koshioka, Y Futsuhara, M Matsuoka, J Yamaguchi

  PLANT CELL 13 5 999 - 1010 2001年05月 [査読無し][通常論文]
   

  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.
• Sugar transporters involved in flowering and grain development of rice

  T Takeda, K Toyofuku, C Matsukura, J Yamaguchi

  JOURNAL OF PLANT PHYSIOLOGY 158 4 465 - 470 2001年04月 [査読無し][通常論文]
   

  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.
• Polymorphism in rice amylases at an early stage of seed germination

  S Mitsunaga, O Kawakami, T Numata, J Yamaguchi, K Fukui, T Mitsui

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 65 3 662 - 665 2001年03月 [査読無し][通常論文]
   

  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.
• Sugar uptake and transport in rice embryo. Expression of companion cell-specific sucrose transporter (OsSUT1) induced by sugar and light

  C Matsukura, T Saitoh, T Hirose, R Ohsugi, P Perata, J Yamaguchi

  PLANT PHYSIOLOGY 124 1 85 - 93 2000年09月 [査読有り][通常論文]
   

  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.
• Characterization and expression of monosaccharide transporters (OsMSTs) in rice

  K Toyofuku, M Kasahara, J Yamaguchi

  PLANT AND CELL PHYSIOLOGY 41 8 940 - 947 2000年08月 [査読有り][通常論文]
   

  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.
• Distribution and characterization of enzymes causing starch degradation in rice (Oryza sativa Cv. Koshihikari)

  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 2000年02月 [査読有り][通常論文]
   

  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 3.2.1.20) and α-amylase (EC 3.2.1.1) 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.
• Glucose modulates the abscisic acid-inducible Rab16A gene in cereal embryos

  K Toyofuku, E Loreti, P Vernieri, A Alpi, P Perata, J Yamaguchi

  PLANT MOLECULAR BIOLOGY 42 3 451 - 460 2000年02月 [査読有り][通常論文]
   

  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.
• Expression of α-amylase isoforms and the RAmy1A gene in rice (Oryza sativa L.) during seed germination, and its relationship with coleoptile length in submerged soil

  J. Huang, K. Toyofuku, J. Yamaguchi, S. Akita

  Plant Production Science 3 1 32 - 37 2000年 [査読有り][通常論文]
   

  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.
• Characterization of isoforms of hexose kinases in rice embryo

  L Guglielminetti, P Perata, A Morita, E Loreti, J Yamaguchi, A Alpi

  PHYTOCHEMISTRY 53 2 195 - 200 2000年01月 [査読有り][通常論文]
   

  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 - 90 2000年01月 [査読有り][通常論文]
  
• Transverse vein differentiation associated with gas space formation - Fate of the middle cell layer in leaf sheath development of rice

  C Matsukura, M Kawai, K Toyofuku, RA Barrero, H Uchimiya, J Yamaguchi

  ANNALS OF BOTANY 85 1 19 - 27 2000年01月 [査読有り][通常論文]
   

  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年12月 [査読有り][通常論文]
  
• オーキシンとイネの根の形態形成 : GASR1遺伝子・タンパク質の解析

  池田 亮, 山口 淳二

  根の研究 = Root research 8 1 9 - 12 Japanese Society for Root Research 1999年03月08日
• Reducing-agents-mediated solubilization and activation of debranching enzyme (pullulanase) in rice flour

  Wakako Takeuchi, Hironori Masui, Junji Yamaguchi

  Bioscience, Biotechnology and Biochemistry 63 3 510 - 514 1999年01月01日 [査読有り][通常論文]
   

  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.
• Changes in α-amylase isoforms during emergence of rice in submerged soil

  Jirong Huang, Junji Yamaguchi, Shigemi Akita

  Plant Production Science 2 1 12 - 13 1999年 [査読有り][通常論文]
  
• 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年01月 [査読無し][通常論文]
  
• Characterization of beta-amylase and its deficiency in various rice cultivars

  J Yamaguchi, S Itoh, T Saitoh, A Ikeda, T Tashiro, Y Nagato

  THEORETICAL AND APPLIED GENETICS 98 1 32 - 38 1999年01月 [査読有り][通常論文]
   

  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.
• Analysis of embryo-specific alpha-amylase using isolated mature rice embryos

  J Yamaguchi

  BREEDING SCIENCE 48 4 365 - 370 1998年12月 [査読有り][通常論文]
   

  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.
• Detection of proteins binding to the promoter region DNA using a nonradioactive gel-retardation assay

  S Mitsunaga, K Fukui, H Ohyama, J Yamaguchi

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 62 9 1812 - 1814 1998年09月 [査読有り][通常論文]
   

  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.
• Promotion of leaf sheath growth by gibberellic acid in a dwarf mutant of rice

  C Matsukura, S Itoh, K Nemoto, E Tanimoto, J Yamaguchi

  PLANTA 205 2 145 - 152 1998年06月 [査読有り][通常論文]
   

  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.
• Promoter elements required for sugar-repression of the RAmy3D gene for alpha-amylase in rice

  K Toyofuku, T Umemura, J Yamaguchi

  FEBS LETTERS 428 3 275 - 280 1998年05月 [査読有り][通常論文]
   

  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.
• Sugar sensing and alpha-amylase gene repression in rice embryos

  T Umemura, P Perata, Y Futsuhara, J Yamaguchi

  PLANTA 204 4 420 - 428 1998年04月 [査読有り][通常論文]
   

  We used a transient expression system to study the mechanism by which carbohydrates repress a lice (Oryza sativa L.) alpha-amylase (EC 3.2.1.1) 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.
• Temporal and spatial expression of the alpha-amylase gene during seed germination in rice and barley

  N Sugimoto, G Takeda, Y Nagato, J Yamaguchi

  PLANT AND CELL PHYSIOLOGY 39 3 323 - 333 1998年03月 [査読有り][通常論文]
   

  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.
• Functional dissection of a sugar-repressed alpha-amylase gene (RAmylA) promoter in rice embryos

  A Morita, T Umemura, M Kuroyanagi, Y Futsuhara, P Perata, J Yamaguchi

  FEBS LETTERS 423 1 81 - 85 1998年02月 [査読有り][通常論文]
   

  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.
• Analysis of embryo-specific α-amylase using isolated mature rice embryos

  J. Yamaguchi

  Breeding Science 48 4 365 - 370 1998年 [査読有り][通常論文]
   

  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.
• Effect of anoxia on gibberellic acid-induced protease and beta-amylase processing in barley seeds

  E Loreti, L Guglielminetti, J Yamaguchi, S Gonzali, A Alpi, P Perata

  JOURNAL OF PLANT PHYSIOLOGY 152 1 44 - 50 1998年01月 [査読有り][通常論文]
   

  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年01月 [査読有り][通常論文]
  
• Sugar repression of a gibberellin-dependent signaling pathway in barley embryos.

  Perata;P, C., Matsukura, P., Vernieri, J., Yamaguchi

  Plant Cell 9 2197-2208 - 2208 1997年01月 [査読有り][通常論文]
  
• 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年01月 [査読有り][通常論文]
  
• Physicochemical and serological characterization of rice alpha-amylase isoforms and identification of their corresponding genes

  T Mitsui, J Yamaguchi, T Akazawa

  PLANT PHYSIOLOGY 110 4 1395 - 1404 1996年04月 [査読有り][通常論文]
   

  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.
• The same nuclear proteins bind to the 5′-flanking regions of genes for the rice seed storage protein: 16 kDa albumin, 13 kDa prolamin and type II glutelin

  Masayuki Nakase, Takehisa Yamada, Takahiro Kira, Junji Yamaguchi, Naohito Aoki, Ryo Nakamura, Tsukasa Matsuda, Takahiro Adachi

  Plant Molecular Biology 32 4 621 - 630 1996年 [査読有り][通常論文]
   

  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.
• Changes in α-amylase activity during plant regeneration from rice calli

  Toshinori Abe, Masumi Kudo, Yasuhiro Oka, Junji Yamaguchi, Takeo Sasahara

  Journal of Plant Physiology 149 5 592 - 598 1996年 [査読有り][通常論文]
   

