CLE多肽在拟南芥根端分生组织激活BAM1/BAM2受体蛋白的分子机制研究

Plants form most of their organs through post-embryogenetic development. As the source of continuously generated new organs, a population of stem cells are constantly maintained at various types of meristems throughout the lifespan of a plant. The CLAVATA3(CLV3)/ESR-related (CLE) peptide signals play a key role in the regulation of stem cell homeostasis, organogenesis and morphogenesis in plants. Previous studies by this applicant indicated that the plasma membrane-localized LRR-receptor-like kinases perceive CLE signals in the form of two receptor complexes, a CLV1/BAM1/BAM2 multimer and a CLV2-CORYNE multimer. How these receptor proteins initiate intracellular signal relay after ligand perception, however, is not clear. In this proposed study, using Arabidopsis root apical meristem as a model system, the mechanisms of receptor activation by CLE peptides will be investigated through 3 different aspects including subcellular localization, phosphorylation status of the receptor proteins and receptor complex formation. To this end, genetic, biochemical and cell biological approaches will be used to analyze changes of the Arabidopsis CLE receptor proteins BAM1 and BAM2 after CLE treatments. Effects of these changes on phenotypic read-outs will be determined to evaluate the significance of the CLE-induced changes on signal transduction. Results from this study will for the first time elucidate the molecular and cellular mechanisms of receptor activation by CLE peptide signals, which will facilitate further understanding of the intracellular signal relay initiated by CLE ligand binding.

植物大部分器官通过胚后发育形成，位于各类分生组织的干细胞是植物能够终生生长、持续发育形成新的器官和组织的基础。CLAVATA3(CLV3)/ESR-related(CLE)多肽信号在维持植物干细胞群动态平衡、器官发生和形态建成中起关键的调控作用。申请人以前的研究表明位于质膜上的LRR受体蛋白激酶CLV1/BAM1/BAM2以及CLV2-CORYNE形成两个受体复合体，分别接收CLE多肽信号。然而这些受体蛋白在接收CLE信号后引发胞内信号级联反应的方式尚不明确。本项目以拟南芥根端干细胞群为研究对象，综合应用遗传学及分子生物学手段，从受体蛋白亚细胞定位、自我磷酸化、受体复合体形成三个方面分析CLE多肽处理后BAM1/BAM2受体蛋白在分子和细胞水平的变化，以及这些变化对CLE信号传导的影响。本研究将阐明CLE多肽受体激活的分子机制，为进一步深入研究CLE途径胞内级联反应打基础。

植物大部分器官通过胚后发育形成，位于各类分生组织的干细胞是植物能够终生生长、持续发育形成新的器官和组织的基础。干细胞群的稳态主要是由CLAVATA3(CLV3)/ESR-related(CLE)多肽信号调节来维持。已知位于质膜上的LRR受体蛋白激酶CLV1/BAM1/BAM2以及CLV2-CORYNE形成两个受体复合体，分别接收CLE多肽信号。然而这些受体蛋白在接收CLE信号后引发胞内信号级联反应的方式尚不明确。在本研究中，我们构建bamclv多重突变体进行遗传分析，发现BAM1和BAM2的突变能够增强clv2对多肽的不敏感性，证明CLV3多肽对根端分生组织发育的调控依赖于受体BAM1/BAM2。通过胞吞抑制剂与多肽处理分析发现，BAM1/BAM2蛋白在CLV3多肽处理下通过胞吞作用向细胞内转运。生化和质谱分析证明，BAM1蛋白在结合CLV3配体后能够发生自我磷酸化作用，并具有多个潜在的自磷酸化位点。ATP结合位点突变(K722E)会使BAM1生物学功能完全丧失，表明蛋白激酶活性对于BAM1行驶CLV信号传导功能是必需的。通过定点突变技术，构建磷酸化突变体并进行表型分析，明确BAM1蛋白S860磷酸化位点是该蛋白参与CLV信号传导的关键磷酸化位点，进一步的细胞学和生化分析表明，该位点模拟非磷酸化点突变能够影响BAM1蛋白的亚细胞定位以及与CLV1蛋白的互作。这些结果暗示着BAM传递CLV3信号的功能依赖于S860位点的磷酸化作用。本研究初步阐明了CLE多肽受体激活的分子机制，为进一步深入研究CLE途径胞内级联反应打下了基础。