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. 2018 May 28;8(6):277. doi: 10.1007/s13205-018-1301-4

The grapevine VvWRKY2 gene enhances salt and osmotic stress tolerance in transgenic Nicotiana tabacum

Rim Mzid 1,✉, Walid Zorrig 1, Rayda Ben Ayed 2, Karim Ben Hamed 1, Mariem Ayadi 2, Yosra Damak 1, Virginie Lauvergeat 3, Mohsen Hanana 1
PMCID: PMC5972082  PMID: 29872608

Abstract

Our study aims to assess the implication of WRKY transcription factor in the molecular mechanisms of grapevine adaptation to salt and water stresses. In this respect, a full-length VvWRKY2 cDNA, isolated from a Vitis vinifera grape berry cDNA library, was constitutively over-expressed in Nicotiana tabacum seedlings. Our results showed that transgenic tobacco plants exhibited higher seed germination rates and better growth, under both salt and osmotic stress treatments, when compared to wild type plants. Furthermore, our analyses demonstrated that, under stress conditions, transgenic plants accumulated more osmolytes, such as soluble sugars and free proline, while no changes were observed regarding electrolyte leakage, H2O2, and malondialdehyde contents. The improvement of osmotic adjustment may be an important mechanism underlying the role of VvWRKY2 in promoting tolerance and adaptation to abiotic stresses. Principal component analysis of our results highlighted a clear partition of plant response to stress. On the other hand, we observed a significant adaptation behaviour response for transgenic lines under stress. Taken together, all our findings suggest that over-expression of VvWRKY2 gene has a compelling role in abiotic stress tolerance and, therefore, would provide a useful strategy to promote abiotic stress tolerance in grape via molecular-assisted breeding and/or new biotechnology tools.

Electronic supplementary material

The online version of this article (10.1007/s13205-018-1301-4) contains supplementary material, which is available to authorized users.

Keywords: Vitis vinifera, WRKY, Over-expression, Salt tolerance, Osmotic regulation

Introduction

Among the abiotic stresses, salinity and drought are two of the most widespread soil degradation factors on the earth. The majority of crops are highly susceptible to these constraints; therefore, there is an urgent need to deal with this problem. Grapevine (Vitis vinifera L.) is considered to be one of the major fruit crops in the world based on the cultivated surface area and economic value with total fruit production in 2013 as 771 Million quintals (FAO 2016: http://faostat.fao.org/beta/en/#data/QC 24/10/2016). Despite the multigenic nature of abiotic stress tolerance, the classical breeding approach for abiotic tolerance in grape is relatively slow (Yamaguchi and Blumwald 2005). For this reason, the transgenic approach seems to be more attractive and appropriate, than the classical breeding method, to improve grapevine species. In fact, the expression of many genes is regulated under these stress constraints, enabling plants to adapt to these conditions (Dorothea and Ramanjulu 2005).There are some studies indicating abiotic stress tolerance improvement by grapevine genes over-expression using Nicotiana tabacum plant model, namely, those related to VvSUC27 (Caiet al. 2017), Vv-α-gal/SIP (Daldoul et al. 2018), VvDhn (Jardak et al. 2017), VvRD22 (Jamoussi et al. 2014), VvbZIP23 (Tak and Mhatre 2013a) and VvSDIR1 (Tak and Mhatre 2013b) as candidate genes. The transcriptional regulation of these genes is under the control of various transcription factors (TFs) (Seki et al. 2002; Zhu 2002); such as the WRKY TFs. The latter belong to a multigenic family characterized by the presence of one or two WRKY conserved domains of about 60 amino acids containing the WRKYGQK sequence and followed by a zinc-binding motif (Eulgem et al. 2000).

The WRKY genes are well-known to be part of various developmental and physiological mechanisms, including disease resistance (Wu et al. 2016), senescence (Rinerson et al. 2015) growth and developmental processes (Guillaumie et al. 2010; Chen et al. 2016). In addition, several genetic and molecular studies demonstrated that WRKY genes are involved in plant response to abiotic stresses (Nuruzzaman et al. 2014; Tripathi et al. 2014; Xi et al. 2016). In fact, dehydration, salt, and cold stresses were reported to enhance the expression of numerous WRKY genes in rice, Arabidopsis thaliana and other crop plants (Seki et al. 2002; Song et al. 2009; Wu et al. 2016; Phukan et al. 2016).

After the release of the whole genomic sequences of highly homozygous and heterozygous grapevine (Jaillon et al. 2007; Troggio et al. 2007; Velasco et al. 2007), a genome-wide identification of WRKY family members and the characterization of their response to cold stress were performed by Wang et al. (2014). Moreover, the evolution study and the expression analysis of this family upon a broad range of abiotic and biotic stresses revealed its key role in the grape adaptation to environmental constraints (Guo et al. 2014). Many genes from this family have been already identified and characterized in different grapevine species such as VvWRKY1 (Marchive et al. 2007) and VvWRKY2 (Mzid et al. 2007) from Vitis vinifera, in addition to VpWRKY1, VpWRKY2 (Li et al. 2010) and VpWRKY3 (Zhu et al. 2012) from Vitis pseudoreticulata.

Particularly, VvWRKY2 is constitutively, yet variably, expressed in the different organs of healthy grapevine plants (supplemental file 1). In leaves, VvWRKY2 is induced by wounding and after infection with Plasmopara viticola. Its over-expression in tobacco plants induced an enhanced tolerance to necrotrophic fungal pathogens (Mzid et al. 2007). However, the role of this gene in improving the tolerance to abiotic stress such as salinity and drought remains unknown.

The current work aimed to investigate the biological role of VvWRKY2 gene in salt and osmotic stress tolerance based on physiological studies of three independent transgenic lines of tobacco, which can be used in genetic programme to improve the tolerance of grapevine to abiotic stress.

Materials and methods

Plant material and growth conditions

Full length of VvWRKY2 coding sequence (AY596466) was amplified by PCR from cDNA library with the primers W2-5# (5#-AAGTAATATTGATCAATGGCTGAA-3#) and W2-3 # (5#-GATATTCTACATCGCCTTGCC-3#) using Pfu polymerase (Promega). This cDNA was first cloned into the pGEM-T vector for sequencing validation and then transferred into the pGREEN vector (http://www.pgreen.ac.uk) under the 35S promoter of the cauliflower mosaic virus using restriction enzymes. The construct was introduced into Agrobacterium GV3010 strain for tobacco (Nicotiana tabacum cv. Xanthi) genetic transformation. Regenerated plants were transferred to soil pots and grown in greenhouse. The genotyping of these plants was carried out by PCR with a specific set of primers.