  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. 3.2.1.1) 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.
• Amylolytic activities in cereal seeds under aerobic and anaerobic conditions

  Lorenzo Guglielminetti, Junji Yamaguchi, Pierdomenico Perata, Amedeo Alpi

  Plant Physiology 109 3 1069 - 1076 1995年 [査読有り][通常論文]
   

  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 3.2.1.1), β-amylase (EC 3.2.1.2), debranching enzyme (EC 3.2.1.41), and α-glucosidase (EC 3.2.1.20) 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.
• DEVELOPMENTAL AND HORMONAL-REGULATION OF RICE ALPHA-AMYLASE(RAMY1A)-GUSA FUSION GENES IN TRANSGENIC RICE SEEDS

  K ITOH, J YAMAGUCHI, N HUANG, RL RODRIGUEZ, T AKAZAWA, K SHIMAMOTO

  PLANT PHYSIOLOGY 107 1 25 - 31 1995年01月 [査読有り][通常論文]
   

  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.
• SEQUENCE-SPECIFIC INTERACTIONS OF A NUCLEAR-PROTEIN FACTOR WITH THE PROMOTER REGION OF A RICE GENE FOR ALPHA-AMYLASE, RAMY3D

  S MITSUNAGA, RL RODRIGUEZ, J YAMAGUCHI

  NUCLEIC ACIDS RESEARCH 22 11 1948 - 1953 1994年06月 [査読有り][通常論文]
   

  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.
• IDENTIFICATION AND CHARACTERIZATION OF GIBBERELLIN-INSENSITIVE MUTANTS SELECTED FROM AMONG DWARF MUTANTS OF RICE

  S MITSUNAGA, T TASHIRO, J YAMAGUCHI

  THEORETICAL AND APPLIED GENETICS 87 6 705 - 712 1994年01月 [査読有り][通常論文]
   

  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.
• EFFECT OF ANOXIA ON THE INDUCTION OF ALPHA-AMYLASE IN CEREAL SEEDS

  P PERATA, N GESHI, J YAMAGUCHI, T AKAZAWA

  PLANTA 191 3 402 - 408 1993年08月 [査読有り][通常論文]
   

  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.
• INDUCTION OF ALPHA-AMYLASE IS REPRESSED BY UNICONAZOLE, AN INHIBITOR OF THE BIOSYNTHESIS OF GIBBERELLIN, IN A DWARF MUTANT OF RICE, WAITO-C

  S MITSUNAGA, J YAMAGUCHI

  PLANT AND CELL PHYSIOLOGY 34 2 243 - 249 1993年03月 [査読有り][通常論文]
   

  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.
• EFFECT OF ANOXIA ON STARCH BREAKDOWN IN RICE AND WHEAT SEEDS

  P PERATA, J POZUETAROMERO, T AKAZAWA, J YAMAGUCHI

  PLANTA 188 4 611 - 618 1992年11月 [査読有り][通常論文]
   

  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 3.2.1.1.) 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.
• COMPLEMENTARY-DNA SEQUENCE OF AN ADENYLATE TRANSLOCATOR FROM ARABIDOPSIS-THALIANA

  A SAINTGUILY, PY LIM, C CHEVALIER, J YAMAGUCHI, T AKAZAWA

  PLANT PHYSIOLOGY 100 2 1069 - 1071 1992年10月 [査読有り][通常論文]
  
• ARTIFACTUAL DETECTION OF ADP-DEPENDENT SUCROSE SYNTHASE IN CRUDE PLANT-EXTRACTS

  P PERATA, J POZUETAROMERO, J YAMAGUCHI, T AKAZAWA

  FEBS LETTERS 309 3 283 - 287 1992年09月 [査読有り][通常論文]
   

  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 2.4.1.13) 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 3.2.1.26) and nucleoside diphosphate kinase (EC 2.7.4.6) activities. We also present data on the partial purification of nucleoside diphosphate kinase from sycamore cells.
• Isolation and Characterization of Nuclei from Rice Embryos

  Junji Yamaguchi, Poh-Yam Lim, Takashi Akazawa, Kazunii Aratani

  Cell Structure and Function 17 2 87 - 92 1992年 [査読有り][通常論文]
   

  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.
• Nucleotide sequence of a cDNA for catalase from Arabidopsis thaliana

  Christian Chevalier, Junji Yamaguchi, Peter McCourt

  Plant Physiology 99 4 1726 - 1728 1992年 [査読有り][通常論文]
  
• The preparation of high-molecular-weight DNA from rice and its analysis by pulsed-field gel electrophoresis

  Shoji Hatano, Junji Yamaguchi, Atsushi Hirai

  Plant Science 83 1 55 - 64 1992年 [査読有り][通常論文]
   

  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.
• ADPG FORMATION BY THE ADP-SPECIFIC CLEAVAGE OF SUCROSE-REASSESSMENT OF SUCROSE SYNTHASE

  J POZUETAROMERO, J YAMAGUCHI, T AKAZAWA

  FEBS LETTERS 291 2 233 - 237 1991年10月 [査読有り][通常論文]
   

  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.
• ANALYTICAL STUDIES ON THE MICROBODY TRANSITION .8. BIOSYNTHESIS AND INTRACELLULAR-TRANSPORT OF GLYOXYSOMAL MALATE-DEHYDROGENASE IN GERMINATING PUMPKIN COTYLEDONS

  J YAMAGUCHI, H MORI, M NISHIMURA

  FEBS LETTERS 213 2 329 - 332 1987年03月 [査読有り][通常論文]
  
• Distribution of 59- and 55-kDa catalase in dark- and light-grown pumpkin and various other plant tissues

  Junji Yamaguchi, Mikio Nishimura, Takashi Akazawa

  Plant and Cell Physiology 28 2 219 - 226 Japanese Society of Plant Physiologists 1987年03月 [査読有り][通常論文]
   

  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).
• PURIFICATION AND CHARACTERIZATION OF HEME-CONTAINING LOW-ACTIVITY FORM OF CATALASE FROM GREENING PUMPKIN COTYLEDONS

  J YAMAGUCHI, M NISHIMURA, T AKAZAWA

  EUROPEAN JOURNAL OF BIOCHEMISTRY 159 2 315 - 322 1986年09月 [査読有り][通常論文]
  
• ANALYTICAL STUDIES ON MICROBODY TRANSITION .5. IMMUNOCYTOCHEMICAL ANALYSIS SHOWS THAT GLYOXYSOMES ARE DIRECTLY TRANSFORMED TO LEAF PEROXISOMES DURING GREENING OF PUMPKIN COTYLEDONS

  M NISHIMURA, J YAMAGUCHI, H MORI, T AKAZAWA, S YOKOTA

  PLANT PHYSIOLOGY 81 1 313 - 316 1986年05月 [査読無し][通常論文]
  
• ANALYTICAL STUDIES ON MICROBODY TRANSITION .3. PURIFICATION OF GLYOXYSOMAL CATALASE AND IMMUNOCHEMICAL COMPARISON OF GLYOXYSOMAL AND LEAF PEROXISOMAL CATALASE IN GERMINATING PUMPKIN COTYLEDONS

  J YAMAGUCHI, M NISHIMURA

  PLANT PHYSIOLOGY 74 2 261 - 267 1984年 [査読無し][通常論文]
  
• ANALYTICAL STUDIES ON THE MICROBODY TRANSITION .4. MATURATION OF CATALASE PRECURSOR PROCEEDS TO A DIFFERENT EXTENT IN GLYOXYSOMES AND LEAF PEROXISOMES OF PUMPKIN COTYLEDONS

  J YAMAGUCHI, M NISHIMURA, T AKAZAWA

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES 81 15 4809 - 4813 1984年 [査読無し][通常論文]
  

MISC

• Methods for elucidation of plant senescence in response to C/N-nutrient balance

  Shoki Aoyama, Junji Yamaguchi, Takeo Sato Methods in Molecular Biology 1744 151 -159 2018年 [査読有り][通常論文]
   

