自噬体在花粉萌发和生长中的生物发生与功能

Autophagic process is an evolutionarily conserved intracellular degradation process, in which cytoplasmic components are degraded in the vacuole to provide raw materials and energy and also to eliminate damaged or toxic components for the maintenance of essential cellular functions. Autophagy is involved in the adaptation of organisms to their environment and to stressful conditions, thereby contributing to cell and organism survival and longevity. In plant cells, autophagy plays roles in recycling of proteins and metabolites including lipids, and is involved in many physiological processes such as abiotic and biotic stress responses. However, the molecular working mechanisms of autophagy in plant fertilization and reproduction had remained largely unknown. In this study, we will use SH3P2 as a maker to study the biogenesis and formation of autophagosome during pollen germination and characterize its roles in regulating pollen tube growth during plant fertilization process. Our preliminary results show the autophagosome is functional involved in pollen germination, pollen tube growth and plant fertilization. Further studies will focus on reveling the biogenesis, formation and functions of autophagosome in pollen germination and plant fertilization. By using inducible transient protein expression and subcellular localization studies, we will reveal the protein dynamics and autophagosome biogenesis during pollen germination. In addition, a combination of genetics, biochemistry and molecular biology, plant fertilization assay and ultrastructure analysis will be used to study the functions of autophagy in regulating pollen tube growth and plant fertilization.

自噬过程是广泛存在于细胞内的物质降解途经,它通过液泡降解细胞中的物质来为细胞的生命活动提供基本物质和能量，同时也降解细胞内有毒或受损的物质来保障细胞的正常生理功能。自噬作为生物物种应对胁迫环境的重要适应机制发挥关键的作用。在植物细胞中，自噬在细胞的生物和非生物胁迫应答过程中发挥着广泛的功能。但是，自噬在植物受精和生殖过程中的作用机制尚未被研究清楚。本项目将以自噬体形成过程中的关键调控蛋白SH3P2作为报告蛋白，来研究自噬体在花粉萌发和生长过程中的生物发生过程和对植物雄配子体受精过程的功能。我们初期的研究结果表明，自噬体存在于花粉的萌发和生长过程并发挥重要调控功能。未来拟通过诱导型蛋白质瞬时表达，蛋白质追踪与动力学分析和亚细胞超微结构解析等，来揭示自噬体在花粉中的生物发生和形成过程。并综合运用遗传学，生物化学与分子生物学以及受精与育性分析等，阐明自噬体在花粉管生长和植物育性中的分子作用机理。

自噬是真核细胞中重要的物质降解与循环再利用途径，它在植物的有性生殖过程中（如雄配子体发育和自交不亲和）也发挥着关键的生物学功能。在被子植物有性生殖过程中，快速生长的花粉管是介导精细胞与卵细胞结合受精的重要桥梁，它可将无运动能力的精细胞由柱头极性运送至含有卵细胞的子房来实现受精。因此，花粉管需要极高的细胞代谢水平以支撑和满足它的快速生长。但是，自噬是否参与调控花粉管的快速生长以及它在其中发挥了怎样的生物学功能都尚未被研究和揭示。在本项目中，我们首先发现自噬水平在花粉萌发和花粉管生长过程中显著升高，一些自噬相关的关键ATG基的表达量也显著上调。其次，我们以一个非ATG蛋白的重要自噬调控蛋白质SH3P2为自噬的报告蛋白，发现它与自噬过程的关键ATG调控蛋白在花粉管中均共定位于自噬体。在拟南芥中通过RNA干涉降低SH3P2的蛋白质含量，发现花粉的体外和体内萌发和生长均受到强烈的抑制，植物育性也大幅降低。再次，对自噬体在花粉管中的细胞器胞动力学研究发现，通过抑制PI3K而抑制自噬能诱导自噬体与液泡前体（PVC）发生关联与和结合。最后，对自噬在花粉管中的功能研究发现，自噬参与了受损线粒体的特异性识别与降解回收，即线粒体自噬的生物发生。此外，超微亚细胞结构分析发现，线粒体自噬体和经典自噬体结构在花粉管细胞共存。综上所述，本项目研究揭示了自噬参与调控拟南芥花粉生长与受精过程，阐明了线粒体自噬和经典自噬调控花粉管生长的分子作用基础和生物学功能。