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Overexpression of a cotton (Gossypium hirsutum) WRKY gene, GhWRKY34, in Arabidopsis enhances salt-tolerance of the transgenic plants
Plant Physiology and Biochemistry 96 (2015) 311e320
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Research article
Overexpression of a cotton (Gossypium hirsutum) WRKY gene, GhWRKY34, in Arabidopsis enhances salt-tolerance of the transgenic plants Li Zhou 1, Na-Na Wang 1, Si-Ying Gong, Rui Lu, Yang Li, Xue-Bao Li* Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 July 2015 Received in revised form 22 August 2015 Accepted 22 August 2015 Available online 28 August 2015
Soil salinity is one of the most serious threats in world agriculture, and often influences cotton growth and development, resulting in a significant loss in cotton crop yield. WRKY transcription factors are involved in plant response to high salinity stress, but little is known about the role of WRKY transcription factors in cotton so far. In this study, a member (GhWRKY34) of cotton WRKY family was functionally characterized. This protein containing a WRKY domain and a zinc-finger motif belongs to group III of cotton WRKY family. Subcellular localization assay indicated that GhWRKY34 is localized to the cell nucleus. Overexpression of GhWRKY34 in Arabidopsis enhanced the transgenic plant tolerance to salt stress. Several parameters (such as seed germination, green cotyledons, root length and chlorophyll content) in the GhWRKY34 transgenic lines were significantly higher than those in wild type under NaCl treatment. On the contrary, the GhWRKY34 transgenic plants exhibited a substantially lower ratio of Naþ/ Kþ in leaves and roots dealing with salt stress, compared with wild type. Growth status of the GhWRKY34 transgenic plants was much better than that of wild type under salt stress. Expressions of the stressrelated genes were remarkably up-regulated in the transgenic plants under salt stress, compared with those in wild type. Based on the data presented in this study, we hypothesize that GhWRKY34 as a positive transcription regulator may function in plant response to high salinity stress through maintaining the Naþ/Kþ homeostasis as well as activating the salt stress-related genes in cells. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Cotton (Gossypium hirsutum) WRKY protein Overexpression High salinity Salt tolerance
1. Introduction Plants constantly encounter various abiotic and biotic stresses throughout the whole life cycle. These stresses can simultaneously affect plant growth and development and/or change the distribution of plant species. Higher plants are sessile organisms that can not move to escape diversely unfavorable environmental factors. To adapt to these challenges, plants have developed a series of complex responsive mechanisms, and have possessed the ability to perceive and respond to diversified external signals via multiple signaling pathways with proper physiological and morphological changes (Tuteja, 2007). Among these mechanisms, a number of stress-related genes directly protect the plants against stress, or either induce or repress the downstream target genes to regulate
* Corresponding author. E-mail address: [email protected] (X.-B. Li). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.plaphy.2015.08.016 0981-9428/© 2015 Elsevier Masson SAS. All rights reserved.
plant development and defense responses synergistically or antagonistically (Skriver and Mundy, 1990). Transcription factors (TFs) interact with cis-elements that are present within the promoter regions of various abiotic stress-related genes and, therefore, regulate the expression of these genes, leading to plant tolerance to abiotic stresses (Agarwal et al., 2006). So far, a large number of TFs belonging to different protein families (such as MYB, DREB, bZIP, NAC and WRKY families) have been identified in plants. WRKY (pronounced ‘worky’) transcription factors are a large family of regulatory proteins in plants. All the WRKY proteins contain one or two conserved WRKY domains composed of about 60 amino acids with the conserved amino acid sequence WRKYGQK at N-terminus and a zinc-finger motif (CeX4e5eCeX22e23eHeX1eH or CeX7eCeX23eHeX1eC) at C-terminus (Eulgem et al., 2000; Ulker and Somssich, 2004). WRKY transcription factors are thought to function by binding their cognate TTGACC/T W-box ciselements in the promoter regions of target genes (Sun et al., 2003). Based on the number of conserved WRKY domains and the structure of the zinc-finger motif, WRKY family is divided into three
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distinct groups (I, II and III). Furthermore, group II can be further classified into five distinct subgroups (IIa to IIe) based on additional conserved structural motifs outside the WRKY domain (Eulgem et al., 2000). It has been reported that some WRKY proteins play vital roles in response to biotic stress (such as pathogen infection) and in regulation of abiotic stress (such as hormone, wounding, high salinity, drought and cold) (Xie et al., 2005; Ryu et al., 2006; Eulgem and Somssich, 2007; Pandey and Somssich, 2009; Sun et al., 2013; Li et al., 2013). Through microarray analysis, two closely related WRKY transcription factors (WRKY25 and WRKY33) were identified in Arabidopsis and their transcripts were accumulated in plants under treatment with NaCl (Jiang and Deyholos, 2009). Overexpression of ZmWRKY33 in Arabidopsis induced expression of RD29A that is known to be involved in stress signaling, and enhanced salt tolerance of the transgenic plants (Li et al., 2013). Overexpression of Musa WRKY71 in the transgenic banana affected its oxidative and salt stress tolerance (Shekhawat and Ganapathi, 2013). Generally, multiple pathways are involved in plant response to high salinity, including ionic and osmotic homeostasis signaling, detoxification and growth regulation. Recently, ten TaWRKYs (designated TaWRKY1eTaWRKY10) were successfully identified. Among these genes, TaWRKY10 was observed to confer plant drought and salt tolerance by regulating osmosis and reducing ROS accumulation (Wang et al., 2013). Alleles of OsWRKY45-1 and OsWRKY45-2 play different roles in ABA signaling and salt tolerance in rice (Tao et al., 2011). WRKY8 antagonistically interacts with VQ9 to modulate salinity tolerance (Hu et al., 2013). Upland cotton (Gossypium hirsutum) is an important fiber crop in the world. However, soil salinity, which is one of the major abiotic stresses in world agriculture, often severely impacts cotton growth and development, leading to a significant loss in cotton crop yield. Therefore, it is important to enhance cotton saltresistance through genetic manipulation. In previous study, we identified 26 cotton WRKY genes, of which GhWRKY34 was induced in cotton under NaCl stress (Zhou et al., 2014). Up to now, however, data associated with cotton WRKY family TFs are still notably limited, and especially, little is known about the role of WRKY TFs in cotton response to salt stress. In present study, ectopic expression of GhWRKY34 in Arabidopsis enhanced the transgenic plant tolerance to high salinity stress. GhWRKY34 as a positive transcription regulator may function in plant response to high salinity stress through maintaining the Naþ/Kþ homeostasis as well as activating the salt stress-related genes in cells.
2. Materials and methods 2.1. DNA and protein sequence analysis The GhWRKY34 cDNA (accession number in GenBank: KJ825876) was identified in cotton cDNA libraries. Its nucleotide sequence and the deduced amino acid sequence were analyzed using DNAstar software (DNAstar Inc., USA). The peptide sequences were aligned with the ClustalW2 program (http://www.ebi.ac.uk). The conserved domains of the GhWRKY34 protein were confirmed at the National Center for Biotechnology Information (NCBI) (http:// blast.ncbi.nlm.nih.gov/Blast.cgi). Identification of protein domains and significant sites was performed with Motifscan (http://myhits. isb-sib.ch/cgi-bin/motif_scan). The phylogenetic tree was constructed by the neighbor-joining (NJ) method in MEGA5.0 program (http://www.megasoftware.net/), based on majority-rule consensus from 1000 bootstrap replicates.
