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. ***
