Insight into the rutin biosynthesis in the unique flavonol synthesis pathway of Tartary buckwheat based on the enzymatic functions of FLSs.

Key message: The homologous genes of FtFLS1-3 are crucial for rutin synthesis in Tartary buckwheat, with FtFLS1 playing a dominant role. The flavonol biosynthesis branch generates the main flavonoids in Tartary buckwheat (TB), with rutin serving as a representative flavonol compound. Flavonol synthase (FLS) is a vital enzyme involved in this metabolic pathway. Out of the five known FLS genes in the TB genome, FtFLS1 is the only gene identified, while information about the remaining four genes is limited. In this study, we cloned the five FtFLS genes from TB and performed molecular identification. The results showed that FtFLS1-3 exhibit high homology and similar molecular characteristics, categorizing them as FLS-like enzymes, while FtFLS4 and FtFLS5 show a certain degree of similarity to other 2-oxoglutarate-dependent dioxygenases. Further investigation revealed a significant correlation between expression of FtFLS1 and the rutin content during the flowering stage of TB (p < 0.05). The promoter sequences of FtFLS1-3 (P_FtFLS1-3) displayed distinctive cis-elements, transcriptional activities, and expression patterns, exhibiting different sensitivities to cold, UV-B, and drought stresses. The overexpression of FtFLS1-3 in Arabidopsis led to a significant elevation in total flavonoid and rutin levels, providing evidence for the FLS activity of FtFLS1-3 in plants. The enzymatic analysis showed that the recombinant FtFLS1-3 were capable of catalyzing the formation of their respective products from dihydroflavanols. FtFLS1 exhibited a superior specific activity, V_max and affinity for dihydroquercetin in terms of enzyme catalytic characteristics compared to FtFLS2 and FtFLS3. In summary, our study establishes the FLS activity of FtFLS1-3 and suggests that the metabolic flow of the flavonol biosynthesis branch in TB involves the conversion from dihydrokaempferol to dihydroquercetin and subsequently to quercetin, ultimately glycosylated to rutin. In this process, FtFLS1 plays a predominant role.