Wild type (WT) tobacco seeds and transgenic tobacco plants obtained from homozygous T2 generations over-expressing VvWRKY2 gene were used in the present study. After performing a semi-quantitative RT-PCR, three transgenic lines (W2-5, W2-11, and W2-12) with different expression levels were analyzed (Mzid et al. 2007). The untransformed WT N. tabacum cv. Xanthi line was used as a control. Tobacco plants were grown in vitro at 25 °C on half strength MS medium (Murashige and Skoog 1962) under a 16 h/8 h photoperiod and a temperature range of 25/20 °C. In all experiments, plant tissues were harvested, frozen in liquid N2 and stored at − 80 °C before analysis.

Performance of transgenic tobacco plants under stress treatment

Seeds of WT and transgenic plants were treated with 70% ethanol for 5 min and then sterilized with 15% bleach. After washing five times with sterile distilled water, the seeds were placed on solidified half strength MS medium. They were stratified at 4 °C for 3 days and then germinated under long-day conditions (15 h light/9 h dark) at 25 °C.

For NaCl or mannitol treatment, 25 seeds from WT and each transgenic line were placed in 120 mm square plastic Petri dishes that were sealed with Parafilm to prevent evaporation. The sensitivity of T2 seed germination to NaCl or mannitol (to mimic a water deficit) was assayed on half-strength MS agar plates including NaCl (0, 100, 200 and 300 mM) or mannitol (0, 100, 200 and 400 mM). Germination kinetic was followed every 3 days up to 21 days. The percentage of germinated seeds was scored as the germination rate.

For physiological analysis, seedlings were transplanted in a magenta box containing fresh medium supplemented with the same concentration of NaCl or mannitol to monitor plantlet growth.

Analysis of relative electrolyte leakage

Membrane damage was assessed by measuring relative electrolyte leakage (REL) from leaf discs. Electrical conductivity was measured (value A), after which the tubes were autoclaved to release all electrolytes for the second determination of the total content of electrolytes in each sample. Ion leakage from the leaf discs was measured as the conductivity of the solution with a pH meter conductimeter (model Jenway). After cooling to room temperature, the conductivity of each sample solution was measured again (value B). For each measurement, REL was expressed as percentage leakage (%), i.e. (value A/value B) ×·100.

Measurement of malondialdehyde (MDA) contents

The level of lipid peroxidation in leaf tissues was determined in terms of the peroxidation by MDA product in samples. The MDA content was determined by the reaction of the thiobarbituric acid (TBA) as described by Hagege et al. (1990). Briefly, 100 mg of plant material were ground in 4 ml of 20% trichloroacetic acid solution (TCA). A volume of 0.1 ml TBA (5 mg/ml) was added to the homogenized samples and the homogenates were incubated at 95 °C for 30 min, centrifuged at 15,000×g for 10 min and 0.55 ml of the supernatant fraction was mixed with 0.55 ml of TBA solution (25% thiobarbituric acid). The mixture was heated at 95 °C for 30 min, chilled on ice, and then centrifuged at 10,000×g for 5 min. The absorbance of the supernatant at 532 nm was read and the value of MDA was estimated using the molar extinction coefficient of 155 mM−1 cm−1.

Hydrogen peroxide assay

H2O2 concentration in leaf samples was determined following the protocol of Velikova et al. (2000). Fresh material (0.1 g) was homogenized with 5 ml of 0.1% TCA and centrifuged at 12,000 rpm for 15 min. To 0.5 ml of the supernatant, 0.5 ml of phosphate buffer (10 mM pH 7), and 1 ml of 1M potassium iodide were added. The absorbance of the solution was taken at 390 nm using a UV visible spectrophotometer. The content of H2O2 was determined using a calibration curve obtained by plotting the rate of intensity increase as a function of H2O2 concentration.

Sugar content analysis

The total soluble sugar contents (sucrose, glucose, fructose and their methylated derivative polysaccharides) were determined using the method of Dubois et al. (1956). Plant materials (100 mg) were extracted using 10 ml of 80% ethanol (V/V) and preheated to 80 °C. The mixture was vigorously vortexed and then placed in a water bath at 70 °C for 30 min. Few drops of ethanol were added occasionally to replace evaporative losses. The alcoholic extract (0.05 ml) was mixed with 5 ml anthrone (100 mg anthrone, 50 ml of concentrated sulphuric acid) and 2.45 ml of ethanol 80% (V/V)). Samples were placed in a boiling water bath for 10 min. The light absorption of the samples was estimated at 640 nm using a PD-303 model spectrophotometer. The concentrations of soluble sugars in samples were calculated by referring to a standard curve of glucose. Soluble sugar contents were expressed as mg glucose g−1 FW of leaves. Finally, the results of the optical densities were reported on the calibration curve of soluble sugars (expressed as glucose).

Determination of proline contents

Proline contents in leaves were determined following the method of Bates (1973) with slight modifications. Fresh plant materials (100 mg) were frozen in liquid N2 and treated with 3 ml of sulphosalicylic acid (3%) for 1 h at 100 °C. About 0.2 ml of the filtered phase was added to 2 ml of solution mixture (composed of 30 ml of glacial acetic acid, 20 ml distilled water and 0.5 g of ninhydrin reagent). The mixture was boiled for one hour. The reaction was stopped by placing tubes in cold water. Samples were vigorously mixed with 4 ml of toluene. The absorbance of toluene phase was estimated at 520 nm against toluene blank. Proline concentration was estimated by referring to a standard curve of proline. Free proline content was expressed as µg g−1 FW of plant sample.

Phylogenetic tree construction

For the phylogenetic analyses, amino acid sequences (for VvWRKY2 protein and some selected AtWRKY proteins) were aligned by CLUSTALW program (http://www.genome.jp/tools/clustalw/) (Higgins et al. 1994) and imported into the Molecular Evolutionary Genetics Analysis (MEGA) package version 6 (http://www.megasoftware.net) (Tamura et al. 2013). All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). Phylogenetic analyses were carried out using the neighbour-joining method (Saitou and Nei 1987), the minimum evolution method (Rzhetsky and Nei 1992), the UPGMA method (Sneath and Sokal 1973), the maximum-likelihood method (Jones et al. 1992) and the maximum parsimony method (Eck and Dayhoff 1966), along with a statistical bootstrapping procedure involving 10,000 replicates. Numbers at the branches with confidence values are based on Felsenstein’s bootstrap method (Felsenstein 1985). Evolutionary distances used to infer the phylogenetic tree were computed using the Poisson correction method (Zuckerkandl and Pauling 1965).

