Genome-wide Identification of WRKY transcription factor family members in sorghum (Sorghum bicolor (L.) moench)

Abstract

WRKY transcription factors regulate diverse biological processes in plants, including abiotic and biotic stress responses, and constitute one of the largest transcription factor families in higher plants. Although the past decade has seen significant progress towards identifying and functionally characterizing WRKY genes in diverse species, little is known about the WRKY family in sorghum (Sorghum bicolor (L.) moench). Here we report the comprehensive identification of 94 putative WRKY transcription factors (SbWRKYs). The SbWRKYs were divided into three groups (I, II, and III), with those in group II further classified into five subgroups (IIa-IIe), based on their conserved domains and zinc finger motif types. WRKYs from the model plant Arabidopsis (Arabidopsis thaliana) were used for the phylogenetic analysis of all SbWRKY genes. Motif analysis showed that all SbWRKYs contained either one or two WRKY domains and that SbWRKYs within the same group had similar motif compositions. SbWRKY genes were located on all 10 sorghum chromosomes, and some gene clusters and two tandem duplications were detected. SbWRKY gene structure analysis showed that they contained 0-7 introns, with most SbWRKY genes consisting of two introns and three exons. Gene ontology (GO) annotation functionally categorized SbWRKYs under cellular components, molecular functions and biological processes. A cis-element analysis showed that all SbWRKYs contain at least one stress response-related cis-element. We exploited publicly available microarray datasets to analyze the expression profiles of 78 SbWRKY genes at different growth stages and in different tissues. The induction of SbWRKYs by different abiotic stresses hinted at their potential involvement in stress responses. qRT-PCR analysis revealed different expression patterns for SbWRKYs during drought stress. Functionally characterized WRKY genes in Arabidopsis and other species will provide clues for the functional characterization of putative orthologs in sorghum. Thus, the present study delivers a solid foundation for future functional studies of SbWRKY genes and their roles in the response to critical stresses such as drought.

Fig 1. Distribution of 94 SbWRKY genes on sorghum chromosomes.

Chr01-Chr10 above the colored bars indicates chromosome (Chr) numbers. The physical location of each SbWRKY gene is shown, and the gene name is indicated on the right side of each bar as SbWRKY#. Red boxes indicate tandem duplications, and green lines denote gene clusters.

Fig 2. Phylogenetic tree of WRKY members in sorghum and Arabidopsis.

SbWRKY and AtWRKY protein sequences were aligned with ClustalW, and a phylogenetic tree was constructed with MEGA7.0 using the neighbour-joining method and 1,000 bootstrap replicates. The members were divided into groups I, II, and III, and group II was further divided into subgroups IIa, IIb, IIc, Ild, and Ile.

Fig 3. Exon–intron structures of SbWRKY genes.

We used the Gene Structure Display Server (GSDS) for gene structure analysis and constructed the phylogenetic tree using MEGA v7.0. In the gene diagrams, blue bars indicate upstream and downstream UTRs, yellow bars indicate coding sequences (CDS), and black lines indicate introns.

Fig 4. Motif analysis of SbWRKYs.

(a) The distribution of 20 conserved motifs identified by MEME in the different groups of SbWRKYs. (b) Each motif is indicated by a different color. (c) Sequence logos for motifs 1–20.

Fig 5. Gene ontology analysis of identified SbWRKYs.

The enrichment analysis shows the involvement of SbWRKY in biological processes, molecular functions, and cellular components.

Fig 6. Heatmaps of SbWRKY gene expression.

SbWRKY expression levels in different tissues (a) and at different growth stages (b). (c) Hierarchical clustering of SWRKY gene expression patterns under different environmental conditions, including drought, ABA, heat, salt and combination stress.

Fig 7. Relative expression of selected SbWRKY genes in response to drought stress.

Relative expression levels of five SbWRKY genes under drought stress. Genes expression was analyzed by RT-qPCR, with the 0 h sample used as untreated control (expression = 1). Error bars represent standard errors; data were calculated using the 2^–ΔΔCt method.