  Carbon (C) and nitrogen (N) are essential elements for metabolism, and the ratio of C to N availability is called the C/N balance. C/N balance is very important for plant growth, but little is known about the detailed mechanisms of plant C/N responses. Previously a method of treating Arabidopsis plants with sugar-supplemented medium for studying C/N responses at early post-germinative growth stages has been developed. This method, however, cannot be used to determine physiological C/N effects in plants of mature growth stages, including senescence. Here we present two methods of analyzing responses to C/N treatments in senescing plants: transient C/N treatment with liquid medium and long-term C/N treatment with elevated atmospheric CO2.
• P022 アントラニル酸および誘導体は不定根特異的発根促進活性を持つ(ポスター発表,植物化学調節学会第50回大会)

  眞木 祐子, 副島 洋, 佐藤 長緒, 綿引 雅昭, 山口 淳二 植物化学調節学会研究発表記録集 50 40 -40 2015年10月01日
• 4-1-7 シロイヌナズナにおける硝酸シグナル応答型転写因子NLPの新規標的遺伝子BT群の機能解析(4-1 植物の多量栄養素,2015年度京都大会)

  前川 修吾, 吉岡 希, 小西 美稲子, 石田 哲也, 加藤 祐樹, 佐々木 勇樹, 佐藤 長緒, 山口 淳二, 柳澤 修一 日本土壌肥料学会講演要旨集 (61) 50 -50 2015年09月09日
• 4-1-9 植物の炭素/窒素栄養バランス応答を制御する膜局在型ユビキチンリガーゼATL31の機能解析(4-1 植物の多量栄養素,2015年度京都大会)

  佐藤 長緒, HUARANCCA REYES Thais, 植村 知博, 深尾 陽一朗, 山口 淳二 日本土壌肥料学会講演要旨集 (61) 50 -50 2015年09月09日
• 4-1-10 大気中二酸化炭素と窒素栄養バランスに応答した老化制御におけるユビキチンリガーゼATL31の機能解析(4-1 植物の多量栄養素,2015年度京都大会)

  青山 翔紀, Reyes Thais Huarancca, Guglielminetti Lorenzo, 陸 宇, 森田 嘉恵, 佐藤 長緒, 山口 淳二 日本土壌肥料学会講演要旨集 (61) 51 -51 2015年09月09日
• 植物免疫における膜局在型ユビキチンリガーゼATL31のリン酸化とその機能解析

  安田盛貴, 長谷川陽子, 門田康弘, 深尾陽一朗, 佐藤長緒, 山口淳二 日本植物生理学会年会要旨集 56th 2015年
• 植物の自然免疫に関わる膜局在型ユビキチンリガーゼの標的タンパク質の探索

  安田盛貴, 長谷川陽子, 門田康弘, 深尾陽一朗, 佐藤長緒, 山口淳二 日本プロテオーム学会大会プログラム・抄録集 2015 (Web) 2015年
• Assay for proteasome-dependent protein degradation and ubiquitinated proteins

  Takeo Sato, Kaori Sako, Junji Yamaguchi Methods in Molecular Biology 1072 655 -663 2014年 [査読有り][通常論文]
   

  The ubiquitin-26S proteasome system (UPS) plays a crucial role in selective removal of short-lived target proteins, archiving fine-tuning of post-translation levels of the target proteins. Recently a number of ubiquitin ligases (E3) have been reported as essential regulators of various plant developmental cues and stress responses. To clarify the detailed biochemical and physiological function of the E3 proteins, identification of their target proteins is of great importance. A transient expression system with tobacco leaves is a powerful method to evaluate E3 function and target degradation via UPS. Here simple methods to assay proteasome-dependent protein degradation combined with a tobacco transient expression system and detection of accumulation of ubiquitinated proteins are presented. © 2014 Springer Science+Business Media, LLC.
• C/Nバランス調節による植物の代謝・成長戦略

  佐藤長緒, 山口淳二 化学と生物 51 (11) 763 -772 2013年 [査読無し][通常論文]
  
• 32. ボカシ肥料中から単離された発根促進物質フェニル乳酸はトリプトファンと協働作用を示す(口頭発表,植物化学調節学会第47回大会)

  眞木 祐子, 副島 洋, 北村 亨, 杉山 民二, 山口 淳二 植物化学調節学会研究発表記録集 47 49 -49 2012年10月05日
• 序論 : 酵母から動植物まで包括するユビキチン-プロテアソーム系の新展開

  山口 淳二, 畠山 鎮次 生化學 84 (6) 407 -408 2012年06月25日 [査読無し][通常論文]
  
• シロイヌナズナ26Sプロテアソームを介したDNAメチル化制御機構の解析

  佐古香織, 金井知行, 加藤絵里子, 綿引雅昭, 山口淳二 日本植物生理学会年会要旨集 52nd 169 2011年03月11日 [査読無し][通常論文]
  
• 低温ストレス応答に関わるmRNA分解速度の調節

  千葉由佳子, 千葉由佳子, 峯田克彦, 平井優美, 鈴木悠也, 山口淳二, GREEN Pamela J, 内藤哲 日本植物生理学会年会要旨集 52nd 246 2011年03月11日 [査読無し][通常論文]
  
• P12-6 植物の低温ストレス応答機構とRNA制御(ポスター紹介,12.植物の代謝成分と農産物の品質,2010年度北海道大会)

  千葉 由佳子, 峯田 克彦, 平井 優美, 山口 淳二, Green Pamela J., 内藤 哲 日本土壌肥料学会講演要旨集 (56) 95 -95 2010年09月07日
• 26Sプロテアソームサブユニットを介した遺伝子発現制御機構の解析

  眞木祐子, 佐古香織, 綿引雅昭, GOTO Derek, 山口淳二 日本植物生理学会年会要旨集 51st 114 2010年03月12日 [査読無し][通常論文]
  
• Towards understanding how plant cell function is changed during root-knot nematode parasitism

  Goto Naoko, Maruyama Yosuke, Noorul Amin Arshana Nor, Asamizu Erika, Ezura Hiroshi, Osaki Mitsuru, Yamaguchi Junji, Goto Derek 日本植物生理学会年会およびシンポジウム 講演要旨集 2010 945 -945 2010年 

  Root-knot nematodes (RKNs) are a major parasite of plants, causing approximately 5% loss of total agriculture world-wide. RKN are sedentary endoparasites that establish a single permanent feeding site within plant roots as a juvenile, and then spend their whole remaining life-cycle at this single site. This is achieved by inducing normal plant cells to change into a new specialised cell-type, a Giant Cell (GC), that functions as a nutrient transfer cell. The pathway for formation of a GC from a normal plant cell remains unclear, mainly due to the difficulties in directly studying the early infection stages hidden within the host root. Using the tomato variety Micro-Tom as a model system, we have initiated a forward genetics approach to identify host genes involved in RKN-induced GC formation and are also studying the early GC cells directly. Lab home page: http://gotolab.cris.hokudai.ac.jp
• タバコの液胞膜型ショ糖トランスポーター(NtSUT4)の同定と機能の生理学的解析

  大窪(栗原) 恵美子, 桧垣匠, 桧垣匠, 栗原志夫, 朽名夏麿, 朽名夏麿, 山口淳二, 馳澤盛一郎, 馳澤盛一郎 日本植物生理学会年会要旨集 51st 2010年
• シロイヌナズナDEAR1はDREBドメインとEARモチーフを持つ転写抑制因子であり,低温応答と病原体抵抗性を制御する

  筒井友和, 加藤航, 矢元奈津子, 浅田裕, 城所聡, 篠崎和子, 玉置雅紀, 池田亮, 山口淳二 日本植物生理学会年会要旨集 49th 2008年
• 植物プロテアソームの機能-19S複合体を中心として

  佐古香織, 園田 裕, 池田 亮, 山口淳二 植物の生長調節 42 (1) 54 -60 2007年 [査読無し][通常論文]
   

