The long-term goal of this research is to understand the cell biology of higher plants' responses to environmental stress. The model system for investigation is the heat shock response of the aleurone layer of barley grains. The cells of this tissue are normally dedicated to protein secretion; however, heat shock dramatically redirects their cellular activities. The synthesis of secretory proteins is abruptly arrested when the tissue is subjected to heat shock, yet the synthesis of nonsecretory proteins continues. This is accomplished by the selective destabilization of messenger RNAs (mRNAs) that code for the secretory proteins and that are otherwise stable. Heat shock also causes a change in the organization of the intracellular membrane compartment that is the site of entry of proteins into the secretory pathway. This compartment, the endoplasmic reticulum (ER), changes from a stacked cisternal lamellar arrangement to a tubular morphology. Ribosome density on the ER also decreases by about half.
The principal distinction between secretory and nonsecretory protein mRNAs is a region encoding an amino terminal signal sequence that directs the translation of secretory protein mRNAs to take place at the ER surface. The signal recognition particle (SRP) is a protein-nucleic acid complex that interacts with the signal sequence of nascent polypeptides and brings the ribosomes that are translating the secretory proteins to the ER. It has been found that heat shock inhibits the release of SRP from the ER following ribosome "docking." This project addresses the question of what mechanisms operate during heat shock to selectively destabilize secretory protein mRNA. Time course experiments will investigate whether degradation of secretory protein mRNA occurs from the ER-bound or free pools. Binding of specific proteins to secretory protein mRNAs has been observed and may be increased as a result of heat shock. Since this protein binding may be of importance in regulating mRNA stability, experiments will be performed to define the binding sequences in the RNA. These candidate sequences will then be either deleted from secretory protein mRNAs or added to normally stable transcripts to determine if they confer heat shock instability. Finally, the genes for the SRP receptor or docking proteins in the ER membrane of barley aleurone will be cloned. The clones will be used to produce antibodies to monitor the effect of heat shock on the expression and distribution of these proteins.
A plant's adaptive responses to environmental stress are crucial to its survival. A fuller understanding of the impact of stress on normal cellular biology and of cellular mechanisms for stress adaptation are important to agricultural efforts aimed at enhancing plant productivity and utility. Most studies on heat shock focus on the induction and function of heat shock-specific proteins. This work focuses instead on the impact of heat shock on normal cellular processes, most specifically the suppression of normal cellular protein expression. Understanding the post-transcriptional mechanisms by which plants regulate gene expression is fundamental to fully realizing the potential that genetic engineering offers.
This project is also significant for its involvement of undergraduates. Knox College has a strong track record for preparing students for graduate study in the sciences. In addition, this project includes a novel teaching postdoctoral position, combining a more traditional research postdoctoral experience with undergraduate teaching experience. This training in the integration of research and teaching is an experience tailored for the preparation of Ph.D. scientists seeking faculty positions at small liberal arts colleges.
