拟南芥中类受体激酶成员LIP1/LIP2的功能及其影响花粉管导向的机制

Successful sexual reproduction in animals and plants requires the communication and coordination between male and female gametes. In flowering plants, unlike in animals, eggs and sperm cells are enclosed in multicellular embryo sacs and pollen grains respectively; guided growth of pollen tube is required to deliver sperm cells into the ovule for successful fertilization. Pollen tube guidance requires accurate perception of ovule-emitted guidance cues by the receptors in pollen tubes. Although several ovule-secreted small peptides that control pollen tube guidance are recently identified, i.e., maize EGG APPARATUS1 (EA1), Torenia LURE1/LURE2 and Arabidopsis CRP810_1/AtLURE1, little is known about their receptors in pollen tubes. In order to identify the receptors involved in perception of female cues in Arabidopsis, we focused on the receptor-like kinase (RLK) family members, which are considered as cell surface receptors to transduce chemical signals in cell-to-cell communications. RNA-Seq analysis showed that 76 out of more than 600 Arabidopsis RLK genes were preferentially expressed in semi in vivo-growth pollen tubes (SIV PT). Among them, we identified two closely-related genes in phylogenetic tree, Lost in Pollen tube guidance 1 (LIP1) and 2 (LIP2), which were involved in guidance control of pollen tubes. Simultaneous inactivation of LIP1 and LIP2 led to significant reduction of male transmission efficiency, and impaired pollen tube guidance into micropyle, suggesting that LIP1/2 may represent the components of receptor complex perceiving female signals in pollen tube guidance. Here, we proposed to further examine the lip1 lip2 pollen tube behavior in response to known female attractants and investigate the molecular function of LIP1/2 through genetic and biochemical assays. Due to the biochemical nature of LIP1/2 as RLKs, we planned to screen their interacting proteins, including other RLKs, and to identify the phosphorylation substrates of LIP1/2. These studies will facilitate to elucidate the molecular mechanisms controlling micropylar pollen tube guidance and to understand the signaling involved in guided pollen tube growth in Arabidopsis.

高等植物的受精需要花粉管通过导向生长把精细胞送至胚囊中，人们认为这一过程是通过花粉管中的受体感受胚珠所释放的吸引信号来实现的。人们在胚珠中已经发现了一些能够导向花粉管生长的小肽，而花粉管中感受这些雌性信号的受体仍不清楚。申请人已利用转录组测序技术，找出了76个在半体内萌发的花粉管中富集表达的类受体激酶基因；将其中在进化树同一支上的两个基因(LIP1和LIP2)同时突变将导致雄性传递率下降，表型分析表明花粉管的导向性生长有缺陷，这为研究花粉管感受雌性信号的机制提供了难得的材料。本申请计划在此基础上，用遗传学、生化以及分子生物学的方法鉴定LIP1/2的功能和作用模式，研究突变体花粉管对已知雌性信号的反应，筛选与LIP1/2相互作用的其它蛋白，寻找其可能的磷酸化底物，揭示LIP1/2蛋白调控花粉管导向的机制，完善受精过程的雌雄识别机理。

双受精是被子植物特有的受精方式。双受精的成功需要雌、雄配子体紧密接触，并完成一系列相互识别过程。在雌、雄配子体识别的过程中，花粉管导向是决定花粉管最终能否进入珠孔的重要过程，而花粉管与柱头之间的相互作用决定着花粉管能否由花柱进入引导组织正常生长。在这两个过程中，雌、雄配子体之间存在着大量的信号交流。在花粉管导向的过程中，已经有人发现由雌配子体分泌的半胱氨酸富集的小肽（CRPs）AtLURE1s能够吸引花粉管。然而，在花粉管中，响应AtLURE1s的受体及其复合体的其他组分却并不清楚。为了寻找雄方表达的响应AtLURE1s的受体，本项目首先利用转录组测序的技术，找出了76个在半体内萌发的花粉管中富集表达的类受体激酶基因。在这76个类受体激酶中，我们发现在演化树上同一支上的两个基因LIP1和LIP2，这两个基因同时突变会导致雄性传递率显著下降，表型分析结果表明lip1lip2双突变体花粉管在植物体内的导向出现严重的缺陷。半体内花粉管吸引实验结果表明，lip1lip2双突变体的花粉管响应AtLURE1s的比例显著下调，说明LIP1和LIP2是AtLURE1s受体复合体的组分之一。这也是拟南芥花粉管中首个发现的响应吸引信号分子AtLURE1的受体复合体的组分成员。之后，我们通过酵母双杂的实验寻找LIP1和LIP2的相互作用蛋白，以寻找其它参与雌雄相互作用的成分。我们发现LIP1和LIP2与参与内膜运输的蛋白VPS41存在相互作用。生物化学的方法发现，LIP1和LIP2可以对VPS41进行磷酸化。同时，我们发现vps41的雄方传递率下降为0，表型分析发现，vps41的花粉管能够在柱头上萌发，但不能穿出柱头。细胞生物学实验结果表明，VPS41介导晚期内吞体到液泡的融合过程。因此，这一结果表明由VPS41介导的晚期内吞体和液泡的融合过程在花粉管与柱头的相互识别过程中起到了十分重要的作用。本项目的研究揭示了参与花粉管导向和花粉管-柱头相互作用两个过程中新成员及其生物学功能。