Characterization of the Brassica napus Flavonol Synthase Gene Family Reveals Bifunctional Flavonol Synthases

Abstract

Flavonol synthase (FLS) is a key enzyme for the formation of flavonols, which are a subclass of the flavonoids. FLS catalyzes the conversion of dihydroflavonols to flavonols. The enzyme belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODD) superfamily. We characterized the FLS gene family of Brassica napus that covers 13 genes, based on the genome sequence of the B. napus cultivar Express 617. The goal was to unravel which BnaFLS genes are relevant for seed flavonol accumulation in the amphidiploid species B. napus. Two BnaFLS1 homeologs were identified and shown to encode bifunctional enzymes. Both exhibit FLS activity as well as flavanone 3-hydroxylase (F3H) activity, which was demonstrated in vivo and in planta. BnaFLS1-1 and -2 are capable of converting flavanones into dihydroflavonols and further into flavonols. Analysis of spatio-temporal transcription patterns revealed similar expression profiles of BnaFLS1 genes. Both are mainly expressed in reproductive organs and co-expressed with the genes encoding early steps of flavonoid biosynthesis. Our results provide novel insights into flavonol biosynthesis in B. napus and contribute information for breeding targets with the aim to modify the flavonol content in rapeseed.

Keywords: 2-oxoglutarate-dependent dioxygenases; bifunctionality; flavanone 3-hydroxylase; flavonoid biosynthesis; gene family; rapeseed; specialized metabolism.

FIGURE 1

Simplified scheme of flavonoid biosynthesis. The flavonol biosynthesis pathway (highlighted via an orange arrow) is part of the flavonoid biosynthesis, which also includes the anthocyanin pathway (highlighted via a violet arrow) (modified after Winkel-Shirley, 2001). The metabolic flux into the flavonol biosynthesis is influenced by dihydroflavonol 4-reductase (DFR) as it competes with FLS for substrates. Enzyme names are abbreviated as follows: chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), UDP-glycosyltransferases (UGTs), anthocyanidin synthase (ANS).

FIGURE 2

Phylogeny of BnaFLS candidates and previously described FLS sequences. Relative bootstrap-values are shown next to relevant nodes. The phylogenetic tree is based on amino acid sequences. FLS family members of B. napus Express 617 are marked with an asterisk. The outgroup comprises the 2-ODD members ANS and F3H, as well as 2-ODD-like sequences (Supplementary Figure 1).

FIGURE 3

Genomic structure of BnaFLS candidates expressed in seeds. The exon-intron structure of BnaFLS candidates is shown. The exons are split into coding sequences (CDS, black) and untranslated regions (UTR, gray) and are displayed by rectangles, introns are displayed as black connecting lines.

FIGURE 4

Multiple sequence alignment of BnaFLS candidates relevant for seed flavonol accumulation. Conserved amino acids and motifs important for FLS functionality were labeled as followed: the “PxxxIRxxxEQP,” “CPQ/RPxLAL,” and “SxxTxLVP” motifs are shown in orange, while residues involved in substrate-, ferrous iron-, and 2-oxoglutarate-binding are marked in green, red, and blue, respectively. Residues important for proper folding and/or highly conserved across 2-ODDs are labeled in violet. Residues relevant for F3H activity are marked with a black star. Black background indicates perfect conservation across all sequences. Secondary structure information is derived from an in silico model of AthFLS1 predicted by I-TASSER. acc = relative accessibility.

FIGURE 5

BnaFLS1-1 and BnaFLS1-2 are bifunctional enzymes exhibiting F3H and FLS activity. (A,B) Bioconversion assay results based on a HPTLC using extracts from E. coli expressing recombinant BnaFLS1-1 or BnaFLS1-2. The substrate of F3H naringenin, as well as the FLS substrate dihydrokaempferol and the product kaempferol were used as standards. AthFLS1 served as positive control and AthFLS5 as negative control. In the last sample no Nargingenin (NA) was supplemented. (C) HPTLC on silica gel-60 plates of methanolic extracts of stem of Col-0, Nö-0, ans/fls1 A. thaliana knock out mutant, and three independent T2 ans/fls1 A. thaliana knock out BnaFLS1-1 and BnaFLS1-2 complementation lines followed by DPBA staining, applied in this order. Pictures were taken under UV illumination. Kaempferol- and quercetin derivatives are green and orange respectively, while sinapate derivates are faint blue, dihydrokaempferol derivates are turquois, and chlorophylls appear red. The following flavonoid derivates are labeled: kaempferol-3-O-rhamnoside-7-O-rhamnoside (K-3R-7R), quercetin-3-O-rhamnoside-7-O-rhamnoside (Q-3R-7R), kaempferol-3-O-glucoside-7-O-rhamnoside (K-3G-7R), quercetin-3-O-glucoside-7-O-rhamnoside (Q-3G-7R), kaempferol-3-O-glucorhamnosid-7-O-rhamnoside (K-3[G-R]-7R), quercetin-3-O-glucorhamnosid-7-O-rhamnoside (Q-3[G-R]-7R), kaempferol-3-O-gentiobioside-7-O-rhamnoside (K-3[G-G]-7R), and quercetin-3-O-gentiobioside-7-O-rhamnoside (Q-3[G-G]-7R). (D,E) Flavonol staining in young seedlings of Col-0, Nö-0, ans/fls1 double and f3h single A. thaliana knock out mutant, as well as representative pictures of three independent T2 ans/fls1 A. thaliana knock out BnaFLS1-1 and BnaFLS1-2 complementation lines and three independent T3 f3h A. thaliana knock out BnaFLS1-1 and BnaFLS1-2 complementation lines. Flavonols in norflurazon-bleached seedlings were stained with DPBA until saturation and imaged by epifluorescence microscopy. Orange color indicates the accumulation of quercetin derivates. Photos of representative seedlings are shown.

FIGURE 6

BnaFLS3-3 and BnaFLS3-4 exhibit F3H activity. See Figure 5 for detailed figure description. (A) Bioconversion assay results of BnaFLS3-3 and (B) BnaFLS3-4. (C) The following flavonoid derivates were additionally labeled: dihydroquercetin-deoxyhexoside (DHQ-DH), dihydrokaempferol-hexoside (DHK-H), dihydroquercetin-hexoside (DHQ-H), quercetin-3-O-rhamnoside-7-O-glucoside (Q-3R-7G). (D,E) Flavonol staining in young seedlings.

FIGURE 7

3D secondary structure models of BnaFLS1s and BnaFLS3s. Homology models of (A) AthFLS1, (B) BnaFLS1-1, (C) BnaFLS1-2, (D) AthF3H, (E) BnaFLS3-3, and (F) BnaFLS3-4 modeled via I-TASSER are shown looking into the center of the jellyroll motif. Ferrous iron-coordinating residues are shown in red, 2-oxoglutarate binding residues are marked in cyan, and the corresponding position of G261 in AthFLS1 is shown in magenta. The N-terminus divergence between BnaFLS1s and BnaFLS3s is marked in yellow (corresponding to amino acids 1-42 in AthFLS1). Orange regions compromise regions postulated to be specific for FLS.

FIGURE 8

Functional activities of the B. napus flavonol synthase family. BnaFLS1-1 and BnaFLS1-2 marked in dark blue, are bifunctional enzyme exhibiting F3H and FLS activity. BnaFLS3-3 and BnaFLS3-4 labeled in light blue possess F3H activity.