Data treatment and statistical analysis

Results presented in all histograms, tables and figures are the means of three independent replicates ± Confidence Interval (CI). Data were subjected to a one-way ANOVA test using XLSTAT software v. 2014 (Addinsoft; http://www.xlstat.com) and means were compared according to Duncan’s multiple-range test at 5% level of significance.

The principal component analysis (PCA) was done using XLSTAT software v. 2014, considering variables centred around their means and normalized with a standard deviation of 1.

Results

Phylogenetic analysis of the VvWRKY2

To investigate the relationships of the VvWRKY2 protein with other WRKYs from A. thaliana, a phylogenetic analysis was performed based on their amino acid sequences using the neighbour-joining method. The results demonstrated that VvWRKY2 was closely related to group I of WRKY family members, including AtWRKY3 and AtWRKY4. In addition, a clear orthology relationship associated VvWRKY2 to AtWRKY3 and AtWRKY4 sequences with, respectively, 58.44 and 54.21% of similarity. These results further confirmed the belonging of VvWRKY2 protein to the group I of WRKY family (Fig. 1).

Fig. 1.

Fig. 1

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The Phylogenetic tree constructed by the MEGA program version 6 (Tamura et al. 2013). Different methods were used; they gave similar results. The presented tree was the optimal tree obtained using the neighbour-joining method (Saitou and Nei 1987) and corresponds to a bootstrap consensus tree inferred from 10,000 replicates. The numbers at the branches are confidence values based on Felsenstein’s bootstrap method (Felsenstein 1985). The tree is drawn to scale, with branch lengths corresponding to the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated, leaving a total of 58 positions in the dataset. The evolutionary distances used to infer the phylogenetic tree were computed using the Poisson correction method (Zuckerkandl and Pauling 1965). Proteins for which a functional role at the plant level has been ascribed are indicated in bold. All positions containing gaps and missing data were eliminated. The analysis involved 66 amino acid sequences

Plants over-expressing VvWRKY2 gene showed improved germination under saline and drought constraints

To investigate the function of VvWRKY2 in the germination process, the seeds of transgenic tobacco lines over-expressing VvWRKY2 were cultivated under saline conditions. The seedling behaviours of three different transgenic lines were compared under different saline concentrations (0, 100, 200 and 300 mM NaCl). Germination rates of WT seeds and transgenic lines under control conditions were similar and did not show any significant difference (Fig. 2a). However, under NaCl concentrations higher than 100 mM, the germination percentages of WT were lower than those of transgenic lines. In particular, transgenic line 5 behaved better than the others, showing a significantly higher germination percentage than control plants under salinity.

Fig. 2.

Fig. 2

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Seed germination of WT (control) and VvWRKY2 transgenic tobacco under salt (a), and water stress conditions (b). Each value is a mean of four separate biological replicates. Error bars are ± CI. For each parameter, bars labelled with different letters are significantly different according to Duncan’s test at P ≤ 0.05

Under 300 mM of NaCl, the germination capacity of WT seeds decreased to less than 5%, whereas for transgenic lines it reached 30%, indicating clearly the involvement of VvWRKY2 in salinity tolerance at the germination stage (Fig. 2a, 3a1, a2).

Fig. 3.

Fig. 3

Fig. 3

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Effect of salt stress (300 mM NaCl) on VvWRKY2 transgenic tobacco plants grown under in vitro (a1, a2) and greenhouse conditions (b1, b2, c1, c2)

For further evaluation of the salt tolerance of transgenic plants, 2-week-old seedlings grown in soil were exposed to 300 mM NaCl solution for 2 weeks. The results showed that, under high salinity, more transgenic plants survived than the control ones and some of them grew vigorously (Fig. 3b, c). Although, in Fig. 3 it seems that transgenic plants grew faster than WT under control condition; this observation needs more significant analyses to be well established.

To assess the implication of VvWRKY2 in osmotic constraint adaptation, tobacco seeds of WT and transgenic lines were germinated under osmotic stress using different concentrations of mannitol (0, 100, 200 and 400 mM). As shown in Fig. 2b, there was no visible difference between WT and transgenic lines on control medium without mannitol. However, starting from 200 mM mannitol, the WT germination rate began to fall up to 50%, whereas the transgenic lines maintained the same level of germination as for 0 mM mannitol. Under higher mannitol concentrations, the transgenic lines displayed better germination rates, particularly under 400 mM where they reached approximately 35% of germination, showing thus the implication of VvWRKY2 in osmotic stress adaptation.

The increased germination in the three independent transgenic (W2-5, W2-11 and W2-12) lines excluded any insertional effects and confirmed the role of VvWRKY2 in the tolerance phenotype. Furthermore, these results indicate that the over-expression of the VvWRKY2 gene improved the germination of seeds under both salt and osmotic constraints. The transgenic lines W2-5, displaying the highest germination rate under both salt and osmotic stresses, had the highest level of accumulation of VvWRKY2 transcripts (Mzid et al. 2007). VvWRKY2 expression patterns analysis (Supplemental File 1a) in 54 tissues/organs samples at various developmental stages of grapevine rootstock (cv Corvina) showed diverse transcription levels, mainly in mid-ripening berries and young leaves. Moreover, expression studies of VvWRKY2 under abiotic stressful conditions (Supplemental File 1b and c) revealed that VvWRKY2 is slightly induced by salt treatment and water deficit. Our results point out the importance of VvWRKY2 transcription levels in plant adaptation.

Oxidative stress evaluation in transgenic VvWRKY2 and WT lines under saline and osmotic stresses

To study whether the over-expression of VvWRKY2 gene increases the tolerance of transgenic plants through better oxidative stress management, REL, MDA and H2O2 contents were analyzed.

The improved stress (osmotic and salt) tolerance of VvWRKY2 over-expressing plants was not correlated with changes in REL, H2O2 or with MDA contents. Indeed, under normal and stress growing conditions, the ion leakage, H2O2 and MDA contents of both WT and VvWRKY2 transgenic lines were similar (Tables 1, 2). These results indicate that the tolerance of transgenic plants was not related to oxidative stress

Table 1.

