Integrative analysis of the metabolome and transcriptome provides insights into the mechanisms of anthocyanins and proanthocyanidins biosynthesis in Trifolium repens

Anthocyanin and proanthocyanidin (PA) are plant natural products beneficial to human and livestock health. In particular, the legume forage with proper PA contents can prevent lethal pasture bloat in ruminant animals. To study the mechanism underlying anthocyanin and PA biosynthesis in legume forages, comparative transcriptomics and metabolomics analyses were performed with ‘Purple’ and ‘Haifa’ white clovers. Metabolic analyses revealed that pelargonidin may be the most important source of color in ‘Purple’. Cyanidin-3-O-(6''-O-p-coumaroyl)-glucoside, petunidin-3-O-(6''-O-p-coumaroyl)-rutinoside, and delphinidin-3-O-rutinoside-7-O-glucoside might contribute to PA biosynthesis. Additionally, 67 differentially expressed genes in anthocyanin and PA biosynthetic pathways were discovered between ‘Purple’ and ‘Haifa’. Among them, DFR (dihydroflavonol-4-reductase), LDOX/ANS (leucoanthocyanidin oxygenase/anthocyanidin synthase), (leucoanthocyanidin reductase), ANR (anthocyanidin reductase), and UFGT (UDPglucose:flavonoid-3-O-glucosyltransferase) may be key determinants of the differences in anthocyanin and PA biosynthesis. Finally, A hypothetical model of gene expression regulating the synthesis of PAs and anthocyanins in white clover was proposed, which could provide a molecular genetic basis for future candidate genes validation and breeding high-PA white clover cultivars.