Genome-Wide Identification and Characterization of WRKY Transcription Factors and Their Expression Profile in Loropetalum chinense var. rubrum

Abstract

The WRKY gene family plays important roles in plant growth and development, as well as in the responses to biotic and abiotic stresses. Loropetalum chinense var. rubrum has high ornamental and medicinal value. However, few WRKY genes have been reported in this plant, and their functions remain unknown. To explore the roles that the WRKY genes play in L. chinense var. rubrum, we identified and characterized 79 LcWRKYs through BLAST homology analysis and renamed them (as LcWRKY1-79) based on their distribution on the chromosomes of L. chinense var. rubrum. In this way, according to their structural characteristics and phylogenetic analysis, they were divided into three groups containing 16 (Group I), 52 (Group II), and 11 (Group III) WRKYs, respectively. LcWRKYs in the same group have similar motifs and gene structures; for instance, Motifs 1, 2, 3, 4, and 10 constitute the WRKY domain and zinc-finger structure. The LcWRKY promoter region contains light response elements (ACE, G-box), stress response elements (TC-rich repeats), hormone response elements (TATC-box, TCA-element), and MYB binding sites (MBS, MBSI). Synteny analysis of LcWRKYs allowed us to establish orthologous relationships among the WRKY gene families of Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum L., Vitis vinifera L., Oryza sativa L., and Zea mays L.; furthermore, analysis of the transcriptomes of mature leaves and flowers from different cultivars demonstrated the cultivar-specific LcWRKY gene expression. The expression levels of certain LcWRKY genes also presented responsive changes from young to mature leaves, based on an analysis of the transcriptome in leaves at different developmental stages. White light treatment led to a significant decrease in the expression of LcWRKY6, 18, 24, 34, 36, 44, 48, 61, 62, and 77 and a significant increase in the expression of LcWRKY41, blue light treatment led to a significant decrease in the expression of LcWRKY18, 34, 50, and 77 and a significant increase in the expression of LcWRKY36 and 48. These results enable a better understanding of LcWRKYs, facilitating the further exploration of their genetic functions and the molecular breeding of L. chinense var. rubrum.

Keywords: Loropetalum chinense var. rubrum; WRKY; expression pattern; genome-wide analysis; light quality.

Figure 1

Protein domains of the 79 LcWRKY protein sequences. Different colored blocks represent different protein domains.

Figure 2

Chromosomal distribution of LcWRKY genes. The left scale determines the position of each LcWRKY gene on the chromosome.

Figure 3

Phylogenetic tree constructed based on the full-length sequences of 72 Arabidopsis WRKYs and 79 LcWRKYs. MEGA11 was used for multi-sequence alignment, and the neighbor-joining (NJ) method was used to construct the phylogenetic tree. To ensure that the structure was credible, the predicted tree was tested using bootstrapping with 1000 replicates. Blue dots and green lines represent Arabidopsis WRKY members, while red dots and yellow lines indicate L. chinense var. rubrum WRKY members. Different sub-groups are distinguished according to different colored lines.

Figure 4

Multiple sequence alignment results for the WRKY domains from LcWRKY members. Alignment was performed using DNAMAN. Different patches of color represent different degrees of similarity in sequences. The red lines represent the WRKY domain, and the red dots represent the zinc-finger domain. A red asterisk indicates special LcWRKYs.

Figure 5

Phylogenetic relationships, conserved motif locations, and gene structures of the LcWRKYs. (A) An ML tree was constructed using MEGA7 to present the protein sequences of all LcWRKY genes. Different colors represent different sub-groups. (B) Motif location distribution of the 79 LcWRKY proteins and (C) gene structure profile of LcWRKY genes.

Figure 6

The number of cis-acting elements in the upstream 2000-bp region with respect to each LcWRKY gene. (A) The number of cis-acting elements in each LcWRKY gene promoter sequence; (B) cis-acting element abbreviations; and (C) explanation of the functional prediction for the cis-acting elements.

Figure 7

Gene duplication events of LcWRKYs and synteny analysis of WRKYs between L. chinense var. rubrum and other plant species. (A) The inter-chromosomal relationships of LcWRKYs. The green lines and heat map represent the density of genes on chromosomes. The red box indicates tandem duplicated gene pairs. Duplicate WRKY gene pairs are linked by red lines, while the gray lines indicates synteny blocks in the L. chinense var. rubrum genome. (B) Collinear genes are shown with gray lines, while WRKY genes are marked with red lines.

Figure 8

Clustered heat map of LcWRKY gene expression patterns, with homogenized rows. The color is indicated from blue to white to red, with the expression ranging from low to high, respectively. (A) Expression profile of LcWRKYs in mature leaves of four different varieties; (B) ‘F’ refers to flower. Expression profile of LcWRKYs in flowers of four different varieties; (C) ‘Ⅰ’ represents young leaves, while ‘Ⅱ’ represents mature leaves.

Figure 9

Differential transcription of LcWRKY genes in L. chinense var. rubrum leaves under white and blue light treatments. Different colors represent different treatment times (day 0 vs. 5). Asterisks represent significant differences (*, p < 0.05; **, p < 0.01).