胞内类受体激酶FUNA调控花粉管珠柄导向的分子机理研究

Pollen tube guidance, the result of combine effect of both female and male gametophytes, is the critical stage of pollination and fertilization in plants. Pollen tube guidance is closely associated with plant reproductive efficiency and crop yield. Pollen tubes are guided at the funiculus to exit the transmission track and at the micropyle to enter the embryo sac, two processes named as funicular guidance and micropylar guidance, respectively. Extensive studies in the past years reveal key male and female components regulating micropylar guidance. However, little is known on the components and genetic pathways regulating funicular guidance..Previously, we characterized an Arabidopsis gene FUNICULAR ADHERENCE (FUNA), Functional loss of FUNA resulted in abnormal pollen tube funicular guidance. Specifically, two or more pollen tubes of funa mutants moved toward the same funiculus..FUNA encodes a receptor-like cytoplasmic kinase (RLCK). Because RLCKs often function as key mediators in cellular signaling during plant growth and responses to environments, FUNA may thus be critical for the understanding of pollen tube machinery during funicular guidance. We thus propose to 1) identify the role of FUNA during funicular guidance; 2) identify genetic epistasis between FUNA and genes involved in pollen tube funicular guidance; 3) identify interacting proteins of FUNA by using protein interaction assays and reverse genetics technique. Results obtained will not only provide novel insights into molecular mechanisms underlying pollen tube funicular guidance, but also supply clues in cellular communication between the female and male gametophytic cells.

花粉管导向是植物授粉受精的关键步骤，是雌雄配子体相互作用的结果，直接关系到植物生殖效率和农作物的产量。花粉管导向胚囊的生长包括珠柄导向和珠孔导向两个过程。近年来对于控制花粉管珠孔导向的研究有了突破性进展。然而，对于控制花粉管珠柄导向的关键因子及遗传途径尚不清楚。申请人前期鉴定了拟南芥胞内类受体激酶FUNA，其突变体花粉管珠柄导向异常，表现在两根或多根突变体花粉管导向同一个珠柄。本项目拟在前期工作基础上，通过对funa进行深入的表型分析，明确FUNA在花粉管珠柄导向中的作用；采用遗传学方法探究FUNA与已知花粉管珠柄导向调控基因的遗传关系；利用互作蛋白筛选结合反向遗传学方法鉴定FUNA的互作蛋白，期望阐明FUNA调控花粉管珠柄导向的分子机理。本项目的实施将为解析花粉管珠柄导向的分子机制提供重要信息，并为揭示雌雄配子体之间细胞信号交流机制提供新的线索。

被子植物有性生殖包括多个复杂过程。其中，花粉萌发涉及细胞间识别和细胞极性建立等科学问题，在雄性不育利用、改变种间生殖屏障等农业生物技术方面也有重要参考价值。花粉萌发受一些蛋白因子调控，且伴随特定的细胞活动，然而调控花粉萌发的主要遗传途径尚不清楚。胞质类受体激酶（receptor-like cytoplasmic kinases，RLCKs）是一类缺乏胞外受体结构或跨膜结构域的蛋白激酶。本研究鉴定了一个拟南芥RLCK-Ⅶ亚家族成员KiTi（Kinase of Tip Growth，申报书中原名为FUNA），GUS染色和亚细胞定位结果表明，KiTi在成熟花粉和花粉管中高量表达，KiTi定位在花粉管顶端质膜。当KiTi功能缺失后雄配子体传代率降低，通过花粉发育和花粉萌发分析发现，KiTi突变导致花粉萌发异常，表明KiTi在花粉萌发过程中发挥重要作用。进一步研究表明，KiTi突变导致花粉中ROS含量降低。BiFC，Pull-down以及体外磷酸化结果表明，KiTi与RBOHH/J互作且磷酸化RBOHH/J，推测KiTi通过磷酸化RBOHH/J介导ROS积累从而调控花粉的萌发和花粉管生长。本研究初步揭示了胞质类受体激酶KiTi在拟南芥花粉萌发和花粉管生长中发挥的功能，为全面和深入解析RLCKs的生物学功能提供了有价值的线索。.此外，在本项目的资助下，课题组在以下3方面取得了研究进展。1.发现了拟南芥ADP核糖基化因子ArfA1s通过调控绒毡层介导花粉发育；2.揭示了拟南芥小G蛋白RAN调控雌雄配子体发育的分子机理；3.揭示了拟南芥COPII复合体组分Sar1调控花粉发育的分子机理。