Protease inhibitor regulation of proteins involved in heat-induced leaf senescence in creeping bentgrass

BACKGROUND AND AIMS: Heat stress accelerates leaf senescence, involving changes in protein metabolism and catabolism. Protease inhibitors may offer a novel strategy for enhancing heat tolerance by reducing heat-induced proteolysis and preserving protein integrity. The objectives were to determine how the serine-class protease inhibitor, aprotinin, regulates protein metabolism and catabolism in perennial grass under heat stress, and to identify the key proteins targeted by aprotinin that help alleviate heat-related damage, as well as the metabolic processes in which these proteins are involved. METHODS: Creeping bentgrass (Agrostis stolonifera 'Penncross') plants were subjected to elevated (38/33 °C, day/night) or optimal (22/17 °C, day/night) temperature conditions for 56 d in environmental growth chambers and foliar-treated every 7 d with 10 µM aprotinin. Visual turf quality, chlorophyll content, and photochemical parameters were evaluated as physiological indicators of leaf senescence, while proteomic methods were implemented to identify proteins that were responsive to aprotinin treatment under non-stress and heat stress conditions. KEY RESULTS: Under heat stress, aprotinin significantly improved turf quality (27.27 and 41.18 % at 42 and 56 d, respectively), chlorophyll content (12.76-18.12 % from 28-56 d), and photosystem II photochemistry (Fv/Fm: 1.57-3.02 % from 28-56 d; ΦPSII: 4.55-11.43 % from 14-56 d; qP: 9.09-19.17 % from 28-56 d) while simultaneously maintaining higher energy dissipation (Rfd: 11.57-17.71 % from 14-56 d; NPQ: 15.0-25.59 % from 42-56 d). Proteomic analysis revealed that aprotinin modulated 40 DEPs under heat stress in several metabolic pathways, including carbohydrate metabolism, nitrogen metabolism, nucleic acid metabolism, energy metabolism, stress response, and proteolysis in plants exposed to heat stress. CONCLUSIONS: The protease inhibitor regulated various proteins that could be involved in mitigating heat-induced leaf senescence. Identification of upstream molecular factors for the specific protease inhibitor-responsive proteins will provide further insight into mechanisms of controlling leaf senescence.