硫化氢和一氧化氮交互作用调节淀粉型种子萌发的生理机制研究

The mechanism of seed germination has underwent extensive research. Hydrogen sufide (H2S) and nitric oxide (NO) were identified as new gaseous signaling molecules in plants involving in seed germination, while their biological functions in seed germination remain obscure. .In this research, starch seeds of rice and wheat will be used to investigate the signaling pathway of H2S and NO in regulating seed germination. In this work, the effects and the relations of the gaseous signals H2S and NO on the rapid mobilization of β-amylase during the early stage of germination will be investigated. The regulatory mechanism of H2S and NO on GA-induced α-amylase synthesis in the aleurone layers will also be studied during the middle stage of germination. Moreover, at the late stage of germination, regulatory effects of H2S and NO signals on the programmed cell death (PCD) of aleurone layer will be examined. Meanwhile, the real-time changes and the subcellular compartmentation of endogenous H2S and NO signals will be monitored along with the subcellular changes in the structures of endosperm cells and aleurone layer cells during the whole process of germination. The modulation pathway of "H2S?NO→β-amylase (early stage of germination)→embryo→GA→aleurone layer→α-amylase→ starch granule hydrolysis (middle stage of germination)→aleurone layer PCD (late stage of germination)" will be clarified, thereby unveiling the physiological roles of H2S and NO in seed germination. .This research will enrich the knowledge on the signalling fuctions of H2S/NO in plants and synthesis and regulation pathway of α-/β-amylase in seeds, and also throw light on the new mechanism of seed germination and agriculture production.

种子萌发机理的探索一直受人们所关注，一氧化氮（NO）和硫化氢（H2S）相继被发现参与了种子萌发过程，但其生理作用尚不十分清晰。.本项目以水稻和小麦淀粉型种子为材料，拟探查萌发早期H2S和NO激活β-淀粉酶启动种子萌发的生理机制；解析萌发中期H2S和NO对依赖于GA信号通路的糊粉层细胞合成α-淀粉酶的影响及调控方式；考查萌发后期H2S和NO对糊粉层细胞程序性死亡（PCD）进程的调节作用；实时监测萌发时序进程中H2S和NO信号的消长及淀粉粒和糊粉层细胞的亚显微结构变化。从而整体构建"H2S?NO→β-淀粉酶（萌发早期）→胚→GA→糊粉层→α-淀粉酶→淀粉粒水解（萌发中期）→糊粉层PCD（萌发后期）"萌发时空模式图，揭示H2S和NO信号调控种子萌发的生理机制。.研究结果不仅为探索植物中H2S与NO的生理功能，充实α/β-淀粉酶调控途径的相关知识，也为种子萌发生理研究和生产实践提供新的理论依据。

依据项目预计研究计划，我们开展了硫化氢和一氧化氮交互作用调节淀粉型种子萌发的生理机制研究。研究表明H2S与NO通过独立的信号途径刺激种子萌发，H2S与NO信号是启动萌发所必须的，且二者具有明显的加成效应。研究了淀粉型种子的萌发早期H2S与NO激活β-淀粉酶快速表达的信号互作关系，发现H2S与NO信号可以以同一类β-淀粉酶的同工酶为作用靶标，协同或单独诱导该类同工酶的活性表达。进一步研究证明，在小麦属种子中，H2S/NO信号激活β-淀粉酶差异表达的调控模式，主要是二者通过解聚结合态β-淀粉酶，释放出活性更高的β-淀粉酶单体，进而行使其促进种子萌发的生理功能。而H2S/NO信号不影响单体β-淀粉酶的活性也不能参与调节糊粉层细胞的β-淀粉酶从头合成。在水稻种子中，H2S/NO信号激活β-淀粉酶差异表达的调控模式，可以通过两种途径，一是解聚结合态β-淀粉酶，二是促进β-淀粉酶的从头合成。项目的研究结果表明，在种子的萌发中期，H2S/NO信号能诱导糊粉层细胞α-淀粉酶的合成与分泌，且不依赖于GA途径。H2S/NO信号介导了种子萌发后期的糊粉层细胞程序性死亡，独立行使延缓细胞凋亡进程的功能，且二者具有明显的加和效应；并发现，H2S信号延缓糊粉层细胞程序性死亡，与其介导了PCD进程中的氧化应答有关。本项目解析了H2S/NO对种子萌发中后期的调控作用及生理贡献。本项目采用激光共聚焦结合特异性荧光探针，建立了一种快速灵敏的H2S信号与NO 信号的亚细胞定位及检测方法，描绘了萌发时序进程中H2S/NO信号的消长规律及其亚细胞定位图谱。本项目整体考察了，“H2S/NO内源信号的动态消长、β/α-淀粉酶的变化规律、糊粉层细胞程序性死亡进程、代表萌发状态的胚乳细胞的亚显微结构变化”之间的相关性，描绘了萌发时空模式图，揭示了H2S/NO对种子萌发的调控作用和生理贡献。此外，基于项目研究发现H2S具有延迟细胞凋亡的功能，我们将研究内容拓展到采后果实的储藏与保鲜领域，发现H2S可以作为内源成熟与衰老的调控因子，延迟果实的采后储藏与保鲜期，为该领域的实践与应用提供了理论参考。通过本项目的实施，项目申请人，以通讯作者发表基金标注的SCI论文14篇，合著外文专著1部（Springer 出版社, Switzerland），申请国际发明专利1项，培养研究生6名，本科生8名。