2.2. RNA isolation and quantitative RT-PCR analysis To analyze the expression of GhWRKY34 and the NaClresponsive genes in the transgenic Arabidopsis plants, total RNA was isolated from two-week-old the transgenic Arabidopsis seedlings and wild type (Columbia ecotype) grown under normal conditions (CK) and under 100 mM NaCl treatments for 72 h. Then, the RNA was reversely transcribed into cDNAs which were used as templates in PCR analysis. Expression of GhWRKY34 and the NaClresponsive genes in the transgenic Arabidopsis plants was analyzed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) with gene-specific primers (Table 1), using the fluorescent intercalating dye SYBR-Green in a detection system (Opticon 2; MJ Research, Waltham, MA, USA). Arabidopsis ACTIN2 gene was used as a standard control in the RT-PCR reactions. A two-step RT-PCR procedure was performed in all experiments using the method described earlier (Li et al., 2005). For all the above quantitative real-time PCR analysis, the assays were repeated three times along with three independent repetitions of the biological experiments, and means of three biological experiments were calculated for estimating gene expression levels. 2.3. Subcellular location of GhWRKY34 protein The coding sequence (without stop codon) of GhWRKY34, amplified by PCR using proofreading Pfu DNA polymerase, was cloned into a pBI121-eGFP vector upstream the eGFP sequence, generating the pBI-WRKY34-eGFP construct with WRKY34:GFP fusion genes under the control of CaMV 35S promoter. Then, the construct was transferred into Agrobacterium tumefaciens strain LBA4404. Arabidopsis thaliana was transformed with Agrobacterium-mediated DNA transfer by the floral dip method. Transgenic seedlings were selected on MS medium containing 50 mg/L kanamycin. Five-day-old WRKY34:GFP transgenic seedlings (T2 generation) were stained with 40 6-diamidino-2-phenylindole (DAPI, a nucleus-spcific dye) for 15 min at room temperature before observation of GFP fluorescence and DAPI staining under the confocal fluorescence microscope (Leica, Germany). The digital images were taken and process by SP5 software (Leica, Germany) (Qin et al., 2014). 2.4. Construction of overexpression vector and Arabidopsis transformation The coding sequence of GhWRKY34 gene, amplified by PCR using proofreading Pfu DNA polymerase, was cloned into pMD vector
Table 1 Gene-specific primers used in real-time quantitative RT-PCR analysis. Genes
Primers
GhWRKY34
50 -ATGTGCAGACTTCGGAATCT-30 50 -GAGCTTCCTAATCGACTGAT-30 50 -TGAAAGGAGGAGGAGGAATGGTTGG-30 50 -ACAAAACACACATAAACATCCAAAGT-30 50 -CCAGATAGCGGAGGGGAAAGGACAT-30 50 -AAGTTCACAAACAGAGGCATCATCATCATAC-30 50 -GGCTTGAAGAAAGTGAGTCTCG-30 50 -GCTACATAGTTCGGAGTTCCACA-30 50 -TCGTTTCAGCCAAATCAGAAAGT-30 50 -TTTGCCTTGTGCTGCTTTCC-30 50 -AACAACTTAGGAGGTGGTGGTCAT-30 50 -TGTAGCAGCTGGCGCAGAAGTCAT-30 50 -GAAATGAAACTGGCTGATGAAACCATAGAG-30 50 -CTCGTGGCAATCTACTCGGTCTTAAACC-30 50 - GAAATCACAGCACTTGCACC-30 50 - AAGCCTTTGATCTTGAGAGC-30
AtRD29A AtRD29B AtSOS2 AtSOS1 AtABF4 AtCBL1 AtACT2
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driven by CaMV 35S promoter. Then, the construct was transferred into Agrobacterium tumefaciens strain LBA4404. A. thaliana was transformed with Agrobacterium-mediated DNA transfer by the floral dip method. Transformed Arabidopsis seeds were selected on MS medium containing 50 mg/L kanamycin. Homozygous lines of T3 generations were used for phenotypic analysis of transgenic plants. 2.5. Phenotypic evaluation of GhWRKY34 overexpression transgenic Arabidopsis To assay the seed germination rate and proportion of seedlings with opened green cotyledons, the surface-sterilized seeds from each transgenic Arabidopsis line and wild type, respectively, were sowed on MS plates supplemented with or without different concentration of NaCl, placed at 4 C for 3 days, and then moved to a growth room under a photoperiod of 16 h light/8 h dark at 22 C. The rate of seed germination (root emergence) was evaluated every day, and the rate of seedlings with opened green cotyledons was counted at the tenth day. Each experiment was repeated three
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times at least with identical results, and at least 300 seeds of each line were selected for analyzing the seed germination rate and cotyledon greening rate. To assay root elongation, five-day-old seedlings of the transgenic lines and wild type were cultured vertically on MS medium supplemented with different concentrations of NaCl for 7 days. Then, the length of roots was measured and calculated. All experiments were repeated at least three times, and 30e60 seedlings of each line were selected for vertical cultivation. For NaCl-irrigation experiment, Arabidopsis seedlings of the transgenic lines and wild type were transplanted into soil for further growing. The transgenic lines and wild type at same developmental stage (i.e. three-week-old wild type and transgenic seedlings, and five-week-old wild type and transgenic seedlings) were used for NaCl treatment, respectively. The transgenic plants and wild type (control) in pots were watered with the same volume 300 mM NaCl for continuous 7 days, and then recovered 7 days. The status of plant tolerance to high salinity (NaCl) were observed and photos were taken. The chlorophyll content was determined by the method
Fig. 1. Sequence analysis of GhWRKY34 protein. (A) Alignment of WRKY domains of GhWRKY34 with the other WRKY proteins. Conserved amino acid residues (WRKYGQK) and zinc-finger are shown in gray. (B) Phylogenetic relationship of GhWRKY34 and the other plant WRKY proteins. All 29 WRKY proteins can be divided into three groups (I, II and III) based on the number of WRKY domain and type of zinc-finger structure. Group II further were divided into five subgroups, including IIa, IIb, IIc, IId and IIe. The plant WRKY proteins used for the phylogenetic tree are AtWRKY8 (NP_199447), AtWRKY9 (NP_176982), AtWRKY10 (NP_175956), AtWRKY11 (NP_567878), AtWRKY14 (NP_564359), AtWRKY18 (NP_001031766), AtWRKY20 (NP_849450), AtWRKY22 (NP_192034), AtWRKY28 (NP_193551), AtWRKY30 (NP_568439), AtWRKY35 (NP_181029), AtWRKY36 (NP_564976), AtWRKY39 (NP_566236), AtWRKY40 (NP_178199), AtWRKY44 (NP_001078015), AtWRKY46 (NP_182163), AtWRKY53 (NP_194112), AtWRKY54 (NP_181607), AtWRKY60 (NP_180072), AtWRKY74 (NP_198217), BnWRKY22-1 (NP_001288962), JcWRKY46 (AGQ04237), GhWRKY31 (AJT43311), TcWRKY35 (EOX95271), TcWRKY46 (XP_007051596), DlWRKY6-1 (AEO31476), VaWRKY30 (AAR92477). At, Arabidopsis thaliana; Bn, Brassica napus; Gh, Gossypium hirsutum; Jc, Jatropha curcas; Tc, Theobroma cacao; Dl, Dimocarpus longan; Va, Vitis aestivalis.
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Fig. 2. Subcellular localization of GhWRKY34 protein. GhWRKY34 fused with eGFP gene under CaMV 35S promoter was expressed in hypocotyl cells of the transgenic Arabidopsis thaliana. GFP fluorescence signals were mainly detected in nuclei of the hypocotyl cells harboring the GhWRKY34:eGFP reporter construct. (A) GFP fluorescent image of the hypocotyl cell; (B) Nuclear DAPI staining of the same cell in A. (C) The transmitted light image of the same cell in image A; (D) The images of A and B were merged over the bright-field image. Bar ¼ 10 mm.
described earlier (Qin et al., 2013). In brief, chlorophyll in 0.2 g leaves of each transgenic line and wild type was extracted with 10 mL of 80% acetone, and chlorophyll content was assayed by measuring absorbance at 645 and 663 nm with a spectrophotometer. The assays were repeated three times along with three independent repetitions of the biological experiments. To measure Na⁺ and K⁺ contents, Arabidopsis leaves and roots of the transgenic lines and wild type grown under normal conditions or 100 mM NaCl for 3 weeks were harvested, rinsed with deionized water, dried at 70 C for at least 48h, and cooled in a desiccation
Fig. 4. Assay of cotyledon greening rate of GhWRKY34 overexpression transgenic Arabidopsis under NaCl stress. Mean values and standard errors (bar) are shown from three independent experiments. One or two asterisks represent there was represent significant difference (P < 0.05) or very significant difference (P < 0.01) in green cotyledon rate between the transgenic lines and wild type. MS, seedlings grew on MS medium without NaCl as control; 100 mM NaCl and 150 mM NaCl, seedlings grew on MS medium with 100 and 150 mM NaCl, respectively. WT, wild type; L13, L22 and L24, GhWRKY34 overexpression transgenic lines 11, 22 and 24.
chamber. Then 100 mg of each sample was digested with 0.1M HNO3 boiling for 30 min, and filtrated through a 0.22 mm filter membrane. The Na⁺ and K⁺ contents in the solution were measured with atomic absorption spectrophotometer. The experiments were performed independently for three times and the results were presented as a average values (Rus et al., 2001). 3. Results 3.1. Sequence analysis of GhWRKY34 protein GhWRKY34 cDNA (KJ825876) encoding a WRKY protein was identified from cotton cDNA libraries (Zhou et al., 2014).
Fig. 3. Assay of seed germination rate of GhWRKY34 overexpression transgenic Arabidopsis under NaCl stress. (A) Quantitative RT-PCR analysis of GhWRKY34 expression in the GhWRKY34 overexpression transgenic lines and wild type. (BeE) Statistical analysis of seed germination rate. Each curve represents an average of three replicates. MS, seeds germinated on MS medium without NaCl as control; 100 mM NaCl, 150 mM NaCl and 200 mM NaCl, seeds germinated on MS medium with 100, 150 and 200 mM NaCl, respectively. WT, wild type; L13, L22 and L24, GhWRKY34 overexpression transgenic lines 11, 22 and 24.