  26Sプロテアソームは,様々な細胞制御に関与する「ユビキチン・プロテアソームシステムによるタンパク質の能動的分解」の実質を担う巨大タンパク質複合体である.シロイヌナズナでは,このユビキチン・プロテアソームシステムに関連する遺伝子群が全ゲノムの5%以上を占める.これは,ゲノムワイドでみた植物の特徴の一つであり,植物の示す優れた環境適応能力の一翼を担う分子機構と考えられている.本稿では,ユビキチン・プロテアソームシステムおよび植物プロテアソームの概要について記した後,19S複合体(19Sプロテアソーム)を構成するサブユニット群の機能の多様性について解説する.植物では,サブユニット間だけでなく,サブユニットのパラログ分子間でも明確な機能の違いが生じる.この具体例として,著者らが行っているRPT2に焦点をあてた研究例を紹介する.RPT2はエンドリデュプリケーション(細胞分裂を伴わないDNA複製)制御に関与することが明らかとなった.細胞サイズ制御の問題とあわせて解説する.
• Salicylic acid and a chitin elicitor both control expression of the CAD1 gene involved in the plant immunity of Arabidopsis

  Tolllokazu Tsutsui, Chlzuko Morita-Yamamuro, Yutaka Asada, Ellchl Minami, Naoto Shibuya, Aklra Ikeda, Junji Yamaguchi BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 70 (9) 2042 -2048 2006年09月 [査読無し][通常論文]
   

  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.
• Loss-of-function mutation of rice hexokinase OsHXK1 does not affect sugar regulation of α-amylase RAmy3D

  Rice Genetics Newsletter 21 90 -92 2004年 [査読無し][通常論文]
  
• Ammonium uptake and assimilation in rice

  Yamaguchi J., Sonoda Y., Iwata A., Ikeda A., Tsutsui T., Morita-Yamamuro C., Yao S.-G., Matsuhashi S., Fujimaki S., Sakamoto K., Arakawa K., Kume T. JAERI-Review 2004 (025) 122 -124 2004年 [査読無し][通常論文]
  
• 炭素源分配の制御機構--C/Nバランスによる制御を中心として (植物の代謝コミュニケーション--植物分子生理学の新展開) -- (クロストーク)

  池田 亮, 園田 裕, 山口 淳二 蛋白質核酸酵素 48 (15) 1980,2103 -2112 2003年11月 [査読無し][通常論文]
  
• 光合成同化産物の細胞間輸送と器官間輸送－糖トランスポーターと維管束機能を中心として－

  山口 淳二 Journa of Applied Glycoscience 50 (4) 91 -92 2003年10月20日 [査読無し][通常論文]
  
• ジベリンシグナル伝達因子 : DELLAファミリーを中心として(<特集>最近の植物ホルモン研究の動向 I 受容と信号伝達)

  池田 亮, 山室 千鶴子, 山口 淳二 植物の生長調節 38 (1) 36 -47 2003年05月30日 [査読無し][通常論文]
  
• 炭素源分配の制御機構－C/Nバランスによる制御を中心として－

  蛋白質核酸酵素 48 2103 -2112 2003年 [査読無し][通常論文]
  
• Negative-feedback regulation of ammonium uptake and translocation in rice under ambient and elevated CO2 conditions

  JAERI-Review 2003 (033) 126 -128 2003年 [査読無し][通常論文]
  
• ジベレリンシグナル伝達因子；DELLAファミリーを中心として

  池田 亮, 山室 千鶴子, 山口 淳二 植物の化学調節 38 (1) 36 -47 2003年 [査読無し][通常論文]
  
• 61 イネアンモニウムトランスポーターOsAMT1;3の機能解析

  齋木 里文, 園田 裕, 池田 亮, 山谷 知行, 山口 淳二 植物化学調節学会研究発表記録集 (37) 131 -131 2002年10月31日 

  We have already isolated two ammonium transporter genes OsAMT1;1,and 1;2 from rice(Oryza sativa L.) and characterized their distinct functions. Furthermore, we isolated an additional ammonium transporter gene OsAMT1;3 and attempted to characterize the gene. OsAMT1;3 gene, as same as OsAMT1;1 and 1;2,was classified into AMT1 gene family, and hydrophobic profile of the OsAMT1;3 -deduced amino acid sequence revealed that the translation product is an integrated protein having 10-11 membrane spanning domains. We examined expression pattern of OsAMT1;3 by RT-PCR method. OsAMT1;3 transcript was detected only in root, and the transcript was higher level under nitrogen deficiency. When rice with pretreatment under nitrogen deficiency was supplied with either NH_4^+ or NO_3^-, OsAMT1;3 expression was repressed by the both treatments compared with that under the nitrogen deficiency. Thus, these results indicate that the OsAMT1;3 gene expression is regulated under nitrogen status in plants and/or rhizosphere, and the protein is involved in the uptake of NH_4^+ under nitrogen deficiency. OsAMT1;3 was functionally complement a yeast mutant which is deficient in NH_4^+ uptake, indicating that the gene encodes functional ammonium transporter.
• イネのアンモニウムトランスポーターの機能解析

  池田 亮, 園田 裕, 斎木 里文, 山口 淳二 根の研究 = Root research 11 (2) 93 -93 2002年06月26日
• 機能性食品としての発芽玄米を用いた糖質とデンプン分解酵素の変動に関するプロファイリング

  山口 淳二, 竹内 若子, 升井 洋至 年報 2002 317 -324 2002年
• 根のアンモニウムイオン取り込みに関与する遺伝子群

  斎木里文, 園田裕, 池田亮, 山口淳二 農業および園芸 77 (1) 39 -47 2002年 [査読無し][通常論文]
  
• Characterization of OsSUT2 cDNA expressed before flowering stage of rice

  Rice Genetics Newsletter 19 49 -52 2002年 [査読無し][通常論文]
  
• 高圧処理米粉におけるデンプン分解酵素について : 近畿支部 第28回研究発表会講演要旨

  升井 洋至, 竹内 若子, 山口 淳二 日本調理科学会誌 34 (4) 116 -116 2001年
• EXPRESSION OF A NOVEL GENE ENCODING Ca^<2+>-BINDING PROTEIN IS INDUCED BY SUGARS :

  OTSUKI Shigeo, MATSUKURA Chiaki, MUTO Shoshi, YAMAGUCHI Junji Plant and cell physiology 42 s102 2001年
• CHARACTERIZATION OF RICE MONOSACCHARIDE TRANSPORTER, OSMST3 :

  TOYOFUKU Kyoko, TANIMOTO Eiichi, FUKUSHIMA Kazuhiko, YAMAGUCHI Junji Plant and cell physiology 42 s211 2001年
• Molecular characterization of sugar transporters involved in flowering and grain development of rice :

  Takeda Taito, Toyofuku Kyoko, Sobolewska Anna, Matsukura Chiaki, Yamaguchi Junji Plant and cell physiology 42 s211 2001年
• 植物の物質輸送と根における窒素源の取り込み

  池田 亮, 園田 裕, 山谷 知行, 山口 淳二 根の研究 = Root research 9 (4) 190 -191 2000年12月20日
• イネにおけるアンモニウムトランスポーターの機能解析

  園田 裕, 池田 亮, 石綿 愛子, 山口 淳二 育種学研究 = Breeding research 2 (2) 62 -62 2000年09月25日
• 種子形成における糖トランスポーターの解析

  武田 泰斗, 豊福 恭子, 山口 淳二 育種学研究 = Breeding research 2 (2) 240 -240 2000年09月25日
• イネの根に特異的なアンモニウムトランスポーター遺伝子の機能解析

  園田 裕, 池田 亮, 山口 淳二 根の研究 = Root research 9 (2) 96 -96 2000年06月22日
• Expression of the α-Amylase Gene RAmy3D in Rice (Oryza sativa L.) under Aerobic, Hypoxic and Anoxic Conditions

  HUANG Jirong, YAMAGUCHI Junji, AKITA Shigemi Plant production science 3 (3) 213 -218 2000年06月01日 

  Expression of the α-amylase gene RAmy3D was studied in aerobic, hypoxic, and anoxic conditions using the rice cultivars Suweon 287 and Nipponbare by in situ hybridization. In the aerobic condition, the initial expression of RAmy3D in the scutellar epithelium and its later expression in the aleurone layer were consistent with the previous reports. However, hypoxia delayed the expression of RAmy3D in the scutellar epithelium, and blocked expression in the aleurone layer. In anoxia, RAmy3D expression in cv.Nipponbare was similar to that in hypoxia, but that in cv.Suweon 287 was significantly suppressed 4 d after seeding. These results suggest that the expression of the RAmy3D gene is also affected by anoxia. Considering the results of our previous report together, RAmy3D-encoded isoforms in anoxia may be mainly regulated at the post-transcriptional level. A possible process in the regulation of RAmy3D expression in germinating seeds is discussed.
• イネ維管束系における糖代謝・糖輸送関連遺伝子群の発現解析

  松倉 千昭, 広瀬 竜郎, 大杉 立, 山口 淳二 育種学研究 = Breeding research 2 (1) 67 -67 2000年04月
• 恒常的ジベレリン反応性突然変異体,"slender rice"の原因遺伝子(SLR1)はコムギ'緑の革命'の遺伝子(RHT-1)と相同性遺伝子である.

