High temperature negatively impacts the yield and quality of fruit crops. Exogenous melatonin (MT) application has shown the capability to enhance heat tolerance, but the response of endogenous MT to heat stress, particularly in perennial fruit trees, remains elusive. This study investigated the effects of high temperatures on transgenic apple plants overexpressing the MT biosynthetic gene N-acetylserotonin methyltransferase 9 ( MdASMT9). Endogenous MT protected transgenic plants from heat stress, scavenging reactive oxygen species (ROS) and increasing soluble carbohydrates and amino acids levels. MdASMT9-overexpressing plants also maintained higher photosynthetic activity by protecting the chloroplasts from damage. Transcriptome sequencing indicates that MdASMT9 overexpression promoting the expression of HSFA1d, HSFA2-like, and HSFA9b, and inhibiting the transcription of HSFB1 and HSFB2b. Application of MT and overexpression of MdASMT9 reduced abscisic acid (ABA) accumulation through promoting MdWRKY33-mediated transcriptional inhibition of MdNCED1 and MdNCED3, thus promoting stomatal opening for better heat dissipation. Furthermore, melatonin enhanced autophagic activity through promoting MdWRKY33-mediated transcriptional enhancement of MdATG18a under heat stress . These findings provide new sight into the regulation of endogenous MT and its role in improving heat tolerance in perennial fruit trees.

Nitrogen (N) is a vital nutrient for crop growth and development which influences both yield and quality. Melatonin (MT), a known enhancer of abiotic stress tolerance, has been extensively studies; however, its relationship with nutrient stress, particularly N, and the underlying regulatory mechanisms of MT on N uptake remain unclear. In this study, exogenous MT treatment was found to improve the tolerance of apple plants to N deficiency. Apple plants overexpressing the MT biosynthetic gene N-acetylserotonin methyltransferase 9 ( MdASMT9) was used to further investigated the effects of endogenous MT on low-N stress. The overexpression of MdASMT9 improved the light harvesting and heat transfer capability of apple plants, thereby mitigating the detrimental effects of N deficiency on the photosynthetic system. Proteomic and physiological data analyses indicated that MdASMT9 overexpression enhanced the trichloroacetic acid (TCA) cycle and positively modulated amino acid metabolism to counteract N-deficiency stress. Additionally, both exogenous and endogenous MT promoted the transcription of MdHY5, which in turn bound to the MdNRT2.1 and MdNRT2.4 promoters and activated their expression. Notably, MT-mediated promotion of MdNRT2.1 and MdNRT2.4 expression in an MdHY5-dependent manner, ultimately enhancing N absorption. Taken together, these results may provide useful insights into the relationship between MdASMT9-mediated MT biosynthesis and N uptake under N-deficiency conditions in apple plants.