Regulation of Phosphoinositide Metabolism and Early Responses to Osmotic Stress

植物的生存取决于它们改变新陈代谢以适应环境变化的能力。虽然对环境胁迫的生理变化有很好的记录，但初始刺激和诱导基因表达之间的信号转导途径尚不清楚。我们有证据表明，由于高渗应激和黄蜂毒液肽（mastoparan）的处理，翻译延伸因子1 α (ef -1 α)的一种异构体的分布和肌醇磷脂代谢发生了快速变化。这一建议的重点是肌醇脂和ef -1 α在启动早期应激反应中的相互作用。ef -1 α是蛋白质合成所必需的，可以调节细胞骨架结构和激活磷脂酰肌醇4-激酶。磷脂酰肌醇4-激酶是合成多磷酸化肌醇磷脂的第一个酶。肌醇磷脂，磷脂酰肌醇-4-单磷酸（PIP）和磷脂酰肌醇-4,5-二磷酸（PIP2）依次影响细胞骨架结构。我们的工作假设是EF-1alpha和PIP和PIP2在细胞响应和适应环境应激的信号转导途径中起着不可或缺的作用。为了验证我们的假设，我们将研究乙醇胺- ef -1 α和PIP合成的分布从质解开始直到细胞达到一个新的稳定状态的协调变化。我们建议对ef -1 α翻译后的甘油酰磷酸乙醇胺修饰进行生化表征；利用定点诱变法制备缺乏乙醇胺附着位点的重组EF-1alpha，并在体外确定这种翻译后修饰对EF-1alpha功能的影响。最后，我们建议获得编码PI 4-激酶的cdna，并使转基因植物过度生产该蛋白。转基因作物对高渗条件的反应将通过显微镜和生物化学方法进行研究，以确定PIP是否参与初始信号事件或恢复到新的稳定状态。与野生型细胞相比，我们期望观察到转基因细胞在质解和恢复的速率或幅度上的差异。这项工作为研究快速生理反应过程中信号转导通路的整合提供了一个令人兴奋的机会。同时，本工作将在两个方面提供新的信息：1)ef -1 α翻译后修饰的甘油酰磷酸乙醇胺的功能意义。2) PIP在高渗应激中的作用。最后，这项工作将通过确定可用于基因改变细胞稳态和细胞对环境刺激反应的调控途径的关键成分，从而导致未来的应用。非技术植物通过改变合成的蛋白质的种类和数量来迅速对环境变化和胁迫做出反应，这些蛋白质的功能是帮助植物适应新的环境。改变的环境如何被感知并在植物细胞内传递从而改变蛋白质合成和细胞代谢是植物生物学的核心问题。蛋白质ef -1 α（延伸因子-1 α）是一种丰富且高度保守的蛋白质，对蛋白质合成至关重要。EF-1alpha调节细胞骨架的胞内网络结构。细胞骨架在确定和维持植物细胞的形状和形态方面起着至关重要的作用。本项目将研究植物胁迫如何诱导EF-1alpha的修饰及其在传递环境胁迫信号中的作用。这个项目很重要，因为它解决了植物生命的一个基本方面，即植物如何响应和适应环境变化。* * *

9604285 Boss Technical The survival of plants depends on their ability to alter metabolism in response to changes in their environment. Although physiological changes in response to environmental stresses are well documented, the signal transduction pathways between the initial stimulus and the induction of gene expression are not well understood. We have evidence for rapid changes in the distribution of one isoform of translational elongation factor 1 alpha (EF-1alpha) and in inositol phospholipid metabolism as a result of hyperosmotic stress and treatment with the wasp venom peptide, mastoparan. This proposal focuses on the interaction of inositol lipids and EF-1alpha in initiating early responses to stress. EF-1alpha is essential for protein synthesis and can regulate cytoskeletal structure and activate phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase is the first committed enzyme in the synthesis of polyphosphorylated inositol phospholipids. The inositol phospholipids, phosphatidylinositol-4-monophosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) in turn can affect cytoskeletal structure. Our working hypothesis is that EF-1alpha and PIP and PIP2 play integral roles in the signal transduction pathway as cells respond and acclimate to environmental stress. To test our hypothesis, we will study the coordinated changes in the distribution of ethanolamine-EF-1alpha and PIP synthesis from the initiation of plasmolysis until the cells reach a new steady state. We propose to biochemically characterize the glycerylphosphoethanolamine posttranslational modification of EF-1alpha; to use site-directed mutagenesis to make recombinant EF-1alpha lacking the ethanolamine attachment site and to determine the effects of this posttranslational modification on the function of EF-1alpha in vitro. Finally, we propose to obtain cDNAs encoding PI 4-kinase and make transgenic plants over producing this protein. The response of the transgenics to hyperosmotic conditions will be stu died microscopically and biochemically to determine whether PIP is involved in the initial signaling event or the recovery to a new steady state. We anticipate observing a difference in the rate or magnitude of plasmolysis and recovery of transgenics compared to wild type cells. The proposed work provides an exciting opportunity for studying the integration of signal transduction pathways during a rapid physiological response. At the same time, the work will contribute new information in two areas: 1) The functional significance of the glycerylphosphoethanolamine posttranslational modification of EF-1alpha. 2) The role of PIP in hyperosmotic stress. Finally, this work will result in future applications by identifying key components of regulatory pathways that can be used to genetically alter cellular homeostasis and cellular responses to environmental stimuli. Nontechnical Plants respond rapidly to environmental changes and stress by changing the types and quantities of proteins synthesized that function to assist to adapt the plant to its new circumstances. How the altered environment is perceived and then transmitted within the plants cell to alter protein synthesis and cellular metabolism is a central problem in plant biology. The protein EF-1alpha (elongation factor-1alpha) is an abundant and highly conserved protein that is essential for protein synthesis. The EF-1alpha regulates the structure of the intracellular network of cytoskeleton. The cytoskeleton has a critical role in defining tand maintaining the shape and morphology of plant cells. This project will examine how plant stress induces the modification of EF-1alpha and its role in transmitting the environmental stress signal. This project is important because it addresses one of the fundamental aspects of plant life, how plants respond and adapt to environmental change. ***