Multi-omics reveals flavonoid biosynthesis and regulation network in colored Amaranthus hypochondriacus seeds with antioxidant benefits

Amaranthus hypochondriacus is a nutritious pseudo-cereal whose metabolic and molecular variations across different seed colors remain largely unexplored. This study integrated ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based metabolomics, flavonoid-targeted metabolomics, and RNA sequencing (RNA-seq)-derived transcriptomics to systematically compare the metabolic and genetic profiles of white, yellow, brown, and black seeds. Among 763 metabolites identified, 484 were differentially accumulated, with flavonoids accounting for 27.4%. White seeds showed elevated amino acids and lipids; yellow and black seeds were enriched in flavonoids, whereas brown seeds accumulated phenolic acids. Yellow seeds showed the strongest antioxidant activity. Eight metabolites, including six flavonoids, were significantly correlated with antioxidant capacity. Integrated omics and weighted gene co-expression network analysis revealed 53 candidate genes associated with flavonoid biosynthesis, among which CHI (chalcone isomerase) was a key gene associated with high flavonoid content in yellow seeds. Furthermore, the transcription factor AhMYBD was experimentally verified to bind the CHI promoter and enhance its expression via yeast one-hybrid and dual-luciferase assays. These results provide important insights into the metabolic pathways and genetic basis associated with seed pigmentation and antioxidant activity, supporting the potential use of amaranth as a functional food ingredient. White (WH), yellow (YL), brown (BR) and black (BL) are the essential color in the grains of A . hypochondriacus . A combined analysis of UHPLC-ESI-MS/MS-based metabolomics, targeted flavonoid metabolomics and RNA-seq based transcriptomics revealed that the flavonoids made up most of the differential accumulated metabolites among different colored grains, and the content of flavonoids was positively correlated with the antioxidant activities of the grains. WGCNA identified a bundle of transcription factors most likely regulating the flavonoid biosynthesis, including MYB/MYB-related, WRKY and bHLH genes. The regulatory relationship between MYB-related genes ( AHMYBD ) and key flavonoid biosynthesis genes (LAR and CHI) was further verified by yeast one-hybrid (Y1H) and dual-luciferase assays. • Distinct metabolomes with 484 differential metabolites across four amaranth seeds. • Six flavonoids strongly linked to higher antioxidant activity in yellow seeds. • White seeds rich in lipids; yellow/black in flavonoids; brown in phenolic acids. • Yellow seeds accumulate high flavonoid levels via MYB-regulated chalcone isomerase.