| Project/Area Number |
12640630
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
植物生理
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| Research Institution | Shinshu University |
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Principal Investigator |
NOZUE Masayuki Shinshu University, Faculty of Textile Science and Technology, Associate professor, 繊維学部, 助教授 (30135165)
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| Co-Investigator(Kenkyū-buntansha) |
SAITO Hideki Shinshu University, Faculty of Textile Science and Technology, Lecturer, 繊維学部, 講師 (30021174)
KOJIMA Mineo Shinshu University, Faculty of Textile Science and Technology, Professor, 繊維学部, 教授 (30023469)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2002: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2001: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2000: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Autophagy / Vacuole / cysteine endopeptidase / plastid / polyphenol oxidase / Ipomoea batatas / cell culture / proteolysis / ポリフェオールオキシダーゼ / 液胞オートファジー / プロテオリシス / システインエンドペプチダーゼ / 懸濁培養細胞 / DAPI / cDNAクローニング / アイソザイム / システインプロテアーゼ |
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| Research Abstract |
Polyphenol oxidase (PPO) that is translated as precursor protein in cytoplasm, then transported and localized in plastids as a latent form. However, PPO's substrates are localized in the other compartment (vacuoles). Plant PPO is well known as one of the most popular enzymes that are involved in browning of the injured or diseased tissues. However, its biological function in the living plant cell is not clear. In the present study, we demonstrate that the plastid-localized latent PPO is most probably activated by proteolytic cleavage involved in autophagy. Main experimental results are shown as follows.
1) We cloned 2 PPO isoform cDNAs from sweet potato cells.
2) Expression of PPO was induced by cell transfer to fresh medium, cell proliferation and treatment with methyl jasmonate.
3) Latent PPO was activated by proteolytic cleavage in the cells by sucrose starvation or methyl jasmonate.
4) A vacuole-localized cysteine endopeptidase (PPOase) that can cleave the latent PPO was partially purified. PPOase showed a high substrate specificity for PPO.
5) Proteolytic cleavage of latent PPO in the sweet potato cells that had been cultured in sucrose-deleted medium was markedly inhibited by E-64 or 3-methyladenine. Plastid number severely decrease by sucrose starvation, but both E-64 and 3-methyladenine also inhibited the breakdown of plastids.
6) Vacuoles were isolated from E-64 treated cells that had been cultured in sucrose-deleted medium. The vacuoles containing many DAPI-stained structures that seemed to be plastids were observed under fluorescent microscopy.
These experimental results indicate that autophagy is induced by sucrose starvation in sweet potato cells, and the active PPO is generated by proteolytic cleavage of the plastid-localized latent PPO involved in cysteine endopeptidase in the vacuoles.
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