使用定量翻译后修饰蛋白组学和分子系统生物学手段揭示丝裂原活化蛋白激酶通路介导的风胁迫响应信号转导网络

Plants constantly sense and respond to mechanical forces derived from both internal and external sources. Wind, as one of the major external sources of mechanical loading, entrains plants to various mechano-responses ranging from cellular events to the morphology of the whole plant. The previous studies in mammalian cells have demonstrated that mechanical forces are perceived by multiple receptors and their signals are transduced through multiple mechanotransduction pathways. Little is known about how plants sense and respond to mechanical stimuli such as wind. To elucidate the molecular mechanism and cell signaling networks underlying plant mechano-response, we intend to employ SILIA-based quantitative PTM proteomics in study of a loss-of-function mutant, mkk1 and/or mkk2, to investigate the substrates of the MAP kinase as well as downstream phosphoproteins and to decipher the MAPK cascade-mediated wind signaling network. Our previous functional and quantitative PTM proteomic study has revealed that the phosphorylation of MKK1/2 kinases is regulated by a mechanical force such as a gentle touch. Thus, the wind mechanical signal may also be transduced through the MKK1/2-dependent kinase cascade. Our recent work has further demonstrated that the mkk1/2 loss-of-function mutations block both mechanical force signaling and Arabidopsis touch response. We, therefore, propose to study the roles of MKK1/2 kinases in a much more complex mechano-signaling network triggered by a major natural force signal, wind, using the in-house established SILIA-based quantitative post-translational modification (PTM) proteomic approach. In the proposed project, we intend to investigate the protein phosphorylation profiles and other PTM profiles of mkk1/2 mutant plants induced by a rapid wind stimulation as we have done before on the touch response and the ethylene response. Molecular systems biology analyses on the phosphoproteome and other PTM proteome resulted from the plants of different wind treatments will allow us to discover the key proteins functioning downstream of MKK1 or MKK2 kinase during wind response. The biological functions of these newly identified wind-specific phosphoproteins and the protein substrates of MKK1/2 will be subjected to molecular genetic study. Those key mutant phosphoproteins resulting in defective wind responses will be studied using reverse genetic and cell biological approaches to substantiate their molecular and cellular functions. The outcomes of this investigation might be of practical implication in molecular breeding, molecular designing, and generation of new cultivars of agricultural and forestry crops.

植物在生长过程中不断感受和响应内外部的机械力信号。作为自然界中存在的主要机械力，风致使植物产生从细胞到个体的独特的应答表型。动物细胞中的多年研究已发现各种机械力信号受体和多条机械力信号转导途径，但对植物细胞，我们了解甚少。我们的研究表明MKK1／2磷酸化水平是受轻轻触摸而增加的，它们的功能缺失突变体抑制拟南芥触摸表型的生成。因此推断，风的机械力信号可能也由此途径转导。为了揭示植物中机械力信号的感受和传递网络，我们将通过定量翻译后修饰蛋白组学方法研究MKK1／2介导的风胁迫信号网络。像我们从前分析触摸和乙烯应答一样，分析短时间风刺激对拟南芥mkk1／2突变体蛋白质磷酸化及其他翻译后修饰水平的改变。然后通过分子系统生物学的分析手段寻找风应答中此激酶下游的关键信号蛋白质。使用分子遗传及细胞生物学手段分析激酶底物与关键磷酸化蛋白质的生物和分子细胞功能。将其成果应用于农林业分子育种，设计和品种培育。

在植物的生命周期中，不断受到各种机械力作用的影响。例如，地球的引力一直作 用于植物细胞。风、雨、雪、雹、动物所施加的机械力在自然环境下影响植物生长 。 又如密林中的林木个体，在随风摇摆的过程中，相互之间用枝条中不断碰撞。植物对这 种机械力刺激具有一定的适应性(Read and Stokes, 2006)。作为一种较柔和的机械力作用，“触摸”可以导致植物的倾触性( thigmonasty)或向触性(thigmotropism)响应(Chehab et al.,2009)。这种轻微的刺激会导致许多可见的植物生长响应。物对“触摸”的适应也呈现短而粗的表型，开花延迟等形态发生的变化，此 现象定义为触摸形态发生(thigmomorphogenesis)。但是目前对于植物细胞感受机械力 刺激的分子机制知道的仍然有限 。 为了揭示植物中机械力信号的感受和传递网络，我们使用定量翻译后修饰蛋白组学方法研究MKK 1/2介导的风胁迫信号网络。同时对生物素化的蛋白研究分析它们的磷酸化位点。再通过分子系统生物学，生物信息学方法寻找MKK1、2的互作蛋白与底物蛋白。结果，从5分钟风刺激下的拟南芥中一共找到11个MKK1互作蛋白与71个MKK2互作蛋白。其中WIRK1激酶是它们两个激酶的共同底物。使用翻译后修饰蛋白组学，我们也找到241个MKK1-ID 和 199个MKK2-ID 转基因植物中的磷酸化寡肽片段。这些位点有些存在于生物素化的蛋白上或理解为MKK1、2激酶的互作蛋白上。因为WIRK1被同时生物素化和磷酸化。加上生物信息学分析，我们预测它是MAPK级联反应中新的磷酸化信号传导因子。为验证此预测，我们做了相关的分子生物实验，证明WIRK1的确调控PATL3蛋白的磷酸化水平。同时也调控触摸基因的表达。当mkk1 和 mkk2 突变体被自动化的头发触摸重复处理后，它们的表型也有变异，主要是延迟开花时间的表型。说明MAPK级联磷酸化反应参与风胁迫的生长表型。