Isolation of Dihydroflavonol 4-Reductase cDNA Clones from Angelonia x angustifolia and Heterologous Expression as GST Fusion Protein in Escherichia coli

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.

Dihydroflavonol 4-Reductase Genes from Freesia hybrida Play Important and Partially Overlapping Roles in the Biosynthesis of Flavonoids

“…Actually, several other plant species, such as Petunia hybrida, Cymbidium hybrida, Angelonia angustifolia, Agapanthus praecox ssp. orientalis (Leighton), have also been found unable to produce pelargonidin derivatives because of the DFR substrate specificities (Mol et al, 1998; Johnson et al, 1999; Gosch et al, 2014; Mori et al, 2014). Therefore, it can be concluded that substrate specificities of FhDFR proteins played crucial role in determination of anthocyanin aglycons in F. hybrdia .…”

Rooting of apical cuttings of Angelonia integerrima Sprengel: concentrations of indole-3-butyric acid and substrates

“…The genus Angelonia originates in the Neotropical region, which covers the area from central Mexico to the southern tip of South America (BOFF et al, 2014). The emergence of different species is not yet fully understood (PLASCHIL and OLBRICHT, 2008;GOSCH et al, 2014), with report of approximately 50 species (TROPICOS, 2018), and differences related to the shape and color of the flower, ranging between blue, violet, white and pink (GOSCH et al, 2014). The genus can be found in all Brazilian biomes and in the most diverse types of vegetation, including Caatinga, Altitude Field, Clean Field, Rupestrian Field, Cerrado, Dryland Forest, Ombrophilous Forest and Restinga, being registered 18 species in Brazil (REFLORA, 2018).…”

In silico study for diversing the molecular pathway of pigment formation: an alternative to manual coloring in cotton fibers

“…To achieve this objective a comparison was made in retrieving sequences of two DFR genes from blue flower species and two from white including sequence alignment for homology and protein modeling. Further more the presence of proline rich region along with positions 12 and 26 was reported to be important in determining substrate specificity for DFR of A. angustifolia (Gosch et al, 2014 ). Other positions emphasizing the 26-amino acid region (132–157) were also mentioned in the literature which played a role in utilizing specific dihydroflovonols: DHK, DHQ, and DHM (Johnson et al, 2001 ; Xie et al, 2004 ).…”