GhWRKY6 Acts as a Negative Regulator in Both Transgenic Arabidopsis and Cotton During Drought and Salt Stress

Abstract

Drought and high salinity are key limiting factors for cotton production. Therefore, research is increasingly focused on the underlying stress response mechanisms of cotton. We first identified and cloned a novel gene encoding the 525 amino acids in cotton, namely GhWRKY6. qRT-PCR analysis indicated that GhWRKY6 was induced by NaCl, PEG 6000 and ABA. Analyses of germination rate and root length indicated that overexpression of GhWRKY6 in Arabidopsis resulted in hypersensitivity to ABA, NaCl, and PEG 6000. In contrast, the loss-of-function mutant wrky6 was insensitive and had slightly longer roots than the wild-type did under these treatment conditions. Furthermore, GhWRKY6 overexpression in Arabidopsis modulated salt- and drought-sensitive phenotypes and stomatal aperture by regulating ABA signaling pathways, and reduced plant tolerance to abiotic stress through reactive oxygen species (ROS) enrichment, reduced proline content, and increased electrolytes and malondialdehyde (MDA). The expression levels of a series of ABA-, salt- and drought-related marker genes were altered in overexpression seedlings. Virus-induced gene silencing (VIGS) technology revealed that down-regulation of GhWRKY6 increased salt tolerance in cotton. These results demonstrate that GhWRKY6 is a negative regulator of plant responses to abiotic stress via the ABA signaling pathway.

Keywords: ABA signaling; Gossypium hirsutum; drought; negative regulation; salt.

Figure 1

Identification of GhWRKY6 as a transcriptional regulator and its response to abiotic stress. (A) Genomic structure of GhWRKY6. (B) Conserved WRKY domain in GhWRKY6. (C) Sequence of the triple tandem repeats of the W-box or mW-box binding elements. (D) W-box or mW-box as bait in the yeast one-hybrid system. Yeast cells carrying pGADT7 or pGADT7-GhWRKY6 were grown on SD/-Leu/-Ura or SD/-Leu/-Ura supplemented with 500 ng/mL AbA. (E–G) Expression patterns of GhWRKY6 in cotton under different abiotic stress conditions. Three-week-old cotton seedlings were subjected to treatment with 100 μM ABA, 400 mM NaCl or 15% PEG 6000. Independent t-tests indicated that there were significant differences at ^∗p < 0.05, ^∗∗p < 0.01.

Figure 2

ABA-sensitivity in GhWRKY6-overexpressing transgenic Arabidopsis lines. (A) Expression of GhWRKY6 in OE lines and wild-type Arabidopsis seedlings during seedling development was analyzed by RT-PCR. The actin2 gene was used as an internal control. (B) Phenotypic comparison of seedlings grown for 10 days on MS medium or MS supplemented with 1 and 1.5 μM ABA. (C) Seeds were grown on MS or MS medium containing 1 or 1.5 μM ABA. The rates of seed germination were calculated from daily recordings over 10 days of cultivation. (D,E) Primary root length was measured in seedlings grown with or without ABA treatment. Seedlings were grown on MS or MS with 0.5 or 1 μM ABA for 10 days, and then photographs were taken so that the length of the roots could be measured. Data are shown as the mean ± SE (n = 3). Independent t-tests indicated that there were significant differences in both seed germination and root elongation among the wild-type, OE lines and wrky6 mutant under ABA treatment. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 3

Analysis of germination and root elongation in GhWRKY6-overexpressing lines, wild-type and wrky6 mutant under mannitol and salt stress conditions. (A) Phenotypic comparison of seedlings grown on MS medium or MS with 100 mM and 150 mM NaCl, or 200 mM and 300 mM mannitol after 10 days. (B) Germination assay of seedlings grown in the conditions described in (A). (C) Root elongation of wild-type, OE lines and wrky6 mutant seedlings after treatment with mannitol and NaCl. (D) Statistical analysis of root lengths of seedlings grown in treatment conditions described in (C). Data in (B,D) represent the means ± SE from three independent experiments An independent t-test indicated that there were significant differences in both seed germination and root elongation among the wild-type, OE lines and wrky6 mutant under NaCl and mannitol treatments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 4

Transgenic Arabidopsis expressing GhWRKY6 display increased ROS levels and open stomata under abiotic stress conditions. (A) Sensitivity of wild-type, OE lines and wrky6 mutant during vegetative growth after 20 days of treatment with 200 mM NaCl. (B) Survival rate of transgenic and wild-type Arabidopsis grown under saline conditions. (C) Proline content, MDA content and electrolyte leakage in wild-type, OE lines and wrky6 mutant lines during salt stress. (D) DAB staining of H₂O₂ in Arabidopsis. (E) Sensitivity of wild-type, OE lines and wrky6 mutant seedlings to drought stress. Drought stress was imposed by not watering the plants for 20 days, after which watering resumed for 1 week. (F) Survival rate of Arabidopsis lines after water stress treatment and rehydration. (G) Stomatal movement in response to drought treatment in transgenic and wild-type Arabidopsis. CK: leaves were placed in PBS until stomata were completely open; PEG 6000: leaves were transferred to 8% PEG 6000 solution for 2.5 h. (H) Measurement of stomatal length: width ratio in plants from panel (G). The data represent the means of 25 stomata pooled from three independent experiments. The different letters above the graph bars in (H) indicate a significant difference (p < 0.05). The data in (B,C,F) represent the means ± SE from three independent experiments. Independent t-tests indicated that there were significant differences among the wild-type, OE lines and wrky6 mutant seedlings at ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 5

qRT-PCR analysis of expression of stress-related genes in transgenic and wild-type Arabidopsis. The 2-week-old wild-type and OE lines seedlings were grown on MS medium and then subjected to 200 mM NaCl treatment condition for 4 h. The seedlings were harvested for qRT-PCR. The values are from three independent experiments. Independent t-tests indicated that there were significant differences in the expression of genes among the wild-type and OE lines seedlings at ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 6

GhWRKY6-VIGS cotton plants show enhanced drought sensitivity. (A) RT-PCR analysis confirming the efficiency of GhWRKY6 silencing. (B) TRV::GhWRKY6 plants had more enhanced salt tolerance than TRV::00 control seedlings did. (C) DAB staining visualized the accumulation of H₂O₂ in cotton plants. (D) Leaves harvested from TRV::GhWRKY6 exhibited less water loss than those from TRV::00 seedlings. (E) Salt damage index of TRV::00 and TRV::GhWRKY6 transgenic lines under saline condition. The data represent the means ± SE from three independent experiments. Independent t-tests indicated that there were significant difference in the water loss between the TRV::GhWRKY6 and TRV::00. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 7

mRNA-Seq analysis for TRV::00 and TRV::GhWRKY6 seedlings. (A) Identification of differentially expressed genes (DEGs) was conducted by adjusted q-values (FDR) ≤ 0.005 and the absolute value of fold change ≥ 2. (B) All of the DEGs were mapped to GO terms using the agriGO database 2.0. (C) Top 20 pathway enrichments in downregulated genes. (D) Top 20 pathway enrichments in upregulated genes. (E) Scanning for W-boxes in the 2,000-bp region of the promoters of identified DEGs. (F) Possible gene families involved in abiotic stress responses.