Genome-Wide Identification and Functional Characterization of the BAHD Acyltransferase Gene Family in Brassica napus L

Abstract

The BAHD acyltransferase family plays a critical role in plant secondary metabolism by catalyzing acyl transfer reactions that are essential for synthesizing metabolites involved in environmental adaptation. However, systematic investigation of this superfamily in Brassica napus has not been reported. In this study, 158 BnaBAHD genes were identified by comprehensive analyses of evolutionary relationships, motif structures, chromosomal distribution, gene collinearity, and selection pressures, and these genes were phylogenetically classified into five clades harboring conserved catalytic domains (HXXXD and DFGWG). Transient overexpression combined with metabolomic profiling demonstrated that two homologous seed-specific Clade V members, BnaBAHD040 and BnaBAHD120, which exhibited elevated expression during late seed development, significantly enhanced the accumulation of acylated metabolites contributing to biotic/abiotic stress resistance. This study provides the first experimental validation of the catalytic functions of BAHD enzymes in B. napus, establishing a theoretical foundation for leveraging this gene family in genetic improvement to develop novel rapeseed cultivars with enhanced stress tolerance and yield.

Keywords: BAHD acyltransferase; Brassica napus L.; acylation; stress resistance.

Figure 1

Maximum-likelihood (ML) phylogenetic tree of the BAHD proteins in Arabidopsis and three Brassica species. The sequences were categorized into five primary clades, which were further subdivided into several subclades. Each clade and subclade were distinguished by specific colors.

Figure 2

Phylogenetic trees, conserved protein motifs, and gene structure pattern in B. napus. (A) Phylogenetic analysis of BnaBAHD proteins. The coloring of each clade is consistent with that presented in Figure 1. (B) Conserved motifs of BnaBAHD proteins. Distinct motifs were visually represented by boxes of different colors. Specifically, motif2 corresponds to the HXXXD domain, and motif3 represents the DFGWG domain. Comprehensive sequence details for each individual motif are provided in Table S2. (C) Gene structure of BAHD gene family. Exons (coding sequences, CDS) and introns are represented by green boxes and gray lines, respectively. Yellow boxes represent upstream or downstream untranslated regions (UTR).

Figure 3

Chromosome distribution and analysis of the BAHD family genes in B. napus.

Figure 4

Gene duplication and collinearity analysis of BAHD genes in B. napus and its parental species. (A) Identification of gene duplication events within the BAHD family of B. napus. (B) Collinearity analysis of BAHD family genes between B. napus and B. rapa. (C) Collinearity analysis of BAHD family genes between B. napus and B. oleracea. The outermost ring displays chromosomes carrying BAHD genes, with distinct colors representing chromosomes from different species. The middle layer, composed of heatmaps, line plots, and bar plots, illustrates the density distribution of genes along these chromosomes. Internally, dark red lines connect homologous gene pairs, highlighting their evolutionary relationships.

Figure 5

Cis-acting elements in the promoters of BnaBAHDs. (A) Spatial distribution of cis-elements (marked with different colored boxes) within the promoters. (B) Functional classification of cis-elements, with categories annotated on the right.

Figure 6

Expression profiles of BnaBAHDs under nitrogen treatment. The expression profiles of each BnaBAHD were normalized using Log₂ transformation (FPKM + 1). Color intensity and area size of tiles correspond to expression levels. L, leaves; R, roots; CK (control), normal nutrient solution with no nitrogen deficiency; LN, low-nitrogen treatment. FPKM, Fragments Per Kilobase of exon model per Million mapped fragments.

Figure 7

Expression profiles of BnaBAHDs under phytohormone treatments. The expression profiles of each BnaBAHD were normalized using Log₂ transformation (FPKM + 1). Color intensity and area size of tiles correspond to expression levels. CK (control), normal nutrient solution with no phytohormone treatments. GA3, gibberellins; 6BA, 6-benzyladenine; IAA, indole-3-acetic acid; ACC, 1-aminocyclopropanecarboxylic acid; ABA, abscisic acid. Time labels (1 h, 3 h, 6 h, 12 h, 24 h) indicate hours post-treatment.

Figure 8

Expression profiles of BnaBAHDs across different tissues and organs. The expression profiles of each BnaBAHD were normalized using Log₂ transformation (FPKM + 1). Ra, radicle; Hy, hypocotyl; Co, cotyledon; Ro, root; St, stem; LeY, Leaf Young; LeO, Leaf Old; Bu, bud; Pe, petal; Pi, pistil; Sta, stamen; At, anther; Cap, capillament; Se, seed; Em, embryo; SC, seed coat; SP, silique pericarp; 48 h, 48 h after seed germination; s_f, seedling stage under field cultivation condition; b, bud stage; f, full-bloom stage; un, unpollinated; i, initial flowering stage.

Figure 9

Functional characterization of BnaBAHD040 through transient expression in N. benthamiana. (A) qRT-PCR analysis of BnaBAHD040 expression levels. (B) Principal component analysis (PCA) of metabolome data from N. benthamiana leaves. The first two principal components accounted for 72.3% of total variance (PC1: 44.9%, PC2: 27.4%). (C) Volcano plot analysis of differentially accumulated metabolites. The red points represent upregulated metabolites and the blue points represent downregulated metabolites. Dashed lines indicate significance thresholds. (D) Relative abundance of seven significantly upregulated acylated metabolites. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (E) Chemical structures of seven significantly upregulated acylated metabolites. Ester (-COO-) and amide (-CONH-) bonds, the characteristic features of acylated metabolites, are highlighted in red.