Metabolome and transcriptome analysis of anthocyanin biosynthesis reveal key metabolites and candidate genes in red-stemmed alfalfa (Medicago sativa)

Alfalfa (Medicago sativa L.) serves as a vital high-quality forage resource, especially in tropical and subtropical regions where there is a deficiency of protein-rich feed. The red pigmentation of stem of space mutated alfalfa was mainly caused by anthocyanin accumulation. However, investigations into the mechanisms governing anthocyanin biosynthesis in alfalfa stems have been scarce. In this study, we conducted combined transcriptome and metabolome analyses on two types of alfalfa stems: space mutation red-stemmed alfalfa and non-space mutation green-stemmed alfalfa (control). Profiling of the anthocyanin metabolome unveiled 45 metabolites linked to anthocyanin biosynthesis, with cyanidin-3-O-glucoside, pelargonidin-3-O-arabinoside, delphinidin-3-O-(6-O-acetyl)-glucoside, and kaempferol-3-O-rutinoside identified as the primary anthocyanins of red-stemmed alfalfa. Transcriptome analysis revealed 72 differentially expressed genes related to anthocyanin biosynthesis pathways, of which 54 genes were highly expressed in red stems, including 12 PALs (phenylalanine ammonia-lyase), 22 4CLs (4-coumaroyl: CoA-ligase), eight CHSs (chalcone synthase), three F3Hs (flavanone 3-hydroxylase), two ANRs (anthocyanidin reductase), three DFRs (dihydroflavonol-4-reductase), three ANSs (anthocyanidin synthase), and one FLS (flavonol synthase) gene. These genes are likely pivotal for anthocyanin biosynthesis in red-stemmed. Co-expression analysis of differentially expressed genes and relative contents of differentially expressed anthocyanin showed that each anthocyanin was closely related to multiple genes, and anthocyanin accumulation process was regulated by multiple genes. The expressions of these genes were significantly positively correlated with the relative contents of cyanidin-3-O-glucoside, pelargonin-3-O-arabinoside, and kaempferol-3-O-rutin. Overall, the expression patterns of PAL, 4CL, CHS, F3H, ANR, DFR, ANS, and FLS structural genes in anthocyanin biosynthesis pathway were closely related to the composition and content of anthocyanins. Different anthocyanins' accumulation patterns may result in the different stem colors of alfalfa. These findings provide comprehensive insights into the molecular mechanisms for anthocyanin biosynthesis in red-stemmed alfalfa.