Overexpression of CDSP32 (GhTRX134) Cotton Gene Enhances Drought, Salt, and Oxidative Stress Tolerance in Arabidopsis

Abstract

Upland cotton (Gossypium hirsutum L.) is the main natural fiber crop worldwide and is an essential source of seed oil and biofuel products. Many abiotic stresses, such as drought and salinity, constrain cotton production. Thioredoxins (TRXs) are a group of small ubiquitous proteins that are widely distributed among organisms. TRXs play a crucial role in regulating diverse functions during plant growth and development. In the present study, a novel GhTRX134 gene was characterized and overexpressed in Arabidopsis and silenced in cotton under drought stress. Furthermore, the proline content and enzyme activity levels were measured in transgenic plants and wild-type (Wt) plants under drought and salt stress. The results revealed that the overexpression of GhTRX134 enhanced abiotic stress tolerance. When GhTRX134 was silenced, cotton plants become more sensitive to drought. Taken together, these findings confirmed that the overexpression of GhTRX134 improved drought and salt tolerance in Arabidopsis plants. Therefore, the GhTRX134 gene can be transformed into cotton plants to obtain transgenic lines for more functional details.

Keywords: Arabidopsis; abiotic stress; gene cloning; thioredoxins; transformation.

Figure 1

Gene structure was analyzed using expasy (A) Schematic structure of the genomic DNA showing exon (black block) and untranslated region (UTR) (grey lines) of GhTRX134. (B) The protein sequence with the two thioredoxins (TRX) domains (PF00085), 76-166 aa and 200-285 aa, are green and yellow highlighted, respectively.

Figure 2

Overexpression of GhTRX134 in Arabidopsis enhanced drought tolerance. Two-week-old plants were treated with 20% polyethylene glycol (PEG). (A) The expression of GhTRX134 in transgenic lines and wild-type (Wt) under drought treatment was monitored by qRT-PCR. (B) Plant height was measured in centimeters (cm), and the percent reduction was calculated in transgenic lines and Wt plants under drought and control (CK) conditions. (C) Phenotypes of untreated (CK) transgenic and Wt plants compared with plants after drought treatment. (D) Differences in dry/wet ratios between transgenic plants and Wt plants after PEG6000 treatment; dry/wet ratio calculated by dry weight/wet weight of plants. The data represent the means of three replicates ±SDs. Asterisks indicate significance (* p < 0.05; ** p < 0.01; and *** p < 0.001) compared to Wt as determined by Student’s t-test.

Figure 3

Overexpression of GhTRX134 in Arabidopsis improved salt tolerance. Two-week-old plants were treated with 200 mM NaCl. (A) The expression of GhTRX134 in transgenic lines and Wt under salt treatment was monitored byqRT-PCR. (B) Plant height was measured, and the percent reduction in transgenic lines and Wt plants was calculated under control and salt conditions. (C) Phenotypes of transgenic and Wt plants under control conditions (CK) compared with plants after salt treatment. (D) Fresh weight of transgenic lines and Wt plants after salt treatment. The data represent the mean of three replicates with ± SDs. Student’s t-test was used to detect significant differences (* p < 0.05; ** p < 0.01; and *** p < 0.001) compared to Wt.

Figure 4

Oxidative stress analysis of transgenic lines and Wt plants (A) Expression level of GhTRX134 in transgenic Arabidopsis and Wt was monitored by qRT-PCR. (B) Phenotypes of GhTRX134-overexpressing and Wt plants after treatment with 20 µM methyl viologen (MV) for 20 days. (C) The survival percentage of transgenic and Wt plants after oxidative stress. Values are the mean of three replicates ± SDs. Student’s t-test was used to determine the significant differences compared to Wt.

Figure 5

Silencing of GhTRX134 using the pCLCrVA-pCLCrVB system inhibited tolerance to drought in cotton plants. (A) The phenotypes of the virus-infected cotton seedlings pCLCrVA::GhTRX134, the control pCLCrVA plants, and the pCLCrVA::PDS indicator plants. (B) The expression of GhTRX134 in the virus-infected plants and the control (pCLCrVA) plants was examined by qRT-PCR. The values represent the means of three replicates ± SD. Student’s t-test was used to determine the significant differences (*** p < 0.001) compared to the control plants.

Figure 6

GhTRX134 shows transcriptional activity. (A) The size of the GhTRX134 fragments. (B) The yeastAH109 strain containing the pGBKT7 empty vector, pGBKT7-GhTRX134FL (full-length GhTRX134), pGBKT7-GhTRX134-N (N-terminal region of GhTRX134), and pGBKT7-GhTRX134-C (C-terminal region of GhTRX134). (C) Transformants were streaked on synthetic dextrose medium lacking tryptophan (SD-Trp) medium (control) and synthetic dextrose medium lacking tryptophan, histidine, and adenine (SD-Trp-His-Ade) medium (selective) to examine HIS3 and ADE2reporter gene activation. The media with yeast cells were incubated for 3 days at 28 °C.

Figure 7

Expression of the AtERD15, AtRAB18, AtRD22, AtRD29A, and AtKIN1 responsive genes in two-week-old transgenic and WtArabidopsis plants under drought, MV, salt, and cold. (A–C) Drought 20% PEG. (D–F) MV 20 µM. (G,H) Salt 200 mM NaCl. (I,J) Cold 4 °C for 72 h stresses was monitored by qRT-PCR. The Arabidopsis thaliana actin gene was used as a housekeeping gene. The comparative threshold cycle (CT) method was used to evaluate the relative expression; each value is the average of three replicates ± SDs. Student’s t-test was used to determine the significant differences (* p < 0.05; ** p < 0.01; and *** p < 0.001) compared to Wt.

Figure 8

GhTRX134 overexpression increases enzyme activities under drought conditions and proline content under salt stress. Two-week-old plants were treated with (A–D) 20% polyethyleneglycol to measurethe proline content, catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) enzyme activity levels. (E–H) 200 mM NaCl to measure proline content, CAT, SOD, and POD enzyme activity levels. (I) Chlorophyll content under salt (200 mM NaCl) and drought (PEG 20%). Proline and chlorophyll content were measured after drought and salt for 5 days using aspectrophotometer. The values represent the means of three replicates ± SDs. Student’s t-test was used to determine significant differences (* p < 0.05; ** p < 0.01; and *** p < 0.001) compared to the control plants.