两组类受体激酶调控拟南芥根生长发育的分子机理

Cell-cell and cell-environment communications are critical for plants to maintain normal growth and development and to adapt diverse and fluctuating environments. Receptor-like protein kinases (RLKs), one of the largest protein families in higher plant genomes, play fundamental roles in these communications. The functions of a majority of RLKs have not yet been elucidated. A typical RLK contains an extracellular domain and a cytoplasmic kinase domain, linked by a single-pass transmembrane segment. An extracellular domain of a RLK is responsible for the perception of an extracellular signaling molecule. Upon perception, the intracellular kinase domain can usually be activated. The signaling cascade is subsequently initiated, leading to corresponding cellular responses. In the previous research, we identified BAK1 as an essential co-receptor for the brassinosteroid signaling pathway. Via genetic analyses, we also discovered that BAK1 functions in several other pathways to regulate plant development and defense. Recently, we reported that BAK1 and its homologs play a significant role in root growth and development, which is mostly independent of the brassinosteroid signaling pathway. Using BAK1 as bait, we identified several LRR-RLKs which can physically interact with BAK1 in yeast two-hybrid analyses. We functionally characterized six LRR-RLKs by genetic approaches. These LRR-RLKs belong to the same subfamily of RLKs. Knocking out three closely related LRR-RLKs resulted in a root elongation phenotype whereas knocking out other three genes showed a short-root phenotype. We hypothesized that these LRR-RLKs may use different CLE peptides as ligands, and use BAK1 as a co-receptor, to trigger novel signaling pathways to regulate root growth and development. We propose to use combined technologies including genetics, cell biology，biochemistry and proteomics to test these hypotheses.

细胞与细胞及细胞与环境的信号交流对植物生长发育与逆境适应极为重要。类受体激酶是一大类位于植物细胞质膜上的单次跨膜蛋白，其胞外结构域负责识别胞外信号分子，激活胞内激酶区来启动信号转导，改变核内基因组的表达，使细胞做出针对性反应。申请人早期曾发现了BAK1作为共受体调控油菜素内酯信号转导，其团队近年来对BAK1的多重生物学功能进行了详细的研究，其中发现BAK1参与调控根的生长发育，且主要不依赖于油菜素内酯的信号途径。利用酵母双杂交技术，本团队发现多个LRR-RLKs能与BAK1互作，对其中位于同一亚家族的两组共六个LRR-RLKs进行了遗传学研究，发现其中一组三个同源基因负向调控根的发育，而与之相邻的另一组三个同源基因正向调控根的发育。本课题将综合遗传学、细胞生物学、生物化学及蛋白组学等方法详细研究这两组LRR-RLKs是否以不同的CLE作为配体，并以BAK1作为共受体，来调控根的生长发育。

根是植物的重要营养器官，具有固定、吸收、合成等生理功能。根尖中的干细胞微环境，包括静止中心细胞和周围的干细胞，对于根的正常生长发育非常重要。静止中心细胞合成并分泌多肽RGFs上调PLT1和PLT2进而促进干细胞分裂，维持干细胞微环境的活力，但RGFs的受体仍然未知。.本实验室前期的证据表明BAK1作为共受体调控油菜素内酯(BRs)的信号转导，还参与不依赖于BRs的根发育途径。我们利用酵母双杂交系统筛选了161个LRR-RLKs，鉴定到83个与BAK1有相互作用，本研究分析了其中两组LRR-RLKs的功能。.第一组类受体蛋白激酶含有五个成员，被命名为RGF1 insensitives（RGIs）。我们构建了一系列RGIs的多重缺失突变体，表现出或多或少的根发育缺陷表型，五重缺失突变体表现出分生区变小的严重根短表型。进一步构建了第二套五重缺失突变体，与第一套表型一致。RGIs五重缺失突变体中PLT1和PLT2表达显著下调，用RGI2的启动子异位表达PLT2能够极大地恢复五重缺失突变体的根尖表型。RGIs的五重缺失突变体对RGF1完全不敏感。斑点印迹和pull-down分析表明RGF1能够与RGI1的胞外结构域结合。施加RGF1能够诱导RGI1和RGI2的磷酸化增强。RGF1能够诱导RGI1的泛素化和降解，还能够诱导BAK1的磷酸化增强。这些结果表明，RGIs作为RGF1的受体，在根尖发育过程中有着不可或缺的作用。第二组类受体蛋白激酶含有三个成员，在根中有不同程度表达。超表达RLK101和RLK219导致初生根变短。三重缺失突变体表现出根生长加快和对BRs略微超敏感的表型。我们还分析了一系列根尖报告基因在三重缺失突变体中的表达情况。.总的来说，我们对RGIs进行了深入的研究，证明了RGIs作为RGF1的受体参与拟南芥根尖干细胞微环境自调控。我们还对第二组RLKs做了一定的研究。