  池田 亮, 上口(田中) 美弥子, 園田 裕, 北野 英己, 蓬原 雄三, 松岡 信, 山口 淳二 育種学研究 = Breeding research 2 (1) 100 -100 2000年04月
• 肥育イネの作出!糖センサーの改変による新しい植物代謝工学

  大槻 茂男, 山口 淳二 バイオサイエンスとインダストリー = Bioscience & industry 58 (4) 38 -39 2000年04月01日 [査読無し][通常論文]
  
• 植物の根に関する諸問題(79)根の側根形成と窒素の取り込みに関する遺伝子群

  池田 亮, 園田 裕, 山口 淳二 農業および園芸 75 (2) 301 -308 2000年02月 [査読無し][通常論文]
  
• イネ登熟種子における糖輸送-デンプン蓄積の制御機構--糖誘導型カルシウム結合性タンパク質OsSUR1の機能解析

  山口 淳二 年報 2000 108 -110 2000年
• FUNCTIONAL ANALYSIS OF HEXOKINASE IN RICE :

  SUNAKO Tomomi, MORITA Akiyoshi, YAMAGUCHI Junji Plant and cell physiology 41 s72 2000年
• MOLECULAR MECHANISMS FOR DETERMINATION OF PLANT HEIGHT BY GIBBERELLINS :

  IKEDA Akira, MATSUKURA Chiaki, YAMAGUCHI Junji Plant and cell physiology 41 s2 2000年
• CONSTITUTIVE GIBBERELLIN RESPONSE MUTANT, SLENDER RICE IS CAUSED BY NULL MUTATION OF SLR GENE, AN ORTHOLOGUE OF GAI/RGA/RHT/D8 :

  IKEDA Akira, TANAKA Miyako, SONODA Yutaka, KITANO Hidemi, FUTSUHARA Yuzo, MATSUOKA Makoto, YAMAGUCHI Junji Plant and cell physiology 41 s197 2000年
• ANALYSES OF SUGAR TRANSPORT VIA VASCULAR SYSTEM IN RICE :

  MATSUKURA Chiaki, HIROSE Tatsuro, OHSUGI Ryu, YAMAGUCHI Junji Plant and cell physiology 41 s28 2000年
• オーキシンとイネの根の形態形成 : GASR1 遺伝子・タンパク質の解析

  池田 亮, 蓬原 雄三, 山口 淳二 根の研究 = Root research 8 (4) 136 -136 1999年12月14日
• 植物の篩管輸送と糖転流 スクローストランスポーターはどのように発現し機能するか

  松倉 千昭, 山口 淳二 化学と生物 37 (10) 639 -642 1999年10月25日 [査読無し][通常論文]
  
• イネの恒常的ジベレリン反応性突然変異体,"slender mutant"の形質発現と遺伝様式

  池田 亮, 田中 繁男, 山口 淳二 名城大学農学部学術報告 (35) 7 -13 1999年03月
• イネの恒常的ジベレリン感受性突然変異体,"slender mutant"におけるジベレリンシグナル伝達系の解析

  池田 亮, 田中 繁男, 山口 淳二 名城大学農学部学術報告 (35) 15 -22 1999年03月
• ISOFORM AND cDNA CHARACTERIZATION OF HEXOSE KINASES IN RICE EMBRYO

  MORITA Akiyoshi, GUGLIELMINETTI Lorenzo, PERATA Pierdomenico, LORETI Elena, ALPI Amedio, YAMAGUCHI Junji Plant and cell physiology 40 s115 -s115 1999年03月
• MOLECULAR MECHANISMS OF SUGAR TRANSPORT 2 -FUNCTIONAL ANALYSIS OF MONOSACCHARIDE TRANSPORTER cDNAs OF RICE USING YEAST MUTAMT-

  TOYOFUKU Kyoko, KASAHARA Michihiro, YAMAGUCHI Junji Plant and cell physiology 40 s170 -s170 1999年03月
• SLENDER RICE, A CONSTITUTIVE GIBBERELLIN RESPONSE MUTANT

  IKEDA Akira, UEGUCHI-TANAKA Miyako, KITANO Hidemi, KOSHIOKA Masaji, FUTSUHARA Yuzo, MATSUOKA Makoto, YAMAGUCHI Junji Plant and cell physiology 40 s50 -s50 1999年03月
• TRANSVERSE VEIN DIFFERENTIATION ASSOCIATED WITH THE AIR SPACE FORMATION - CELL FATE OF MIDDLE LAYER IN LEAF SHEATH DEVELOPMENT OF RICE -

  MATSUKURA Chiaki, KAWAI Maki, TOYOFUKU Kyoko, BARRERO Robert A., UCHIMIYA Hirofumi, YAMAGUCHI Junji Plant and cell physiology 40 s15 -s15 1999年03月
• CHARACTERIZATION OF SLENDER RICE, A CONSTITUTIVE GIBBERELLIN RESPONSE MUTANT OF RICE

  IKEDA Akira, KITANO Hidemi, FUTSUHARA Yuzo, YAMAGUCHI Junji Plant and cell physiology 39 S70 -S70 1998年05月
• REGULATORY MECHANISMS OF SUGAR TRANSPORT -EXPRESSION OF SUGAR TRANSPORTERS USING ISOLATED RICE EMBRYOS-

  TOYOFUKU Kyoko, SAITOH Toshikazu, MATSUKURA Chiaki, HIROSE Tatsuro, OHSUGI Ryu, YAMAGUCHI Junji Plant and cell physiology 39 S131 -S131 1998年05月
• CHARACTERIZATION OF GASR1 GENE RELATED TO MODE OF ACTION OF AUXIN FOR ROOT MORPHOGENESIS IN RICE

  IKEDA Akira, FUTSUHARA Yuzo, YAMAGUCHI Junji Plant and cell physiology 39 S42 -S42 1998年05月
• 低温・湛水直播条件下における水稲発芽の時α-アミラーゼアイソザイムの変化

  黄 継栄, 山口 淳二, 秋田 重誠 日本作物學會紀事 67 94 -95 1998年04月02日
• 高等植物における細胞分裂と生理機能の制御機構の分子遺伝学的解析

  安積 良隆, 鈴木 秀穂, 山口 淳二, 川上 直人 年報 97 9 -13 1998年03月 

  生き物は成長するために細胞分裂し細胞増殖しなければならない。しかしそれも植物の場合にはその時期が適切でなければならない。移動能力のある動物の場合は受精と同時に発生を開始し、できるだけ速やかに成長することが生き残るためにもっとも大事なことといえる。しかし移動能力のない植物の場合は適切な時期に発生や成長をしなければ、枯死する運命が待ち受けている。成長に不利な時期を耐え、生き残るために、植物は発生や成長の時期を制御したり、種子を形成して休眠する。そして適当な時期が到来すると発芽し発生を開始する。生殖を行うために花を形成するのにも時期が大切である。さらに植物に特有な現象であるがセネッセンスと呼ばれる現象がある。これは環境がその植物の成長に不適切で個体を維持するのが困難であったり不利であったりする場合に、その個体の一部あるいは大部分の生理機能を低下させたり枯死させたりし、環境が回復するのを待つというものである。このように植物はその生活環の様々な場面で細胞分裂や生理機能の調節を行っている。我々は i) 種子の発芽の誘導機構、ii) 栄養成長から生殖成長への転換(花器官形成)の誘導機構、iii) 緑葉におけるセネッセンスの誘導機構の 3 つの現象に着目し分子遺伝学の手法を用いて解析を行う。
• α-アミラーゼ遺伝子の発現制御 : -植物ホルモンと糖によるシグナル伝達過程を解明するための分子指標として-

