淹水胁迫诱导小麦胚乳PCD进程提前的ROS信号和细胞骨架动态研究

In our recent research, we have confirmed and reported that the endosperm cells of wheat undergo a special programmed cell death (PCD) with shorten and hasty course under waterlogging stress. Multidiscipline techniques in physiology and molecular cell biology will be used in this research to deeply probe the regulatory mechanism about this early endosperm PCD and the molecular cytological reasons about the differences of resist-waterlogging tolerances between wheat cultivars. It primarily includes the following contents: ①The ultrastructure features of mitochondrial and cytoskeleton will be studied during endosperm PCD course. Especially, the impacts of mitochondrial membrane damage on cytochrome c releasing and PCD course will be deeply analyzed. ② The dynamic changes of ROS and cytochrome c signal will be detected to charify their functions in triggering and controlling PCD course. ③ The localization of antioxidant enzymes (SOD and CAT et al.), PCD course regulating proteins (caspase-like proteases), cytoskeleton and cytoskeleton binding proteins, and the activity and gene expression levels of some functional proteins will be researched to find out how antioxidant enzymes regulate ROS and affect the cytoskeleton assemble and PCD course, and to figure out how caspases-like proteases regulate PCD course. ④ The ROS signaling pathways and signal response mechanisms in wheat endosperm under waterlogging stress will be preliminary illustrated. ⑤Theoretical basis of the differences of endosperm PCD course between wheat cultivars with different waterlogging tolerances will be explored. The results will enrich the stress theory about wheat cultivation and breeding.

前期研究表明淹水胁迫导致小麦胚乳细胞编程性死亡进程提前。项目利用生理学和分子细胞生物学技术手段，探寻淹水胁迫导致胚乳细胞PCD进程提前的理论机制和品种耐湿性差异的分子细胞学原因。包括：①研究胚乳细胞PCD中线粒体和细胞骨架的超微结构变化，重点分析线粒体结构损伤对细胞色素c释放和PCD进程的影响；②动态检测ROS和细胞色素c等信号变化，分析其在PCD启动和进程调控中的作用；③进行抗氧化酶SOD和CAT、PCD调控蛋白（如caspases-like蛋白酶）和细胞骨架及结合蛋白的定位；进行部分功能蛋白活性检测和基因表达研究，弄清抗氧化酶是如何调控ROS，进而影响细胞骨架的结构和PCD进程的；弄明Caspases-like蛋白酶调控PCD进程机理。④初步探明对淹水胁迫响应的ROS信号转导途径和作用机制；⑤探寻不同耐湿性品种胚乳PCD进程差异的理论基础，丰富小麦抗逆境栽培和育种理论。

本项目利用多种技术手段，探寻淹水胁迫导致小麦胚乳细胞PCD进程提前和品种耐湿性差异的生理和细胞学机制。包括：①研究胚乳PCD过程中线粒体和细胞骨架的超微结构变化，分析线粒体结构损伤对细胞色素c释放和PCD进程的影响；②检测ROS和细胞色素c等信号变化，分析其在PCD启动和进程调控中的作用；③进行主要功能蛋白定位、活性检测和基因表达研究，分析ROS、caspases-like蛋白酶调控PCD进程机理。④探明响应淹水胁迫的ROS信号转导途径和作用机制；⑤探寻不同品种耐湿性差异的理论基础。. 主要结果如下：①在正常胚乳发育过程中也会积累少量ROS，后期线粒体结构被破坏，在15-20 DAF会从线粒体向细胞质中释放细胞色素c，启动胚乳PCD进程。淹水会导致胚乳中积累ROS数量增加，且均12 DAF达到峰值；ROS积累加重线粒体内膜损伤，导致其膜透性增强，细胞色素c释放时间早，释放量增多，提前启动胚乳PCD进程。抗氧化酶直接影响胚乳细胞中ROS积累；线粒体膜通透性转换孔(MPTP)可能参与对胚乳PCD的调节。②淹水胁迫可刺激胚乳细胞中微管蛋白和微丝蛋白的合成，它们共同参与胚乳淹水胁迫的应急反应。淹水胁迫能促进华麦8号微管结合蛋白MAPA65的合成，减少微丝结合蛋白profilin（抑制微丝合成）合成，但对华麦9号的影响均较弱。③正常情况下，两个小麦品种胚乳中均可检测到caspase1, 2, 3, 4, 6, 8, 9-like蛋白酶活性；淹水使大多数caspase-like蛋白酶活性整体高于对照组，其中caspase3，6，8-like蛋白酶可能在控制胚乳PCD进程中发挥关键作用。淹水胁迫导致metcaspase相关基因TaMCA1和TaMCA4表达量增加。④华麦9号与华麦8号上述主要指标之间有明显差异，但华麦9号胚乳发育对淹水胁迫更为敏感。上述研究结果可丰富作物抗逆境理论。