Cellular signaling mechanisms during pollen-pistil interactions in Brassicaceae

植物通过适当选择遗传多样性的配偶同时拒绝相关配偶来保持其产量、活力和遗传多样性。鉴于作物产量是最重要的农艺性状，了解植物繁殖背后的分子机制是一个主要的研究重点。成功的植物繁殖取决于花粉/花粉管和雌蕊之间的兼容相互作用。在以干燥柱头为特征的十字花科植物中，只有相容的花粉才能诱导雌蕊为其水合和渗透到柱头组织中提供所有必要的资源。相比之下，在芸苔属植物（油菜、西兰花和卷心菜）自交不亲和性（SI）（一种防止近交的选择机制）期间，对自体花粉的识别会诱导高度有序的信号通路，拒绝自体花粉（阻止分配用于花粉水合/发芽的雌蕊资源）。在芸苔属 SI 反应期间，花粉和雌蕊之间的相互作用导致 ARC1 E3 连接酶募集到蛋白酶体（蛋白质降解机制）。 ARC1 的目标被认为是相容性因素，当 ARC1 对其进行降解时，会导致花粉排斥。我的长期目标是全面了解植物繁殖过程中调节相容和不相容授粉事件的信号机制。使用蛋白质组学方法来鉴定甘蓝柱头 SI 反应后目标破坏的蛋白质，我们鉴定了各种代谢、细胞骨架和信号蛋白。我们已经研究了其中一些候选蛋白（乙二醛酶 1 (GLO1)、微管蛋白），并获得了令人兴奋的数据，表明这些蛋白质是成功授粉所需的相容性因子。该拟议研究计划的第一个主题将集中于确定相容性因子 GLO1 如何通过 SI 响应途径靶向蛋白酶体进行降解。此外，甲基乙二醛（GLO1 解毒的细胞毒性代谢物）在 SI 过程中的作用也将通过生化和分子方法来解决。第二个主题是确定 MAPK 的上游调节因子，我们已确定这些调节因子参与介导相容授粉。这些 MAPK 激活剂的鉴定将使我们能够构建兼容的信号传导途径。第三个主题是识别和表征控制 MAPK 磷酸状态的磷酸酶及其可能的靶标之一 EXO70A1。磷酸酶的鉴定可以揭示除蛋白酶体介导的降解途径之外的 SI 反应发挥作用的另一种机制。这项研究将显着增进我们对花粉-雌蕊相互作用过程中早期信号传导的理解，这是成功繁殖的关键事件。

Plants maintain their yield, vigour and genetic diversity through appropriate selection of genetically diverse mates while rejecting related partners. Given the fact that crop-yield is the most important agronomic trait, understanding the molecular mechanism behind plant reproduction is a major research priority. Successful plant reproduction is dependent on a compatible interaction between the pollen/pollen tube and the pistil. In plant species of Brassicaceae, which are characterized by dry stigmas, only compatible pollen can induce the pistil to provide all the necessary resources for its hydration and penetration into the stigmatic tissue. In contrast, during Brassica (canola, broccoli and cabbage) self-incompatibility (SI) (a selection mechanism to prevent inbreeding), recognition of the self-pollen, induces a highly ordered signaling pathway that rejects the self-pollen (blocks allocation of pistil resources for pollen hydration/germination). During SI response in Brassica, interaction between the pollen and the pistil, leads to recruitment of ARC1 E3 ligase to the proteasome (protein degradation machinery). The targets of ARC1 are proposed to be compatibility factors, which when targeted for degradation by ARC1 results in pollen rejection. My long-term goal is to gain a thorough understanding of the signaling mechanisms that regulate compatible and incompatible pollination events during plant reproduction. Using proteomics approach to identify proteins targeted for destruction following SI response in Brassica stigmas, we identified various metabolic, cytoskeletal and signaling proteins. We have pursued a few of these candidates (Glyoxalase 1 (GLO1), tubulin) and have exciting data that suggest that these proteins are compatibility factors required for successful pollination. The first theme of this proposed research program will focus on determining how the compatibility factor GLO1 is targeted to the proteasome for degradation by the SI response pathway. In addition, the role of methylglyoxal, the cytotoxic metabolite detoxified by GLO1, during SI, will also be addressed using biochemical and molecular approaches. The second major theme is to identify the upstream regulators of MAPKs that we have identified to be involved in mediating compatible pollination. Identification of the activator of these MAPKs would allow us to build the compatible signaling pathway. The third theme is to identify and characterize the phosphatase that controls the phospho-status of MAPKs and one of its likely targets, EXO70A1. Identification of the phosphatase could reveal another mechanism through which SI response could be functioning other than the proteasome-mediated degradation pathway. This research will significantly advance our understanding of the early signaling during pollen-pistil interactions, a key event for successful reproduction.