Relative electrolyte leakage, free H2O2 and MDA contents in seedlings of WT and VvWRKY2 tobacco lines (W2-5, W2-11 and W2-12) under control and salt stress

NaCl (mM) Genotype Relative electrolyte leakage (%) H2O2 (nmol/g FW) MDA (nmol/g FW)
0 WT 28.00 ± 5.67 g 260.24 ± 59.32 f 3.52 ± 1.02 cd
W2-5 33.69 ± 3.30 g 310.52 ± 10.15 def 3.12 ± 0.59 d
W2-11 32.43 ± 2.90 g 303.57 ± 8.55 ef 4.41 ± 1.26 cd
W2-12 36.41 ± 2.40 fg 280.24 ± 7.85 f 3.87 ± 1.08 d
100 WT 44.74 ± 1.52 def 375.00 ± 11.32 bc 7.94 ± 0.90 cd
W2-5 40.20 ± 1.92 efg 351.86 ± 19.38 cde 5.91 ± 1.79 cd
W2-11 48.61 ± 1.36 de 367.14 ± 12.44 cd 11.27 ± 7.30 bc
W2-12 46.6 ± 2.15 de 378.57 ± 53.97 bc 7.92 ± 2.94 cd
200 WT 58.33 ± 10.99 c 432.62 ± 17.75 b 17.00 ± 0.74 b
W2-5 51.90 ± 0.88 cd 370.48 ± 9.74 bcd 15.31 ± 1.18 b
W2-11 57.3 ± 6.21 c 430.38 ± 8.91 b 17.37 ± 10.49 b
W2-12 59.4 ± 1.50 c 500.14 ± 6.47 a 15.17 ± 1.26 b
300 WT 68.98 ± 8.42 b 517.86 ± 4.04 a 27.56 ± 5.49 a
W2-5 73.96 ± 10.64 ab 403.57 ± 31.31 bc 24.73 ± 3.04 a
W2-11 75.33 ± 8.49 ab 515.00 ± 2.14 a 23.52 ± 2.71 a
W2-12 79.47 ± 4.00 a 516.19 ± 20.47 a 24.15 ± 3.57 a
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Values represent the mean ± CI of three experiments. For each parameter, values labelled with different letters are significantly different according to Duncan’s test at P ≤ 0.05

Table 2.

Relative electrolyte leakage, free H2O2 and MDA contents in WT and VvWRKY2 tobacco (W2-5, W2-11 and W2-12) seedlings under control and osmotic stress

Mannitol (mM) Genotype Relative electrolyte leakage (%) H2O2 (nmol/g FW) MDA (nmol/g FW)
0 WT 28.24 ± 0.85 h 326.19 ± 7.85 f 5.82 ± 2.25 de
W2-5 34.75 ± 2.24 g 331.43 ± 15.61 f 3.22 ± 1.25 e
W2-11 31.11 ± 1.44 h 350.48 ± 6.48 ef 4.60 ± 2.05 e
W2-12 36.14 ± 1.17 g 322.14 ± 5.30 f 7.32 ± 2.19 de
100 WT 44.79 ± 0.56 f 375.00 ± 11.32 e 11.56 ± 1.66 cd
W2-5 43.82 ± 3.59 f 455.24 ± 32.39 cd 15.39 ± 1.37 bc
W2-11 47.57 ± 2.60 ef 428.10 ± 8.30 d 16.05 ± 2.27 bc
W2-12 48.94 ± 2.46 e 316.86 ± 10.93 f 12.94 ± 2.99 bc
200 WT 59.43 ± 1.40 d 432.62 ± 17.75 d 17.52 ± 7.32 b
W2-5 62.73 ± 1.22 cd 537.14 ± 19.67 b 16.43 ± 6.02 bc
W2-11 60.30 ± 3.45 d 468.81 ± 2.84 c 17.72 ± 3.12 b
W2-12 64.59 ± 3.23 c 427.57 ± 6.15 d 17.33 ± 2.56 b
400 WT 71.82 ± 1.85 b 517.86 ± 4.04 b 28.38 ± 2.28 a
W2-5 72.85 ± 1.16 b 581.43 ± 60.77 a 28.06 ± 1.88 a
W2-11 72.88 ± 4.47 b 542.14 ± 5.66 b 27.46 ± 2.44 a
W2-12 77.03 ± 2.57 a 514.29 ± 42.77 b 29.41 ± 4.24 a
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Values represent a mean ± CI of three experiments. For each parameter, values labelled with different letters are significantly different according to Duncan’s test at P ≤ 0.05

Over-expression of the VvWRKY2 increases proline and sugar accumulation under saline and osmotic stresses

To elucidate and understand the physiological changes of VvWRKY2 transgenic lines, contents of free proline and soluble sugars were measured upon application of salt and osmotic stresses.

Proline accumulation in the leaves was determined in the WT and the VvWRKY2 over-expressing plants. Under control condition, proline levels were similar in transgenic and wild type plants (Fig. 4a, b). However, under osmotic and salt conditions, when compared with the WT plants, the transgenic lines showed remarkably higher levels of both prolines (Fig. 4a, b) and soluble sugars (Fig. 5a, b), highlighting their role in osmotic stress adaptation through osmo-regulation mechanism.

Fig. 4.

Fig. 4

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Changes of free proline contents in WT and VvWRKY2 tobacco plants (W2-5, W2-11 and W2-12) under salt (a) and water stress conditions (b). Values are means of three replicates. For each parameter, bars labelled with different letters are significantly different according to Duncan’s test at P ≤ 0.05

Fig. 5.

Fig. 5

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Changes of sugar contents in WT and VvWRKY2 tobacco plants (W2-5, W2-11 and W2-12) under salt (a), and water stress conditions (b). Values are means of three replicates. For each parameter, bars labelled with different letters are significantly different according to Duncan’s test at P ≤ 0.05

Thus, the results of physiological characterization suggest that over-expression of VvWRKY2 increased proline and sugar contents, with no change in MDA, H2O2 and ion leakage accumulation under salt and dehydration conditions.

Principal component analysis (PCA)

The trait-by-trait analyses were completed by Principal Component Analysis (PCA) which took into account traits characterizing transgenic plants that were cultivated under stress conditions.

Taking into account the PCA performed for NaCl treatments, by considering six parameters and two plants lines (WT and transgenic tobacco plant over-expressing VvWRKY2), the first two components (F1 and F2) explained 92.63% of the total variation. The first component (axis 1) explained 78.2% of the variation, followed by 14.43% for the second component (axis 2) (Fig. 6).

Fig. 6.

Fig. 6

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Principal component analysis. The significance of parameters used to study the response of transgenic and WT tobacco plants under salt stress conditions. These parameters, as well as the different growth conditions, are projected onto the F1–F2 principal factorial plane that explains 92.63% of the variation. Plants were exposed to saline conditions with increasing NaCl concentrations (0, 100, 200 and 300 mM). Black circles represent different analysis parameters. Black diamonds represent the different growth conditions, and white circles represent the different study lines (wild type and the different transgenic lines)

Likewise, we found that NaCl was correlated with the studied parameters. In fact, we noticed that all studied parameters showed high correlation coefficients with saline treatments (R2 = − 0.87 for germination rate, 0.94 for leaf relative electrolyte leakage, 0.81 for sugar content, 0.76 for proline content, 0.88 for H2O2 content and 0.92 for MDA content) (Table 3). The analysis showed also statistically significant positive correlations between transgenic tobacco plants grown under salt treatments and the contents of sugar and proline (R2 = 0.39 and 0.35, respectively) (Table 3).

