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Heat stress causes premature leaf senescence in cool-season grass species. The objective of this study was to identify proteins regulated by nitrogen, cytokinins, and ethylene inhibitor in relation to heat-induced leaf senescence in creeping bentgrass (Agrostis stolonifera). Plants (cv. Penncross) were foliar sprayed with 18 mM carbonyldiamide (N source), 25 μM aminoethoxyvinylglycine (AVG, ethylene inhibitor), 25 μM zeatin riboside (ZR, cytokinin), or a water control, and then exposed to 20/15°C (day/night) or 35/30°C (heat stress) in growth chambers. All treatments suppressed heat-induced leaf senescence, as shown by higher turf quality and chlorophyll content, and lower electrolyte leakage in treated plants compared to the untreated control. A total of 49 proteins were responsive to N, AVG, or ZR under heat stress. The abundance of proteins in photosynthesis increased, with ribulose-1,5-bisphosphate carboxylase/oxygenase affected by all three treatments, chlorophyll a/b-binding protein by AVG and N or Rubisco activase by AVG. Proteins for amino acid metabolism were upregulated, including alanine aminotransferase by three treatments and ferredoxin-dependent glutamate synthase by AVG and N. Upregulated proteins also included catalase by AVG and N and heat shock protein by ZR. Exogenous applications of AVG, ZR, or N downregulated proteins in respiration (enolase, glyceraldehyde 3-phosphate dehydrogenase, and succinate dehygrogenase) under heat stress. Alleviation of heat-induced senescence by N, AVG, or ZR was associated with enhanced protein abundance in photosynthesis and amino acid metabolism and stress defense systems (heat shock protection and antioxidants), as well as suppression of those imparting respiration metabolism.

Leaf senescence is characterized by decreased chlorophyll content in leaves. The objectives of this study were to determine whether heat‐induced chlorophyll decline is due to inhibited chlorophyll synthesis or accelerated chlorophyll degradation and to determine whether genetic variations in heat tolerance of bentgrass (Agrostis spp.) species were associated with differential chlorophyll‐enzymatic responses to heat stress. Five turfgrass lines, including two transgenic creeping bentgrass (A. stolonifera L.) lines overexpressing isopentenyl transferase (ipt) gene ligated to a senescence‐activated promoter (SAG12) or heat shock promoter (HSP18.2) for controlling cytokinin synthesis, two thermal bentgrass (A. scabra Willd.) lines, and a wild‐type (WT) creeping bentgrass (‘Penncross’) were subjected to heat (38/33°C, day/night) or optimal temperature (22/18°C, day/night) (nonstress) for 42 d in growth chambers. The physiological parameters of turf quality, chlorophyll content, chlorophyll index, and dark green color index were measured. The data suggested significant genetic variations in the level of heat‐induced leaf senescence among the bentgrass lines. The enzyme activity of a key chlorophyll‐synthesizing enzyme, porphobilinogen deaminase, did not differ significantly across all the lines after 42 d of heat stress. The activities of chlorophyll‐degrading enzymes, including chlorophyllase and chlorophyll‐degrading peroxidase, increased significantly after heat stress, whereas pheophytinase activity was unchanged. Heat‐tolerant transgenic lines and thermal bentgrass maintained significantly lower activities of chlorophyll‐degrading enzymes than the WT under heat stress. Heat‐induced chlorophyll loss in bentgrass could be mainly due to accelerated chlorophyll degradation. Selecting for low chlorophyll degradation enzyme activity will help to facilitate the development of elite stay‐green bentgrass lines.

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