Role of PID Kinase in Auxin Regulation

植物激素生长素在植物发育过程中控制着多种有丝分裂和形态发生事件。生长素信号转导调节内源性模式过程，包括分生组织分配和植物维管系统形成的早期步骤。生长素还调节对外部刺激（如光和重力）的热带反应。基因筛选已经确定了参与生长素运输、生长素调节基因转录和靶向蛋白降解的基因，这些基因是生长素信号传导的关键因素。然而，这些不同成分相互作用和协调调节的机制尚不清楚。本文提出的研究是基于我们最近分离和鉴定的拟南芥PINOID （PID）基因，该基因编码丝氨酸-苏氨酸蛋白激酶。多效性PID功能丧失和过表达表型类似于已知的生长素信号和运输突变体，与该蛋白在生长素调节中的特定作用一致。PID是第一个与生长素特异性表型相关的激酶。因此，PID为探索蛋白质磷酸化对生长素介导过程的影响提供了一个独特而有力的工具。对PID序列的分析表明，PID是一类新的植物丝氨酸-苏氨酸激酶的成员。我们将利用遗传，分子和生化策略来详细剖析PID及其同源物在拟南芥中调控生长素活性的作用。本研究的主要目的是：(1)利用遗传方法鉴定生长素信号中PID上游或下游的通路成分；(2)利用双杂交分析鉴定与PID直接相互作用的蛋白；(3)确定拟南芥中是否有其他pid样基因也调节生长素活性；(4)定量测量PID错表达对全局基因表达和生长素分布的影响。我们将使用体外和体内的方法来表征候选相互作用的基因和蛋白质，并确定相互作用的特异性。提出的实验将有助于对生长素介导的信号转导机制的基本理解。这代表了向阐明内源性和环境信号如何与植物激素相互作用以引发特定发育反应以及将这些机制与其他真核生物的信号传导过程联系起来的长期目标迈出的第一步。

The plant hormone auxin controls diverse mitogenic and morphogenic events during plant development. Auxin signal- transduction regulates endogenous patterning processes, including early steps in meristem partitioning and formation of the plant vascular system. Auxin also regulates tropic responses to external stimuli such as light and gravity. Genetic screens have identified genes involved in auxin transport, auxin regulated gene transcription, and targeted protein degradation as critical elements in auxin signaling. However, the mechanisms by which these various components interact and are coordinately regulated is not yet known. The research proposed here is based on our recent isolation and characterization of the PINOID (PID) gene of Arabidopsis, that encodes a serine-threonine protein kinase. The pleiotropic PID loss-of-function and over-expression phenotypes resemble those of known auxin signaling and transport mutants, consistent with a specific role for this protein in auxin regulation. PID is the first kinase associated with auxin specific phenotypes. Therefore, PID offers a unique and powerful tool to explore the effects of protein phosphorylation on auxin- mediated processes. Analysis of the PID sequence indicates that PID is a member of a novel class of plant serine-threonine kinases. We will utilized genetic, molecular and biochemical strategies to initiate a detailed dissection of the role of PID and its homologues in the regulation of auxin activity in Arabidopsis. The primary aims of this study are (1) to use a genetic approach to identify pathway components that function upstream or downstream of PID in auxin signaling; (2) to use two- hybrid analysis to identify proteins that interact directly with PID; (3) to determine whether other PID-like genes in Arabidopsis also regulate auxin activity; and (4) to quantitatively measure the effects of PID mis-expression on global gene expression and auxin distribution. We will use in vitro and in vivo approaches to characterize candidate interacting genes and proteins and determine the specificity of the interactions. The proposed experiments will contribute to a fundamental understanding of the mechanisms behind auxin-mediated signal-transduction. This represents the first step toward the long-term objective of elucidating how endogenous and environmental signals interact with plant hormones to elicit specific developmental responses and to relate these mechanisms to signaling processes in other eukaryotic organisms.