Table 3.

Pearson’s correlation matrix analyzing the effect of salt stress on the germination rate, relative electrolyte leakage, sugar content, proline content, H2O2 content, MDA content in WT and VvWRK2 tobacco plants lines

Variables Germination rate Relative electrolyte leakage Sugar content Proline content H2O2 content MDA content NaCl WT Transgenic lines
Germination rate 1 − 0.8437 − 0.5292 − 0.4976 − 0.8201 − 0.8537 − 0.8704 − 0.2528 0.2528
Leaf relative electrolyte leakage 1 0.7638 0.7276 0.8625 0.8815 0.9441 − 0.0771 0.0771
Sugar content 1 0.9498 0.5970 0.7271 0.8112 − 0.3850 0.3850
Proline content 1 0.5032 0.6737 0.7636 − 0.3452 0.3452
H2O2 content 1 0.8037 0.8810 0.0126 − 0.0126
MDA content 1 0.9245 0.0470 − 0.0470
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Variables were centred around their means and normalized with a standard deviation of 1

Bold values represent significant correlation levels at 0.05

Congruous results were showed based on PCA carried out for mannitol treatments. In fact, the first two components explained 92.54% of the total variation. The first component (axis 1) explained 79.72% of the variation, followed by 12.82% for the second component (axis 2) (Fig. 7).

Fig. 7.

Fig. 7

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Principal component analysis. The significance of parameters used to study the response of transgenic and WT tobacco plants under mannitol conditions. These parameters, as well as the different growth conditions, are projected onto the F1–F2 principal factorial plane that explains 92.54% of the variation. Plants were exposed to saline conditions with increasing mannitol concentrations (0, 100, 200 and 400 mM). Black circles represent different analysis parameters. White diamonds represent the different growth conditions, and white circles represent the different study lines (wild type and the different transgenic lines)

Similarly to results shown by NaCl, mannitol was correlated with all studied parameters (R2 = − 0.85 for germination rate, 0.96 for leaf relative electrolyte leakage, 0.68 for sugar content, 0.84 for proline content, 0.87 for H2O2 content and 0.94 for MDA content) (Table 4). Transgenic plants grown under mannitol treatments showed positive correlations (significant at 0.05 level) with germination rate (R2 = 0.35), sugar content (R2 = 0.47) and proline content (R2 = 0.33) (Table 4).

Table 4.

Pearson’s correlation matrix analyzing the effect of mannitol on the germination rate, relative electrolyte leakage, sugar content, proline content, H2O2 content, MDA content in WT and VvWRKY2 tobacco plants lines

Variables Germination rate Relative electrolyte leakage Sugar content Proline content H2O2 content MDA content Mannitol WT Transgenic lines
Germination rate 1 − 0.8290 − 0.3543 − 0.5158 − 0.6883 − 0.7816 − 0.8460 − 0.3450 0.3450
Leaf relative electrolyte leakage 1 0.7554 0.8279 0.8496 0.9103 0.9569 − 0.0911 0.0911
Sugar content 1 0.8738 0.7115 0.6945 0.6813 − 0.4688 0.4688
Proline content 1 0.8477 0.7941 0.8401 − 0.3256 0.3256
H2O2 content 1 0.8352 0.8737 − 0.1323 0.1323
MDA content 1 0.9412 − 0.0263 0.0263
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Variables were centred around their means and normalized with a standard deviation of 1. Bold values represent significant correlations at 0.05 level

PCA analyses confirm clearly the positive effects of VvWRKY2 gene over-expression on the tolerance of tobacco plants to salt and osmotic stresses, particularly throughout the adjustment of sugar and proline contents.

Discussion

Numerous studies demonstrated that several members of WRKY TFs family are involved in the responses of many plants to multi-environmental stresses (Zhao et al. 2015; Song et al. 2016; Jiang et al. 2017). However, their functions in the response of grapevine to stress remain unclear. To go further, our previous study on VvWRKY2 biological roles (Mzid et al. 2007), we explored the putative involvement of this gene in the response to salinity and dehydration stresses.

Amino acid comparisons and phylogenetic analyses showed a clear orthologous relationship of VvWRKY2 with AtWRKY3 and AtWRKY4, all of which belong to the group I of WRKY family. The AtWRKY3 was induced by SA and pathogens (Lai et al. 2008; Rushton et al. 2010), while the AtWRKY4 was regulated by JA, SA, pathogens, sucrose, senescence, cold and salinity (Hammargren et al. 2008).

Ramamoorthy et al. (2008) demonstrated that WRKY proteins play a crucial role in abiotic stress tolerance alongside their roles in biotic stress tolerance. Guo et al. (2014) indicated that the expression of VvWRKY2 was induced by salt and by drought after 48 and 144 h, respectively. This result was in line with the finding of Wang et al. (2012) and Liu et al. (2013) who revealed the induction of BcWRKY46 (Brassica campestris ssp. chinensis) and DgWRKY3 (Dendranthema grandiflorum) by the salt and drought treatments. Consequently, it is clear that VvWRKY2 is a functional WRKY TF involved in the response to salt and water stresses. Being induced by NaCl treatments, we performed a functional analysis on VvWRKY2 to confirm its implication in salinity tolerance and response. In the current study, we demonstrated that the over-expression of VvWRKY2 in tobacco improved the tolerance of transgenic plants to salt and water-deficit stresses at germination and seedling stages. In fact, the germination under salt and drought stress conditions was higher in transgenic lines than for WT plants. Likewise, many previous studies demonstrated that the constitutive expression of some grapevine WRKY genes, including VvWRKY11, VpWRKY2 and VpWRKY3, could enhance tolerance to abiotic stress in transgenic lines (Li et al. 2010; Liu et al. 2011). These results are similar to those previously obtained for tobacco plants over-expressing the wheat TaWRKY10 (Wang et al. 2013) and the cotton GhWRKY41 genes (Chu et al. 2015), which showed an increased germination rate under salt and water stress conditions. More recently, Zhou et al. (2015) revealed that over-expression of GhWRKY34 in the model plant A. thaliana led to a significantly higher germination rate in transgenic lines, as compared with WT in presence of NaCl. Other authors demonstrated that the over-expression of (Reaumuria trigyna) RtWRKY1 gene in Arabidopsis was responsible for the salt tolerance improvement by regulating plant growth, osmotic balance, Na+/K+ homeostasis and the antioxidant system (Du et al. 2017). Nevertheless, when over-expression a cotton GhWRKY25 gene in Arabidopsis, a low tolerance to drought was obtained, while better salt tolerance was observed (Liu et al. 2016).