  豊福 恭子, 光永 伸一郎, 山口 淳二 植物の化学調節 32 (2) 132 -143 1997年12月25日 [査読無し][通常論文]
  
• 11 イネα-アミラーゼ遺伝子RAmy1Aの転写調節機構

  森田 章義, 豊福 恭子, Perata Pierdomenico, 山口 淳二 植物化学調節学会研究発表記録集 (32) 27 -27 1997年11月28日
• 12 ジベレリンによる根の細胞壁伸展性の制御:ジベレリン・オーキシンによる茎・葉鞘の伸長および根のジベレリン・酸誘導伸長とのクリープ粘弾性の相互比較

  谷本 英一, 山本 良一, 山口 淳二, 松倉 千昭 植物化学調節学会研究発表記録集 (32) 29 -30 1997年11月28日
• 10 オオムギ種子におけるジベレリン誘導生α-アミラーゼ遺伝子の糖による抑制 : 植物ホルモンと糖シグナル伝達経路のクロストーク現象について

  Perata Pierdomenico, 松倉 千昭, Vernieri Paolo, 山口 淳二 植物化学調節学会研究発表記録集 (32) 25 -25 1997年11月28日
• 78 低温 低酸素条件下において出芽中の稲種子のα-およびβ-アミラーゼ遺伝子の発現

  尹 炳星, 根本 圭介, 山口 淳二, 秋田 重誠 日本作物學會紀事 66 (1) 156 -157 1997年04月02日
• SUGAR SENSING AND α-AMYLASE GENE REPRESSION IN RICE EMBRYOS

  UEMURA Taka-aki, PERATA Pierdomenico, FUTSUHARA Yuzo, YAMAGUCHI Junji Plant and cell physiology 38 s137 1997年03月
• 1 ジベレリンによるイネ葉鞘伸長促進現象の解析

  松倉 千昭, 伊藤 伸一, 池田 亮, Perata Pierdomenico, 山口 淳二 植物化学調節学会研究発表記録集 (31) 13 -14 1996年10月04日
• ANALYSIS OF GIBBERELLIN-INDUCED ELONGATION OF LEAF SHEATH OF RICE

  ITOH Shin-ichi, MATSUOKA Makoto, YAMAGUCHI Junji Plant and cell physiology 37 144 -144 1996年03月
• 出芽性とデンプン分解酵素活性の関連性にみられる水稲品種間差

  尹炳星, 山口 淳二, 秋田 重誠 日作紀 65 (2) 55 -56 1996年
• パルスフィールド電気泳動の染色パターンにより検出されるイネ核DNAの多型性

  秦野 彰二, 山口 淳二, 堤 伸浩, 平井 篤志 Breeding science 45 (2) 205 -209 1995年06月01日 

  イネの緑葉から核由来の巨大DNAを調製する新しい方法を開発した.この方法では子葉のみならず成熟葉も材料として用いることができ,多くの品種や系統について,少量ずつ巨大DNAを調製して比較することが容易になった.このため,従来の胚芽を材料として用いた方法では,多くの種子が必要であったが,多量の種子の入手が困難な系統からも巨大DNAを調製できるようになった. smaIを用いてイネ核巨大DNAを切断し,パルスフィールド電気泳動法で分離し,染色すると,一様なバックグランド上に微細な明暗のバンドパターンが観察される.このパターンについて,イネの品種間で多型性が見られるかどうかを検討した.
• ANALYTICAL STUDIES ON STARCH BREAKDOWNIN CEREAL SEEDINGS - STARCH DEGRADING ENZYMES DURING ANOXICGERMINATION OF RICE, BARLEY AND WHEAT

  YAMAGUCHI Junji, GUGLIELMINETTI Lorenzo, MASUI Hironori, ALPI Amedeo, PERATA Pierdomenico Plant and cell physiology 36 S20 1995年03月
• ANALYSIS ON MODE OF ACTION OF GIBBERELLIN USING SPECIFIC INDEX, INDUCTION OF ALPHA-AMYLASE

  S MITSUNAGA, J YAMAGUCHI NIPPON NOGEIKAGAKU KAISHI-JOURNAL OF THE JAPAN SOCIETY FOR BIOSCIENCE BIOTECHNOLOGY AND AGROCHEMISTRY 67 (2) 156 -159 1993年02月 [査読無し][通常論文]
  
• α-アミラーゼ誘導を指標としたジベレリン機能の解析

  光永伸一郎, 山口淳二 日本農芸化学会誌 67 (2) 156 -159 1993年 [査読無し][通常論文]
  
• 種子貯蔵デンプン分解系

  山口 淳二 植物細胞工学 5 184 -192 1993年 [査読無し][通常論文]
  
• ENZYMATIC MECHANISMS OF STARCH SYNTHESIS IN RIPENING RICE GRAINS .8. DISTINCT PROFILES OF ADP-SPECIFIC AND UDP-SPECIFIC SUCROSE SYNTHASES IN DEVELOPING RICE GRAINS

  PY LIM, P PERATA, J POZUETAROMERO, T AKAZAWA, J YAMAGUCHI BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 56 (4) 695 -696 1992年04月 [査読無し][通常論文]
  
• 発芽過程-ジベレリンによって発現される遺伝子群

  山口淳二, 光永伸一郎 蛋白質核酸酵素 37 243 -252 1992年 [査読無し][通常論文]
  
• 遺伝子工学実験講座-18-植物細胞における遺伝子クロ-ンの発現

  山口 淳二, 赤沢 尭 Radioisotopes 38 (4) p229 -236 1989年04月 [査読無し][通常論文]
  
• 2Ap02 イネ α-アミラーゼ遺伝子の転写調節機構 イネ形質転換系を用いたシス活性化因子の検索

  山口 淳二, 梅村 賢司, 林 誠, 赤沢 堯 日本植物生理学会年会およびシンポジウム : 講演要旨集 29 307 -307 1989年
• 1Dp-8 マイクロボディ変換機構の解析 : 組織化学的アプローチ

  山口 淳二, 森 仁志, 西村 幹夫, 赤沢 尭, 横田 貞記 日本植物生理学会年会とシンポジウム講演要旨集 26 127 -127 1986年
• 脂肪性種子子葉におけるマイクロボディ変換機構の解析

  山口 淳二 化学と生物 23 491 -493 1985年 [査読無し][通常論文]
  
• 1Ba-5 カタラーゼ生合成機構 : カタラーゼ前駆体の精製とその諸性質

  山口 淳二, 西村 幹夫, 赤沢 堯 日本植物生理学会年会とシンポジウム講演要旨集 24 74 -74 1984年
• 3Dp-1 マイクロボディ変換機構の解析 (4)カボチャ子葉におけるグリコール酸酸化酵素の___- ___-生合成

  西村 幹夫, 山口 淳二, 赤沢 尭 日本植物生理学会年会とシンポジウム講演要旨集 23 301 -301 1983年
• 3Dp-2 マイクロボディ変換機構の解析 (5)カボチャ子葉グリオキシゾームおよびパーオキシゾームカタラーゼのタンパク化学的比較

  山口 淳二, 西村 幹夫, 赤沢 尭 日本植物生理学会年会とシンポジウム講演要旨集 23 302 -302 1983年
• 3Ba-7 マイクロボディ変換機構の解析 (2)カボチャ子葉カタラーゼの精製と免疫化学的性質

  山口 淳二, 西村 幹夫, 赤沢 堯 日本植物生理学会年会およびシンポジウム : 講演要旨集 22 214 -214 1982年

書籍等出版物

• Plant Proteomics: Methods in Molecular Biology, vol. 1072

  Sato T, Sako K, Yamaguchi J (担当:分担執筆範囲:Chapter 45 Assay for proteasome-dependent protein degradation and ubiquitinated proteins)
  Humana Press 2013年 (ISBN: 9781627036306) 655-663
• Frontiers in Agriculture Proteome Research

