液泡加工酶/液泡介导水稻糊粉层PCD的细胞形态学特征及其作用机制

The programmed cell death (PCD) of aleurone layers is a key event in cereal seed germination. It is not only related to seed normal germination, healthy seedling and complete plantlet, but also related to the subsequent crop yield. Our previous study showed that vacuole play an important role in the PCD of rice aleurone layers. At the same time, recent studies suggest that vacuolar processing enzyme (VPE) is a proteinase responsible for the maturation of vacuolar proteins, and recent studies have shown that VPEs are involved in plant PCD, in that they disrupt the vacuole, resulting in the release of degradation of cell contents. Although, known that the VPEs constitute a gene family in rice, the functions of the different family members in the PCD of rice seed germination are still poorly undrstood. Therefore, it is important significance in theory and practice that exploring the regulatory mechanism of VPE and vacuole underlying the PCD of aleurone layer in germinating rice seeds. In this project, after researching the VPE closely related to the PCD of aleurone layer, and the critical VPE similar to those of Caspase in function is determined. Tonoplast rupture is necessary in mediated-vacuole PCD, according to this, the regulation of VPE on actin microfilaments and vacuolar marker hydrolase are carried out. Futhermore, the characteristics of vacuole in morphology, structure, integration, transformation, rupture of membranes and collapse with disintegration of cytoplasm and order of occurrence during the PCD of aleurone layer in germinating rice seeds. All above investigations will be done by means of cell biology, plant anatomy, cell morphology, biochemistry and molecular biology experiments and various microscopic techniques. Taken together, these results will reveal the regulating mechanisms of VPE in the vacuole-mediated PCD of aleurone layers in germinating rice seeds, and lay the foundation for studying the downstream signaling molecules, the potential substrate and the more detailed molecular mechanism of VPE in the regulation of plant PCD. In short, all proved a reference model for the study of plant PCD and a basis for the quality improvement of germination on rice seeds in agriculture production.

糊粉层PCD是禾谷类种子萌发过程的重要事件，种子能否正常萌发关系到能否壮苗、全苗，从而影响到作物的产量。液泡是执行植物PCD的关键细胞器。在液泡介导的PCD中，VPE的调控和激活是PCD的关键环节。因此，研究液泡/VPE系统调控水稻糊粉层PCD发生的机制具有重要的理论和实践意义。本项目通过借助细胞生物学、植物解剖学、细胞形态学、生物化学和分子生物学等实验手段及各种显微技术，研究与糊粉层PCD密切相关的VPE，确定关键VPE与Caspase类似的作用，围绕液泡介导PCD发生的关键环节“液泡膜破裂”，开展VPE对微丝、液泡标志性水解酶的调节研究，并结合糊粉层细胞死亡中液泡出现的形态、结构、融合、转化、膜破裂、崩溃及细胞核解体、细胞质溶解消失等细胞形态学特征及发生顺序，阐明液泡加工酶通过液泡介导调控水稻糊粉层PCD的作用机制，为研究植物PCD提供参考模式，也为生产上提高水稻种子萌发品质提供依据

已证实，糊粉层PCD是禾谷类种子萌发的关键性事件。在此类细胞死亡过程中，液泡形态发生显著变化，而液泡加工酶（vacuolar processing enzyme，VPE）在液泡的形态变化及液泡化进程中起着重要的作用。本研究以水稻（Oryza sativa L.）种子糊粉层为实验材料，通过药理学、生物化学、细胞形态学和分子生物学等手段，采用荧光标记及荧光染色等方法，并结合实时荧光定量PCR技术和显微技术，解析萌发水稻种子糊粉层PCD中液泡的形态变化特征，并揭示液泡加工酶、微丝、H2O2等在液泡介导的水稻糊粉层PCD中的协同调控机制。研究结果表明：（1）萌发水稻糊粉层细胞中存在蛋白贮藏液泡（PSV）和溶解型液泡（LV）两种类型液泡的融合及转化，且中央大液泡是PCD中的一个显著特征；（2）中央大液泡的破裂消失引发细胞核及细胞质的降解消失，最终细胞死亡；（3）抑制液泡融合阻缓了糊粉层PCD进程，从而影响水稻种子的萌发；（4）OsVPE3在水稻糊粉层PCD中起着关键的作用，且其具有类似caspase-1的活性，可能通过类似caspase-3起作用；（5）GA可上调OsVPE3转录水平及VPE活性水平，促进水稻糊粉层PCD进程，且ABA与GA存在拮抗作用；（6）在整个液泡融合过程中伴随着微丝的解聚与聚合，VPE可与微丝协同调控水稻糊粉层液泡的融合及PCD的发生；（7）OsNOX8是水稻糊粉层中关键的NOX基因；（8）GA通过诱导OsNOX8的基因表达和H2O2的产生，促进糊粉层PCD进程；（9）NADPH氧化酶途径产生的H2O2可与OsVPE3协同调控水稻糊粉层液泡化进程及PCD的发生。本项目的研究结果不仅有助于深入了解水稻糊粉层细胞死亡的特点及其作用机制，为研究植物发育过程细胞程序性死亡增添新的内容，而且在指导水稻等禾谷类种子的播种生产方面也具有重要的实践价值。