Integrated metabolomic and transcriptomic analysis of anthocyanin accumulation mechanisms in maize kernels of different colors

Introduction: Anthocyanins are important natural pigments and bioactive compounds in colored maize kernels. Although colored fresh-eating maize has attracted increasing attention because of its nutritional and functional value, the molecular basis underlying anthocyanin accumulation in maize kernels with different colors remains incompletely understood. Methods: In this study, four fresh-eating maize cultivars with contrasting kernel colors, including white (WN 2000), yellow (HN), multicolored (CN1), and black (HTN188), were used to investigate the mechanism of anthocyanin accumulation. Total anthocyanin content was measured across developmental stages, and integrated metabolomic and transcriptomic analyses were performed to identify anthocyanin-related metabolites, differentially expressed genes, and candidate regulatory factors associated with kernel pigmentation. Results: Metabolomic profiling identified 49 anthocyanin-related metabolites/features, with marked enrichment of cyanidin-, peonidin-, and pelargonidin-related compounds in the black maize cultivar HTN188. Transcriptomic analysis identified 12,557 differentially expressed genes among the four cultivars, which were mainly enriched in phenylpropanoid biosynthesis, flavonoid biosynthesis, glutathione metabolism, fructose and mannose metabolism, and glyoxylate and dicarboxylate metabolism pathways. Several anthocyanin biosynthetic genes and regulatory transcription factor candidates were upregulated in anthocyanin-rich kernels, including DFR (Zm00001d011438), MYB (Zm00001d003052), bHLH (Zm00001d015990), and NAC (Zm00001d012508). In particular, the expression of DFR (Zm00001d011438) was strongly positively correlated with total anthocyanin content. Weighted gene co-expression network analysis further identified gene modules and candidate hub regulators associated with anthocyanin accumulation. Discussion: These findings provide a comparative multi-omics perspective on anthocyanin accumulation in fresh-eating maize kernels of different colors. The results suggest that the high anthocyanin accumulation in black maize is associated with the coordinated enrichment of anthocyanin-related metabolites and the upregulation of key structural genes and transcription factors. This study provides candidate genes and regulatory information for future functional studies and anthocyanin-oriented breeding in fresh-eating maize.