The large soybean (Glycine max) WRKY TF family expanded by segmental duplication events and subsequent divergent selection among subgroups

Abstract

Background: WRKY genes encode one of the most abundant groups of transcription factors in higher plants, and its members regulate important biological process such as growth, development, and responses to biotic and abiotic stresses. Although the soybean genome sequence has been published, functional studies on soybean genes still lag behind those of other species.

Results: We identified a total of 133 WRKY members in the soybean genome. According to structural features of their encoded proteins and to the phylogenetic tree, the soybean WRKY family could be classified into three groups (groups I, II, and III). A majority of WRKY genes (76.7%; 102 of 133) were segmentally duplicated and 13.5% (18 of 133) of the genes were tandemly duplicated. This pattern was not apparent in Arabidopsis or rice. The transcriptome atlas revealed notable differential expression in either transcript abundance or in expression patterns under normal growth conditions, which indicated wide functional divergence in this family. Furthermore, some critical amino acids were detected using DIVERGE v2.0 in specific comparisons, suggesting that these sites have contributed to functional divergence among groups or subgroups. In addition, site model and branch-site model analyses of positive Darwinian selection (PDS) showed that different selection regimes could have affected the evolution of these groups. Sites with high probabilities of having been under PDS were found in groups I, II c, II e, and III. Together, these results contribute to a detailed understanding of the molecular evolution of the WRKY gene family in soybean.

Conclusions: In this work, all the WRKY genes, which were generated mainly through segmental duplication, were identified in the soybean genome. Moreover, differential expression and functional divergence of the duplicated WRKY genes were two major features of this family throughout their evolutionary history. Positive selection analysis revealed that the different groups have different evolutionary rates. Together, these results contribute to a detailed understanding of the molecular evolution of the WRKY gene family in soybean.

Figure 1

Chromosome distribution of soybean (Glycine max) WRKY genes. The size of a chromosome is indicated by its relative length. Red outlined boxes represent segmentally duplicated genes. Tandem duplicated genes are indicated with vertical green lines. The location information and chromosome information were obtained from Phytozome. The figure was produced using the MapInspector program.

Figure 2

Alignment of multiple soybean WRKY genes and selectedAtWRKY domain amino acid sequences. Alignment was performed using Clustal W. The suffixes ‘N’ or ‘C’ denote the N-terminal and C-terminal WRKY domains from Group I WRKY proteins, respectively. The amino acids forming the zinc-finger motif are highlighted in yellow. The conserved WRKY amino acid signature is highlighted in blue. The four β-strands are shown in red. The position of a conserved intron is indicated by an arrowhead.

Figure 3

Phylogenetic tree of WRKY domains among soybean andArabidopsis. The amino acid sequences of the WRKY domain of soybean and Arabidopsis were aligned with Clustal W and the phylogenetic tree was constructed using the maximum likelihood method in MEGA 5.0. Group 1 proteins with the suffix ‘N’ or ‘C’ indicate the N-terminal WRKY domains or the C-terminal WRKY domains, respectively. Genes with similar functions clustered together are indicated by filled green circles. Gene expansion in soybean and Arabidopsis are indicated by coloring the subclade with the same color as the leaf label. The red arcs indicate different groups (or subgroups) of WRKY domains.

Figure 4

Expression profiles of 127 soybean WRKY genes. The hierarchical cluster color code: the largest values are displayed as the reddest (hot), the smallest values are displayed as the bluest (cool), and the intermediate values are a lighter color of either blue or red. Pearson correlation clustering was used to group the developmentally regulated genes. Six genes were excluded from the analysis due to no expression in an organ or a period.

Figure 5

Model building of the 3D structure of the soybean WRKY protein (Glyma13g00380) based on similarity to theAtWRKY4-C domain (Protein Data Bank (PDB) code: 2lexA). The ensemble of the selected structures in stereo view (A), (B), (C), and (D) positive selection sites detected by the branch-site model presented in group I, group II c, group II e, and group III, respectively. The sites with red color indicate amino acid residues under statistically significant (p < 0.05) positive selection, as calculated by Bayes Empirical Bayes estimation methods. The presented region is Asp247–Pro306, excluding the N-terminal region. The figure was produced using the Swiss-model and pyMOL programs.