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Article|22 Apr 2022|OPEN
Functional analysis of the dihydroflavonol 4-reductase family of Camellia sinensis: exploiting key amino acids to reconstruct reduction activity
Haixiang Ruan1,2 ,† , Xingxing Shi1,3 ,† , Liping Gao2 ,† , Arif Rashid2 , Yan Li2 , Ting Lei2 and Xinlong Dai3 , Tao Xia1 , , Yunsheng Wang,1,2 ,
1State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui 230036, China
2School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, China
3College of Tea Science, Guizhou University, Guiyang Guizhou 550025, China
*Corresponding author. E-mail: xiatao62@126.com,wangyunsheng@ahau.edu.cn
Haixiang Ruan and Xingxing Shi,Liping Gao contributed equally to the study.

Horticulture Research 9,
Article number: uhac098 (2022)
doi: https://doi.org/10.1093/hr/uhac098
Views: 156

Received: 21 Jan 2022
Accepted: 15 Apr 2022
Published online: 22 Apr 2022

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 CsDFR genes were isolated from Camellia sinensis, and their overexpression was analyzed in vitro and in vivo. Based on transcription and metabolic analyses, CsDFR 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 in vitro, but CsDFRb1 and CsDFRb3 did not. CsDFRa and CsDFRc overexpression in AtDFR mutants (tt3) revealed that CsDFRs are involved in the biosynthesis of anthocyanins and PAs, as CsDFRa and CsDFRc 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 CsDFRb1N120S and CsDFRb1C126T exhibited a higher affinity for DHM. Our findings comprehensively characterize the DFRs from C. sinensis and shed light on their critical role in metabolic engineering.