Flowering is a necessary condition and basis for yield in the life cycle of woody fruit trees.  Although there has been considerable interest in the regulatory mechanisms underlying floral induction and flowering, the associated epigenetic modifications remain poorly characterized.  We identified genome-wide DNA methylation changes and the transcriptional responses in axillary buds of ‘Qinguan’ (QA) and ‘Fuji’ (FA) varieties with contrasting flowering behaviors.  The DNA methylation levels were 19.35, 62.96 and 17.68% in FA, and 19.64, 62.49 and 17.86% in QA in the CG, CHG and CHH contexts, respectively.  The number of hypermethylated and hypomethylated differentially methylated regions (DMRs) in different regions contributed to significantly up- and downregulated gene expression.  DNA methylation can positively or negatively regulate gene expression depending on the CG, CHG and CHH contexts and their locations in different regions.  Additionally, the huge differences in transcription of MIKC^c-type MADS-box genes, and multiple flowering genes in multiple flowering pathways (i.e., light, aging, GA and sugar) by changing DNA methylation, contributed to contrasting flowering behaviors in both QA and FA.  Specifically, the floral meristem identity genes (i.e., FT, LEAFY, AP1 and SOC1) exhibited significantly higher expression in QA than FA, but the floral repressors (i.e., SVP, AGL15, and AGL18) showed the opposite trend.  Significant differences in multiple hormone levels were due to differentially expressed genes (DEGs) and their DMRs in hormone synthesis pathways, leading to both contrasting axillary bud outgrowth and flowering behaviors.  These findings reflect the diversity in the epigenetic regulation of gene expression and may be helpful for elucidating the epigenetic regulatory mechanism underlying the axillary bud flowering in apple.

Bud dormancy is a key adaptive strategy in perennial plants, enabling them to survive in adverse environmental conditions. However, it generates challenges in crop cultivation, especially in fruit crops like apple, where synchronized bud break is crucial for consistent growth and yield. The synthetic cytokinin 6-benzylaminopurine (6-BA) promotes dormancy release, but its molecular and metabolic mechanisms remain poorly understood. This study investigates dormancy release in Xianheng 01 apple rootstock nursery plants through integrated transcriptomic, metabolomics, and hormonal analyses. Dormant buds were treated with 6-BA, followed by morphological, biochemical, and molecular profiling conducted over 30 days. 6-BA treatment increased plant height and leaf emergence by increasing the level of endogenous hormones such as cytokinins (DHZR/IPA) and decrease in level of abscisic acid (ABA). Transcriptomics analysis identified 7,009 differentially expressed genes (DEGs) in response to 6-BA treatment. The cytokinin-responsive gene A-ARR8 exhibited a distinct expression pattern, remained upregulated at 1, 3 and 6 d post-treatment but downregulated at 11 d. In contrast ABA related genes SnRK2a/b, PP2C and ABF3 were consistently downregulated throughout the treatment period. Metabolomics analysis identified 2,053 metabolites, showing early-phase dominance of phenylpropanoids and flavonoids, followed by a shift towards ABC transporter-mediated nutrient mobilization. Conjoint analysis highlighted coordinated activation of secondary metabolite biosynthesis and cytokinin signaling. These results demonstrate that 6-BA induces dormancy release through cytokinin-ABA antagonism and phased metabolic reprogramming from stress protection to growth promotion. Our findings provide a comprehensive framework for optimizing dormancy management in apple cultivation and highlight 6-BA as an effective agrochemical for enhancing temperate fruit production.