Several studies reported that seed germination is regulated by WRKY TFs through different mechanisms. For example, AtWRKY2-mediated seed germination and post-germination stages are inhibited by ABA, and OsWRKY78, which might proceed as a regulator in stem elongation and seed development (Jiang and Yu 2009; Zhang et al. 2011). Besides, Lagacé and Matton (2004) revealed the implication of WRKY proteins in the embryogenesis of the wild potato species (Solanum chacoense). In fact, they demonstrated the expression of ScWRKY1 in ovules bearing late torpedo-staged embryos. Additionally, and more recently, Raineri et al. (2016) showed that the sunflower transcription factor HaWRKY10 could improve the germination of A. thaliana through the stimulation of reserve mobilization. Other studies highlighted that the WRKY TF over-expression can inhibit the germination process under stress conditions. For instance, Yan et al. (2014) reported that GhWRKY17 played a negative role in regulating ABA-mediated seed germination and seedling growth in Nicotiana benthamiana transgenic plants. This was already pointed out by Jia et al. (2015) for the GhWRKY68.

After 4 weeks of drought or salt stress conditions, transgenic lines accumulated high levels of proline and soluble sugars (Figs. 4, 5). This result is in accordance with others demonstrating that the accumulation of compatible solutes such as proline and a variety of sugars is effective in improving tolerance of plants to salt and osmotic stresses (Huang et al. 2009; Petronia et al. 2011), playing highly protective roles under stress constraints (Hayat et al. 2012; Murakeozy et al. 2003). Thus, our results indicated that the transgenic lines display drought and salt resistances through a better mechanism of osmo-regulation. Similarly, constitutive expression of different WRKY genes, including TaWRKY20 and TaWRKY44, resulted in a greater accumulation of proline and sugars in transgenic lines (Wang et al. 2013; Wang et al. 2015).

Electrolyte leakage represents a rapid, sensitive and quantitative method to evaluate the effect of stress on plant cells (Verslues et al. 2006). Moreover, the MDA was used to analyse lipid peroxidation (Loreto and Velikova 2001). We thus, evaluated the accumulation of H2O2 in the leaf after stress conditions. We found that no difference was observed within these parameters for both control (WT) and transgenic (VvWRKY2) plants under salt stress conditions.

Maintenance of membrane functionality due to unchanged oxidative stress indicators in grapevine under salt stress may be attributed to the adequate response of the antioxidant system to salt stress-induced oxidative damage, as also observed in some other salt- or drought-treated plants such as Pisum sativum (Noreen and Ashraf 2009), maize (Jiang and Zhang 2002), sorghum (Zhang and Kirkham 1996) and Plantago maritima (Sekmen et al. 2007).

The over-expression of VvWRKY2 gene did not change the response of the oxidative indicators observed in wild plants under salt stress. These results are in contrast with those of Liu et al. (2013) and Liang et al. (2017) showing reduced accumulation of MDA and H2O2 in transgenic chrysanthemum over-expressing DgWRKY genes under salt stress, when compared with control plants (WT). In another study, the GhWRKY68-over-expressing plants have a reduced resistance to drought and salt. They exhibited reduced tolerance to oxidative stress after drought and salt stress treatments. It was correlated with the accumulation of ROS, reduced enzyme activities, elevated MDA content and altered ROS-related gene expression (Jia et al. 2015). All these results clearly showed that the relationship between WRKY genes and salt-induced oxidative stress is variable among plant species. In our study, the improvement of the tolerance to drought and salt stress in transgenic WRKY plants cannot be associated with an improved ROS detoxification process.

In conclusion, our results indicated that over-expression of VvWRKY2 in transgenic tobacco enhanced salt and drought stress tolerance when compared with wild types. On one hand, these functions are achieved by accumulating proline and soluble sugar contents, but on the other hand, the plant ROS amount and MDA content remain unchanged. Hence, based on overall results, it could be presumed that VvWRKY2 over-expression in grapevine would improve its salt and drought stress tolerance. Moreover, the mechanism of osmotic stress tolerance must be further investigated. Therefore, the role of VvWRKY2 in grapevine and its target genes in various signalling pathways must be studied to better understand and reveal the exact mechanisms of plant responses to these constraints.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PPTX 216 KB) (216KB, pptx)

Acknowledgements

We gratefully thank Mrs Sana Louati, Mrs Leila Hjaiej and Mr. Mounir Triki for proofreading the present paper.

Abbreviations

CI

Confidence interval

FW

Fresh weight

H2O2

Hydrogen peroxide

PCA

Principal component analysis

ROS

Reactive oxygen species

TBA

Thiobarbituric acid

TF

Transcription factor

WT

Wild type

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Electronic supplementary material

The online version of this article (10.1007/s13205-018-1301-4) contains supplementary material, which is available to authorized users.

