Stress Hormone Signal Transduction in Arabidopsis

项目摘要 脱落酸（阿坝）是拟南芥中的一种中心应激激素，其下调细胞增殖， 导致细胞周期停滞。下调细胞增殖的早期信号转导网络是关键 控制有丝分裂发生及其错误调节的重要性与许多人类疾病有关。很长的- 本研究的长期目标是实现对早期介导的事件网络的定量理解 脱落酸信号转导利用有效的拟南芥和保卫细胞系统。我们将描述 新发现的关键细胞信号机制假设控制早期阿坝信号模块 由阿坝受体、PP 2C蛋白磷酸酶和SnRK 2和Raf-like蛋白激酶组成。目的： 艾姆岛PP 2C磷酸酶去磷酸化并关闭SnRK 2蛋白激酶，从而关闭阿坝信号传导。 SnRK 2激酶的再激活是通过自身磷酸化还是通过其他蛋白激酶完成的 仍然未知。我们最近的研究结果表明，去磷酸化的SnRK 2激酶不能重新激活 自己这些发现揭示了SnRK 2激酶的重新激活，其协调下游阿坝 信号传导需要SnRK 2激酶中特定位点的磷酸化。通过基因冗余筛选，我们 已经确定了长期寻求的SnRK 2再激活机制为Raf-like-kinases（Raf-Ks）。我们将 确定Raf-Ks整合入阿坝受体-PP 2C-SnRK 2和渗透胁迫的机制 使用跨学科的方法，包括新的动态SNRK 2-FRET活性纳米传感器的信号。 Aim II.通过我们最近开发的一种创新的全基因组人工microRNA筛选平台， 设计用于沉默多余的同源基因，我们已经确定了以前未表征的E3 阿坝信号转导所需的连接酶F-Box蛋白。候选F-Box目标已被 通过F盒诱饵的方法分离出来的已鉴定的F-Box蛋白的功能及其与 靶点，包括GASA信号肽，将在动态阿坝信号网络中通过 保卫细胞信号传导、遗传、蛋白质组学、生物化学和泛素化分析的组合。 Aim III.通过正向遗传学筛选，我们已经确定了染色质重塑因子SYD， 阿坝的反应。我们将研究快速ABA触发的染色质重塑在细胞凋亡中起作用的假说。 在指导大量ABA诱导的SnRK 2驱动的转录反应中起关键作用，基于新的 结果，包括基因组规模的快速差异ABA诱导的染色质可及性变化。机制 哪些Snf 2染色质重塑因子介导ABA诱导的染色质可及性反应以及如何介导 将确定SYD在ABA诱导的SnRK 2驱动的转录重编程的特异性中的功能。 从这些综合方法的结果将提供新的和机械的见解，一个高度发达的 细胞应激信号网络蛋白激酶和PP 2C磷酸酶调节的原理， 下游转录组重编程与理解多种疾病状态有关。

PROJECT SUMMARY Abscisic acid (ABA) is a central stress hormone in Arabidopsis that down-regulates cell proliferation and causes cell cycle arrest. Early signal transduction networks that down-regulate cell proliferation are of key importance for controlling mitogenesis and their mis-regulation is linked to many human diseases. The long- term goal of this research is to achieve a quantitative understanding of the network of events that mediate early abscisic acid signaling making use of the potent Arabidopsis and guard cell systems. We will characterize newly identified key cellular signaling mechanisms hypothesized to control the early ABA signaling module consisting of ABA receptors, PP2C protein phosphatases and SnRK2 and Raf-like protein kinases. Aims: Aim I. PP2C phosphatases dephosphorylate and shut off SnRK2 protein kinases and thus ABA signaling. Whether SnRK2 kinase re-activation is accomplished by auto-phosphorylation or by other protein kinases remained unknown. Our recent findings show that dephosphorylated SnRK2 kinases cannot re-activate themselves. These findings reveal that re-activation of the SnRK2 kinases, which orchestrate downstream ABA signaling, requires phosphorylation of a specific site in SnRK2 kinases. Via a genetic redundancy screen, we have identified the long-sought SnRK2 re-activation mechanism as Raf-like-kinases (Raf-Ks). We will determine the mechanisms by which Raf-Ks integrate within ABA receptor - PP2C - SnRK2 and osmotic stress signaling using interdisciplinary approaches, including new dynamic SNRK2-FRET activity nano-sensors. Aim II. Through our recent development of an innovative genome-wide artificial microRNA-screening platform, that is designed to silence redundant homologous genes, we have identified previously uncharacterized E3 ligase F-Box proteins that are required for ABA signal transduction. Candidate F-Box targets have been isolated through a dominant F-Box decoy approach. The functions of the identified F-Box proteins and their targets, including GASA signaling peptides, will be determined in the dynamic ABA signaling network through a combination of guard cell signaling, genetic, proteomic, biochemical and ubiquitination analyses. Aim III. Through a forward genetics screen, we have identified a chromatin remodeling factor SYD, as required for ABA responses. We will investigate the hypothesis that rapid ABA-triggered chromatin remodeling plays a critical role in directing the massive ABA-induced SnRK2-driven transcriptional response, based on new findings, including genomic scale rapid differential ABA-induced chromatin accessibility shifts. Mechanisms by which Snf2 chromatin remodeling factors mediate the ABA-induced chromatin accessibility response and how SYD functions in specificity of ABA-induced SnRK2-driven transcriptional reprogramming will be determined. Results from these integrated approaches will provide new and mechanistic insights into a highly-developed cellular stress-signaling network. The principles for protein kinase and PP2C phosphatase regulation and downstream transcriptome reprogramming are relevant to understanding multiple disease states.