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GhWRKY34 protein contains an approximate 60-amino acid WRKY domain that is composed of the conserved amino acid sequence WRKYGQK. However, a zinc-finger motif in GhWRKY34 protein is C-X-C-X23-HeT-C, instead of C-X-C-X22e23-HeX-H, indicating that this protein is a member of WRKY group III. GhWRKY34 shares relatively high identity with TcWRKY46 (XP_007051596) (74%), VaWRKY30 (AAR92477) (58%), DlWRKY6-1 (AEO31476) (60%), and AtWRKY46 (NP_182163.1) (41%) in the WRKY domain and zincfinger motif (Fig. 1A). To investigate the evolutionary relationships among WRKYs from different species, phylogenetic analysis was performed with MEGA program, based on the amino acid sequences of GhWRKY34 and other plant WRKY proteins. Generally, plant WRKY proteins can be classified into three groups (I, II and III), of which group II is further divided into five subgroups (IIa e IIe). As shown in Fig. 1B, GhWRKY34 with TcWRKY46 forms a cluster in the tree, suggesting that GhWRKY34 is a member of group III of cotton WRKY family. 3.2. Subcellular localization of GhWRKY34 protein By PSORT program, we predicted that GhWRKY34 protein is located in the cell nucleus due to the existence of the putative nuclear localization signal (NLS, a short polypeptide consisting of four amino acid residues PKRR). To confirm the real location of GhWRKY34 protein, we constructed GhWRKY34:GFP fusion gene controlled by CaMV 35S promoter. Then the fusion gene was transferred into A. thaliana by Agrobacterium-mediated transformation. The GhWRKY34:GFP transgenic seedlings (T2 generation) were stained with DAPI, and then GFP fluorescence and DAPI staining in hypocotyl cells of the GhWRKY34:GFP transgenic
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seedlings was observed by confocal microscopy. As shown in Fig. 2, both the GFP fluorescence and DAPI staining were strongly accumulated in nuclei of hypocotyl cells, suggesting that GhWRKY34 protein was localized in the cell nucleus. 3.3. Overexpression of GhWRKY34 in Arabidopsis enhances plant tolerance to high salinity We have been already known that GhWRKY34 transcript was largely accumulated in leaves, and expression of GhWRKY34 was strongly up-regulated in cotton seedlings under NaCl treatment, compared with that in cotton seedlings without NaCl treatment, suggesting that GhWRKY34 may be involved in plant response to salt stress (Zhou et al., 2014). To examine the role of GhWRKY34 response to salt, GhWRKY34 gene under the control of CaMV 35S promoter was introduced into Arabidopsis via Agrobacterium mediated DNA transfer. More than thirty independent transgenic lines were obtained, and the homozygous transgenic progenies (T3 generations) were selected through kanamycin-resistance assay and PCR analysis. GhWRKY34 expression levels in transgenic lines were examined by quantitative RT-PCR analysis. The representative GhWRKY34 overexpression transgenic lines L13, L22 and L24, which exhibited different GhWRKY34 expression levels, were selected for the following experiments (Fig. 3A). To confirm the response of GhWRKY34 to salt, we tested the seed germination capacity on MS medium with 0 (control), 100, 150 and 200 mM NaCl. Under normal conditions (without NaCl treatment), there was no significant difference between GhWRKY34 transgenic lines and wild type. Although GhWRKY34 transgenic lines and wild type showed little difference in seed germination on MS medium with 100 mM NaCl,
Fig. 5. Assay of root length of GhWRKY34 overexpression transgenic Arabidopsis under NaCl stress. (A) Wild type and GhWRKY34 overexpression seedlings grew on MS medium. (B) Wild type and GhWRKY34 overexpression seedlings grew on MS medium with 100 mM NaCl. (C) Statistical analysis of root length of wild type and GhWRKY34 overexpression seedlings grown on MS medium without or with 100 mM NaCl. Seeds germinated on MS medium for 7 days and then the seedlings were transferred onto MS medium supplemented with 0 (control) and 100 mM NaCl for 7 days. Mean values and standard errors (bar) are shown from three independent experiments. One asterisk represents there was represent significant difference (P < 0.05) in root length between the transgenic lines and wild type. MS, seedlings grew on MS medium without NaCl as control; 100 mM NaCl, seedlings grew on MS medium with 100 mM NaCl. WT, wild type; L13, L22 and L24, GhWRKY34 overexpression transgenic lines 11, 22 and 24.
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however, the GhWRKY34 transgenic lines displayed higher tolerance to salt stress than wild type under 150 and 200 mM NaCl treatments. On MS medium with 200 mM NaCl, seed germination rates of three GhWRKY34 transgenic lines were 91% (L13), 89% (L22) and 92% (L24), respectively, while wild type was only 52% (Fig. 3BeE). Statistical analysis revealed there was significant difference in seed germination between the transgenic lines and wild type. Meanwhile, we also calculated the rates of cotyledon greening of the transgenic lines and wild type. On MS medium, we found the rates of cotyledon greening of the GhWRKY34 transgenic lines were similar to that of wild type. Under 100 and 150 mM NaCl treatments, however, the number of seedlings with opened green cotyledons of GhWRKY34 transgenic lines was much more than that of wild type, especially on MS with 150 mM NaCl (Fig. 4). In addition, seedlings of the transgenic lines and wild type were cultured vertically on MS medium with different NaCl concentrations for one week, and then the root length of these seedlings was measured. As shown in Fig. 5A, root length of the GhWRKY34 transgenic seedlings was little altered on MS medium, compared with that of wild type. On the contrary, root length of the GhWRKY34 transgenic seedlings were remarkably longer than that of wild type under 100 mM NaCl treatment (Fig. 5B). Measurement and statistical analysis revealed that there was significant difference in root length between the transgenic lines and wild type under NaCl stress (Fig. 5C). These results indicated that GhWRKY34 enhance plant tolerance to high salinity stress during early plant development. To further investigate the role of GhWRKY34 gene in plant tolerance to high salinity at the late developmental stages, wild type and the transgenic plants (T2 and T3 generations) grown in soil were watered with NaCl solution (see Methods). As shown in Fig. 6, wild type and GhWRKY34 overexpression transgenic plants grown under salt stress occurred growth retardation, compared with those plants grown in normal conditions. The 3-week-old Arabidopsis plants grown in pots were watered with 300 mM NaCl solution. After several days, wild type plants became yellow and wilting, whereas the transgenic plants were still green (Fig. 6B). Likewise, the five-week-old transgenic plants after watering with 300 mM NaCl solution for several days also displayed higher salttolerance than wild type (Fig. 6D). Environmental stressors (such as salt and drought stresses) may cause a change in chlorophyll content in plants. To determine whether chlorophyll content is altered in the transgenic plants under salt stress, we detected the chlorophyll content in leaves of the GhWRKY34 transgenic seedlings and wild type grown under normal conditions (control) or under NaCl stress. As shown in Fig. 6E, there was no significant difference in chlorophyll cotent between the transgenic lines and wild type in normal growth conditions. Under NaCl treatment, however, chlorophyll content in leaves of the GhWRKY34 transgenic lines was remarkably higher than that in wild type although chlorophyll content was declined in both transgenic plants and wild type. These results suggested overexpression of GhWRKY34 in Arabidopsis confers higher salt-tolerance on transgenic plants and GhWRKY34 may play a positive role in response to salt stress during cotton growth and development. 3.4. Overexpression of GhWRKY34 in Arabidopsis influences Naþ/ Kþ homeostasis in plant cells under salt stress Plant salt tolerance is closely related to the ability of a plant to maintain a low cytosolic Naþ/Kþ ratio (Shabala and Cuin, 2007). Thus, to analyze the mechanism of enhancing salt tolerance in GhWRKY34 overexpression transgenic Arabidopsis, the Naþ and Kþ contents in leaves and roots of wild type and the transgenic plants with or without 100 mM NaCl treatment were determined by
Fig. 6. Phenotypic assay of GhWRKY34 transgenic Arabidopsis plants under NaCl stress. (A) Five-week-old seedlings grew in soil under normal conditions. (B) Five-week-old seedlings grew in soil watered with 300 mM NaCl. (C) Seven-week-old plants grew in soil under normal conditions. (D) Seven-week-old seedlings grew in soil watered with 300 mM NaCl. (E) Assay of chlorophyll content in leaves of four-week-old GhWRKY34 overexpression transgenic lines and wild type under normal conditions and under NaCl stress. Mean values and standard errors (bar) are shown from three independent experiments. One and two asterisks represent there was represent significant difference (P < 0.05) and very significant difference (P < 0.01) in leaf chlorophyll content between the transgenic lines and wild type, respectively. CK, seedlings grew in soil under normal conditions (without NaCl) as controls; NaCl, seedlings grew in soil under NaCl treatment; WT, wild type; L13, L22 and L24, GhWRKY34 overexpression transgenic lines 11, 22 and 24.
atomic absorption spectrophotometer. As shown in Fig. 7, there was no significant difference in Naþ content, Kþ content and Naþ/Kþ ratio in leaves and roots between wild type and the transgenic lines under normal conditions. However, Naþ content in both leaves and roots of wild type under salt stress was remarkably increased and significant higher than that in the transgenic lines, although Naþ content in the transgenic lines was also increased in the same conditions (Fig. 7A and 7C). Meanwhile, Kþ content was decresed in leaves and roots of wild type, but dramatically increased in leaves and roots of the GhWRKY34 overexpression transgenic lines (Fig. 7B and 7D). Therefore, GhWRKY34 overexpression transgenic plants displayed significant lower Naþ/Kþ ratio in leaves and roots than wild type under salt stress (Fig. 7E and 7F). These results suggested
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Fig. 7. Changes in Naþ content, Kþ content and Naþ to Kþ ratio in leaves and roots of GhWRKY34 overexpression transgenic Arabidopsis under NaCl stress. (A, B) Naþ and Kþ contents in leaves of the transgenic lines and wild type under normal conditions or 100 mM NaCl for 3 weeks. (C, D) Naþ and Kþ contents in roots of the transgenic lines and wild type under normal conditions or 100 mM NaCl for 3 weeks. (E) Naþ/Kþ ratio in leaves calculated from the data in A and B. (F) Naþ/Kþ ratio in roots calculated from the data in C and D. Each column is the mean of three independent experiments and bars represent standard errors. One and two asterisks represent there was represent significant difference (P < 0.05) and very significant difference (P < 0.01) between the transgenic lines and wild type, respectively. CK, seedlings grew under normal conditions (without NaCl); 100 mM NaCl, seedlings grew under 100 mM NaCl treatment for 3 weeks; WT, wild type; L13, L22 and L24, GhWRKY34 transgenic lines 13, 22 and 24.
