Mungbean DIRIGENT Gene Subfamilies and Their Expression Profiles Under Salt and Drought Stresses

Abstract

DIRIGENT (DIR) genes are key players in environmental stress responses that have been identified in many vascular plant species. However, few studies have examined the VrDIR genes in mungbean. In this study, we characterized 37 VrDIR genes in mungbean using a genome-wide identification method. VrDIRs were distributed on seven of the 11 mungbean chromosomes, and chromosome three contained the most VrDIR genes, with seven members. Thirty-two of the 37 VrDIRs contained a typical DIR gene structure, with one exon; the conserved DIR domain (i.e., Pfam domain) occupied most of the protein in 33 of the 37 VrDIRs. The gene structures of VrDIR genes were analyzed, and a total of 19 distinct motifs were detected. VrDIR genes were classified into five groups based on their phylogenetic relationships, and 13 duplicated gene pairs were identified. In addition, a total of 92 cis-acting elements were detected in all 37 VrDIR promoter regions, and VrDIR genes contained different numbers and types of cis-acting elements. As a result, VrDIR genes showed distinct expression patterns in different tissues and in response to salt and drought stress.

Keywords: VrDIR; drought stress; gene expression; mungbean; salt stress.

FIGURE 1

Analysis of the evolutionary relationships of DIR proteins from mungbean and other species. The amino acid sequences of DIR proteins from mungbean, Arabidopsis, and other species reported by Corbin et al. (2018) were used to conduct the phylogenetic analysis in MEGA 7.0 using the neighbor-joining method. DIR proteins were classified into six groups based on their phylogenetic relationships, DIR-a to DIR-f, which are indicated by different colors in the phylogenetic tree.

FIGURE 2

Phylogenetic and gene structure analyses of VrDIR genes. (A) Phylogenetic analysis of VrDIR proteins. The phylogenetic tree was constructed in MEGA 7.0 using the neighbor-joining method. (B) Exon-intron organization of VrDIR genes. The light blue, pink, and black boxes indicate UTRs, exons, and introns, respectively. The genomic lengths of VrDIR genes are indicated.

FIGURE 3

Conserved domain and motif analyses in VrDIR proteins. (A) The positions and lengths of the conserved DIR domains in VrDIR proteins. The light blue boxes indicate the conserved DIR domains. (B) The conserved motifs in VrDIR proteins. The 19 motifs are indicated using different colored boxes. The length of each DIR protein is indicated.

FIGURE 4

Duplication analysis of VrDIR genes. The positions of VrDIR genes in each chromosome are presented, and the duplicated gene pairs are connected using red lines. The length of each chromosome is indicated, and CHR indicates chromosome.

FIGURE 5

Cis-acting element analysis in VrDIR promoter regions. The cis-acting elements were classified into six groups based on their predicted functions as described by Jin et al. (2019). Different cis-acting elements were presented using different colored boxes, and the Y-axis indicates the number of cis-acting elements in VrDIR promoters.

FIGURE 6

Relative expression levels of VrDIR genes in different tissues. Eight tissues, including flowers, pods, leaves, seeds, nodule roots, stems, roots, and shoot apices, were used for analysis. The expression level of VrDIR37 in nodule roots was set as one, and the others were adjusted accordingly. The gene expression results were visualized using a heatmap generated with Multiple Experiment Viewer 4.9.0 (Saeed et al., 2003). The expression levels from 0 to one are indicated by different colors.

FIGURE 7

Expression levels of VrDIR genes in response to drought stress. The expression of VrDIR genes in shoots and roots grown under normal and drought conditions was analyzed using qRT-PCR. Each sample was analyzed using three biological replicates and normalized to an Actin-expressing gene in mungbean.

FIGURE 8

Expression levels of VrDIR genes in response to salt stress. The expression of VrDIR genes in shoots and roots grown under normal and salt conditions was analyzed using qRT-PCR. Each sample was analyzed using three biological replicates and normalized to an Actin-expressing gene in mungbean.