  Yasuda S, Maekawa S, Sato T, Yamaguchi J (担当:分担執筆範囲:Proteomics approach to study metabolic regulation mediated by ubiquitin-proteasome system in Arabidopsis)
  NARO Institute of Crop Science 2012年 (ISBN: 9784904633991) 39-44
• 植物の百科事典

  山口淳二 (担当:分担執筆範囲:炭素代謝)
  朝倉書店 2009年 (ISBN: 9784254171372) 9
• Botanical encyclopedia

  Asakura 2009年
• 種子の科学とバイオテクノロジー

  佐藤長緒, 山口淳二 (担当:分担執筆範囲:種子デンプンの分解)
  学会出版センター 2009年 (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 (担当:分担執筆範囲: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年 239-255
• 朝倉植物生理学講座第２巻代謝

  武田泰斗, 山口淳二 (担当:分担執筆範囲:デンプンの合成と分解)
  朝倉書店 2001年 (ISBN: 4254176562) 94-102
• 新農学大事典

  山口淳二, 松倉千昭 (担当:分担執筆範囲:発芽)
  養賢堂 2001年
• 植物ホルモンと細胞の形

  山口淳二, 松倉千昭 (担当:分担執筆範囲:α-アミラーゼの誘導)
  学会出版センター 1998年 (ISBN: 4762218707) 43-52
• 植物化学調節実験マニュアル

  山口 淳二 (担当:分担執筆範囲: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年 (ISBN: 4881370634) 2-7
• 種子のバイオサイエンス

  山口 淳二 (担当:分担執筆範囲: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年 (ISBN: 476228789X) 87-93
• 酵素実験法（１）

  山口淳二, 赤沢堯 (担当:分担執筆範囲:植物酵素の精製)
  1993年 (ISBN: 4567243609) 80-87
• 遺伝子工学実験

  山口 淳二 (担当:分担執筆範囲: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年 143-150
• 新生化学実験講座 Vol 1

  山口淳二, 赤沢堯 (担当:共著範囲:植物組織と抽出法)
  東京化学同人 1990年 (ISBN: 4807910477) 20-27

所属学協会

• 植物細胞分子生物学会   日本植物生理学会   

共同研究・競争的資金等の研究課題

• 栄養シグナルによる膜交通制御と植物成長最適化機構の解明

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2021年04月 -2024年03月 

  代表者 : 山口 淳二, 佐藤 長緒, 高木 純平
• ユビキチン修飾による病原体の認識受容と機能発現制御機構の全容解明

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2018年04月 -2021年03月 

  代表者 : 山口 淳二, 佐藤 長緒

   

  ATL31は，N末に膜貫通領域を有する膜局在RING型ユビキチンリガーゼ（E3）である。野生型ATL31に比べ，E3活性を欠損したATL31C143Sは高いタンパク質安定性を示す。興味深いことに，E3活性の有無によってATL31自体の細胞内局在性はダイナミックに変化する。すなわち，野生型ATL31は主に内膜系-特にTGN（trans-Golgi network）に局在するのに対し，ATL31C143Sは主に細胞膜に局在する。これらの知見から，ATL31は，自己ユビキチン（Ub）修飾を引き金として，細胞膜からTGNへと局在性が変化すると予想される。 ATL31の自己ユビキチン化による局在性の変化にATL31の相互作用因子であるTGN局在型SNAREタンパク質SYP61が関わることを明らかにした。また，植物体内においてSYP61がユビキチンK63鎖による修飾を受けていることや，in vitroにおいてATL31がSYP61のSNAREドメインのリジン残基をユビキチン化することを示した。最終的に，一過的発現系を用いてこのATL31によるSYP61のK63タイプのユビキチン化が植物体内でも起こることを証明した。これらの成果をまとめ，現在論文投稿中である。 C/N応答時のリン酸化プロテオームを実施し，広範なシグナル伝達にかかわるタンパク質のリン酸化状態を明らかにした。特に植物のエネルギーセンサーとして注目されているT6P-SnRK1がこの制御に深く関与することを証明した。上記リン酸化プロテオームより同定された新規受容体様キナーゼLMK1に関する解析を進めた。LMK1はLRR（ロイシンリッチリピート）とMalectinマレクチン様ドメインを持ち，細胞死制御に関与する因子であることを実験的に証明した。マレクチンドメインの機能を含めた今後の研究を進める予定である。
• 植物膜タンパク質のユビキチン修飾と機能発現制御機構の解明

  文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))

  研究期間 : 2015年 -2016年 

  代表者 : 山口 淳二

   

  本研究では「植物膜タンパク質のユビキチン修飾と機能発現制御機構の解明」を目指し，3研究課題に取組んでいる。H28年度は以下のような成果を得た。 植物に特有の膜局在型ユビキチンリガーゼATL遺伝子族は，標的となる膜タンパク質をユビキチン化することで，膜タンパク質の安定化，細胞内局在等の制御に関与する。また，その性質から，細胞膜を介した環境シグナルの受容・伝達制御に深く関わる。申請者は，基幹代謝制御に関与するATL31の発見以来，一貫してATL族の機能解明を進めてきた。本申請では，ATL31による細胞膜上の環境シグナル受容と伝達制御機構の全容解明を目指す。また， ATL31の膜局在性に着目し，細胞の内と外の情報受容・伝達制御にATL31がどのように関与するかについて検討した。 ATL31はユビキチンリガーゼ活性に依存的してC/N応答を制御するが，これまでの解析でATL31の細胞内局在性がユビキチンリガーゼ活性依存的に変化することを発見し，ATL31の細胞内局在性がその機能と密接に関係することが示唆されていた。ATL31のユビキチン化標的を調べ得るためIP-MS解析による相互作用因子の探索を行ったところ，SNAREタンパク質が複数同定された。SNAREタンパク質群はSANRE複合体を形成することで膜融合を促進する膜交通制御の鍵因子である。その中でもSYP61は重要で，受容体やトランスポーター，細胞壁合成装置などの様々なタンパク質の小胞を介した輸送制御の中心を担っている。興味深いことに，ATL31はこのSYP61と相互作用し，ユビキチンリガーゼ活性依存的にSYP61の細胞内局在性を変化させることを発見した。これらの結果から，ATL31がSYP61を介して膜交通に作用することでC/N応答を制御する可能性が示唆された。
• 膜局在型植物ユビキチンリガーゼＡＴＬ３１による環境シグナル伝達調節の包括的解明

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2014年 -2016年 

  代表者 : 山口 淳二

   

  シロイヌナズナATL31は膜在型のユビキチンリガーゼであり，栄養素応答だけでなく防御応答にも関与する。ATL31の相互作用因SYP121は，うどんこ病菌の侵入を阻止するためのパピラと呼ばれる植物細胞壁形成の制御因子である。最終的にATL31がSYP121と共にパピラ形成による防御応答に関与することを明らかにした。一方，C/N栄養素応答の鍵制御因子として３種類のCIPKタンパク質を同定した。CIPKはATL31をリン酸化することで安定化し，これがC/N応答に関係する14-3-3タンパク質の分解を制御していることを明らかにした。これはリン酸化を介した栄養素シグナル伝達の新たな仕組みの発見である。
• 栄養素シグナルによる植物の花成制御機構の解明

  文部科学省：科学研究費補助金(挑戦的萌芽研究)

  研究期間 : 2014年 -2015年 

  代表者 : 山口 淳二

   

  炭素（C）と窒素（N）は代謝の必須要素であり，その利用可能な相対量比（C/Nバランスと称される）は植物の成長の最適化機構と密接な関係がある。特に花成はC/Nの影響は顕著である。環境シグナルに応答した花成制御に関しては多くの報告があるにもかかわらず，花成とC/Nのような栄養素状態に関する関連性は今でも不明の点が多い。そこでC/N依存的な花成の分子基盤の解明を目指して，大気中CO2濃度と水耕栽培によるN濃度制御システムを確立し，これを用いたC/N依存性花成解析の研究を実施した。長日条件下の高C/低N条件においてシロイニナズナの花成が促進することを明らかとし，その分子基盤の解析を実施した。
• ユビキチン修飾による膜交通と葉緑体形成制御機構の解明