that overexpression of GhWRKY34 in Arabidopsis may enhance plant ability of selectively uptaking Naþ and Kþ, and thereby maintain a low Naþ/Kþ ratio in the leaves and roots, conferring salt tolerance on transgenic plants. 3.5. Expression of the salt stress-related genes is upregulated in the GhWRKY34 transgenic plants To further investigate the role of GhWRKY34 in response to salt, the expression levels of salt stress-related genes, including RD29A, RD29B, ABF4, SOS1, SOS2 and CBL1 that act as markers for monitoring salt stress response pathways in Arabidopsis, were examined in the transgenic seedlings under both normal condition and NaCl treatment. As shown in Fig. 8, expressions of four genes (RD29A, ABF4, SOS1 and SOS2) were remarkably enhanced in the transgenic plants compared with those in wild type. Furthermore, all of the six genes were up-regulated in both GhWRKY34 transgenic lines and wild type after NaCl treatment. However, expression levels of these genes in the transgenic plants were dramatically higher than those in wild type under salt stress. Statistical analysis revealed there were significant differences in expression levels of these genes between the transgenic lines and wild type, suggesting that GhWRKY34 may participate in response to salt stress by regulating expressions of the stress-related genes during plant growth and
development. 4. Discussion WRKY transcription factors are a large family of regulatory proteins in plants. Although some researchers indicated that plant WRKY transcription factors are involved in biotic and abiotic stresses, the most of these studies mainly focus on the model plants, such as A. thaliana and rice, and little is known about the role of WRKY transcription factors in cotton. Previously, we identified 26 WRKY genes in cotton (Zhou et al., 2014). In this study, our data revealed that GhWRKY34 is classified into group III of cotton WRKY family. By means of PSORT program we found a predicted nuclear localization signal (NLS) sequence KKRK in GhWRKY34 protein. Further subcellular localization assay indicated that GhWRKY34 was localized to the cell nucleus, suggesting that GhWRKY34 may be a transcription factor in cotton. Soil salinity is one of the most serious threats in world agriculture. It is expected that soil salinity will result in the loss of up to 50% fertile land by the middle of the 21st century (Manchanda and Garg, 2008). High salinity imposes imbalance in osmotic, ionic and secondary oxidative stresses upon plant cells, and ultimately affects plant growth and development (Zhu, 2001). It has been demonstrated that a large number of transcription factors, including basic
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Fig. 8. Quantitative RT-PCR analysis of expressions of the NaCl stress-related genes in GhWRKY34 overexpression transgenic Arabidopsis. Total RNA was isolated from two-week-old Arabidopsis seedlings grown under normal conditions (CK) and under 100 mM NaCl treatments for 72 h. Relative value of the expression of RD29A, RD29B, ABF4, SOS2, SOS1 and CBL1 in Arabidopsis was shown as percentage of AtACTIN2 expression activity. Mean values and standard errors (bar) were shown from three independent experiments. One and two asterisks represent there was represent significant difference (P < 0.05) and very significant difference (P < 0.01) in gene expression level between the transgenic lines and wild type, respectively. CK, seedlings grew under normal conditions (without NaCl); þNaCl, seedlings grew under NaCl treatment; WT, wild type; L13, L22 and L24, GhWRKY34 transgenic lines 13, 22 and 24.
leucine zipper (bZIP), WRKY, APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF), MYB, basic helixeloopehelix (bHLH) and NAC families, are integral in linking salt sensory pathways for plant stress-tolerance responses (Kasuga et al., 1999; Tran et al., 2004; Yang et al., 2009; Jiang and Deyholos, 2009; Cui et al., 2013; Cheng et al., 2013). So far, there are increasing evidence that WRKY proteins regulate salt stress and even multiple abiotic stress responses. For example, overexpression of ZmWRKY33 in Arabidopsis induced expression of RD29 which is known to be involved in stress signaling, and enhanced salt tolerance of the transgenic plants (Li et al., 2013). GhWRKY15 transcripts were accumulated in cotton seedlings for responding to biotic stress modulators (such as salicylic acid and methyl jasmonate) and could impart the transgenic tobacco plants tolerance to fungal pathogens (Yu et al., 2012). MusaWRKY71 transcripts in banana plants were found to be upregulated by cold, dehydration, salt, ABA, H2O2, ethylene, salicylic acid (SA) and methyl jasmonate (MJ) (Shekhawat et al., 2011). Similarly, we found GhWRKY34 was induced by NaCl (Zhou et al., 2014). In this study, our data revealed that overexpression of GhWRKY34 in Arabidopsis conferred higher salt-tolerance on the transgenic plants, compared with wild type. The seed germination rate, cotyledon greening and root length of GhWRKY34 transgenic seedlings at early developmental stages were significantly higher than those of wild type in the presence of NaCl. Furthermore, the transgenic plants still dispayed more salt-tolerance and higher chlorophyll content than wild type at the stages of adult development. These results suggested that GhWRKY34 protein may function as a positive modulator in plant response to high salinity stress. To get further insight to the mechanism of enhancing salt tolerance in GhWRKY34 overexpression transgenic Arabidopsis
under salt stress, the Naþ and Kþ contents in leaves and roots of the transgenic plants were analyzed. Under salt stress, the Naþ/Kþ homeostasis in plants was usually disrupted. Plants absorb the large numbers of Naþ ions which could lead to growth cessation or cell death instead of Kþ ions which have vital functions in maintaining many enzyme activities and osmotic regulation, so the Naþ/ Kþ ratio is closely related to plant salt tolerance (Zhu, 2003). Therefore, an important strategy for plants against salt stress is inhibiting Naþ influx, and increasing Kþ uptake and Naþ extrusion for maintaining a low Naþ/Kþ ratio in cells under high salinity environment (Yokoi et al., 2002). In our study, compared to wild type, the Naþ/Kþ ratio maintained relatively stable in both leaves and roots of GhWRKY34 overexpression transgenic plants grown under normal conditions or under salt stress. The Naþ content in the GhWRKY34 overexpression transgenic plants was significantly lower, whereas the Kþ content was higher than those in wild type under salt stress conditions, indicating that GhWRKY34 transgenic plants may keep relatively strong ability to maintain Naþ/Kþ homeostasis in cells, thereby enhancing plant salt tolerance. To investigate if GhWRKY34 affects the expression of salt stressrelated genes in plants, some marker genes were analyzed in the GhWRKY34 transgenic Arabidopsis lines and wild type. The results indicated that the expression levels of RD29A, RD29B, ABF4, SOS2, SOS1 and CBL1 genes increased remarkably in the transgenic plants, compared with those in wild type, after NaCl treatment. It has been demonstrated that calcineurin B-like protein1 (CBL1) is a member of calcineurin B-like calcium sensor proteins family and interacts with CBL-interacting protein kinase23 (CIPK23). CBL1 may act as a regulatory subunit of a plant calcineurin-like activity mediating calcium signaling under certain stress conditions. The CBL1
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expression level was increased in response to specific stresses such as high salinity, drought, cold and wounding (Hashimoto et al., 2012; Gao et al., 2008). The ABRE-binding factor 4 (ABF4) bZIP transcription factor specifically interacts with abscisic acid (ABA)responsive elements (ABREs) and functions as transcriptional activator in ABRE-dependent transcription in Arabidopsis. The expression of ABF4 in Arabidopsis was enhanced by dehydration, salt treatment, and endogenous ABA treatment (Uno et al., 2000). In this study, we found that overexpression of GhWRKY34 promoted ABF4 and CBL1 expression in the transgenic plants under normal growth conditions and under NaCl treatment, suggesting that GhWRKY34 as a positive regulator regulates these genes in the transgenic Arabidopsis. It was reported that RD29A (responsive to dessication 29A) and RD29B (responsive to dessication 29A) were induced by conditions of dehydration, low temperature, high salinity or by treatment with exogenous ABA. The promoter region of RD29A contains the cis-acting dehydration-responsive element (DRE) that is involved in expression of RD29A rapidly responding to dehydration and high salinity stresses in Arabidopsis. Similarly, the promoter region of RD29B contains two ABA-responsive elements (ABREs) that are required for the dehydration-responsive expression of RD29B in Arabidopsis (Kazuko and Kazuo, 1994). Here, the transcription levels of RD29A and RD29B were dramatically increased in the GhWRKY34 transgenic plants under high salinity stress. This may also help to explain the observation that GhWRKY34 transgenic plants had stronger salt-tolerance than wild type. In addition, the SOS pathway is an important regulatory system activated by salt stress. SOS2 is an important signal transducer which could activate the downstream target gene, SOS1, which encodes a plasma membrane Na⁺/H⁺ antiporter that regulates Na⁺/K⁺ homeostasis under saline conditions (Xiong et al., 2002). In present study, overexpression of GhWRKY34 promoted expression of SOS1 and SOS2 genes in the transgenic plants under both normal conditions and salt stress, implying that GhWRKY34 may be involved in regulation of Naþ and Kþ homeostasis through activating the SOS pathway with enhancing activity of the Naþ/Hþ antiporter, SOS1. Besides, owing to WRKY transcription factors are thought to function by binding their cognate TTGACC/T W-box ciselements in the promoter regions of target genes, the promoter sequences of RD29A, RD29B, AtSOS1, AtSOS2, ATABF4 and ATCBL1 were analyzed with the PlantCARE analysis database (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/). We found that AtSOS2 contains four W-box cis-elements and AtABF4 has one W-box cis-element in their promoter regions, respectively. Therefore, we speculate that GhWRKY34 may bind to the W-box ciselements in the promoters of AtSOS2 and AtABF4 in response to salt stress. In brief, based on the data presented in this study, we hypothesize that GhWRKY34 may be a positive transcription regulator in plant response to high salinity stress through maintaining the Naþ/Kþ homeostasis as well as activating expression of the salt stress-related genes in cotton. Authors' contributions XBL conceived and designed the experiments. LZ, NNW, SYG, RL and YL performed the experiments. LZ, NNW and XBL analyzed the data and wrote the paper. Acknowledgment This work was supported by National Natural Sciences Foundation of China (Grant No. 31471542) and the project from the Ministry of Agriculture of China for transgenic research (Grant No. 2014ZX08009-27B).
