Metabolomic and physiological analysis of bud differentiation in dense apple (Malus×domestica Borkh.) orchards following thinning and reshaping.

In China, a majority of apple orchards were initially planted under arboriculture. The branch number increases, causing a deterioration of the canopy light conditions as the trees age. This phenomenon blocks bud differentiation, resulting in yield decrease and deterioration of fruit quality. Thinning and reshaping are effective strategies for addressing these issues. However, the impact of thinning and reshaping remains unclear. This study analyzed the physiological and metabolic aspects of flower bud differentiation following thinning and reshaping of an overcrowded orchard. Methods: Physiological and metabolomic analyses were conducted on terminal flower bud samples collected during early (T1) and late (T2) bud differentiation following thinning and reshaping alongside controls CK1 and CK2. Results: Sucrose, glucose, fructose, and sorbitol contents in T1 and T2 groups were significantly higher than in CK1 and CK2. Metabolomic analysis revealed significant differences: 60 metabolites (32 up-regulated, 28 down-regulated) in T1 vs CK1 and 51 metabolites (26 up-regulated, 25 down-regulated) in T2 vs CK2. KEGG enrichment analysis revealed that the biosynthesis of secondary metabolites was the most enriched pathway, with the crucial compounds associated with flower bud differentiation. There was an accumulation of coumarin (C05851), eriodictyol (C05631), and histidine (C00135) during both the early and late stages of flower bud differentiation, underscoring their promoting role. In T1 vs CK1, 2-isopropylmalic acid (C02504), (+)-catechin (C09727), and trans-ferulic acid (C01494) were significantly up-regulated, highlighting their potential as metabolic activators. Conversely, 3',5'-dimethoxy-3,5,7,4'-tetrahydroxyflavone (C11620) and 4-hydroxyphenylpyruvic acid (C01179) decreased, implying divergent regulatory mechanisms. Temporal specificity emerged in T2 vs CK2, with significant upregulation of myricetin (C10107) and isopimpinellin (C02162). In contrast, umbelliferone (C09315) and trans-caffeic acid (C01197) exhibited opposing trends. Moreover, jasmonic acid (C08491) and 6-phosphogluconic acid (C00345) increased sharply in T2, while trans-traumatic acid (C16308) declined. Conclusions: Thinning and reshaping improved light penetration and increased the accumulation of nutrients and secondary metabolites. Sucrose, glucose, fructose, and sorbitol contents and the relative levels of coumarin, eriodictyol, and histidine increased during early and late flower bud differentiation stages, which suggested their positive roles during these stages. 2-isopropylmalic acid, (+)-catechin, and trans-ferulic acid exhibited metabolic activation potential, while 3',5'-dimethoxyflavone and 4-hydroxyphenylpyruvic acid were distinctly regulated in T1 vs CK1. Myricetin, isopimpinellin, jasmonic acid, and 6-phosphogluconic acid exhibited activation potential, while trans-traumatic acid was distinctly regulated in T2 vs CK2. Metabolic changes and pathway-specific activation/inhibition patterns underlying flower bud differentiation exhibited a direct-indirect regulatory network in secondary metabolism.