  文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))

  研究期間 : 2013年 -2014年 

  代表者 : 山口 淳二

   

  本研究では「ユビキチン修飾による膜交通と葉緑体形成制御機構の解明」を目指し，3研究課題に取組んでいる。H26年度は以下のような成果を得た。 本年度の研究実施計画 計画２）ユビキチン修飾による膜交通-植物免疫制御機構の解析： ATL31と相互作用するSNAREタンパク質SYP121は，植物の病害抵抗性と深いかかわりを持つ。糸状菌であるうどん粉病菌を植物に感染させると，SYP121欠損変異植物syp121-1では野生型（WT）と較べて抵抗性が劇的に減少するのに対し，ATL31過剰発現植物ATL31oxでは，抵抗性が上昇する。この抵抗性には，パピラという細胞壁の肥厚化が関与している。パピラは，カロース等の細胞壁成分がゴルジ体からの小胞輸送により感染箇所に分泌されることで形成される。解析の結果，i) 膜局在型ATL31によるSNAREタンパク質のユビキチン化→ii) パピラ形成部位の決定→iii) 細胞壁の肥厚化（パピラ形成）→iv) 病原体抵抗性の亢進，という一連の膜交通-植物免疫シグナル伝達制御反応に関する仮説を証明し，論文発表した（Plant Physiol. 2014）。 計画３）ユビキチン修飾による葉緑体形成制御機構の解析: 植物プロテアソームと相互作用するタンパク質のプロテオーム解析により，複数の葉緑体局体タンパク質が同定された。詳細な研究解析の結果，i) プロテアソームサブユニットRPT2aは，複数の葉緑体タンパク質移行トランジットペプチド（以下cTP）と相互作用すること，また，ii) LTA2のような葉緑体タンパク質は，ユビキチンリガーゼAtCHIPと相互作用すること，等を証明し，論文発表した（J. Proteome Res, 2014）。これにより，ユビキチン修飾による新たな制御機構-タンパク質のオルガネラ移行制御-の可能性を示唆した。
• ユビキチンカスケードが関わるC/Nバランスと高CO2応答の全容解明

  文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))

  研究期間 : 2012年 -2012年 

  代表者 : 山口 淳二

   

  本研究計画では，高CO2条件下におけるC/N応答制御の分子ネットワーク全容解明を目指している。今年度は，計画1），2）のタンパク質相互作用に着目したC/Nシグナル伝達の分子基盤解析に加えて，3）CO2/Nによる植物の成長制御に関しても「花成」および「老化」に着目した生理学的解析を進めた。 1)詳細な生化学的解析と形質転換植物の解析を実施し，ATL31と14-3-3との相互作用と機能にリン酸化が必須であることを証明し，また，そのキナーゼの同定を進めた[J. Bio. Chem. 2014]。 2)ATL31がSNAREタンパク質SYP121との相互作用によりパピラ形成を制御し，うどん粉病耐性に関与することを証明した[Plant Physiol. 2014]。 3) ATL31と14-3-3等関連遺伝子導入植物を用いて，高CO2条件下で応答実験を実施した。高CO2（700 ppm）条件下では，野生型において老化の促進が観察されるが，ATL31過剰発現体ではその抑制が，またATL31KO変異体ではその亢進が観察できた。これにより，ATL31がC/N応答だけでなく，CO2/N応答にも関与することを証明した。また，その老化に関与する転写因子の同定に成功した[Plant Cell Physiol. 2014; Plant Signal. Behav. 2014]。
• 高等植物における糖蛋白質の生合成輸送、分泌機構の細胞生化学的解析

  日本学術振興会：科学研究費助成事業

  研究期間 : 1987年 -1988年 

  代表者 : 赤沢 堯, 小林 裕和, 高倍 鉄子, 山口 淳二

   

  イネαーアシラーゼは植物における典型的な分泌性糖タンパク質として研究が進められてきた。これまでの研究の結果、イネ種子胚盤組織から分泌されたαーアミラーゼには、ハイマンノース型糖鎖を持つものとコンプレックス型糖鎖を持つものが1:1の割合で含まれることが明らかになった。そこで、糖鎖構造の異なる2種類のαーアミラーゼアイソフォームの分泌機構について検討してきたが、種子胚盤組織を用いた実験系では詳しい解析が技術的に困難であった。本研究では、新たに種子胚盤組織に由来する液体培養細胞を確立し、この細胞を用いて研究を進めた。その結果、液体培養細胞は、種子胚盤上皮組織とは異なり、コンプレックス型糖鎖を持つαーアミラーゼのみを分泌することが明らかになった。そこで、種々の培養条件下で液体培養細胞が分泌するαーアミラーゼの糖鎖の詳細な解析を行ったところ、イネαーアミラーゼの分泌と糖鎖修飾に関して以下の様な知見が得られた。 1.細胞外へ分泌されたイネαーアミラーゼには、(GlcNAc)_2Man_3(Xyl)GlcNAc_2またはGlcNAcMan_3(Xyl)GlcNAc_2の構造を持った植物糖タンパク質に特有のコンプレックス型糖鎖が付加されている。 2.イネαーアミラーゼのコンプレックス型糖鎖は、ハイマンノース型の糖鎖を持つ前駆体が小胞体およびゴルジ体における糖鎖の修飾作用を受けることによって形成される。 3.培養条件によって糖鎖修飾阻害されると、ハイマンノース型糖鎖からコンプレックス型糖鎖の変換が起こらず、ハイマンノース型の糖鎖を持ったままαーアミラーゼが分泌される。 以上の結果から、イネαーアミラーゼの糖鎖形成と動物の分泌性糖タンパク質の糖鎖形成が大筋において同一であることが示唆された。

産業財産権

• 特開2017-178852:不定根発生誘導剤及び根系発達促進剤  2017年10月05日

  眞木 祐子, 副島 洋, 山口 淳二, 谷野 圭持, 綿引 雅昭, 佐藤 長緒  雪印種苗株式会社, 国立大学法人北海道大学  201703019308771890
• WO2016-035685:不定根発生誘導剤及び根系発達促進剤  2016年03月10日

  眞木 祐子, 副島 洋, 山口 淳二, 綿引 雅昭, 佐藤 長緒  雪印種苗株式会社, 国立大学法人北海道大学  201703011248659459
• 特許第5343193号:生物挙動コントロール方法及びその装置  2013年08月23日

  門出 健次, 山口 淳二, 深澤 知行  国立大学法人北海道大学, 日本分光株式会社  201403039448597376  

  特開2008-228688(P2008-228688A)
• 特開2008-228688:生物挙動コントロール方法及びその装置  2008年10月02日

  門出 健次, 山口 淳二, 深澤 知行  国立大学法人 北海道大学, 日本分光株式会社  200903011148119063
• 特開2007-000107:イネの根特異的プロモーターおよびその利用  2007年01月11日

  山口 淳二, 池田 亮, 園田 裕, 市川 裕章, 中村 英光  国立大学法人 北海道大学, 独立行政法人農業生物資源研究所  200903029726454756  

  特開2007-000107
• 特開2006-020605:アンモニウム吸収能が強化された形質転換イネ  2006年01月26日

  山口 淳二, 池田 亮, 園田 裕  国立大学法人 北海道大学  200903085413777179  

  特開2006-020605
• 特開2005-295954:植物の免疫機構の活性化抑制剤  2005年10月27日

  山口 淳二, 森田 千鶴子, 筒井 友和, 斉藤 和季  独立行政法人科学技術振興機構  200903008902457581  

  特開2005-295954
• WO2002-055689:イネスクローストランスポーター遺伝子プロモーター  2002年07月18日

  山口 淳二, 松倉 千昭, 武田 泰斗  日本たばこ産業株式会社, シンジェンタ リミテッド  200903090081724290
• 特許6679490:不定根発生誘導在剤及び根系発達促進剤