Differential Gene Expression is Partially Associated with Differential DNA Methylation During the Vegetative Phase Change in Apple

The vegetative phase change (VPC) is a typical epigenetic event in anthophytes. DNA methylation exerts vital functions in the modulation of plant ontogenetic and biological processes. The microRNA miR156 is established as the core regulatory hub of plant VPC and the levels of miR156 are regulated at both transcriptional, post-transcriptional, and epigenetic levels. Nevertheless, whether and how genomic DNA methylation participates in the regulation of miR156 precursor genes during the VPC remain largely ambiguous in perennial woody plants including apple (Malus domestica Borkh.). In this study, whole‑genome bisulfite sequencing and RNA‑seq were used to explore how DNA methylation influences gene expression during apple VPC. Genome‑wide CG, CHG, and CHH methylation levels were stable across developmental stages. Intergenic regions were more highly methylated than gene regions. And in gene regions all three methylation contexts were significantly higher in the adult phase (nodes 61–180 on the trunk of a tree) than in the juvenile phase (nodes 0–60). Genes upregulated in the adult phase, enriched in photosynthesis, carbohydrate/lipid metabolism, and energy pathways, were related to hypomethylation. No differential methylation was detected in miR156 precursors. However, transcription factors co‑expressed with miR156 and MdMIR156a5, including brassinosteroid biosynthesis genes and MdRVE1‑1/MdRVE1‑2 were hypermethylated in adult phase. The 5‑azacytidine treatment reduced methylation of MdRVEs and increased expression of MdMIR156a5 and miR156. Overall, 57.1% of differentially expressed genes showed a negative correlation between DNA methylation and expression. Hypermethylation of MdRVEs in the adult phase was associated with repressed expression of MdMIR156a5 and miR156.