水稻雄蕊原基早期发育过程中光合基因表达抑制的分子机制及其生物学意义

Photosynthesis is a core process in plant energy and material metabolism. Previously, the structural basis and regulatory mechanism were mainly investigated with photosynthetic organs such as leaves. However, some shoot apical meristem derived organs, such as rice stamens, do not differentiated into photosynthetic, rather into non-photosynthetic. The regulatory mechanisms of the organ developmental fates switching between photosynthetic and non-photosynthetic would provide a new perspective to understand how the machinery of photosynthesis were established and regulated. We found to our surprise in our previous studies that OsMADS58, a protein encoded by a gene annotated as a C-class floral organ identity gene, can systematically bind photosynthetic genes, inhibit their expression and chloroplast development in early development of rice stamen. In addition, this protein affects redox status in stamen cells and differentiation of germ cell (collectively refers to archesporial and microspore mother cells). These findings provided a novel experimental system for understanding how the cells of stamen primordia are canalized their fate into non-photosynthetic, rather than photosynthetic, the default fate for a photoautotroph organism. Furthermore, these findings reveal an essentially antagonistic relationship between photosynthesis and germ cell initiation, which provides a new clue to understand how germ cells initiated in rice stamen from somatic cells. Based on above mentioned findings, we would decipher in this project the molecular mechanism of OsMADS58 that binds photosynthetic genes and inhibits their expression, through a comparative investigation with that of OsMADS3, a closely related protein encoded by another annotated C-class MADS gene. Also, we would systematically investigate whether there is a causal relationship between the establishment of photosynthetic machinery and the redox status in cells of early rice stamen. Furthermore, we would systematically describe the details of differentiation process from somatic cells to germ cells in rice stamen and investigate if such a differentiation is affected by redox status during early development.

光合作用是植物物质和能量代谢的核心。长期以来，对光合作用结构基础和调控机制的研究主要以光合器官如叶片为对象。但了解原基如何不向光合器官分化，而向非光合器官分化的调控机制，必将对理解光合作用系统的构建提供全新的视角。本实验室发现，在水稻雄蕊发育过程中，被注释为花器官特征决定ABC类基因的C类MADS基因OsMADS58（OM58）所编码的蛋白会大规模地结合光合基因、抑制其表达及叶绿体发育。还发现，该蛋白影响雄蕊细胞的氧化还原状态及生殖细胞的分化。这不仅为理解原基如何向非光合器官分化提供了一个全新的实验系统，还为揭示水稻雄蕊中出现光合机制的抑制与生殖细胞的分化之间可能存在的因果关系提供了一个全新的契机。围绕上述问题，本项目将通过OM58及其同源基因OM3的比较研究，揭示OM58作用的分子机制；同时，系统分析水稻雄蕊光合机制构建与细胞氧化还原状态的关系，以及氧化还原状态与生殖细胞分化之间的关系。

本项目通过对以CRISPR技术所构建的OsMADS58（OM58）基因敲除突变体的系统研究，发现该基因的缺失所引发的表型与之前的转座子插入突变体不同，可以造成减数分裂异常和绒毡层发育异常，并最终表现为雄性不育。虽然在该基因敲除突变体中证实了之前所发现的OM58蛋白结合光合作用光系统基因并抑制其表达的现象，但后续的影响雄蕊细胞内氧化还原状态的效应却出现了与之前转座子插入突变体的不同。为寻找OM58基因敲除突变体表型背后的分子机制，我们选择了全基因组转录组分析及基因调控网络分析的策略，发现相比于野生型和转座子插入的弱表型突变体，CRISPR基因敲除突变体的基因调控网络，尤其是与叶绿体发育、氧化还原状态、减数分裂相关的基因调控网络出现了显著的改变。更为有趣的是，尽管目前已知的减数分裂相关基因都不是OM58蛋白的结合对象，这些减数分裂相关基因的表达水平在CRISPR基因敲除突变体中都出现了或高或低的改变。由于OM58基因敲除突变体中减数分裂异常的表型与已知一些减数分裂基因缺失突变体类似，我们认为OM58基因的功能，很可能是稳定雄蕊早期发育过程中的基因调控网络，为雄蕊发育过程中后续分化事件提供合适的基因表达条件。一旦该基因缺失，基因调控网络改变，关键细胞分化事件如减数分裂无法正常完成。此外，这个工作所发现的现象，即同样的减数分裂行为异常既可以因单个基因缺失所致，也可以因减数分裂相关基因调控网络改变所致，为探索基因与表型之间的关系提供了一个全新的视角：在基因与表型的关系上，不仅存在过去已知的在基因表达过程中特定基因与特定蛋白之间的“点对点”和以“点对点”为基础的级联反应（或路径/pathway）的调控关系，还存在更大范围内的以网络为中介的调控关系。因缺失突变而造成表型的基因，其对表型的影响未必是直接调控与已知表型相关的基因，而是改变与表型相关的基因网络结构。这一发现应该反映了一类过去没有被关注的情况，大大拓展了理解和解析表型形成分子机制的探索空间，从而具有非常重要的科学意义。