Identification, Expression, and Functional Analysis of the Group IId WRKY Subfamily in Upland Cotton (Gossypium hirsutum L.)

Abstract

WRKY transcription factors have diverse functions in regulating stress response, leaf senescence, and plant growth and development. However, knowledge of the group IId WRKY subfamily in cotton is largely absent. This study identified 34 group IId WRKY genes in the Gossypium hirsutum genome, and their genomic loci were investigated. Members clustered together in the phylogenetic tree had similar motif compositions and gene structural features, revealing similarity and conservation within group IId WRKY genes. During the evolutionary process, 14 duplicated genes appeared to undergo purification selection. Public RNA-seq data were used to examine the expression patterns of group IId WRKY genes in various tissues and under drought and salt stress conditions. Ten highly expressed genes were identified, and the ten candidate genes revealed distinct expression patterns under drought and salt treatments by qRT-PCR analysis. Among them, Gh_A11G1801 was used for functional characterization. GUS activity was differentially induced by various stresses in Gh_A11G1801p::GUS transgenic Arabidopsis plants. The virus-induced gene silencing (VIGS) of Gh_A11G1801 resulted in drought sensitivity in cotton plants, which was accompanied by elevated malondialdehyde (MDA) content and reduced catalase (CAT) content. Taken together, these findings obtained in this study provide valuable resources for further studying group IId WRKY genes in cotton. Our results also enrich the gene resources for the genetic improvements of cotton varieties that are suitable for growth in stressful conditions.

Keywords: Gh_A11G1801; expression patterns; group IId WRKY genes; stress response; upland cotton.

Figure 1

Chromosomal locations of group IId WRKY genes in the Gossypium hirsutum genome.

Figure 2

Phylogenetic tree and motif composition of group IId WRKY members from different species. (A) Phylogenetic tree of group IId WRKY members from different species. A phylogenetic tree was constructed by using MEGA 7 software with the neighbor-joining method. (B) Motif composition of group IId WRKY members from different species. The motifs were predicted by MEME software.

Figure 3

Phylogenetic tree and exon-intron structure analysis of group IId WRKY genes in cotton. (A) Phylogenetic analysis of 34 group IId WRKY genes. A phylogenetic tree was constructed by using MEGA 7 software with the neighbor-joining method. (B) Exon-intron structure of 34 group IId WRKY genes. The exon-intron structure was generated on GSDS software online. Red indicates exons, and black indicates introns.

Figure 4

Expression patterns of group IId WRKY genes in different tissues from RNA-seq data. The scale represents the FPKM values. The tissues were from root, stem, leaf, calycle, torus, petal, stamen, pistil, fiber at 10 days post-anthesis and ovule at 10 days post-anthesis.

Figure 5

Expression patterns of group IId WRKY genes under drought and salt stresses from RNA-seq data. (A) The expression patterns of group IId WRKY genes under drought stress. (B) The expression patterns of group IId WRKY genes under salt stress. The scale represents the FPKM values.

Figure 6

The relative transcript abundances of ten candidate group IId WRKY genes under drought and salt treatments were examined by qRT-PCR. (A) qRT-PCR analysis of ten candidate group IId WRKY genes under drought treatment. (B) qRT-PCR analysis of ten candidate group IId WRKY genes under salt treatment. Ten-day-old cotton seedlings were subjected to either 15% PEG6000 or 200 mM NaCl. The cotyledon samples were collected at 0, 2, 4, 6, 8, and 12 h. Three biological repeats and three technical repetitions were performed. GhActin was used as an internal reference. The data are presented as the means ± standard errors (SEs) from three biological replicates.

Figure 7

GUS activity analysis of Gh_A11G1801p::GUS transgenic Arabidopsis under various stresses. GUS staining of Gh_A11G1801p::GUS transgenic Arabidopsis under (A) CK, (B) 200 mM D-mannitol, (C) 150 mM NaCl, (D) 100 μM ABA, (E) 500 μM SA, and (F) 100 μM JA treatments. The 10-day-old seedlings grown on 1/2 MS solid medium were transferred to 1/2 MS liquid medium supplemented with 200 mM D-mannitol, 150 mM NaCl, 100 μM ABA, 500 μM SA, or 100 μM JA. After 3 h of treatment, the seedling samples were used for GUS staining. Arabidopsis seedlings that had been placed in 1/2 MS liquid medium without any stress treatment were used as controls. (G) The transcript levels of the GUS gene in Gh_A11G1801p::GUS transgenic Arabidopsis under various treatments. AtActin2 was used as the reference control. The data are presented as the means ± SEs from three biological replicates. ** and * indicate statistical significance at the 0.01 and 0.05 probability levels, respectively.

Figure 8

Silencing Gh_A11G1801 via VIGS increased sensitivity to drought stress in cotton. (A) Plant phenotypes of positive control plants. (B) Expression level of Gh_A11G1801 in empty control and VIGS plants. (C) Phenotype of empty control and VIGS plants under 15% PEG6000 treatment. After three weeks of injection, the VIGS plants were treated with 15% PEG6000 for 8 days. (D) Phenotype of empty control and VIGS plants under water deficiency. After three weeks of injection, the VIGS plants were treated with water shortage for 7 days. (E) The MDA content of empty control and VIGS plants under normal growth and water-withholding conditions. (F) The CAT content of empty control and VIGS plants under normal growth and water-withholding conditions. CK-pYL156 and CK-VIGS indicate plants under normal growth conditions. Drought-pYL156 and Drought-VIGS indicate plants under water-withholding conditions. GhActin was used as the reference control. The bars represent the means ± SEs from three independent experiments. ** and * indicate statistical significance at the 0.01 and 0.05 probability levels, respectively.