Integrated metabolome and transcriptome reveal the molecular basis of flavonoid pathway-mediated fruit firmness variation in sweet cherry (Prunus avium L.).

Sweet cherry is rich in diverse bioactive compounds and functional nutrients. Fruit firmness is a key determinant of postharvest storability, shelf life, and commercial value. Secondary metabolism is one of the important factors regulating sweet cherry fruit firmness, yet the molecular mechanisms underlying the regulation of firmness by secondary metabolism remain unclear. We performed integrated transcriptomic and untargeted metabolomic analyses at four key developmental stages using two cultivars with contrasting firmness: soft-fleshed 'Shangnan2808' and hard-fleshed 'Wanhongzhu'. We also conducted bulked metabolomic analysis on extreme firmness phenotypes from an F1 segregating population derived from these two parents. Fruit firmness differed significantly from color break to full ripeness stage, with epidermal cell thickness and mesocarp cell packing density identified as major structural contributors. KEGG enrichment revealed that flavonoid biosynthesis was the most significantly enriched pathway among six pathways containing 41 stable differential metabolites. Multi-omics integration identified 186 differentially expressed genes involved in these pathways. Among them, the flavanone 3-hydroxylase-like gene FUN_006793 (F3HL) exhibited an expression pattern highly consistent with kaempferol accumulation, suggesting it may be a core candidate regulating fruit firmness. Bulked metabolomics further validated seven key metabolites, including kaempferol and chlorogenic acid, both involved in flavonoid biosynthesis, confirming the central role of this pathway. WGCNA identified three co-expression modules significantly correlated with firmness and allowed the construction of a flavonoid-mediated regulatory network. This study systematically reveals the potential mechanism by which flavonoid biosynthesis may modulate sweet cherry fruit firmness, and provides a theoretical basis for investigating firmness regulatory networks and offer valuable candidate genes, metabolic biomarkers, and functional markers for molecular breeding of high-quality, long-storability sweet cherry cultivars.