The Cotton WRKY Gene GhWRKY41 Positively Regulates Salt and Drought Stress Tolerance in Transgenic Nicotiana benthamiana

Abstract

WRKY transcription factors constitute a very large family of proteins in plants and participate in modulating plant biological processes, such as growth, development and stress responses. However, the exact roles of WRKY proteins are unclear, particularly in non-model plants. In this study, Gossypium hirsutum WRKY41 (GhWRKY41) was isolated and transformed into Nicotiana benthamiana. Our results showed that overexpression of GhWRKY41 enhanced the drought and salt stress tolerance of transgenic Nicotiana benthamiana. The transgenic plants exhibited lower malondialdehyde content and higher antioxidant enzyme activity, and the expression of antioxidant genes was upregulated in transgenic plants exposed to osmotic stress. A β-glucuronidase (GUS) staining assay showed that GhWRKY41 was highly expressed in the stomata when plants were exposed to osmotic stress, and plants overexpressing GhWRKY41 exhibited enhanced stomatal closure when they were exposed to osmotic stress. Taken together, our findings demonstrate that GhWRKY41 may enhance plant tolerance to stress by functioning as a positive regulator of stoma closure and by regulating reactive oxygen species (ROS) scavenging and the expression of antioxidant genes.

Fig 1. Characterization of GhWRKY41 as a transcriptional regulator.

(A) Schematic diagrams of the p35S::GhWRKY41-GFP fusion construct and the control p35S::GFP construct. (B) Transient expression of the p35S::GhWRKY41-GFP and p35S::GFP constructs in N. benthamiana. Green fluorescence was observed using a confocal microscope five days after transformation. (C) GhWRKY41 demonstrates transactivation activity. The full-length ORF of GhWRKY41 was subcloned into pGBKT7, and transformed yeast was selected on both SD-Trp and SD-Trp-His-Ade media. Positive transformants were further identified by spotting serial yeast dilutions (1/1, 1/10 and 1/100). The triangle indicates the dilutions from 1 to 100. (D) Sequences of three tandem W-boxes (TGAC) and mW-boxes. (E) Yeast one-hybrid assays. (1) pAbAi-Wbox + pGADT7. (2) pAbAi-Wbox + pGAD-GhWRKY41. (3) pAbAi-mWbox + pGADT7. (4) pAbAi-mWbox + pGAD-GhWRKY41.

Fig 2. Spatiotemporal expression patterns of ProGhWRKY41::GUS in transgenic Arabidopsis.

(A) Leaf, (B) stem, (C) bottom of bud, (D) root, (E) flower and (F) young silique flower. Bars are presented in the lower left corner.

Fig 3. Relative expression of GhWRKY41 in different tissues and in response to different stress factors.

Quantification of GhWRKY41 band intensity based on the expression of GhUbiquitin rRNA genes using Quantity One software. The roots, stems and cotyledons of 7-day-old cotton seedlings were used to assess the tissue-specific expression of GhWRKY41 (A). The seedlings were treated with 200 mM NaCl (B), with 15% PEG (C), at 4°C (D), at 37°C (E), with 2 mM salicylic acid (SA) (F), with 5 mM ethylene (ET) released from ethephon (G), with 10 μM abscisic acid (ABA) (H) or with 10 mM H₂O₂ (I).

Fig 4. Drought tolerance test comparing wild-type and GhWRKY41-overexpressing N. benthamiana plants.

(A, B) Seed germination assay. (C) Post-germination seedling development of the WT and OE lines on MS supplemented with different concentrations of mannitol. (D) Primary root lengths of the seedlings 20 days after germination in the presence of different concentrations of mannitol. (E) Photograph of representative 8-week-old WT and OE plants grown in soil under drought conditions for 7 days and then watered for 3 days to allow them to recover.

Fig 5. Drought tolerance of WT and GhWRKY41-overexpressing N. benthamiana plants in the vegetative stage.

(A) Expression levels of antioxidant enzyme genes. (B-D) Analysis of antioxidant enzyme activity. (E) Drought-induced MDA accumulation in the WT and OE lines. (F) Drought-induced H₂O₂ accumulation detected via DAB staining.

Fig 6. Salt tolerance of WT and GhWRKY41-overexpressing N. benthamiana plants.

(A, B) Seed germination assay. (C) Photograph of representative 8-week-old WT and OE plants watered with 200 mM NaCl for 1 month.

Fig 7. Salt tolerance of WT and GhWRKY41-overexpressing N. benthamiana plants in the vegetative stage.

(A) Leaf discs from WT and OE plants were incubated with NaCl at different concentrations (0, 600 or 1,200 mM) under greenhouse conditions. (B) Relative chlorophyll content was determined in the leaf discs of WT and OE plants following NaCl treatments. Disks floated in water were used as controls. The presented data are the means ± SE of three independent experiments (n = 6). (C-E) Analysis of stress-related enzyme activity. (F) Expression levels of stress-related genes. WT⁺ indicates the WT lines after NaCl treatment. OE1⁺ indicates the OE1 lines after NaCl treatment. (G) Salt-induced H₂O₂ accumulation detected via DAB staining.

Fig 8. GhWRKY41 regulates stomatal movement.

(A) Comparison of stomatal aperture in response to salt and drought. (B) Stomatal aperture data were calculated from 50 stomata from the leaves of three different plants. Values are the mean ± SD. (C) GUS staining of leaves from transgenic Arabidopsis exposed to salt and drought treatments.

Fig 9. GhWRKY41 regulates stomatal movement in an ABA-dependent manner.

(A, B) Comparison of stomatal aperture in response to ABA treatment. (C) Expression pattern of rbohA and rbohB genes in WT and OE plants following drought stress. Data were calculated from 50 stomata from the leaves of three different plants. Values are the mean ± SD.

Fig 10. The expression of genes associated with ABA signaling in WT and OE plants following drought or salt stress.