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Overexpression of a cotton (Gossypium hirsutum) WRKY gene, GhWRKY34, in Arabidopsis enhances salt-tolerance of the transgenic plants Li Zhou 1, Na-Na Wang 1, Si-Ying Gong, Rui Lu, Yang Li, Xue-Bao Li* Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 July 2015 Received in revised form 22 August 2015 Accepted 22 August 2015 Available online 28 August 2015
Soil salinity is one of the most serious threats in world agriculture, and often influences cotton growth and development, resulting in a significant loss in cotton crop yield. WRKY transcription factors are involved in plant response to high salinity stress, but little is known about the role of WRKY transcription factors in cotton so far. In this study, a member (GhWRKY34) of cotton WRKY family was functionally characterized. This protein containing a WRKY domain and a zinc-finger motif belongs to group III of cotton WRKY family. Subcellular localization assay indicated that GhWRKY34 is localized to the cell nucleus. Overexpression of GhWRKY34 in Arabidopsis enhanced the transgenic plant tolerance to salt stress. Several parameters (such as seed germination, green cotyledons, root length and chlorophyll content) in the GhWRKY34 transgenic lines were significantly higher than those in wild type under NaCl treatment. On the contrary, the GhWRKY34 transgenic plants exhibited a substantially lower ratio of Naþ/ Kþ in leaves and roots dealing with salt stress, compared with wild type. Growth status of the GhWRKY34 transgenic plants was much better than that of wild type under salt stress. Expressions of the stressrelated genes were remarkably up-regulated in the transgenic plants under salt stress, compared with those in wild type. Based on the data presented in this study, we hypothesize that GhWRKY34 as a positive transcription regulator may function in plant response to high salinity stress through maintaining the Naþ/Kþ homeostasis as well as activating the salt stress-related genes in cells. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Cotton (Gossypium hirsutum) WRKY protein Overexpression High salinity Salt tolerance
1. Introduction Plants constantly encounter various abiotic and biotic stresses throughout the whole life cycle. These stresses can simultaneously affect plant growth and development and/or change the distribution of plant species. Higher plants are sessile organisms that can not move to escape diversely unfavorable environmental factors. To adapt to these challenges, plants have developed a series of complex responsive mechanisms, and have possessed the ability to perceive and respond to diversified external signals via multiple signaling pathways with proper physiological and morphological changes (Tuteja, 2007). Among these mechanisms, a number of stress-related genes directly protect the plants against stress, or either induce or repress the downstream target genes to regulate
* Corresponding author. E-mail address: [email protected] (X.-B. Li). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.plaphy.2015.08.016 0981-9428/© 2015 Elsevier Masson SAS. All rights reserved.
plant development and defense responses synergistically or antagonistically (Skriver and Mundy, 1990). Transcription factors (TFs) interact with cis-elements that are present within the promoter regions of various abiotic stress-related genes and, therefore, regulate the expression of these genes, leading to plant tolerance to abiotic stresses (Agarwal et al., 2006). So far, a large number of TFs belonging to different protein families (such as MYB, DREB, bZIP, NAC and WRKY families) have been identified in plants. WRKY (pronounced ‘worky’) transcription factors are a large family of regulatory proteins in plants. All the WRKY proteins contain one or two conserved WRKY domains composed of about 60 amino acids with the conserved amino acid sequence WRKYGQK at N-terminus and a zinc-finger motif (CeX4e5eCeX22e23eHeX1eH or CeX7eCeX23eHeX1eC) at C-terminus (Eulgem et al., 2000; Ulker and Somssich, 2004). WRKY transcription factors are thought to function by binding their cognate TTGACC/T W-box ciselements in the promoter regions of target genes (Sun et al., 2003). Based on the number of conserved WRKY domains and the structure of the zinc-finger motif, WRKY family is divided into three
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distinct groups (I, II and III). Furthermore, group II can be further classified into five distinct subgroups (IIa to IIe) based on additional conserved structural motifs outside the WRKY domain (Eulgem et al., 2000). It has been reported that some WRKY proteins play vital roles in response to biotic stress (such as pathogen infection) and in regulation of abiotic stress (such as hormone, wounding, high salinity, drought and cold) (Xie et al., 2005; Ryu et al., 2006; Eulgem and Somssich, 2007; Pandey and Somssich, 2009; Sun et al., 2013; Li et al., 2013). Through microarray analysis, two closely related WRKY transcription factors (WRKY25 and WRKY33) were identified in Arabidopsis and their transcripts were accumulated in plants under treatment with NaCl (Jiang and Deyholos, 2009). Overexpression of ZmWRKY33 in Arabidopsis induced expression of RD29A that is known to be involved in stress signaling, and enhanced salt tolerance of the transgenic plants (Li et al., 2013). Overexpression of Musa WRKY71 in the transgenic banana affected its oxidative and salt stress tolerance (Shekhawat and Ganapathi, 2013). Generally, multiple pathways are involved in plant response to high salinity, including ionic and osmotic homeostasis signaling, detoxification and growth regulation. Recently, ten TaWRKYs (designated TaWRKY1eTaWRKY10) were successfully identified. Among these genes, TaWRKY10 was observed to confer plant drought and salt tolerance by regulating osmosis and reducing ROS accumulation (Wang et al., 2013). Alleles of OsWRKY45-1 and OsWRKY45-2 play different roles in ABA signaling and salt tolerance in rice (Tao et al., 2011). WRKY8 antagonistically interacts with VQ9 to modulate salinity tolerance (Hu et al., 2013). Upland cotton (Gossypium hirsutum) is an important fiber crop in the world. However, soil salinity, which is one of the major abiotic stresses in world agriculture, often severely impacts cotton growth and development, leading to a significant loss in cotton crop yield. Therefore, it is important to enhance cotton saltresistance through genetic manipulation. In previous study, we identified 26 cotton WRKY genes, of which GhWRKY34 was induced in cotton under NaCl stress (Zhou et al., 2014). Up to now, however, data associated with cotton WRKY family TFs are still notably limited, and especially, little is known about the role of WRKY TFs in cotton response to salt stress. In present study, ectopic expression of GhWRKY34 in Arabidopsis enhanced the transgenic plant tolerance to high salinity stress. GhWRKY34 as a positive transcription regulator may function in plant response to high salinity stress through maintaining the Naþ/Kþ homeostasis as well as activating the salt stress-related genes in cells.
2. Materials and methods 2.1. DNA and protein sequence analysis The GhWRKY34 cDNA (accession number in GenBank: KJ825876) was identified in cotton cDNA libraries. Its nucleotide sequence and the deduced amino acid sequence were analyzed using DNAstar software (DNAstar Inc., USA). The peptide sequences were aligned with the ClustalW2 program (http://www.ebi.ac.uk). The conserved domains of the GhWRKY34 protein were confirmed at the National Center for Biotechnology Information (NCBI) (http:// blast.ncbi.nlm.nih.gov/Blast.cgi). Identification of protein domains and significant sites was performed with Motifscan (http://myhits. isb-sib.ch/cgi-bin/motif_scan). The phylogenetic tree was constructed by the neighbor-joining (NJ) method in MEGA5.0 program (http://www.megasoftware.net/), based on majority-rule consensus from 1000 bootstrap replicates.
