Targeted metabolomic and transcriptomic analyses provide insights into flavonoid biosynthesis in the grain of Foxtail millet

Foxtail millet (Setaria italica L.), a traditional Chinese crop, is valued for its rich abundance of health-beneficial compounds (e.g., flavonoids). Despite the nutritional significance of flavonoids, their biosynthetic pathways in foxtail millet remain largely uncharacterized. In this study, we integrated targeted metabolomic and transcriptomic analyses to comprehensively dissect the flavonoid biosynthesis pathway and identify genes encoding key enzymes. Quantitative profiling of a foxtail millet recombinant inbred line (RIL) population revealed significant variations in grain flavonoid content, with levels in the high-flavonoid (HF) group exceeding those in the low-flavonoid (LF) group by five-fold. Quantitative analysis of a foxtail millet recombinant inbred line (RIL) population showed striking variations in grain flavonoid content. The high-flavonoid (HF) group exhibited flavonoid levels five-fold higher than those of the low-flavonoid (LF) group. Through integrated targeted metabolomic and transcriptomic analyses, we elucidated the pivotal regulatory networks governing flavonoid biosynthesis in foxtail millet. Comparative transcriptomic analysis demonstrated significant differences in the expression patterns of flavonoid biosynthesis-associated genes between the HF and LF groups. Targeted metabolomic quantification further identified ten specific flavonoids with significantly elevated levels in the HF group. Integrative omics analyses revealed that genes encoding shikimate O-hydroxycinnamoyl transferase (HCT), phenylalanine ammonia-lyase (PAL), and phenylalanine/tyrosine ammonia-lyase (PTAL) are key determinants of the differential flavonoid accumulation observed between genotypes. Our study provides novel insights into the genetic regulatory mechanisms governing flavonoid metabolism in foxtail millet. The identified candidate genes, including those encoding HCT, PAL, and PTAL, represent valuable molecular targets for breeding programs focused on improving the nutritional profile of foxtail millet cultivars.