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.

Genome-Wide Identification and Expression Profiles of 13 Key Structural Gene Families Involved in the Biosynthesis of Rice Flavonoid Scaffolds

“…In addition, the substrate binding motifs (F142, F144, K213, F304, E306) were only found in OsFLS1 , which was consistent with recent findings [ 44 ]. Furthermore, the two FLS-specific motifs were also found in the three OsFLSs , except OsFLS2 only included one motif due to the truncation of the C-end [ 113 ]. Interestingly, 12 residues relevant for F3H activity existed in almost all rice F3H , FLS , LDOX and ANS genes ( Supplementary Figure S5 ) [ 114 ], indicating that the F3H gene may be the ancestor of FLS , LDOX and ANS genes [ 57 ].…”

Mapping-by-Sequencing Reveals Genomic Regions Associated with Seed Quality Parameters in Brassica napus

“…Seeds and leaves (28 days after flowering (DAF)) RNA-Seq samples of Janetzkis Schlesischer (P2) and seeds and leaves (23 and 35 DAF) RNA-Seq samples of B. napus SGDH14 (medium–high seed GSL content) [ 63 ] were prepared and sequenced according to Schilbert et al 2021 [ 64 ]. Additionally, RNA-Seq reads derived from seeds and leaves of B. napus Express 617 [ 64 ] and public RNA-Seq data sets ( File S23 ) were mapped to the B. napus Darmor-bzh v4.1 and Zheyou7 assemblies using STAR v.2.7.1a [ 65 ]. STAR was run in basic mode allowing maximal 5% mismatches and requiring an alignment spanning at least 90% of the read length.…”

Analysis of flavonol regulator evolution in the Brassicaceae reveals MYB12, MYB111 and MYB21 duplications associated with MYB11 and MYB24 gene loss

“…Moreover, the expression pattern of SG7 members in B. napus differs from the ones described for A. thaliana seedlings: BnaMYB12 are predominantly expressed in reproductive tissues and BnaMYB111 in anthers and buds. One of the main target genes of the SG7 members, flavonol synthase ( FLS ), is also mainly expressed in reproductive tissues in B. napus [58] indicating the relevance of the transcriptional activation of flavonol accumulation in reproductive tissues. Reduced flavonol levels were linked with decreased pollen viability and germination, as e.g.…”

“…read counts and transcripts per millions (TPMs), was calculated via kallisto v. 0.44 [78] using default parameters and the transcript file of the B. napus cultivar Express 617 [79]. The heatmap was constructed with a customized python script calculating mean TPMs per tissue using 276 paired-end RNA-Seq data sets from B. napus as previously described [58]. Conditionindependent co-expression analysis was performed as described before [58] to identify co-expressed genes using Spearman’s correlation coefficient by incorporating 696 B. napus RNA-Seq data sets.…”