Stage-resolved multi-omics links flavonoid accumulation to antioxidant capacity in Isatis indigotica fruit

Isatis indigotica fruit is an underutilized source of natural antioxidant ingredients. However, the developmental basis of its bioactive metabolites remains unclear. We integrated widely targeted metabolomics, transcriptomics, and antioxidant phenotyping across five fruit developmental stages (S1-S5) to characterize metabolic remodeling, identify flavonoid-related metabolite features associated with antioxidant capacity, and infer candidate regulatory modules. We detected 2413 metabolites, revealing pronounced developmental reprogramming and late-stage enrichment of flavonoid-related metabolites, particularly anthocyanin-associated features. Consistent with these changes, DPPH 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, FRAP (ferric reducing antioxidant power), ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging activity, and total flavonoid content increased markedly during maturation, reaching 4.5-, 7.1-, 4.6-, and 13.0-fold of the S1 levels, respectively. Correlation analysis prioritized 12 flavonoid-related metabolite features strongly associated with antioxidant indices. Integrated transcriptomic analysis and weighted gene co-expression network analysis further identified a co-expression module linked to these features and highlighted Iin17052 (NAC family), Iin08518 (C3H family), and Iin25007 (MYB family) transcription factors as candidate regulators associated with late flavonoid biosynthetic genes, including DIHYDROFLAVONOL 4-REDUCTASE and ANTHOCYANIDIN SYNTHASE. Together, these findings provide a stage-resolved framework for harvest-stage selection and support the valorization of I. indigotica fruit as a potential source of plant-derived antioxidant ingredients for functional food development.