Isolation of dihydroflavonol 4-reductase cDNA clones from Angelonia x angustifolia and heterologous expression as GST fusion protein in Escherichia coli.

Keith R. Davis,Malvina Milosevic,Karl Stich,Klaus Olbricht,Karthik Mudigere Nagesh,Heidi Halbwirth,Shaghef Ejaz,Sylvia Plaschil,Jana Thill,Annette Rompel,Christian Gosch,Silvija Miosic

doi:10.1371/journal.pone.0107755

Abstract

Blue Angelonia × angustifolia flowers can show spontaneous mutations resulting in white/blue and white flower colourations. In such a white line, a loss of dihydroflavonol 4-reductase (DFR) activity was observed whereas chalcone synthase and flavanone 3-hydroxylase activity remained unchanged. Thus, cloning and characterization of a DFR of Angelonia flowers was carried out for the first time. Two full length DFR cDNA clones, Ang.DFR1 and Ang.DFR2, were obtained from a diploid chimeral white/blue Angelonia × angustifolia which demonstrated a 99% identity in their translated amino acid sequence. In comparison to Ang.DFR2, Ang.DFR1 was shown to contain an extra proline in a proline-rich region at the N-terminus along with two exchanges at the amino acids 12 and 26 in the translated amino acid sequence. The recombinant Ang.DFR2 obtained by heterologous expression in yeast was functionally active catalyzing the NADPH dependent reduction of dihydroquercetin (DHQ) and dihydromyricetin (DHM) to leucocyanidin and leucomyricetin, respectively. Dihydrokaempferol (DHK) in contrast was not accepted as a substrate despite the presence of asparagine in a position assumed to determine DHK acceptance. We show that substrate acceptance testing of DFRs provides biased results for DHM conversion if products are extracted with ethyl acetate. Recombinant Ang.DFR1 was inactive and functional activity could only be restored via exchanges of the amino acids in position 12 and 26 as well as the deletion of the extra proline. E. coli transformation of the pGEX-6P-1 vector harbouring the Ang.DFR2 and heterologous expression in E. coli resulted in functionally active enzymes before and after GST tag removal. Both the GST fusion protein and purified DFR minus the GST tag could be stored at −80°C for several months without loss of enzyme activity and demonstrated identical substrate specificity as the recombinant enzyme obtained from heterologous expression in yeast.

Highlights

The genus Angelonia is a perennial plant which originates from South America
All three enzymes could be detected with preparations from flowers which were obtained according to a relatively simple extraction procedure with quartz sand and buffer, with only moderate activity (Table 1)
chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR) activity were slightly higher when the preparations were obtained via a protocol developed for polyphenol rich tissues [31,32]

Summary

Introduction

The genus Angelonia is a perennial plant which originates from South America. It belongs to the family Plantaginaceae (formerly Scrophulariaceae) [1], which comprises about 30 species, displaying a great diversity in form and colour ranging from blue to violet, white and pink (Figure 1) [2,3]. In species which usually accumulate anthocyanins as plant pigments, white flowering genotypes result from a deficiency in at least one step of the anthocyanin pathway [8]. Such blockage of the anthocyanin pathway leading to white flower colour may occur at several levels. We analyzed cyanic and acyanic flowers of Angelonia for the absence of enzymes from the anthocyanin pathway and demonstrated that the acyanic line is DFR deficient. For future studies into the molecular basis behind flower colour variegation in Angelonia we isolated for the first time DFR cDNA clones of this plant species. This study provides important clues for the investigation of DFR characteristics in general

Results and Discussion

Conclusions

Materials and Methods