Functional analysis of the dihydroflavonol 4-reductase family of Camellia sinensis: exploiting key amino acids to reconstruct reduction activity

Anthocyanin and proanthocyanidins (PAs) are important components of flavonoids, secondary metabolites in plants with a wide range of industrial and pharmaceutical applications. DFR (dihydroflavonol 4-reductase) is a pivotal enzyme which plays an important role in the flavonoid pathway. Here, four CsDFRs genes were isolated from Camellia sinensis and their overexpression were analyzed in vitro and in vivo. Based on transcription and metabolic analyses, the CsDFRs expression is closely consistent with catechins and PAs accumulation. Moreover, the enzyme activity analyses revealed that the two recombinant proteins CsDFRa and CsDFRc exhibited DFR activity, which converts dihydroflavonols into leucocyanidin in vitro, but not CsDFRb1 and CsDFRb3. CsDFRa and CsDFRc overexpression in AtDFR mutants (tt3) revealed that CsDFRs are involved in the biosynthesis of anthocyanins and PAs, as CsDFRa and CsDFRc not only restored the petiole purple phenotype, but also the seed coat color. Site-directed mutagenesis revealed that the two amino acid residues S117 and T123 of CsDFRa play a prominent role in controlling DFR reductase activity. Enzymatic assays indicated that CsDFRa and CsDFRc exhibited a higher affinity for DHQ and DHK, respectively, whereas CsDFRb1N120S and CsDFRb1C126T exhibited a higher affinity for DHM. Our findings present comprehensive characterization of the DFRs from C. sinensis and shed light on their critical role in metabolic engineering.