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

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.

Fig 1. Alignment of the deduced amino acid sequences of DFRs.

Identical residues are highlighted in black, and similar residues are highlighted in gray. A putative NAD(P)-binding domain and a putative substrate binding domain are underlined. The red asterisk indicates the 133rd amino acid residue, which is particularly important for substrate specificity. PhDFR: Petunia hybrid; NtDFR: Nicotiana tabacum; VvDFR: Vitis vinifera; AtDFR: Arabidopsis thaliana; ZmDFR: Zea mays; GbDFR: Ginkgo biloba; DeDFR1-2: Dryopteris erythrosora; AfDFR1-6: Azolla filiculoides; ScDFR1-4: Salvinia cucullata.

Fig 2. Alignment of the deduced amino acid sequences of DFRs.

A phylogenetic tree of DFRs constructed by the neighbor-joining method with 1000 bootstrap replicates. Asn-type (no label), Asp-type (labeled with a filled triangle), Arg-type (labeled with a filled circle) and non-Asn/Asp/Arg-type (labeled with a square) DFRs are labeled with different shapes in front of the species. For accession numbers of the DFR sequences, see Materials and Methods.

Fig 3. Expression of DeDFRs and anthocyanidin accumulation during frond development in D. erythrosora.

(a) Frond development in D. erythrosora: rolled immature fronds (RF), young fronds (YF) and mature fronds (MF). (b) HPLC chromatogram of anthocyanidin standards. Peak 1, delphinidin; Peak 2, cyanidin; Peak 3, pelargonidin. (c) The HPLC profile of anthocyanidin levels in young fronds of D. erythrosora shows the elution times for delphinidin and cyanidin. (d) Accumulation of delphinidin and cyanidin in fronds. Bars represent the mean ± SD. For the delphinidin content, A, B, and C above the bars indicate a significant difference between the samples at p<0.01. For the cyanidin content, A' and B' above the bars indicate a significant difference between the samples at p<0.01. (e) Relative gene expression levels of DeDFRs normalized to the level of DeGAPDH. Three replicates were used for each sample. Bars represent the mean ± SD. Different lowercase letters above the bars indicate a significant difference between the samples at p<0.05.

Fig 4. The product peak area of HPLC for DeDFR1, DeDFR2 and AtDFR proteins with DHK, DHQ, and DHM as substrates.

Bars represent the mean ± SD. *Statistically significant difference compared with AtDFR at 0.01<p<0.05. **Statistically significant difference compared with AtDFR at p<0.01.

Fig 5. The product peak area of HPLC for modified AtDFR proteins with DHK, DHQ, and DHM as substrates.

Bars represent the mean ± SD. *Statistically significant difference compared with AtDFR at 0.01<p<0.05. **Statistically significant difference compared with AtDFR at p<0.01.

Fig 6. Overexpression of DeDFR1 and DeDFR2 in Arabidopsis tt3-1 mutant.

(a) Phenotype of the wild type (WT, Col), tt3-1 mutant and transgenic lines seed coats. (b) HPLC analysis of cyanidin content in the seeds of wild-type, tt3-1 mutant and transgenic lines. Three replicates were used for each sample. Bars represent the mean ± SD. Different capital letters above the bars indicate a significant difference between the samples at p<0.01. (c) Expressional analysis of DeDFR1 and DeDFR2 by RT-PCR in the leaves of wild-type, tt3-1 mutant and transgenic lines.

Fig 7. Proposed pathway for anthocyanin biosynthesis in D. erythrosora.

The enzymes studied herein are represented in red font. Abbreviations: CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol 4-reductase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; FLS, flavonol synthase; DHK, dihydrokaempferol; DHQ, dihydroquercetin; DHM, dihydromyricetin.