The long-term goal of this research is to understand the cell biology of higher plant 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 otherwise stable secretory protein mRNAs. Heat shock also causes the loss of endoplasmic reticulum lamellar structures upon which secretory protein mRNAs are translated; only small fragments of ER remain. This appears to provide the discriminatory mechanism for selectively stopping the synthesis of secretory proteins, as nonsecretory protein mRNAs are not translated by ER-bound ribosomes. The work proposed investigates the molecular and cellular basis for these heat shock-induced events. In vitro translation reactions supplemented with barley microsomes will be used to investigate whether the ER fragments of heat-shocked aleurone cells can serve as sites for the translation and translocation of proteins. The ER lamellae are supported by a cytoskeleton. In other organisms, the cytoskeleton has been shown to be unstable during heat shock. Immunofluorescence microscopy will be used to investigate the effect of heat shock on cytoskeletal elements of barley aleurone cells. Heat-induced shifts in the buoyant densities of ER membranes have been observed; these will be investigated by assaying protein:lipid ratios of microsomes isolated on sucrose density gradients. Secretory protein mRNAs contain secretory signals that direct them to be translated on ER lamellae. Aleurone cells transformed with signal sequence - reporter gene constructs will be used to determine of the presence of such a signal is sufficient to render the mRNA unstable during heat shock. These experiments should provide insights into the cell biology of ER, as well as reveal mechanisms for regulating protein expression during heat shock. These efforts will ultimately build a better understanding of how plants respond and adapt to stress.
