Arg-type dihydroflavonol 4-reductase genes from the fern Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins.

Shaojun Dai,Xuefei Chen,Huanhuan Fu,Quanxi Wang,Xianyan Huang,Wenli Liu,Youfang Wang,Jianguo Cao

doi:10.1371/journal.pone.0232090

Abstract

Dihydroflavonol 4-reductase (DFR), a key enzyme involved in the biosynthesis of anthocyanins, has been cloned from various species. However, little research has been conducted on this enzyme in ferns, which occupy a unique evolutionary position. In this study, we isolated two novel DFR genes from the fern Dryopteris erythrosora. In vitro enzymatic analysis revealed that DeDFR1 and DeDFR2 enzymes can catalyze dihydrokaempferol and dihydroquercetin but cannot catalyze dihydromyricetin. Amino acid sequence analysis showed that DeDFR1 and DeDFR2 have an arginine at the same substrate-specificity-determining site as that in the ferns Salvinia cucullata and Azolla filiculoides. Thus, we speculate that the Arg-type DFR is a new DFR functional type. To further verify the substrate preferences of the Arg-type DFR, an amino acid substitution assay was conducted. When N133 was mutated to R133, Arabidopsis DFR protein completely lost its catalytic activity for dihydromyricetin, as observed for DeDFR1 and DeDFR2. Additionally, heterologous expression of DeDFR2 in the Arabidopsis tt3-1 mutant resulted in increasing anthocyanin accumulation. In summary, DeDFR1 and DeDFR2 are considered to be a new type of DFR with unique structures and functions. The discovery of the Arg-type DFR provides new insights into the anthocyanin biosynthesis pathway in ferns.

Highlights

Anthocyanins represent an important subgroup of flavonoids, which are important secondary metabolites produced by plants to adapt to the terrestrial environment
The alignment showed that DeDFR1 and DeDFR2 belonged to the SDR superfamily, including a conserved NAD(P)-binding domain and a substrate-binding domain
The newly identified DeDFR1 and DeDFR2 genes were associated with the accumulation of anthocyanins in D. erythrosora

Summary

Introduction

Anthocyanins represent an important subgroup of flavonoids, which are important secondary metabolites produced by plants to adapt to the terrestrial environment. Anthocyanins in food plants are potentially valuable components of the human diet. Some studies have indicated that amino acid residue 133 plays an important role in substrate specificity and classified DFRs into different types based on this site. DFRs in most plants are Asn-type DFRs, which can catalyze three types of substrates (DHK, DHQ, DHM) Some species, such as Petunia and Cymbidium species, have Asp-type DFRs, which do not have the ability to catalyze DHK. These species do not produce brick red/orange flowers with pelargonidin-based anthocyanins [26, 27]. Most genes have been isolated from seed plants, and few studies on the function of DFR genes in spore plants

Methods

Results

Discussion

Conclusion