Molecular Regulatory Mechanisms of Populus Deltoides under Combined High Temperature and Short-Photoperiod Stress Associated with DNA Methylation.

Populus deltoides is a key species for industrial timber and ecological construction in temperate regions, where increasingly frequent and persistent heat waves pose serious challenges to its survival. However, the epigenetic mechanisms by which DNA methylation regulates environmental responses remain poorly understood. Here, whole-genome bisulfite sequencing and RNA-seq were performed on five P. deltoides genotypes grown in temperate and tropical regions. Results revealed that CG/CHG methylation stability is closely correlated with environmental sensitivity. Significant CG/CHG methylation variations may occur specifically in sensitive genotypes with large provenance-environment differences, thereby threatening the survival of P. deltoides by inhibiting the expression of key genes involved in life processes. CHH methylation variation may act as a potential epigenetic regulator of environmental adaptation. Promoter CHH-hypermethylation appears to represent a general response of P. deltoides under high-temperature and short-photoperiod (HS) stress, potentially regulating the expression of 56 genes to activate Ca2+ influx and heat shock proteins, repressing auxin, cytokinin, and cell cycle pathways, thereby initiating stress-protective responses. PdeCNGC13, PdeARF6, PdeLOG3, and PdeHSP15.7 were identified as potential regulatory genes of HS adaptation that are associated with DNA methylation. The mechanism of PdeLOG3 may involve HS-induced CHH-hypermethylation at its PdeLOG3 promoter, which may be associated with suppressed expression, reduced dihydrozeatin levels, and growth. This effect was partially reversed by 5-Azacytidine administration, accompanying increased cytokinin synthesis, enhanced antioxidant capacity, and coinciding with alleviation of HS stress. Our work preliminarily reveals the molecular mechanisms underlying DNA methylation-associated environmental adaptation in poplar, providing potential genetic targets for breeding climate-resilient trees.