2.2. RNA isolation and quantitative RT-PCR analysis To analyze the expression of GhWRKY34 and the NaClresponsive genes in the transgenic Arabidopsis plants, total RNA was isolated from two-week-old the transgenic Arabidopsis seedlings and wild type (Columbia ecotype) grown under normal conditions (CK) and under 100 mM NaCl treatments for 72 h. Then, the RNA was reversely transcribed into cDNAs which were used as templates in PCR analysis. Expression of GhWRKY34 and the NaClresponsive genes in the transgenic Arabidopsis plants was analyzed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) with gene-specific primers (Table 1), using the fluorescent intercalating dye SYBR-Green in a detection system (Opticon 2; MJ Research, Waltham, MA, USA). Arabidopsis ACTIN2 gene was used as a standard control in the RT-PCR reactions. A two-step RT-PCR procedure was performed in all experiments using the method described earlier (Li et al., 2005). For all the above quantitative real-time PCR analysis, the assays were repeated three times along with three independent repetitions of the biological experiments, and means of three biological experiments were calculated for estimating gene expression levels. 2.3. Subcellular location of GhWRKY34 protein The coding sequence (without stop codon) of GhWRKY34, amplified by PCR using proofreading Pfu DNA polymerase, was cloned into a pBI121-eGFP vector upstream the eGFP sequence, generating the pBI-WRKY34-eGFP construct with WRKY34:GFP fusion genes under the control of CaMV 35S promoter. Then, the construct was transferred into Agrobacterium tumefaciens strain LBA4404. Arabidopsis thaliana was transformed with Agrobacterium-mediated DNA transfer by the floral dip method. Transgenic seedlings were selected on MS medium containing 50 mg/L kanamycin. Five-day-old WRKY34:GFP transgenic seedlings (T2 generation) were stained with 40 6-diamidino-2-phenylindole (DAPI, a nucleus-spcific dye) for 15 min at room temperature before observation of GFP fluorescence and DAPI staining under the confocal fluorescence microscope (Leica, Germany). The digital images were taken and process by SP5 software (Leica, Germany) (Qin et al., 2014). 2.4. Construction of overexpression vector and Arabidopsis transformation The coding sequence of GhWRKY34 gene, amplified by PCR using proofreading Pfu DNA polymerase, was cloned into pMD vector
Table 1 Gene-specific primers used in real-time quantitative RT-PCR analysis. Genes
Primers
GhWRKY34
50 -ATGTGCAGACTTCGGAATCT-30 50 -GAGCTTCCTAATCGACTGAT-30 50 -TGAAAGGAGGAGGAGGAATGGTTGG-30 50 -ACAAAACACACATAAACATCCAAAGT-30 50 -CCAGATAGCGGAGGGGAAAGGACAT-30 50 -AAGTTCACAAACAGAGGCATCATCATCATAC-30 50 -GGCTTGAAGAAAGTGAGTCTCG-30 50 -GCTACATAGTTCGGAGTTCCACA-30 50 -TCGTTTCAGCCAAATCAGAAAGT-30 50 -TTTGCCTTGTGCTGCTTTCC-30 50 -AACAACTTAGGAGGTGGTGGTCAT-30 50 -TGTAGCAGCTGGCGCAGAAGTCAT-30 50 -GAAATGAAACTGGCTGATGAAACCATAGAG-30 50 -CTCGTGGCAATCTACTCGGTCTTAAACC-30 50 - GAAATCACAGCACTTGCACC-30 50 - AAGCCTTTGATCTTGAGAGC-30
AtRD29A AtRD29B AtSOS2 AtSOS1 AtABF4 AtCBL1 AtACT2
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driven by CaMV 35S promoter. Then, the construct was transferred into Agrobacterium tumefaciens strain LBA4404. A. thaliana was transformed with Agrobacterium-mediated DNA transfer by the floral dip method. Transformed Arabidopsis seeds were selected on MS medium containing 50 mg/L kanamycin. Homozygous lines of T3 generations were used for phenotypic analysis of transgenic plants. 2.5. Phenotypic evaluation of GhWRKY34 overexpression transgenic Arabidopsis To assay the seed germination rate and proportion of seedlings with opened green cotyledons, the surface-sterilized seeds from each transgenic Arabidopsis line and wild type, respectively, were sowed on MS plates supplemented with or without different concentration of NaCl, placed at 4 C for 3 days, and then moved to a growth room under a photoperiod of 16 h light/8 h dark at 22 C. The rate of seed germination (root emergence) was evaluated every day, and the rate of seedlings with opened green cotyledons was counted at the tenth day. Each experiment was repeated three
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times at least with identical results, and at least 300 seeds of each line were selected for analyzing the seed germination rate and cotyledon greening rate. To assay root elongation, five-day-old seedlings of the transgenic lines and wild type were cultured vertically on MS medium supplemented with different concentrations of NaCl for 7 days. Then, the length of roots was measured and calculated. All experiments were repeated at least three times, and 30e60 seedlings of each line were selected for vertical cultivation. For NaCl-irrigation experiment, Arabidopsis seedlings of the transgenic lines and wild type were transplanted into soil for further growing. The transgenic lines and wild type at same developmental stage (i.e. three-week-old wild type and transgenic seedlings, and five-week-old wild type and transgenic seedlings) were used for NaCl treatment, respectively. The transgenic plants and wild type (control) in pots were watered with the same volume 300 mM NaCl for continuous 7 days, and then recovered 7 days. The status of plant tolerance to high salinity (NaCl) were observed and photos were taken. The chlorophyll content was determined by the method
Fig. 1. Sequence analysis of GhWRKY34 protein. (A) Alignment of WRKY domains of GhWRKY34 with the other WRKY proteins. Conserved amino acid residues (WRKYGQK) and zinc-finger are shown in gray. (B) Phylogenetic relationship of GhWRKY34 and the other plant WRKY proteins. All 29 WRKY proteins can be divided into three groups (I, II and III) based on the number of WRKY domain and type of zinc-finger structure. Group II further were divided into five subgroups, including IIa, IIb, IIc, IId and IIe. The plant WRKY proteins used for the phylogenetic tree are AtWRKY8 (NP_199447), AtWRKY9 (NP_176982), AtWRKY10 (NP_175956), AtWRKY11 (NP_567878), AtWRKY14 (NP_564359), AtWRKY18 (NP_001031766), AtWRKY20 (NP_849450), AtWRKY22 (NP_192034), AtWRKY28 (NP_193551), AtWRKY30 (NP_568439), AtWRKY35 (NP_181029), AtWRKY36 (NP_564976), AtWRKY39 (NP_566236), AtWRKY40 (NP_178199), AtWRKY44 (NP_001078015), AtWRKY46 (NP_182163), AtWRKY53 (NP_194112), AtWRKY54 (NP_181607), AtWRKY60 (NP_180072), AtWRKY74 (NP_198217), BnWRKY22-1 (NP_001288962), JcWRKY46 (AGQ04237), GhWRKY31 (AJT43311), TcWRKY35 (EOX95271), TcWRKY46 (XP_007051596), DlWRKY6-1 (AEO31476), VaWRKY30 (AAR92477). At, Arabidopsis thaliana; Bn, Brassica napus; Gh, Gossypium hirsutum; Jc, Jatropha curcas; Tc, Theobroma cacao; Dl, Dimocarpus longan; Va, Vitis aestivalis.
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Fig. 2. Subcellular localization of GhWRKY34 protein. GhWRKY34 fused with eGFP gene under CaMV 35S promoter was expressed in hypocotyl cells of the transgenic Arabidopsis thaliana. GFP fluorescence signals were mainly detected in nuclei of the hypocotyl cells harboring the GhWRKY34:eGFP reporter construct. (A) GFP fluorescent image of the hypocotyl cell; (B) Nuclear DAPI staining of the same cell in A. (C) The transmitted light image of the same cell in image A; (D) The images of A and B were merged over the bright-field image. Bar ¼ 10 mm.
described earlier (Qin et al., 2013). In brief, chlorophyll in 0.2 g leaves of each transgenic line and wild type was extracted with 10 mL of 80% acetone, and chlorophyll content was assayed by measuring absorbance at 645 and 663 nm with a spectrophotometer. The assays were repeated three times along with three independent repetitions of the biological experiments. To measure Na⁺ and K⁺ contents, Arabidopsis leaves and roots of the transgenic lines and wild type grown under normal conditions or 100 mM NaCl for 3 weeks were harvested, rinsed with deionized water, dried at 70 C for at least 48h, and cooled in a desiccation
Fig. 4. Assay of cotyledon greening rate of GhWRKY34 overexpression transgenic Arabidopsis under NaCl stress. Mean values and standard errors (bar) are shown from three independent experiments. One or two asterisks represent there was represent significant difference (P < 0.05) or very significant difference (P < 0.01) in green cotyledon rate between the transgenic lines and wild type. MS, seedlings grew on MS medium without NaCl as control; 100 mM NaCl and 150 mM NaCl, seedlings grew on MS medium with 100 and 150 mM NaCl, respectively. WT, wild type; L13, L22 and L24, GhWRKY34 overexpression transgenic lines 11, 22 and 24.
chamber. Then 100 mg of each sample was digested with 0.1M HNO3 boiling for 30 min, and filtrated through a 0.22 mm filter membrane. The Na⁺ and K⁺ contents in the solution were measured with atomic absorption spectrophotometer. The experiments were performed independently for three times and the results were presented as a average values (Rus et al., 2001). 3. Results 3.1. Sequence analysis of GhWRKY34 protein GhWRKY34 cDNA (KJ825876) encoding a WRKY protein was identified from cotton cDNA libraries (Zhou et al., 2014).
Fig. 3. Assay of seed germination rate of GhWRKY34 overexpression transgenic Arabidopsis under NaCl stress. (A) Quantitative RT-PCR analysis of GhWRKY34 expression in the GhWRKY34 overexpression transgenic lines and wild type. (BeE) Statistical analysis of seed germination rate. Each curve represents an average of three replicates. MS, seeds germinated on MS medium without NaCl as control; 100 mM NaCl, 150 mM NaCl and 200 mM NaCl, seeds germinated on MS medium with 100, 150 and 200 mM NaCl, respectively. WT, wild type; L13, L22 and L24, GhWRKY34 overexpression transgenic lines 11, 22 and 24.