References

  1. Cai Y, Tu W, Zu Y, Jing Y, Xu Z, Lu J, Zhang Y. Overexpression of a grapevine sucrose transporter (VvSUC27) in tobacco improves plant growth rate in the presence of sucrose in vitro. Front Plant Sci. 2017;8:1069. doi: 10.3389/fpls.2017.01069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chen M, Tan Q, Sun M, Li D, Fu X, Chen X, Xiao W, Li L, Gao D. Genome-wide identification of WRKY family genes in peach and analysis of WRKY expression during bud dormancy. Mol Genet Genom. 2016;291(3):1319–1332. doi: 10.1007/s00438-016-1171-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chu X, Wang C, Chen X, Lu W, Li H, Wang X, Hao L, Guo X. The cotton WRKY gene GhWRKY41 positively regulates salt and drought stress tolerance in transgenic Nicotiana benthamiana. PLoS ONE. 2015;10(11):e0143022. doi: 10.1371/journal.pone.0143022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Daldoul S, Amar AB, Gargouri M, Mliki A, Wetzel TA. Grapevine-inducible gene Vv-α-gal/SIP confers salt and desiccation tolerance in Escherichia coli and tobacco at germinative stage. Biochem Genet. 2018;56(1–2):78–92. doi: 10.1007/s10528-017-9831-8. [DOI] [PubMed] [Google Scholar]
  5. Dorothea B, Ramanjulu S. Drought and salt tolerance in plants. Crit Rev Plant Sci. 2005;24:23–58. doi: 10.1080/07352680590910410. [DOI] [Google Scholar]
  6. Du C, Zhao P, Zhang H, Li N, Zheng L, Wang Y. The Reaumuria trigyna transcription factor RtWRKY1 confers tolerance to salt stress in transgenic Arabidopsis. J Plant Physiol. 2017;215:48–58. doi: 10.1016/j.jplph.2017.05.002. [DOI] [PubMed] [Google Scholar]
  7. Dubois M, Gilles KA, Hamilton JK, Rebecs PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem. 1956;28(3):350–356. doi: 10.1021/ac60111a017. [DOI] [Google Scholar]
  8. Eck RV, Dayhoff MO. Atlas of Protein Sequence and Structure. Silver Springs: National Biomedical Research Foundation; 1966. [Google Scholar]
  9. Eulgem T, Rushton PJ, Robatzek S, Somssich IE. The WRKY superfamily of plant transcription factors. Trends Plant Sci. 2000;5:199–206. doi: 10.1016/S1360-1385(00)01600-9. [DOI] [PubMed] [Google Scholar]
  10. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39:783–791. doi: 10.1111/j.1558-5646.1985.tb00420.x. [DOI] [PubMed] [Google Scholar]
  11. Guillaumie S, Mzid R, Mechin V, Leon C, Hichri I, Destrac-Irvine A, Trossat-Magnin C, Delrot S, Lauvergeat V. The grapevine transcription factor WRKY2 influences the lignin pathway and xylem development in tobacco. Plant Mol Biol. 2010;72:215–234. doi: 10.1007/s11103-009-9563-1. [DOI] [PubMed] [Google Scholar]
  12. Guo C, Guo R, Xu X, Gao M, Li X, Song J, Zheng Y, Wang X. Evolution and expression analysis of the grape (Vitis vinifera L.) WRKY gene family. J Exp Bot. 2014;65(6):1513–1528. doi: 10.1093/jxb/eru007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hagege D, Nouvelot A, Boucaud J, Gaspar T. Malondialdehyde titration with thiobarbiturate in plant extracts: avoidance of pigment interference. Phytochem Anal. 1990;1:86–89. doi: 10.1002/pca.2800010208. [DOI] [Google Scholar]
  14. Hammargren J, et al. A novel connection between nucleotide and carbohydrate metabolism in mitochondria: sugar regulation of the Arabidopsis nucleoside diphosphate kinase 3a gene. Plant Cell Rep. 2008;27(3):529–534. doi: 10.1007/s00299-007-0486-5. [DOI] [PubMed] [Google Scholar]
  15. Hayat S, Hayat Q, Alyemeni MN, Wani AS, Pichtel J, Ahmad A. Role of proline under changing environments: a review. Plant Signal Behav. 2012;7(11):1456–1466. doi: 10.4161/psb.21949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Higgins D, Thompson J, Gibson T, Thompson JD, Higgins DG, Gibson T. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huang Y, Bie Z, Liu Z, Zhen A, Wang W. Protective role of proline against salt stress is partially related to the improvement of water status and peroxidase enzyme activity in cucumber. Soil Sci Plant Nutr. 2009;55:698–704. doi: 10.1111/j.1747-0765.2009.00412.x. [DOI] [Google Scholar]
  18. Jaillon O, Aury JM, Noel B, Policrit A, Clepet, et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature. 2007;449:463–467. doi: 10.1038/nature06148. [DOI] [PubMed] [Google Scholar]
  19. Jamoussi RJ, Elabbassi MM, Ben Jouira H, Ghorbel A, Mliki A. Physiological responses of transgenic tobacco plants expressing the dehydration responsive RD22 gene of Vitis vinifera to salt stress. Turk J Bot. 2014;38(2):268–280. doi: 10.3906/bot-1301-53. [DOI] [Google Scholar]
  20. Jardak R, El Abbassi MM, Ben Jouira H, Mliki A, Ghorbel A. Ex vitro assessment of increased salt tolerance in tobacco constitutively expressing the Vitis vinifera dehydration responsive gene. Acta Hort. 2017;1157:223–230. doi: 10.17660/ActaHortic.2017.1157.33. [DOI] [Google Scholar]
  21. Jia H, Wang C, Wang F, Liu S, Li G, Guo X. GhWRKY68 reduces resistance to salt and drought in transgenic Nicotiana benthamiana. PLoS ONE. 2015;10(3):e0120646. doi: 10.1371/journal.pone.0120646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jiang W, Yu D. Arabidopsis WRKY2 transcription factor mediates seed germination and postgermination arrest of development by abscisic acid. BMC Plant Biol. 2009;9:96. doi: 10.1186/1471-2229-9-96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jiang M, Zhang J. Involvement of plasma membrane NADPH oxidase in abscicic acid and water stress induced antioxidant defense in leaves of maize seedlings. Planta. 2002;215:1022–1030. doi: 10.1007/s00425-002-0829-y. [DOI] [PubMed] [Google Scholar]
  24. Jiang J, Ma S, Ye N, Jiang M, Cao J, Zhang J. WRKY transcription factors in plant responses to stresses. J Integr Plant Biol. 2017;59(2):86–101. doi: 10.1111/jipb.12513. [DOI] [PubMed] [Google Scholar]
  25. Jones DT, Taylor WR, Thornton JM. The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci. 1992;8(3):275–282. doi: 10.1093/bioinformatics/8.3.275. [DOI] [PubMed] [Google Scholar]
  26. Lagace M, Matton DP. Characterization of a WRKY transcription factor expressed in late torpedo-stage embryos of Solanum chacoense. Planta. 2004;219:185–189. doi: 10.1007/s00425-004-1253-2. [DOI] [PubMed] [Google Scholar]
  27. Lai Z, et al. Roles of Arabidopsis WRKY3 and WRKY4 transcription factors in plant responses to pathogens. BMC Plant Biol. 2008;8:68. doi: 10.1186/1471-2229-8-68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Li H, Xu Y, Xiao Y, Zhu Z, Xie X, Zhao H, Wang Y. Expression and functional analysis of two genes encoding transcription factors, VpWRKY1 and VpWRKY2, isolated from Chinese wild Vitis pseudoreticulata. Planta. 2010;232(6):1325–1337. doi: 10.1007/s00425-010-1258-y. [DOI] [PubMed] [Google Scholar]
