GhDof1.7, a Dof Transcription Factor, Plays Positive Regulatory Role under Salinity Stress in Upland Cotton

Abstract

Salt stress is a major abiotic stressor that can severely limit plant growth, distribution, and crop yield. DNA-binding with one finger (Dof) is a plant-specific transcription factor that plays a crucial role in plant growth, development, and stress response. In this study, the function of a Dof transcription factor, GhDof1.7, was investigated in upland cotton. The GhDof1.7 gene has a coding sequence length of 759 base pairs, encoding 252 amino acids, and is mainly expressed in roots, stems, leaves, and inflorescences. Salt and abscisic acid (ABA) treatments significantly induced the expression of GhDof1.7. The presence of GhDof1.7 in Arabidopsis may have resulted in potential improvements in salt tolerance, as suggested by a decrease in H₂O₂ content and an increase in catalase (CAT) and superoxide dismutase (SOD) activities. The GhDof1.7 protein was found to interact with GhCAR4 (C2-domain ABA-related 4), and the silencing of either GhDof1.7 or GhCAR4 resulted in reduced salt tolerance in cotton plants. These findings demonstrate that GhDof1.7 plays a crucial role in improving the salt tolerance of upland cotton and provide insight into the regulation of abiotic stress response by Dof transcription factors.

Keywords: ABA; DNA-binding with one finger; H2O2; salt stress; upland cotton.

Figure 1

Gene structure, protein sequence, and phylogenetic analysis of GhDof1.7. (A) Gene structure of GhDof1.7. (B) Phylogenetic relationship between Arabidopsis Dof proteins and GhDof1.7 protein. (C) Protein sequence alignment of the GhDof1.7 protein with closely related proteins. The abbreviations before the gene names are as follows: Pt, Populus tomentosa; Gm, Glycine max; Rs, Raphanus sativus; Dz, Durio zibethinus; Hs, Hibiscus syriacus; At, Arabidopsis thaliana; Th, Theobroma cacao.

Figure 2

Subcellular localization assays showing that 35S::GhDof1.7-GFP fusion protein is located in the nucleus. Scale bars, 20 μm. The experiments were replicated at least three times.

Figure 3

Expression of GhDof1.7 gene in various tissues of upland cotton. (A) Relative expression levels of GhDof1.7 gene in different tissues. Data are means ± SD (n = 3). Bars followed by different letters indicate significant difference at 5%. (B) GUS activity in different tissues of Arabidopsis thaliana overexpressing the GhDof1.7 promoter fragment. Error bars show the standard deviation of three biological repeats. The experiments were replicated at least three times.

Figure 4

The expression of the GhDof1.7 gene was regulated by external stress. (A) Expression pattern of GhDof1.7 under ABA and GA stress. Data are means ± SD (n = 3). Bars followed by different letters indicate significant difference at 5%. (B) Expression patterns of GhDof1.7 genes under PEG, NaCl, and cold stress. Data are means ± SD (n = 3). Bars followed by different letters indicate significant difference at 5%. (C) GUS activity of Arabidopsis overexpressing GhDof1.7 promoter fragment under ABA stress conditions. The experiments were replicated at least three times.

Figure 5

Salt tolerance of transgenic Arabidopsis at germination stage. (A) Transcript levels of the GhDof1.7 gene in wild-type and transgenic plants; (B) germination of wild-type and transgenic plants in 1/2 MS medium and in salt-containing medium; (C) germination rates of wild-type and transgenic plants in 1/2 MS medium and in salt-containing medium. Error bars indicate SD (** p < 0.01, n = 3).

Figure 6

Salt tolerance of transgenic Arabidopsis at seedling stage. (A) Phenotypes after salt treatment in wild-type and overexpressing GhDof1.7 Arabidopsis; (B) detection of H₂O₂ content in wild-type and transgenic Arabidopsis before and after salt treatment; (C,D) detection of SOD and CAT activity in wild-type and transgenic Arabidopsis before and after salt treatment; (E,F) expression analysis of ABA synthesis and signaling-related genes in wild-type and GhDof1.7 transgenic Arabidopsis. Error bars indicate SD (** p < 0.01, 0.01 < * p < 0.05, n = 3).

Figure 7

Auto-activation detection of GhDof1.7 in yeast cells and its interaction with candidate proteins. (A) Self-activation test result; (B) yeast two-hybrid point-to-point validation results; positive control group: pGADT7-largeT + pGBKT7-p53; negative control group: pGADT7-largeT + pGBKT7-laminC; (C) BiFC validation mutual interaction results in tobacco.

Figure 8

Phenotypic identification and analysis of VIGS cotton. (A) Positive control phenotype and phenotypes of plants silenced for GhCAR4 and GhDof1.7 under salt stress; (B) the expression levels of GhCAR4 and GhDof1.7 in pYL156 and VIGS plants; (C,D) H₂O₂ content, SOD activity, and CAT activity in pYL156 and VIGS plants after salt treatment. Error bars indicate SD (** p < 0.01, n = 3).