Genome-Wide Identification and Expression Analysis of bHLH-MYC Family Genes from Mustard That May Be Important in Trichome Formation

Abstract

The trichomes of mustard leaves have significance due to their ability to combat unfavorable external conditions and enhance disease resistance. It was demonstrated that the MYB-bHLH-WD40 (MBW) ternary complex consists of MYB, basic Helix-Loop-Helix (bHLH), and WD40-repeat (WD40) family proteins and plays a key role in regulating trichome formation and density. The bHLH gene family, particularly the Myelocytomatosis (MYC) proteins that possess the structural bHLH domain (termed bHLH-MYC), are crucial to the formation and development of leaf trichomes in plants. bHLH constitutes one of the largest families of transcription factors in eukaryotes, of which MYC is a subfamily member. However, studies on bHLH-MYC transcription factors in mustard have yet to be reported. In this study, a total of 45 bHLH-MYC transcription factors were identified within the Brassica juncea genome, and a comprehensive series of bioinformatic analyses were conducted on their structures and properties: an examination of protein physicochemical properties, an exploration of conserved structural domains, an assessment of chromosomal positional distributions, an analysis of the conserved motifs, an evaluation of the gene structures, microsynteny analyses, three-dimensional structure prediction, and an analysis of sequence signatures. Finally, transcriptome analyses and a subcellular localization examination were performed. The results revealed that these transcription factors were unevenly distributed across 18 chromosomes, showing relatively consistent conserved motifs and gene structures and high homology. The final results of the transcriptome analysis and gene annotation showed a high degree of variability in the expression of bHLH-MYC transcription factors. Five genes that may be associated with trichome development (BjuVA09G22490, BjuVA09G13750, BjuVB04G14560, BjuVA05G24810, and BjuVA06G44820) were identified. The subcellular localization results indicated that the transcription and translation products of these five genes were expressed in the same organelle: the nucleus. This finding provides a basis for elucidating the roles of bHLH-MYC family members in plant growth and development, and the molecular mechanisms underlying trichome development in mustard leaves.

Keywords: MBW; MYC; bHLH; leaf trichome; mustard.

Figure 1

The phylogenetic trees of Arabidopsis thaliana, Chinese cabbage, and mustard were constructed using the maximum likelihood method for the binding domains of the bHLH transcription factors. The proteins clustered into 16 distinct groups designated by a group number, B1 to B16, and labeled with different colored branches. Multiple sequence comparisons were performed in accordance with MUSCLE3.8.31 software with the use of default parameters. Phylogenetic trees were constructed using MEGA7, and the results are based on 1000 bootstrap values.

Figure 2

An analysis of the conserved structural domains within the mustard bHLH gene family was conducted. The structural domain information was sourced from NCBI-CDD. Following selection, the final dataset comprised six structural domains, which exhibited comparable amino acid lengths. Among these domains, bHLH-MYC occupied a similar position within the protein sequence.

Figure 3

The chromosomal distribution of 45 bHLH-MYC genes in mustard. Chromosome numbers are indicated in green font. Notably, AA_Chr02, AA_Chr08, and BB_Chr07 do not harbor any of these genes, while 15 chromosomes are depicted in the figure as carrying them. The bHLH-MYC family members are denoted in red font. Specifically, AA_Chr09 contains 7 members, AA_Chr07 and AA_Chr10 each have 1 member, and the remaining members are more evenly distributed across the other chromosomes.

Figure 4

Conservation analysis of 45 bHLH-MYC family genes, including cluster analysis, amino acid sequence motif analysis, and exon and intron evaluation. Motif number is defined as 10, and other parameters are specified as default.

Figure 5

The collinearity analysis of bHLH-MYC family genes between B. rapa, B. juncea, and A. thaliana. A01–A10 represent 10 chromosomes in B. rapa, A01–B08 represent 18 chromosomes in B. rapa, and Chr1 to Chr5 represent 5 chromosomes in A. thaliana.

Figure 6

The collinearity analysis of mustard bHLH-MYC genes. The red line indicates the linear correlation of genes in the mustard bHLH-MYC family. More red lines for the same gene show highly conserved homology.

Figure 7

The expression levels of the 45 bHLH-MYC family members. Material denoted as w possesses trichome, whereas material denoted as y is glabrous. Significant differences in the expression of these family members were observed between the two types of material. Following selection, BjuVB04G14560, BjuVA05G24810, BjuVA06G44820, BjuVA09G22490, and BjuVA09G13750 were identified as being potentially associated with trichome development.

Figure 8

Three-dimensional (3D) structure analyses of the bHLH-MYC protein ((A–E) depict the protein structures of mustard bHLH-MYC, corresponding to the genes BjuVA09G22490, BjuVA09G13750, BjuVB04G14560, BjuVA05G24810 and BjuVA06G44820).

Figure 9

The sequence logo analyses of the bHLH-MYC of mustard. In the sequence logo analysis images, the height of the letter indicates how often that amino acid occurs at that position, implying that the type of amino acid at that position is relatively fixed in the set of sequences being analyzed and may be important for the function or structure of the sequence.

Figure 10

Potential functions of mustard bHLH-MYC. Subcellular localization of five mustard bHLH-MYCs that were transiently expressed in N. benthamiana leaves using eGFP as a control. Bright field indicates bright field microscopy image, eGFP indicates green fluorescence image, and merge indicates merged bright field and green fluorescence image.