活性氧调控花药绒毡层细胞程序性死亡的分子机理研究

Male sterility in angiosperms has been extensively studied due to its wide applications in the agricultural industry. Tapetum is a layer of cells adjacent and of vital importance to developing microspores by providing constructing materials through a programmed cell death (PCD) process, in which transcriptional cascades and proteolytic activities play key roles. As universal signals for developmental PCD, reactive oxygen species (ROS) were involved in this process since failures in ROS scavenging led to male sterility. However, it is unclear from what sources ROS are generated, whether there is temporal ROS amplitude, how the ROS-producing system interacts with the tapetal transcriptional network, and its downstream signaling pathway contributing to tapetal PCD. Preliminary studies in our laboratory showed that two Arabidopsis RESPIRATORY-BURST OXIDASE HOMOLOG (RBOH) genes are spatiotemporally expressed in tapetum during anther development stage 6-10. Functional loss of these RBOH genes resulted in pollen abortion due to delayed tapetal degeneration. By contrast, overexpression of RBOH under a tapetum-specific promoter led to pollen abortion due to precocious tapetal PCD. These results indicated that spatiotemporal ROS in tapetum was mainly controlled by NADPH oxidases. We propose to fine dissect the molecular and cellular mechanisms underlying ROS-mediated PCD. Results obtained from this study not only provide answers to pressing questions as mentioned above, but also contribute to the generation of new germplasms useful for hybrid production.

植物雄性不育广泛应用于杂交制种、杂种优势利用、增加遗传多样性等方面。花药绒毡层细胞程序性死亡（PCD）异常是雄性不育的一个主要成因。近年来的研究揭示了调控花药绒毡层PCD的关键转录因子以及转录调控网络。然而，对于这些转录因子调控的下游信号途径并不清楚。此外，最新的研究表明活性氧（ROS）参与水稻花药绒毡层PCD，但并不清楚花药绒毡层ROS的来源、ROS与绒毡层转录调控网络间的关系、以及绒毡层ROS的下游信号途径。申请人发现拟南芥编码NADPH氧化酶的RBOH基因在花药绒毡层细胞中表达。其功能缺失造成绒毡层PCD延迟、花粉败育，而过量表达RBOH基因导致绒毡层PCD提前，同样造成花粉败育。本课题拟通过综合使用分子生物学、遗传学和细胞学等手段对其表达及活性的调控机制以及ROS的下游信号途径进行深入解析，以期探究上述的几个关键科学问题，并且有助于利用遗传工程创制雄性不育系。

活性氧（ROS）作为关键信号分子，调控动物细胞的程序性死亡过程。前人在对水稻雄性不育的研究中，发现花药绒毡层中ROS清除机制异常导致花粉败育、雄性不育。然而，ROS可由多种细胞器产生，其在绒毡层中的产生机制如何？ROS受到怎样的精确调控并保证花粉正常发育？这些都是亟待进一步解决的问题。我们通过发掘转录组数据，结合反向遗传学手段，证明拟南芥RBOHE及RBOHC这两个编码质膜NADPH氧化酶的基因在绒毡层发育过程中呈现动态的表达，其表达丰度与绒毡层程序性细胞死亡的启动与终止直接相关。继而通过药剂学及细胞学手段，揭示了这两个时空特异性表达的NADPH氧化酶产生的活性氧控制绒毡层程序性死亡的启动及进程。RBOHE及RBOHC的功能获得性或丧失性突变体分别提高或降低绒毡层ROS水平，导致绒毡层程序性死亡异常。异常的绒毡层程序性死亡引发花粉败育、雄性不育。这一研究结果对于认识雄配子体花粉发育过程中孢子体与配子体间的细胞交流机制具有重要意义。相关研究结果发表在Plant Cell上(Xie et al., 2014)。此外，这一研究结果表明，可以通过对特定RBOH基因的遗传突变创制雄性不育种质，在生产上用于经济便捷地创制杂交种。我们近期已在烟草及西红柿中证明这一手段的可行性(Yu et al., 2017; BMC Plant Biology)。该研究成果获得2016年山东省优秀博士学位论文“拟南芥NADPH氧化酶介导动态活性氧的水平从而调控绒毡层程序性细胞死亡及花粉发育的研究”；获得2015年山东省研究生优秀科技创新成果一等奖“活性氧调控绒毡层细胞凋亡的分子机理”；获得2015年山东高等学校优秀科研成果奖“活性氧调控花药绒毡层程序性死亡的分子机制”；被Trends in Plant Science，Current Opinion in Plant Biology，Plant Cell，PNAS等有重要影响的学术刊物引用。