FLS调控水稻灌浆期叶绿素降解的分子机理研究

Premature senescence of functional leaves has significant effect on yield and quality during grain filling stage. Therefore，research on the genes related to chlorophyll degradation is of great significance to increase assimilatory capacity and grain yield potential during grain filling stage. A leaf early senescence mutant fls (functional leaf senescence) was obtained by T-DNA insertion. The symptoms of the premature senescence mutant started at 5 days after heading, with leaves turning yellow and the activity of photosystem II decline gradually during filling stage，and ultimately affect the grain setting rate. Our previous study showed that the phenotype of leaves turning yellow during grain filling was due to the expression of a gene encoding a putative aldo-keto reductase increase more than 7 times comparing to the wild type. Further, the phenotypes were complemented by knock outing FLS in fls mutant using CRISPR/Cas9 system. These results indicated that FLS may involve in regulating the degradation of chlorophyll, thereby reducing grain setting rate, while its the molecular mechanism is still unknown. In this study, functions of FLS will be characterized by analyzing subcellular localization, temporal and spatial expression patterns, cis-acting elements in FLS promoter and so on. To illustrate the molecular roles of FLS in regulating chlorophyll degradation, we will compare ultra-structure of chloroplasts, thylakoid membrane proteomics, photosynthesis characteristic, gene expression related to chlorophyll metabolism and leaf senescence among wild type rice Zhonghua 11, fls mutant and its complemented strains.

水稻灌浆期功能叶早衰是影响产量和品质的重要因素。开展灌浆期叶绿素降解相关基因研究对提高水稻叶片光合同化力，实现产量突破意义重大。我们发现了一个灌浆期功能叶黄化的T-DNA突变体fls（functional leaf senescence），该突变体在营养生长期表现正常，但抽穗后叶色退绿变黄，光系统II活性降低，最终影响籽粒结实。前期研究已表明fls突变体叶色黄化是受一个醛-酮还原酶基因家族成员基因表达上调所致。利用CRISPR/Cas9系统在突变体中敲除FLS能够互补突变体叶色黄化表型，暗示FLS基因可能参与叶绿素降解的调节，但其分子机理仍不清楚。本项目拟进一步通过亚细胞定位，时空表达模式，启动子区分析等解析FLS基因的功能；通过比较野生型、fls突变体和互补转基因在叶绿体超微结构、内囊体膜蛋白组学、光合参数、叶绿素代谢和衰老相关基因表达等方面的差异探索FLS基因调控叶绿素降解的分子机理。

水稻灌浆期功能叶早衰是影响产量和品质的重要因素。开展灌浆期叶绿素降解相关基因研究对提高水稻叶片光合同化力，实现产量突破意义重大。本项目以发现的一个灌浆期功能叶黄化的T-DNA突变体fls（functional leaf senescence）为研究材料。该突变体在营养生长期表现正常，但抽穗后叶色退绿变黄。遗传分析表明，fls受单个隐性核基因控制。通过对T-DNA侧翼系列的Tail-PCR鉴定和T-DNA插入位点附件基因的表达分析发现fls突变体叶色黄化是受一个醛-酮还原酶基因家族成员基因表达上调所致。利用CRISPR/Cas9系统在突变体中敲除FLS能够互补突变体叶色黄化表型，进一步表明FLS基因参与调控叶绿素的降解。亚细胞定位的结果显示FLS定位于叶绿体。与野生型比较，fls类囊体片层结构抽穗后快速松散，光系统II活性逐步降低，暗示FLS表达上调致使定位于叶绿体的FLS蛋白增多，可能参与调控了叶绿素的降解，进而影响光合蛋白复合体解离，内囊体片层结构松散，光合活性下降。