使用4C定量翻译后修饰蛋白组学和蛋白互作组学研究揭示触摸型态发生中的信号传导通道和机理

机械力信号刺激可以诱导形成特异的植物株型或触摸型态发生。对拟南芥触摸型态发生的初步蛋白质组学和转录组学研究，我们发现了几十个触摸调控的磷酸化蛋白质和四百多个触摸诱导的基因。为了揭示植物机械力信号传递网络，我们将采用翻译后修饰蛋白组学和近靠生物素标记蛋白质互作学相结合的手段，进一步研究触摸型态发生中的信号传导网络。首先我们把机械力诱导的磷酸化钙依赖性激酶CPK1和CPK6和生物素标转移酶重组，再把重组基因转入拟南芥。通过定量翻译后修饰蛋白组学方法分析同时被融合蛋白磷酸化和生物素化的底物蛋白质。 再通过活细胞化学交联的互作组学方法寻找多聚蛋白复合受体的亚基或下游信号传导重要组件。进而使用反向遗传学，生理生化以及分子细胞生物学手段分析与机械力信号传导相关的激酶和磷酸化蛋白在触摸型态发生中的作用。用分子系统生物学分析手段建立分子模型。将其成果应用于农林业抗风，雨，雹的新品种开发。

On earth, the environmental factors directly and profoundly influences the evolutionary progress, adaptation, growth, development and morphogenesis of plants. These external factors include salt, water, light, electromagnetic waves, temperature and natural mechanical forces, among which the mechanical stimuli can be further divided into the forces of wind, rain, hail, gravity, wound, animal and plant touch. In the past years, in the field of plant molecular biology research, people have acquired tremendous amount of knowledge about the osmosis, light and temperature sensing receptors and signaling components. However, little is known about the plant force-sensing receptors and signaling components during the plant touch response. Under the influences of various environmental force loadings, plant cells, tissues and organs usually respond to these signals and develop unique mechanical responses, such as thigmotropism and thigmomorphogenesis. Because our current understanding of receptors and signaling components of the plant force responses is still limited to the availability of homologs of force receptors and signaling components derived from the animal and bacteria force response studies. How plant senses and responds to the environmental forces is still by and large an untouched plant touch response research field. ..Given the scarce knowledge on the force sensing and its signaling networks in plant thigmomorphogenesis, we have conducted a preliminary quantitative phosphoproteomic and transcriptomic studies on Arabidopsis thigmomorphogenesis and discovered dozens of touch-regulated phosphoproteins and more than 400 touch-induced genes. To further unravel the molecular mechanism underlying the force sensing and signaling network in the plant force response, in this project, we plan to integrate the stable isotope labeling in Arabidopsis (SILIA) and 4C quantitative PTM proteomics with the proximity-dependent biotinylation protein-protein interactomics to map out the signaling network mediating the plant thigmomorphogenesis. To that end, we plan first to confirm the mutated thigmomorphogenesis phenotype of cpk1 and cpk6 mutants. Secondly, we will fuse the touch-regulated phosphoprotein CPK1 and CPK6 with the TurboID, a recombinant biotinylation enzyme, and transfer these recombinant fusion enzymes into Arabidopsis subsequently. The plant force response of these transgenic plants expressing the fusion enzymes will be investigated upon repetitive force stimulation, followed by quantitative PTM proteomics to identify protein substrates that will be simultaneously modified by phosphorylation and biotinylation upon the mechanical stimulation. At the same time, we will perform IPQCX-MS-based interactomics to identify components of mechanical signalosome and downstream force signaling components using the biotinylation enzyme, TurboID, as an affinity enrichment tag. These protein substrates of the fusion enzyme and CPK complex-interacting proteins may serve as the key signaling components in the plant thigmomorphogenesis. The biological roles of CPK1, CPK6 and the downstream key phosphoproteins in plant force response will be investigated and validated using molecular and cellular biology as well as transgenics approaches. The multi-omics data will be further analyzed using molecular systems biology methods and used to establish force signaling network in Arabidopsis. The outcome of this research may be applied in the molecular breeding of novel agricultural crops and forestry cultivars of mechanical stress, such as wind, rain and hail stress, tolerance.