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

Abstract

Anthocyanins and proanthocyanidins (PAs) are important types of flavonoids, plant secondary metabolites with a wide range of industrial and pharmaceutical applications. DFR (dihydroflavonol 4-reductase) is a pivotal enzyme that plays an important role in the flavonoid pathway. Here, four <i>CsDFR</i> genes were isolated from <i>Camellia sinensis</i>, and their overexpression was analyzed <i>in vitro</i> and <i>in vivo.</i> Based on transcription and metabolic analyses, <i>CsDFR</i> expression was closely consistent with catechins and PAs accumulation. Moreover, enzyme activity analyses revealed that the two recombinant proteins CsDFRa and CsDFRc exhibited DFR activity, converting dihydroflavonols into leucoanthocyanins <i>in vitro</i>, but CsDFRb1 and CsDFRb3 did not. <i>CsDFRa</i> and <i>CsDFRc</i> overexpression in <i>AtDFR</i> mutants (<i>tt3</i>) revealed that <i>CsDFRs</i> are involved in the biosynthesis of anthocyanins and PAs, as <i>CsDFRa</i> and <i>CsDFRc</i> restored not only the purple petiole 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 CsDFRb1^N120S and CsDFRb1^C126T exhibited a higher affinity for DHM. Our findings comprehensively characterize the DFRs from <i>C. sinensis</i> and shed light on their critical role in metabolic engineering.