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GhWRKY34 protein contains an approximate 60-amino acid WRKY domain that is composed of the conserved amino acid sequence WRKYGQK. However, a zinc-finger motif in GhWRKY34 protein is C-X-C-X23-HeT-C, instead of C-X-C-X22e23-HeX-H, indicating that this protein is a member of WRKY group III. GhWRKY34 shares relatively high identity with TcWRKY46 (XP_007051596) (74%), VaWRKY30 (AAR92477) (58%), DlWRKY6-1 (AEO31476) (60%), and AtWRKY46 (NP_182163.1) (41%) in the WRKY domain and zincfinger motif (Fig. 1A). To investigate the evolutionary relationships among WRKYs from different species, phylogenetic analysis was performed with MEGA program, based on the amino acid sequences of GhWRKY34 and other plant WRKY proteins. Generally, plant WRKY proteins can be classified into three groups (I, II and III), of which group II is further divided into five subgroups (IIa e IIe). As shown in Fig. 1B, GhWRKY34 with TcWRKY46 forms a cluster in the tree, suggesting that GhWRKY34 is a member of group III of cotton WRKY family. 3.2. Subcellular localization of GhWRKY34 protein By PSORT program, we predicted that GhWRKY34 protein is located in the cell nucleus due to the existence of the putative nuclear localization signal (NLS, a short polypeptide consisting of four amino acid residues PKRR). To confirm the real location of GhWRKY34 protein, we constructed GhWRKY34:GFP fusion gene controlled by CaMV 35S promoter. Then the fusion gene was transferred into A. thaliana by Agrobacterium-mediated transformation. The GhWRKY34:GFP transgenic seedlings (T2 generation) were stained with DAPI, and then GFP fluorescence and DAPI staining in hypocotyl cells of the GhWRKY34:GFP transgenic
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seedlings was observed by confocal microscopy. As shown in Fig. 2, both the GFP fluorescence and DAPI staining were strongly accumulated in nuclei of hypocotyl cells, suggesting that GhWRKY34 protein was localized in the cell nucleus. 3.3. Overexpression of GhWRKY34 in Arabidopsis enhances plant tolerance to high salinity We have been already known that GhWRKY34 transcript was largely accumulated in leaves, and expression of GhWRKY34 was strongly up-regulated in cotton seedlings under NaCl treatment, compared with that in cotton seedlings without NaCl treatment, suggesting that GhWRKY34 may be involved in plant response to salt stress (Zhou et al., 2014). To examine the role of GhWRKY34 response to salt, GhWRKY34 gene under the control of CaMV 35S promoter was introduced into Arabidopsis via Agrobacterium mediated DNA transfer. More than thirty independent transgenic lines were obtained, and the homozygous transgenic progenies (T3 generations) were selected through kanamycin-resistance assay and PCR analysis. GhWRKY34 expression levels in transgenic lines were examined by quantitative RT-PCR analysis. The representative GhWRKY34 overexpression transgenic lines L13, L22 and L24, which exhibited different GhWRKY34 expression levels, were selected for the following experiments (Fig. 3A). To confirm the response of GhWRKY34 to salt, we tested the seed germination capacity on MS medium with 0 (control), 100, 150 and 200 mM NaCl. Under normal conditions (without NaCl treatment), there was no significant difference between GhWRKY34 transgenic lines and wild type. Although GhWRKY34 transgenic lines and wild type showed little difference in seed germination on MS medium with 100 mM NaCl,
Fig. 5. Assay of root length of GhWRKY34 overexpression transgenic Arabidopsis under NaCl stress. (A) Wild type and GhWRKY34 overexpression seedlings grew on MS medium. (B) Wild type and GhWRKY34 overexpression seedlings grew on MS medium with 100 mM NaCl. (C) Statistical analysis of root length of wild type and GhWRKY34 overexpression seedlings grown on MS medium without or with 100 mM NaCl. Seeds germinated on MS medium for 7 days and then the seedlings were transferred onto MS medium supplemented with 0 (control) and 100 mM NaCl for 7 days. Mean values and standard errors (bar) are shown from three independent experiments. One asterisk represents there was represent significant difference (P < 0.05) in root length between the transgenic lines and wild type. MS, seedlings grew on MS medium without NaCl as control; 100 mM NaCl, seedlings grew on MS medium with 100 mM NaCl. WT, wild type; L13, L22 and L24, GhWRKY34 overexpression transgenic lines 11, 22 and 24.
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however, the GhWRKY34 transgenic lines displayed higher tolerance to salt stress than wild type under 150 and 200 mM NaCl treatments. On MS medium with 200 mM NaCl, seed germination rates of three GhWRKY34 transgenic lines were 91% (L13), 89% (L22) and 92% (L24), respectively, while wild type was only 52% (Fig. 3BeE). Statistical analysis revealed there was significant difference in seed germination between the transgenic lines and wild type. Meanwhile, we also calculated the rates of cotyledon greening of the transgenic lines and wild type. On MS medium, we found the rates of cotyledon greening of the GhWRKY34 transgenic lines were similar to that of wild type. Under 100 and 150 mM NaCl treatments, however, the number of seedlings with opened green cotyledons of GhWRKY34 transgenic lines was much more than that of wild type, especially on MS with 150 mM NaCl (Fig. 4). In addition, seedlings of the transgenic lines and wild type were cultured vertically on MS medium with different NaCl concentrations for one week, and then the root length of these seedlings was measured. As shown in Fig. 5A, root length of the GhWRKY34 transgenic seedlings was little altered on MS medium, compared with that of wild type. On the contrary, root length of the GhWRKY34 transgenic seedlings were remarkably longer than that of wild type under 100 mM NaCl treatment (Fig. 5B). Measurement and statistical analysis revealed that there was significant difference in root length between the transgenic lines and wild type under NaCl stress (Fig. 5C). These results indicated that GhWRKY34 enhance plant tolerance to high salinity stress during early plant development. To further investigate the role of GhWRKY34 gene in plant tolerance to high salinity at the late developmental stages, wild type and the transgenic plants (T2 and T3 generations) grown in soil were watered with NaCl solution (see Methods). As shown in Fig. 6, wild type and GhWRKY34 overexpression transgenic plants grown under salt stress occurred growth retardation, compared with those plants grown in normal conditions. The 3-week-old Arabidopsis plants grown in pots were watered with 300 mM NaCl solution. After several days, wild type plants became yellow and wilting, whereas the transgenic plants were still green (Fig. 6B). Likewise, the five-week-old transgenic plants after watering with 300 mM NaCl solution for several days also displayed higher salttolerance than wild type (Fig. 6D). Environmental stressors (such as salt and drought stresses) may cause a change in chlorophyll content in plants. To determine whether chlorophyll content is altered in the transgenic plants under salt stress, we detected the chlorophyll content in leaves of the GhWRKY34 transgenic seedlings and wild type grown under normal conditions (control) or under NaCl stress. As shown in Fig. 6E, there was no significant difference in chlorophyll cotent between the transgenic lines and wild type in normal growth conditions. Under NaCl treatment, however, chlorophyll content in leaves of the GhWRKY34 transgenic lines was remarkably higher than that in wild type although chlorophyll content was declined in both transgenic plants and wild type. These results suggested overexpression of GhWRKY34 in Arabidopsis confers higher salt-tolerance on transgenic plants and GhWRKY34 may play a positive role in response to salt stress during cotton growth and development. 3.4. Overexpression of GhWRKY34 in Arabidopsis influences Naþ/ Kþ homeostasis in plant cells under salt stress Plant salt tolerance is closely related to the ability of a plant to maintain a low cytosolic Naþ/Kþ ratio (Shabala and Cuin, 2007). Thus, to analyze the mechanism of enhancing salt tolerance in GhWRKY34 overexpression transgenic Arabidopsis, the Naþ and Kþ contents in leaves and roots of wild type and the transgenic plants with or without 100 mM NaCl treatment were determined by
Fig. 6. Phenotypic assay of GhWRKY34 transgenic Arabidopsis plants under NaCl stress. (A) Five-week-old seedlings grew in soil under normal conditions. (B) Five-week-old seedlings grew in soil watered with 300 mM NaCl. (C) Seven-week-old plants grew in soil under normal conditions. (D) Seven-week-old seedlings grew in soil watered with 300 mM NaCl. (E) Assay of chlorophyll content in leaves of four-week-old GhWRKY34 overexpression transgenic lines and wild type under normal conditions and under NaCl stress. Mean values and standard errors (bar) are shown from three independent experiments. One and two asterisks represent there was represent significant difference (P < 0.05) and very significant difference (P < 0.01) in leaf chlorophyll content between the transgenic lines and wild type, respectively. CK, seedlings grew in soil under normal conditions (without NaCl) as controls; NaCl, seedlings grew in soil under NaCl treatment; WT, wild type; L13, L22 and L24, GhWRKY34 overexpression transgenic lines 11, 22 and 24.
atomic absorption spectrophotometer. As shown in Fig. 7, there was no significant difference in Naþ content, Kþ content and Naþ/Kþ ratio in leaves and roots between wild type and the transgenic lines under normal conditions. However, Naþ content in both leaves and roots of wild type under salt stress was remarkably increased and significant higher than that in the transgenic lines, although Naþ content in the transgenic lines was also increased in the same conditions (Fig. 7A and 7C). Meanwhile, Kþ content was decresed in leaves and roots of wild type, but dramatically increased in leaves and roots of the GhWRKY34 overexpression transgenic lines (Fig. 7B and 7D). Therefore, GhWRKY34 overexpression transgenic plants displayed significant lower Naþ/Kþ ratio in leaves and roots than wild type under salt stress (Fig. 7E and 7F). These results suggested
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Fig. 7. Changes in Naþ content, Kþ content and Naþ to Kþ ratio in leaves and roots of GhWRKY34 overexpression transgenic Arabidopsis under NaCl stress. (A, B) Naþ and Kþ contents in leaves of the transgenic lines and wild type under normal conditions or 100 mM NaCl for 3 weeks. (C, D) Naþ and Kþ contents in roots of the transgenic lines and wild type under normal conditions or 100 mM NaCl for 3 weeks. (E) Naþ/Kþ ratio in leaves calculated from the data in A and B. (F) Naþ/Kþ ratio in roots calculated from the data in C and D. Each column is the mean of three independent experiments and bars represent standard errors. One and two asterisks represent there was represent significant difference (P < 0.05) and very significant difference (P < 0.01) between the transgenic lines and wild type, respectively. CK, seedlings grew under normal conditions (without NaCl); 100 mM NaCl, seedlings grew under 100 mM NaCl treatment for 3 weeks; WT, wild type; L13, L22 and L24, GhWRKY34 transgenic lines 13, 22 and 24.
