拟南芥黄酮醇合成酶基因FLS1调控叶片衰老的分子机制研究

The leaf is where photosynthesis occurs in a plant, leaf senescence therefore has a great impact on growth, nutrient accumulation and yield of crop plants. Studies on the mechanisms of leaf senescence have theoretical and practical significance in agriculture since yield increase can be achieved through controlling or delaying leaf senescence in crops. Recent studies showed that flavonols are involved in regulating several aspects of plant growth and development as signal molecules, but whether they are involved in the regulation of leaf senescence has not been reported. Our earlier study showed that contents of flavonols in Arabidopsis increased with leaf age and that a gene encoding the key enzyme Flavonol Synthase 1 (FLS1) was up-regulated at early stages of leaf senescence. The fls1-3 mutant displayed a significant delay in leaf senescence, suggesting that FLS1 may be involved in the regulation of leaf senescence. To further investigate the role of flavonols in leaf senescence, this proposed study will obtain FLS1 inducible overexpression lines and apply exogenous flavonols on Arabidopsis to confirm the senescence-promoting role of flavonols in Arabidopsis leaves. Transcriptomic analysis and exogenous flavonols application on different senescence mutants will be conducted to reveal flavonol-mediated regulatory genes and related signaling pathways. Results from this study will for the first time elucidate the role of flavonols in regulating leaf senescence as signaling molecules.

叶片是植物光合作用的主要器官，叶片的衰老影响作物生长、营养积累和产量的形成。深入研究叶片衰老的调控机理，对生产中有计划地控制或延缓衰老从而提高产量具有重要的理论和实践意义。近年来研究表明黄酮醇作为信号因子参与植物的生长发育调控，但它是否参与调控叶片衰老进程尚无报道。前期研究发现，黄酮醇含量随着叶龄的增长明显升高，编码黄酮醇合成的关键酶基因FLS1在拟南芥叶片衰老初期表达量上调，且缺失突变体fls1-3相对于野生型具有延缓叶片衰老的表型，暗示FLS1可能参与拟南芥的叶片衰老调控。本项目将在此基础上进一步证实黄酮醇在叶片衰老过程中的调控作用，通过诱导过表达FLS1以及外源黄酮醇处理植株表型分析等方法明确黄酮醇及FLS1对叶片衰老的促进作用，进一步通过转录组测序和黄酮醇处理现有衰老突变体等方法筛选受黄酮醇调控表达的基因和信号通路。本研究将首次明确黄酮醇作为信号因子对叶片衰老的调控作用。

叶片是植物光合作用的主要器官，叶片的衰老影响作物生长、营养积累和产量的形成。黄酮醇作为一类重要的次生代谢产物，在动物细胞生命活动和植物生长发育过程中起着非常重要的作用。本项目研究发现，黄酮醇含量随着拟南芥叶龄的增长明显升高，编码黄酮醇合成的关键酶基因FLS1在衰老叶片中上调表达，且黄酮醇突变体fls1-3较野生型相比具有延缓叶片衰老的表型，暗示黄酮醇可能参与拟南芥的叶片衰老调控。通过功能互补实验，构建FLS1自身启动子驱动该基因的表达载体并转化fls1-3突变体，回补株系衰老表型得到恢复，生长状态接近于野生型。同时，诱导过表达和衰老特异时期过表达株系较对照材料相比，衰老过程明显提前。此外，外源黄酮醇处理离体叶片实验表明，黄酮醇能够加速离体叶片衰老进程，处理6天的拟南芥离体叶绿素含量明显低于对照，衰老marker基因SAG12上调表达，以上结果证明黄酮醇作为正调控因子参与调控叶片衰老过程，通过转录组测序和qRT-PCR分析，结果显示与细胞自噬相关的基因PI3K、ATGs在fls1-3突变体中均上调表达，通过创制fls1-3/pi3k和fls1-3/atg8e双突变体材料，发现fls1-3/atg8e抑制了fls1-3晚衰的表型，推测黄酮醇可能通过细胞自噬途径调控叶片衰老过程。目前我们正利用细胞学方法证实黄酮醇调控细胞自噬作用的发生。综合上述结果，本研究为深入探讨黄酮醇在调控植物叶片衰老中作用的分子机制奠定基础。