拟南芥HOPS复合体成员AtVPS18的功能及其影响雄配子传递和胚胎发育的机制

Vesicle trafficking plays a critical role in Arabidopsis male transmission and embryo development, however, the underlying mechanisms are poorly understood. Previously we found that AtVPS41-mediated late stage of endocytic pathway is essential for Arabidopsis male transmission. Studies in yeast and mammalian cells showed that VPS41 belongs to a hexamer protein complex-HOPS (Homotypic Fusion and Vacuole Protein Sorting) complex and VPS18 is central to HOPS assembly. Putative homologs of all six HOPS subunits are present in Arabidopsis genome. Our preliminary results showed that Arabidopsis HOPS complex is also existent and it is essential for Arabidopsis male transmission and (or) embryo development. In this project, we will focus on the functional characterization of AtVPS18. Here, we proposed to further examine the phenotype of vps18 mutant, detect its expression pattern in the embryo sac and subcellular localization in the pollen tube, clarify endomembrane trafficking process it takes part in, study the function of each domain, identify its interacting proteins and find out putative cargo proteins of AtVPS18. These studies will facilitate to elucidate the mechanisms under which AtVPS18 mediates male transmission and embryo development and further to decipher the role of HOPS complex during Arabidopsis sexual reproduction.

雄配子传递和胚胎发育是被子植物有性生殖的重要环节，囊泡运输在其中扮演重要角色，但是其具体作用机制仍不清楚。申请者前期研究表明，AtVPS41介导的内吞途径晚期过程在雄配子传递中发挥关键作用。在酵母和哺乳动物细胞中，VPS41隶属于六亚基HOPS复合体，而VPS18是该复合体组装的核心。本项目前期结果表明，拟南芥中也存在HOPS复合体，并且其各亚基对拟南芥雄配子传递和（或）胚胎发育均十分重要。本项目将以AtVPS18为研究对象，综合运用分子遗传学、细胞生物学和生物化学的方法，对其展开功能研究。并通过进一步分析vps18突变体表型、明确其在胚囊中的表达模式和花粉管中的亚细胞定位、鉴定其参与的内膜运输过程、检测其各结构域的功能、筛选其互作蛋白、寻找其可能的运输底物等实验，探究AtVPS18可能的作用机制。该研究将为进一步揭示HOPS复合体参与拟南芥雄配子传递和胚胎发育的分子机制奠定基础。

双受精是被子植物特有的受精方式，该过程中精细胞需要经由花粉管的极性生长被运送至胚囊中完成受精。花粉管的顶端生长依赖于其活跃的内膜运输系统。HOPS复合体通过介导内膜系统中不同组分间膜的融合而在细胞内膜运输过程中发挥重要作用。但是关于HOPS复合体是否以及如何参与调控植物双受精过程，特别是花粉管的极性生长，其具体机制尚不清晰。本研究发现HOPS复合体核心亚基AtVPS18的缺失会导致花粉管体内生长变缓，从而大大减弱vps18花粉管的竞争力，造成vps18雄配子传递率显著降低。细胞生物学研究显示，AtVPS18定位于花粉管中晚期内吞体和液泡膜上，并介导了花粉管中从晚期内吞体到液泡的内膜运输。BioID实验结果显示，HOPS复合体可能通过与胞吐复合体互作，参与花粉管生长过程中果胶甲酯酶和脂酶的分泌。进一步的研究揭示，AtVPS18的缺失会反馈式加强花粉管中的分泌活动，导致花粉管细胞壁中果胶和果胶甲酯酶的过量积累，从而减缓花粉管的生长速度。本研究为未来深入解析HOPS复合体调控植物生殖过程的机制奠定了扎实的理论基础。