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. 2013 Jun 10;8(6):e65120.
doi: 10.1371/journal.pone.0065120. Print 2013.

A Wheat WRKY Transcription Factor TaWRKY10 Confers Tolerance to Multiple Abiotic Stresses in Transgenic Tobacco

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Free PMC article

A Wheat WRKY Transcription Factor TaWRKY10 Confers Tolerance to Multiple Abiotic Stresses in Transgenic Tobacco

Chen Wang et al. PLoS One. .
Free PMC article

Abstract

WRKY transcription factors are reported to be involved in defense regulation, stress response and plant growth and development. However, the precise role of WRKY transcription factors in abiotic stress tolerance is not completely understood, especially in crops. In this study, we identified and cloned 10 WRKY genes from genome of wheat (Triticum aestivum L.). TaWRKY10, a gene induced by multiple stresses, was selected for further investigation. TaWRKY10 was upregulated by treatment with polyethylene glycol, NaCl, cold and H2O2. Result of Southern blot indicates that the wheat genome contains three copies of TaWRKY10. The TaWRKY10 protein is localized in the nucleus and functions as a transcriptional activator. Overexpression of TaWRKY10 in tobacco (Nicotiana tabacum L.) resulted in enhanced drought and salt stress tolerance, mainly demonstrated by the transgenic plants exhibiting of increased germination rate, root length, survival rate, and relative water content under these stress conditions. Further investigation showed that transgenic plants also retained higher proline and soluble sugar contents, and lower reactive oxygen species and malonaldehyde contents. Moreover, overexpression of the TaWRKY10 regulated the expression of a series of stress related genes. Taken together, our results indicate that TaWRKY10 functions as a positive factor under drought and salt stresses by regulating the osmotic balance, ROS scavenging and transcription of stress related genes.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Predicted domains in the TaWRKY1 - TaWRKY10 protein sequences and phylogenetic tree.
(A) Predicted domains in the WRKY protein. The conserved domains were carried out by MEME using the protein sequences of TaWRKYs and other known WRKYs. This online software was used to create the logo representations of the WRKY domain and the zinc finger motif. On the y axis (measured in bits), depicts the overall height of the stack indicating the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino or nucleic acid at that position. (B) Phylogenetic tree of the TaWRKYs domains from various plants. The multiple alignments were generated by CLUSTAL W and the phylogenetic tree was constructed with MEGA4.0 using a bootstrap test of phylogeny with minimum evolution test and default parameters. GenBank accession numbers of WRKY proteins used for drawing phylogenetic tree are shown in Table S5.
Figure 2
Figure 2. Expression patterns of wheat TaWRKY10 in different tissues and under different treatments.
The wheat seedlings were treated with 20% PEG6000, 200 mM NaCl, cold (4°C) or 10 mM H2O2. 200 ng Poly(A)+ mRNA was subjected to reverse transcription, and served as the qRT-PCR template. The y axis indicates the relative expression difference in mRNA level and the data were calculated using the 2–ΔΔCt formula. The transcripts of TaActin in the same samples was using as a reference. Transcript levels of the TaWRKY10 gene in untreated wheat were taken as 1. At least three biological experiments were carried out, which produced similar results.
Figure 3
Figure 3. The genomic organization, protein localization and transcriptional activation activity of the TaWRKY10 gene.
(A) Southern blot analysis of the TaWRKY10 gene. 10 µg genomic DNA of hexaploid wheat cv. Chinese Spring was digested completely with the restriction enzymes. The ORF of TaWRKY10 was used as the hybrid probe. The TaWRKY10 overexpressing vector was used as control. The 1 kb DNA Ladder (MBI Fermentas) are indicated on the left. (B) Subcellular localization of TaWRKY10 in onion epidermal cells. Onion epidermal cells were transferred with vector carrying GFP or TaWRKY10-GFP using bombardment method. Free GFP and TaWRKY10-GFP fusion proteins were transiently expressed in onion epidermal cells and observed with an inverted fluorescence microscope. (C) Transactivation activity of the TaWRKY10 protein in Yeast. The schematic diagram demonstrating the TaWRKY10 cDNA fragments encoding different portions of TaWRKY10 that were fused to the yeast vector pGBKT7 (pBD). Transactivation activity analysis of TaWRKY10 was performed using yeast strain AH109. The transformants were streaked on the SD/−Trp or on SD/−His medium. The transformants were examined for growth in the presence of 3-AT and X-α-D-gal. Three biological experiments were carried out, which produced similar results.
Figure 4
Figure 4. Identification and the early development assay of the TaWRKY10 transformed tobacco plants.
(A) Southern blot confirmation of the TaWRKY10 copy number. The 1 kb DNA Ladder (MBI Fermentas) is indicated on the left. (B) Western blot confirmation of TaWRKY10 protein expression. (C) Germination rate of tobacco overexpressing the TaWRKY10 gene. (D) Root lengths of tobacco plants overexpressing the TaWRKY10 gene. The data present means ± SD of three experiments performed. Significant differences between the TG and control lines are indicated as *p<0.05; **p<0.01.
Figure 5
Figure 5. Analysis of the enhanced drought tolerance in transgenic tobacco lines.
(A) Phenotype of WT and TG tobacco lines after 3 weeks of drought treatment. (B) Rate of leaf yellowing. (C) Survival rate. (D) RWC content. (E) Proline content. (F) Soluble sugar content. (G) MDA content. (H) Tissue localization of O2 generation by NBT staining and tissue localization of H2O2 generation by DAB staining. The data present means ± SD of three experiments performed. Significant differences between the TG and control lines are indicated as *p<0.05; **p<0.01.
Figure 6
Figure 6. Analysis of the enhanced salt tolerance in transgenic tobacco lines.
(A) Phenotype of WT and TG tobacco after 3 weeks of 400 mM NaCl treatment; (B) Rate of leaf yellowing. (C) Survival rate. (D) RWC content. (E) Proline content. (F) Soluble sugar content. (G) MDA content. (H) Tissue localization of O2 generation by NBT staining and tissue localization of H2O2 generation by DAB staining. The data present means ± SD of three experiments performed. Significant differences between the TG and control lines are indicated as *p<0.05; **p<0.01.
Figure 7
Figure 7. Overexpression of the TaWRKY10 gene in tobacco enhances the expression of stress related genes.
The y axis indicates the relative expression difference in mRNA levels of these genes in TG tobacco, and the data were calculated using the 2–ΔΔCt formula. The transcripts of NtUbiquitin in the same samples was using as a reference. Three biological experiments were performed, which produced similar results.

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Grant support

This work was supported by the International Science and Technology Cooperation Key Projects of the Ministry of Science and Technology of China (Grant No. 2009DFB30340), the National Genetically Modified New Varieties of Major Projects of China (2011ZX08002-004, 2011ZX08010-004), the Key Projects of Science and Technology Research of the Ministry of Education of China (Grant No. 109105) and the Wuhan Municipal Science and Technology research project (Grant No. 201120922286). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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