ELECTRON TRANSPORT PATHWAY MEDIATED BY CHLOROPLASTIC NAD(P)H DEHYDROGENASE

叶绿体 NAD(P)H 脱氢酶介导的电子传输途径

• 批准号: 13640646
• 负责人: ENDO Tsuyoshi
• 金额: $ 1.92万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13640646/
• 关键词: NADPH dehydrogenase; Cyclic electron transport; Photosynthetic electron transport; Chloroplast transformation

Purification of NDHFirst, I tried to isolate chloroplastic NDH from spinach leaves. The content of NDH in the crude extract was very much lower than expected thus I concluded that it would be very difficult to purify enough amount of NDH for enzymological study. Next, I screened more than 200 plant species for high NDH activity using a newly developed assay method with chlorophyll fluorescence. As a results I found that some C4 plants have very high NDH activity. Therefore I purified NDH from a few C4 plant species which could be commercially available. Intact chloroplasts were isolated from leaves, and membrane associated protein complexes were solubilized by detergents. Then NDH was purified through anion exchange and gel filtration chromatographies.Physiological analysisResponses to very high light was investigated using newly generated chloroplast transformants in which NDH K-J or C-K-J subunits were inactivated. Redox states in stroma and the intersystem chain were monitored with P700 redox and chlorophyll fluorescence, respectively. A crucial role of NDH in preventing over-reduction induced after exposure to high light was proposed.

NDH的纯化首先，我试图从菠菜叶中分离叶绿体NDH。由于粗提物中NDH的含量远低于预期，因此很难纯化出足够量的NDH用于酶学研究。接下来，我用一种新开发的叶绿素荧光测定方法筛选了200多种植物的高NDH活性。结果发现，一些C4植物具有很高的NDH活性。因此，我从一些C4植物中纯化了NDH，这些植物可以在市场上买到。从叶片中分离出完整的叶绿体，用去污剂溶解膜相关蛋白复合物。生理分析用NDH K-J和C-K-J亚基失活的叶绿体转化子研究了NDH对强光的反应。基质和系统间链的氧化还原状态分别用P700氧化还原和叶绿素荧光进行监测。提出了NDH在防止强光照射后诱导的过度还原中的关键作用。

项目成果

Ikezawa, N.: "Inhibition of photosystem II of spinach by the respiration inhibitors piricidin A and thenoyltrifluoroacetone"Biosci. Biotechnol. Biochem.. 66. 1925-1929 (2002)

Ikezawa, N.：“呼吸抑制剂哌啶甲素 A 和酚酰三氟丙酮对菠菜光系统 II 的抑制”Biosci。

Munekage, Y. et al.: "PGR5 is involved in cyclic electron flow around photosytem I and is essential for photoprotecion in Arabidopsis"Cell. 100. 361-371 (2002)

Munekage, Y. 等人：“PGR5 参与光系统 I 周围的循环电子流，对于拟南芥”细胞的光保护至关重要。

Takabayashi, A: "Post-illumination reduction of the plastoquinone pool in chioroplast transformants in which chiroplastic NAD(P)H was inactivated"Biosci. Biotechnol. biochem.. 66. 2107-2111 (2002)

Takabayashi, A：“手形体 NAD(P)H 失活的手形体转化体中质体醌池的光照后减少”Biosci。

遠藤 剛: "PAMクロロフィル蛍光計による光合成測定の原理と応用"植物の生長調節. 37. 69-75 (2002)

Tsuyoshi Endo：“利用 PAM 叶绿素荧光计进行光合作用测量的原理和应用”植物生长调节。 37. 69-75 (2002)。

Takabayashi, A. 他: "Post-illumination reduction of plasto quinore pool in NDH deficient tobacco mutants"Proceedings of International Congress of Photosynthesis. S14-O17 (2001)

Takabayashi, A. 等人：“NDH 缺陷烟草突变体中质体 quinore 池的光照后减少”国际光合作用大会论文集 S14-O17 (2001)。