叶绿体硫氧还蛋白系统氧化还原调控叶绿素合成关键酶GSAAT活性的机制

The mechanism underlying activity regulation of Glutamate-1-semialdehyde aminotransferase (GSAAT), which is possibly related to chloroplast redox state, remains unclear. Previous and our published results showed that chloroplast thioredoxin system plays an important role in regulating chloroplast redox state. Furthermore, our latest results showed that the redox state of GSAAT in pea leaves closely associated with chloroplast thioredoxin system, suggesting that GSAAT was the target protein of chloroplast thioredoxin system. Therefore, we speculated that chloroplast thioredoxin system redox-regulated the GSAAT activity. However, the underlying mechanism is required to be further elucidated. In this study, combining with the methods examining protein-protein interaction such as ultra-high resolution microscopy-based bimolecular fluorescence complementation, the means measuring redox state and enzyme activity of proteins and the technology of virus-induced gene silencing, we aimed to: 1) verify the interactions between the members of chloroplast thioredoxin system and GSAAT; 2) determine that by which pathways chloroplast thioredoxin system redox-regulates GSAAT activity in vitro; 3) elucidate the underlying mechanism of redox regulation of GSAAT activity by chloroplast thioredoxin system in vivo. The results of the project will not only supplement and improve the regulatory network of the chloroplast thioredoxin system, but also provide insights for the study of the regulation of chlorophyll biosynthesis.

叶绿素合成关键酶谷氨酸-1-半醛氨基转移酶（GSAAT）的活性调控机制不明，可能与叶绿体氧化还原状态有关。前人和我们已发表的结果均表明叶绿体硫氧还蛋白系统在调控叶绿体氧化还原状态中扮演重要角色；且申请人最新结果显示：豌豆叶片中GSAAT氧化还原状态与叶绿体硫氧还蛋白系统密切相关。因此，我们推测叶绿体硫氧还蛋白系统参与氧化还原调控GSAAT活性，但具体机制有待阐明。结合基于超高分辨显微技术的双分子荧光互补等蛋白相互作用检测手段、氧化还原状态和活性测定以及病毒介导的基因沉默技术等方法，本项目拟：1）验证叶绿体硫氧还蛋白系统成员与GSAAT的相互作用；2）体外探明叶绿体硫氧还蛋白系统通过何种途径氧化还原调控GSAAT活性；3）体内进一步揭示叶绿体硫氧还蛋白系统氧化还原调控GSAAT活性的机制。本项目结果将不仅补充和完善叶绿体硫氧还蛋白系统的调控网络，而且为叶绿素合成调控研究提供新思路。

叶绿素合成关键酶谷氨酸-1-半醛氨基转移酶（GSAAT）的活性调控机制不明，可能与叶绿体硫氧还蛋白系统密切相关。本课题结合酵母双杂交等蛋白相互作用检测手段、氧化还原状态和酶活性测定以及病毒介导的基因沉默技术等方法研究了叶绿体硫氧还蛋白对GSAAT的氧化还原调控机制。发现了叶绿体硫氧化蛋白TRX-F、M和NTRC形成复合物。其中，TRX-F和M锚定GSAAT，便于TRX-F/TRX-M/NTRC复合物氧化还原调控GSAAT。此外，TRX-F/TRX-M/NTRC共沉默植株与NTRC单独沉默植株均出现黄化表型，叶绿素含量下降、光合作用受损、叶绿体结构异常、叶片中大量活性氧物质积累、GSAAT大部分处于氧化失活态且酶活性降低。综上所述，该项目揭示了TRX-F、TRX-M和NTRC形成一个复合物，共同氧化还原调控GSAAT的酶活性。以上部分结果发表在Frontiers in Plant Science期刊，后续将进一步在体外验证叶绿体硫氧还蛋白各成员对GSAAT的氧化还原状态和酶活性调控，这些工作将不仅补充和完善叶绿体硫氧还蛋白系统的调控网络，而且为叶绿素合成调控研究提供新思路。