Proteomic Responses to Exogenous Application of Nitrogen, Cytokinin, and Ethylene Inhibitors in Relation to Heat-Induced Senescence in Creeping Bentgrass.

Heat is a major abiotic stress inducing premature senescence in the widely used turfgrass creeping bentgrass (Agrostis stolonifera). Previous studies have demonstrated that application of  nitrogen, cytokinin, and ethylene inhibitor delayed leaf senescence and increased heat tolerance in creeping bentgrass, but the mechanisms of this improved heat tolerance is not fully understood. The objective of this study was to examine what specific proteins and related metabolic processes may be altered by the exogenous application of nitrogen, cytokinins, and ethylene inhibitor that lead to the suppression of heat-induced leaf senescence and the improvement of whole-plant heat tolerance in creeping bentgrass.   'Penncross' plants were exposed to heat stress conditions (35/30 C day/night temperatures) and optimal temperature (20/15 C day/night) in controlled environment growth chambers.  Exogenous applications of nitrogen, cytokinin, an ethylene inhibitor, and a combination of all three treatments were applied at previously determined effective rates three days prior to the stress treatment and then at weekly intervals during the stress period. To assess treatment effects and the progress of heat induced senescence, chlorophyll content, membrane stability, and overall quality ratings were taken every seven days. Leaf tissues were sampled  for proteomic analysis.  At three weeks of heat stress all treatments had declined in chlorophyll content, membrane stability and quality;  however, the plants treated with nitrogen, cytokinin, ethylene inhibitor or a combination of all three had significantly less decline in these parameters, demonstrating a delay in senescence.  A two-dimentional polyacrylamide gel electrophoresis allows for the separation and quantification of a wide range of expressed proteins allowing for the identification of proteins with altered abundances. This proteomic analysis highlights important proteins in various pathways including, photosynthesis, energy production, and stress defense which may be responsible for the tolerance conferring effects seen by these treatments.  Comparison of these protein changes will allow for a greater understanding into both the underlying mechanisms of heat tolerance as well as specific pathways induced by the exogenous applications of these compounds.