This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The correlation between proline accumulation and stress tolerance is well established in plants and fungi but most of the attention has been on the ability of proline to function as an osmoprotectant. Evidence for an obscure role for proline and potentially a more broad-based function is from our recent work with the fungal pathogen of alfalfa, Collectotrichum trifolii. Dominant active mutations in Ras from C. trifolii, a small G protein, caused oncogenic phenotypes in mice and displayed a nutrient(starvation)-dependent phenotype in the fungus resulting in abnormal hyphal proliferation, defects in polarized growth and an inability to differentiate. Various carbon sources, nitrogen sources, vitamins, pH, heat, osmoticum and combinations of these factors failed to revert the phenotype. Remarkably, restoration of the wild type phenotype required only proline. It was noted that these activated Ras strains had abnormally high levels of reactive oxygen species (ROS). When these strains were treated with proline or antioxidants, the activated Ras phenotype reverted and was directly coupled to significant decreases in ROS levels, indicating proline is a scavenger for ROS. Furthermore the high ROS levels induced by activated Ras caused a programmed-like cell death in C. trifolii that shares features with mammalian apoptosis including DNA condensation and fragmentation as well as externalization of phosphatidylserine. These features are absent when the fungus is supplemented with proline. Since ROS levels increase in plants, fungi, and animals during a variety of environmental stresses and ROS is a mediator of apoptosis, the ability of proline to scavenge ROS may be an important function of this amino acid in organisms responding to cellular stress. The central hypothesis of our studies is that proline functions as an antioxidant cytoprotectant during stress by scavenging ROS and maintaining redox homeostasis. The major goals of this proposal are to characterize and determine the extent and range of proline cytoportection from ROS mediated stress and to identify plant genes that modulate ROS mediated programmed cell death. Both of these topics are relatively unexplored.The connection between proline and ROS has scarcely been examined and biochemical characterization of proline metabolic enzymes is incomplete. Knowledge gained from this study on the versatile roles of proline in cellular events will contribute to the understanding of proline accumulation and stress adaptation.
The specific aims of this proposal are: (1) Determine the effect of proline on the REDOX status in yeast; (2) Identify plant and yeast genes that regulate ROS-induced cell death; and (3) Determine whether the antioxidant effects of proline extend to mammalian cells.
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