Genome-Wide Analysis of the BAHD Family in Welsh Onion and CER2-LIKEs Involved in Wax Metabolism

Abstract

BAHD acyltransferases (BAHDs), especially those present in plant epidermal wax metabolism, are crucial for environmental adaptation. Epidermal waxes primarily comprise very-long-chain fatty acids (VLCFAs) and their derivatives, serving as significant components of aboveground plant organs. These waxes play an essential role in resisting biotic and abiotic stresses. In this study, we identified the BAHD family in Welsh onion (Allium fistulosum). Our analysis revealed the presence of AfBAHDs in all chromosomes, with a distinct concentration in Chr3. Furthermore, the cis-acting elements of AfBAHDs were associated with abiotic/biotic stress, hormones, and light. The motif of Welsh onion BAHDs indicated the presence of a specific BAHDs motif. We also established the phylogenetic relationships of AfBAHDs, identifying three homologous genes of CER2. Subsequently, we characterized the expression of AfCER2-LIKEs in a Welsh onion mutant deficient in wax and found that AfCER2-LIKE1 plays a critical role in leaf wax metabolism, while all AfCER2-LIKEs respond to abiotic stress. Our findings provide new insights into the BAHD family and lay a foundation for future studies on the regulation of wax metabolism in Welsh onion.

Keywords: BAHD acyltransferase; CER2-LIKEs; abiotic stress; epicuticular wax; genes family.

Figure 1

The Location of AfBAHDs in Welsh onion chromosomes. The distribution of the AfBAHDs gene on eight chromosomes. The number of chromosomes is displayed at the top of each chromosome. The AfBAHDs gene name is displayed on the left side of each chromosome. The scale of the genome size is given on the left.

Figure 2

Phylogenetic relationships of AfBAHD proteins. A phylogenetic tree was constructed in MEGA6.0 on the basis of the full-length amino-acid sequences of previously identified BAHD proteins, with the accession numbers of sequences used in the alignment provided in the methods. ClustalW was used with 10,000 bootstrap replicates, and the tree was built using the neighbor-joining (NJ) method. All BAHDs belong to seven different clades; different clade members are labeled different colors.

Figure 3

Gene structure and multiple motifs analysis of BAHD family in Welsh onion. (A) The evolutionary relationship analyses of BAHDs based on phylogenetic relationships. (B) The motif analyses of AfBAHDs. Conservation motifs of BAHDs were analyzed using the MEME online tool, by setting the upper limit at 10 for identifying conservation motifs. Each box represents a conserved motif, as shown in the legend on the right. (C) The exon–intron analyses of AfBAHDs. Exon and intron information was extracted from the genome annotation file. The green box represents noncoding regions, and the yellow box represents coding regions. Legends are shown on the right.

Figure 4

Analysis of cis-regulatory elements and expression of the AfBAHD genes. (A) The number of cis-regulatory elements of AfBAHDs. (B) The gene expression of AfBAHDs in Welsh onion leaves and scape. W1L, W2L, and W3L denote Welsh onion leaves. W1, W2, and W3 denote Welsh onion scape. (C) KEGG enrichment analysis of AfBAHDs. The analyses were performed using BMKCloud (www.biocloud.net, accessed on 3 May 2022).

Figure 5

Sequence analysis of CER2-LIKEs in Welsh onion. Using DNAMAN, a multiple sequence alignment of AfCER2-LIKEs was performed with the homologous genes of CER2 previously reported. The motif of HXXXD is marked by a red line.

Figure 6

The 3D models of AfCER-LIKEs constructed using the online tools of Phyre. (A) The 3D model of AfCER2-LIKE1. (B) The 3D model of AfCER2-LIKE2. (C) The 3D model of AfCER2-LIKE3.

Figure 7

Relative expression of AfCER2-LIKEs in different tissue. Analysis of AfCER2 expression in the roots, stems, leaves, flowers, and scape of Welsh onion using quantitative real-time PCR. (A) Wildtype Welsh onion “BG” and wax-deficient mutant Welsh onion “GLBG”. (B) Expression of AfCER2-LIKE1 in different tissues of Welsh onion. (C) Expression of AfCER2-LIKE2 in different tissues of Welsh onion. (D) Expression of AfCER2-LIKE3 in different tissues of Welsh onion. Each experiment was performed with three biological replicates.

Figure 8

The expression of AfCER2-LIKEs under different stress. (A) CER2-LIKEs of Welsh onion under heat stress. (B) CER2-LIKEs of Welsh onion under drought stress. (C) CER2-LIKEs of Welsh onion under salt stress. (D) CER2-LIKEs of Welsh onion under cold stress. Each experiment was performed with three biological replicates.