Regulation of Phosphoinositide Metabolism and Early Responses to Osmotic Stress

9604285 Boss Technical植物的生存取决于它们改变新陈代谢以应对环境变化的能力。虽然响应于环境胁迫的生理变化被充分记录，但是初始刺激和诱导基因表达之间的信号转导途径还没有被很好地理解。我们有证据表明，由于高渗应激和黄蜂毒肽mastoparan治疗，翻译延伸因子1 α（EF-1 α）的一种亚型的分布和肌醇磷脂代谢发生了快速变化。这项建议的重点是肌醇脂质和EF-1 α在启动早期应激反应的相互作用。EF-1 α是蛋白质合成所必需的，可以调节细胞骨架结构并激活磷脂酰肌醇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关键酶。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（PIP 2）进而可以影响细胞骨架结构。我们的工作假设是，EF-1 α和PIP和PIP 2在信号转导途径中发挥不可或缺的作用，因为细胞响应和适应环境应激。 为了验证我们的假设，我们将研究从细胞浆形成开始直到细胞达到新的稳态，乙醇胺-EF-1 α和PIP合成的分布的协调变化。我们建议的生化特征的甘油磷酸乙醇胺翻译后修饰EF-1 α，使用定点突变，使重组EF-1 α缺乏乙醇胺附着位点，并确定这种翻译后修饰的EF-1 α在体外的功能的影响。最后，我们提出了获得编码PI 4-激酶的cDNA，并使转基因植物过量产生这种蛋白。 转基因对高渗条件的反应将在显微镜下和生化上进行研究，以确定PIP是否参与初始信号传导事件或恢复到新的稳态。我们预期观察到与野生型细胞相比，转基因细胞的原生质体形成和恢复的速率或幅度的差异。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，本研究还将在以下两个方面提供新的信息：1）EF-1 α的甘油磷酸乙醇胺翻译后修饰的功能意义。2)PIP在高渗应激中的作用。最后，这项工作将导致在未来的应用中，通过确定可用于遗传改变细胞稳态和细胞对环境刺激的反应的调控途径的关键组成部分。 非技术性植物通过改变合成的蛋白质的类型和数量来快速响应环境变化和压力，这些蛋白质的功能是帮助植物适应新的环境。植物如何感知环境的改变并将其传递到细胞内，从而改变蛋白质的合成和细胞的代谢，是植物生物学的一个核心问题。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-1 α调节细胞骨架的细胞内网络的结构。细胞骨架在决定植物细胞的形态和结构中起着至关重要的作用。本项目将研究植物胁迫如何诱导EF-1 α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，因为它涉及植物生命的一个基本方面，即植物如何应对和适应环境变化。 ***

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. ***