Leaf senescence is a prominent symptom of heat injury observed in cool-season plants. The application of melatonin (MT) and inoculation with mycorrhizal fungi can mitigate heat-induced leaf senescence. The current research aims to examine the individual and combined effects of arbuscular mycorrhizal (AM) inoculation and MT application on heat-related leaf senescence in perennial ryegrass (Lolium perenne L.). In addition, whether the effects of these treatments were in any way associated with abscisic acid (ABA), gibberellin (GA), and cytokinin (CTK) for exerting the bioactivity. According to the results, the suppression of heat-mediated leaf senescence using MT treatment and AM inoculation manifested as increased photosynthesis, turf quality (TQ), and plant growth, and reduced membrane lipid peroxidation levels as well as the chlorophyll catabolic genes (CCGs) and senescence-associated genes (LpSAG12.1 and Lph36) transcript levels in the heat-stressed plants. MT application and AM inoculation elevated the internal MT, GA, and CTK levels, while reducing the ABA levels in the heat-stressed plants. In addition, the MT biosynthesis genes (LpTDC1, LpTDC2, LpCOMT1, LpASMT1, and LpASMT3), the ABA catabolic gene, the GA biosynthesis genes, CTK biosynthesis genes, along with the associated signal transduction response transcription factors (TFs) (type-B ARRs) exhibited increased levels, whereas the expressions of the GA and CTK catabolic genes and the biosynthesis and signal transduction genes related to ABA exhibited decreased levels after MT application and AM inoculation in the heat-stressed plants. Furthermore, the application of exogenous MT notably elevated the AM colonization rate at high temperature (HT) condition. After 28 days of heat stress, compared to the treatment with neither AMF nor MT application, AM combined with MT achieved a synergistic effect, resulting in a 25.32%, 87.21%, 58.69%, 57.17%, and 28.46% increase in TQ, leaf chlorophyll content, net photosynthesis (Pn), leaf photochemical efficiency (Fv/Fm), and relative water content (RWC), respectively, in perennial ryegrass. Additionally, this treatment significantly reduced the elec-trolyte leakage (EL), hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels by 30.76%, 38.19%, and 31.95%, respectively, and also significantly reduced the transcription levels of CCGs and senescence-associated genes. Consequently, it was inferred that a combination of AM and MT treatments promotes plant growth and development and suppresses heat-induced leaf senescence in plants.