that overexpression of GhWRKY34 in Arabidopsis may enhance plant ability of selectively uptaking Naþ and Kþ, and thereby maintain a low Naþ/Kþ ratio in the leaves and roots, conferring salt tolerance on transgenic plants. 3.5. Expression of the salt stress-related genes is upregulated in the GhWRKY34 transgenic plants To further investigate the role of GhWRKY34 in response to salt, the expression levels of salt stress-related genes, including RD29A, RD29B, ABF4, SOS1, SOS2 and CBL1 that act as markers for monitoring salt stress response pathways in Arabidopsis, were examined in the transgenic seedlings under both normal condition and NaCl treatment. As shown in Fig. 8, expressions of four genes (RD29A, ABF4, SOS1 and SOS2) were remarkably enhanced in the transgenic plants compared with those in wild type. Furthermore, all of the six genes were up-regulated in both GhWRKY34 transgenic lines and wild type after NaCl treatment. However, expression levels of these genes in the transgenic plants were dramatically higher than those in wild type under salt stress. Statistical analysis revealed there were significant differences in expression levels of these genes between the transgenic lines and wild type, suggesting that GhWRKY34 may participate in response to salt stress by regulating expressions of the stress-related genes during plant growth and
development. 4. Discussion WRKY transcription factors are a large family of regulatory proteins in plants. Although some researchers indicated that plant WRKY transcription factors are involved in biotic and abiotic stresses, the most of these studies mainly focus on the model plants, such as A. thaliana and rice, and little is known about the role of WRKY transcription factors in cotton. Previously, we identified 26 WRKY genes in cotton (Zhou et al., 2014). In this study, our data revealed that GhWRKY34 is classified into group III of cotton WRKY family. By means of PSORT program we found a predicted nuclear localization signal (NLS) sequence KKRK in GhWRKY34 protein. Further subcellular localization assay indicated that GhWRKY34 was localized to the cell nucleus, suggesting that GhWRKY34 may be a transcription factor in cotton. Soil salinity is one of the most serious threats in world agriculture. It is expected that soil salinity will result in the loss of up to 50% fertile land by the middle of the 21st century (Manchanda and Garg, 2008). High salinity imposes imbalance in osmotic, ionic and secondary oxidative stresses upon plant cells, and ultimately affects plant growth and development (Zhu, 2001). It has been demonstrated that a large number of transcription factors, including basic
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Fig. 8. Quantitative RT-PCR analysis of expressions of the NaCl stress-related genes in GhWRKY34 overexpression transgenic Arabidopsis. Total RNA was isolated from two-week-old Arabidopsis seedlings grown under normal conditions (CK) and under 100 mM NaCl treatments for 72 h. Relative value of the expression of RD29A, RD29B, ABF4, SOS2, SOS1 and CBL1 in Arabidopsis was shown as percentage of AtACTIN2 expression activity. Mean values and standard errors (bar) were shown from three independent experiments. One and two asterisks represent there was represent significant difference (P < 0.05) and very significant difference (P < 0.01) in gene expression level between the transgenic lines and wild type, respectively. CK, seedlings grew under normal conditions (without NaCl); þNaCl, seedlings grew under NaCl treatment; WT, wild type; L13, L22 and L24, GhWRKY34 transgenic lines 13, 22 and 24.
leucine zipper (bZIP), WRKY, APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF), MYB, basic helixeloopehelix (bHLH) and NAC families, are integral in linking salt sensory pathways for plant stress-tolerance responses (Kasuga et al., 1999; Tran et al., 2004; Yang et al., 2009; Jiang and Deyholos, 2009; Cui et al., 2013; Cheng et al., 2013). So far, there are increasing evidence that WRKY proteins regulate salt stress and even multiple abiotic stress responses. For example, overexpression of ZmWRKY33 in Arabidopsis induced expression of RD29 which is known to be involved in stress signaling, and enhanced salt tolerance of the transgenic plants (Li et al., 2013). GhWRKY15 transcripts were accumulated in cotton seedlings for responding to biotic stress modulators (such as salicylic acid and methyl jasmonate) and could impart the transgenic tobacco plants tolerance to fungal pathogens (Yu et al., 2012). MusaWRKY71 transcripts in banana plants were found to be upregulated by cold, dehydration, salt, ABA, H2O2, ethylene, salicylic acid (SA) and methyl jasmonate (MJ) (Shekhawat et al., 2011). Similarly, we found GhWRKY34 was induced by NaCl (Zhou et al., 2014). In this study, our data revealed that overexpression of GhWRKY34 in Arabidopsis conferred higher salt-tolerance on the transgenic plants, compared with wild type. The seed germination rate, cotyledon greening and root length of GhWRKY34 transgenic seedlings at early developmental stages were significantly higher than those of wild type in the presence of NaCl. Furthermore, the transgenic plants still dispayed more salt-tolerance and higher chlorophyll content than wild type at the stages of adult development. These results suggested that GhWRKY34 protein may function as a positive modulator in plant response to high salinity stress. To get further insight to the mechanism of enhancing salt tolerance in GhWRKY34 overexpression transgenic Arabidopsis
under salt stress, the Naþ and Kþ contents in leaves and roots of the transgenic plants were analyzed. Under salt stress, the Naþ/Kþ homeostasis in plants was usually disrupted. Plants absorb the large numbers of Naþ ions which could lead to growth cessation or cell death instead of Kþ ions which have vital functions in maintaining many enzyme activities and osmotic regulation, so the Naþ/ Kþ ratio is closely related to plant salt tolerance (Zhu, 2003). Therefore, an important strategy for plants against salt stress is inhibiting Naþ influx, and increasing Kþ uptake and Naþ extrusion for maintaining a low Naþ/Kþ ratio in cells under high salinity environment (Yokoi et al., 2002). In our study, compared to wild type, the Naþ/Kþ ratio maintained relatively stable in both leaves and roots of GhWRKY34 overexpression transgenic plants grown under normal conditions or under salt stress. The Naþ content in the GhWRKY34 overexpression transgenic plants was significantly lower, whereas the Kþ content was higher than those in wild type under salt stress conditions, indicating that GhWRKY34 transgenic plants may keep relatively strong ability to maintain Naþ/Kþ homeostasis in cells, thereby enhancing plant salt tolerance. To investigate if GhWRKY34 affects the expression of salt stressrelated genes in plants, some marker genes were analyzed in the GhWRKY34 transgenic Arabidopsis lines and wild type. The results indicated that the expression levels of RD29A, RD29B, ABF4, SOS2, SOS1 and CBL1 genes increased remarkably in the transgenic plants, compared with those in wild type, after NaCl treatment. It has been demonstrated that calcineurin B-like protein1 (CBL1) is a member of calcineurin B-like calcium sensor proteins family and interacts with CBL-interacting protein kinase23 (CIPK23). CBL1 may act as a regulatory subunit of a plant calcineurin-like activity mediating calcium signaling under certain stress conditions. The CBL1
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expression level was increased in response to specific stresses such as high salinity, drought, cold and wounding (Hashimoto et al., 2012; Gao et al., 2008). The ABRE-binding factor 4 (ABF4) bZIP transcription factor specifically interacts with abscisic acid (ABA)responsive elements (ABREs) and functions as transcriptional activator in ABRE-dependent transcription in Arabidopsis. The expression of ABF4 in Arabidopsis was enhanced by dehydration, salt treatment, and endogenous ABA treatment (Uno et al., 2000). In this study, we found that overexpression of GhWRKY34 promoted ABF4 and CBL1 expression in the transgenic plants under normal growth conditions and under NaCl treatment, suggesting that GhWRKY34 as a positive regulator regulates these genes in the transgenic Arabidopsis. It was reported that RD29A (responsive to dessication 29A) and RD29B (responsive to dessication 29A) were induced by conditions of dehydration, low temperature, high salinity or by treatment with exogenous ABA. The promoter region of RD29A contains the cis-acting dehydration-responsive element (DRE) that is involved in expression of RD29A rapidly responding to dehydration and high salinity stresses in Arabidopsis. Similarly, the promoter region of RD29B contains two ABA-responsive elements (ABREs) that are required for the dehydration-responsive expression of RD29B in Arabidopsis (Kazuko and Kazuo, 1994). Here, the transcription levels of RD29A and RD29B were dramatically increased in the GhWRKY34 transgenic plants under high salinity stress. This may also help to explain the observation that GhWRKY34 transgenic plants had stronger salt-tolerance than wild type. In addition, the SOS pathway is an important regulatory system activated by salt stress. SOS2 is an important signal transducer which could activate the downstream target gene, SOS1, which encodes a plasma membrane Na⁺/H⁺ antiporter that regulates Na⁺/K⁺ homeostasis under saline conditions (Xiong et al., 2002). In present study, overexpression of GhWRKY34 promoted expression of SOS1 and SOS2 genes in the transgenic plants under both normal conditions and salt stress, implying that GhWRKY34 may be involved in regulation of Naþ and Kþ homeostasis through activating the SOS pathway with enhancing activity of the Naþ/Hþ antiporter, SOS1. Besides, owing to WRKY transcription factors are thought to function by binding their cognate TTGACC/T W-box ciselements in the promoter regions of target genes, the promoter sequences of RD29A, RD29B, AtSOS1, AtSOS2, ATABF4 and ATCBL1 were analyzed with the PlantCARE analysis database (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/). We found that AtSOS2 contains four W-box cis-elements and AtABF4 has one W-box cis-element in their promoter regions, respectively. Therefore, we speculate that GhWRKY34 may bind to the W-box ciselements in the promoters of AtSOS2 and AtABF4 in response to salt stress. In brief, based on the data presented in this study, we hypothesize that GhWRKY34 may be a positive transcription regulator in plant response to high salinity stress through maintaining the Naþ/Kþ homeostasis as well as activating expression of the salt stress-related genes in cotton. Authors' contributions XBL conceived and designed the experiments. LZ, NNW, SYG, RL and YL performed the experiments. LZ, NNW and XBL analyzed the data and wrote the paper. Acknowledgment This work was supported by National Natural Sciences Foundation of China (Grant No. 31471542) and the project from the Ministry of Agriculture of China for transgenic research (Grant No. 2014ZX08009-27B).
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