  29. Liang QY, Wu YH, Wang K, Bai ZY, Liu QL, Pan YZ, Jiang BB. Chrysanthemum WRKY gene DgWRKY5 enhances tolerance to salt stress in transgenic chrysanthemum. Sci Rep. 2017;7:4799. doi: 10.1038/s41598-017-05170-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Liu H, Yang W, Liu D, Han Y, Zhang A, Li S. Ectopic expression of a grapevine transcription factor VvWRKY11 contributes to osmotic stress tolerance in Arabidopsis. Mol Biol Rep. 2011;38(1):417–427. doi: 10.1007/s11033-010-0124-0. [DOI] [PubMed] [Google Scholar]
  31. Liu QL, Zhong M, Li S, Pan YZ, Jiang BB, Jia Y, et al. Overexpression of a chrysanthemum transcription factor gene, DgWRKY3, in tobacco enhances tolerance to salt stress. Plant Physiol Biochem. 2013;69:27–33. doi: 10.1016/j.plaphy.2013.04.016. [DOI] [PubMed] [Google Scholar]
  32. Liu X, Song Y, Xing F, Wang N, Wen F, Zhu C. GhWRKY25, a group I WRKY gene from cotton, confers differential tolerance to abiotic and biotic stresses in transgenic Nicotiana benthamiana. Protoplasma. 2016;253(5):1265–1281. doi: 10.1007/s00709-015-0885-3. [DOI] [PubMed] [Google Scholar]
  33. Loreto F, Velikova V. Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol. 2001;127(4):1781–1787. doi: 10.1104/pp.010497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Marchive C, Mzid R, Deluc L, Barrieu F, Pirrello J, Gauthier A, Corio-Costet MF, Regad F, Cailleteau B, Hamdi S, Lauvergeat V. Isolation and characterization of a Vitis vinifera transcription factor, VvWRKY1, and its effect on responses to fungal pathogens in transgenic tobacco plants. J Exp Bot. 2007;58:1999–2010. doi: 10.1093/jxb/erm062. [DOI] [PubMed] [Google Scholar]
  35. Murakeozy EP, Nagy Z, Duhaze C, Bouchereau A, Tuba Z. Seasonal changes in the levels of compatible osmolytes in three halophytic species of inland saline vegetation in Hungary. J Plant Physiol. 2003;160:395–401. doi: 10.1078/0176-1617-00790. [DOI] [PubMed] [Google Scholar]
  36. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant. 1962;15:473–477. doi: 10.1111/j.1399-3054.1962.tb08052.x. [DOI] [Google Scholar]
  37. Mzid R, Marchive C, Blancard D, Deluc L, Barrieu F, Corio-Costet MF, Drira N, Hamdi S, Lauvergeat V. Overexpression of VvWRKY2 in tobacco enhances broad resistance to necrotrophic fungal pathogens. Physiol Plant. 2007;131:434–447. doi: 10.1111/j.1399-3054.2007.00975.x. [DOI] [PubMed] [Google Scholar]
  38. Noreen Z, Ashraf M. Assessment of variation in antioxidative defense system in salt-treated pea (Pisum sativum) cultivars and its putative use as salinity tolerance markers. J Plant Physiol. 2009;166:1764–1774. doi: 10.1016/j.jplph.2009.05.005. [DOI] [PubMed] [Google Scholar]
  39. Nuruzzaman M, Sharoni AM, Satoh K, Kumar A, Leung H, Kikuchi S. Comparative transcriptome profiles of the WRKY gene family under control, hormone-treated, and drought conditions in near-isogenic rice lines reveal differential, tissue specific gene activation. J Plant Physiol. 2014;171:2–13. doi: 10.1016/j.jplph.2013.09.010. [DOI] [PubMed] [Google Scholar]
  40. Petronia C, Maria Grazia A, Giovanni P, Amodio F, Pasqualina W (2011) Salinity stress and salt tolerance. In: Shanker A (ed) Abiotic stress in plants—mechanisms and adaptations. InTech, pp 21–38. 10.5772/22331 (ISBN 978-953-307-394-1)
  41. Phukan UJ, Jeena GS, Shukla RK. WRKY transcription factors: molecular regulation and stress responses in plants. Front Plant Sci. 2016 doi: 10.3389/fpls.2016.00760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ramamoorthy R, Jiang SY, Kumar N, Venkatesh PN, Ramachandran SA. Comprehensive transcriptional profiling of the WRKY gene family in rice under various abiotic and phytohormone treatments. Plant Cell Physiol. 2008;49:865–879. doi: 10.1093/pcp/pcn061. [DOI] [PubMed] [Google Scholar]
  43. Rinerson CI, Scully ED, Palmer NA, Donze-Reiner T, Rabara RC, Tripathi P, Shen QJ, Sattler SE, Rohila JS, Sarath G, Rushton PJ. The WRKY transcription factor family and senescence in switchgrass. BMC Genom. 2015;16:912. doi: 10.1186/s12864-015-2057-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rzhetsky A, Nei M. A simple method for estimating and testing minimum evolution trees. Mol Biol Evol. 1992;9:945–967. [Google Scholar]
  45. Saitou N, Nei M. The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]
  46. Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T, Satou M, Akiyama K, Taji T, Yamaguchi-Shinozaki K, Carninci P, Kawai J, Hayashizaki Y, Shinozaki K. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J. 2002;31:279–292. doi: 10.1046/j.1365-313X.2002.01359.x. [DOI] [PubMed] [Google Scholar]
  47. Sekmen AH, Turkan I, Takio S. Differential responses of antioxidative enzymes and lipid peroxidation to salt stress in salt-tolerant Plantago maritima and salt sensitive Plantago media. Physiol Plant. 2007;131:399–411. doi: 10.1111/j.1399-3054.2007.00970.x. [DOI] [PubMed] [Google Scholar]
  48. Sneath PHA, Sokal RR. Numerical taxonomy. San Francisco: Freeman; 1973. [Google Scholar]
  49. Song Y, Jing S, Yu D. Overexpression of the stress-induced OsWRKY08 improves osmotic stress tolerance in Arabidopsis. Chin Sci Bull. 2009;54:4671–4678. [Google Scholar]
  50. Song H, Wang P, Hou L, Zhao S, Zhao C, Xia H, Li P, Zhang Y, Bian X, Wang X. Global analysis of WRKY genes and their response to dehydration and salt stressin soybean. Front Plant Sci. 2016;7:9. doi: 10.3389/fpls.2016.00009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tak H, Mhatre M. Cloning and molecular characterization of a putative bZIP transcription factor VvbZIP23 from Vitis vinifera. Protoplasma. 2013;250(1):333–345. doi: 10.1007/s00709-012-0417-3. [DOI] [PubMed] [Google Scholar]
  52. Tak H, Mhatre M. Molecular characterization of VvSDIR1 from Vitis vinifera andits functional analysis by heterologous expression in Nicotiana tabacum. Protoplasma. 2013;250(2):565–576. doi: 10.1007/s00709-012-0442-2. [DOI] [PubMed] [Google Scholar]
  53. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725–2729. doi: 10.1093/molbev/mst197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tripathi P, Rabara RC, Rushton PJ. A systems biology perspective on the role of WRKY transcription factors in drought responses in plants. Planta. 2014;239:255–266. doi: 10.1007/s00425-013-1985-y. [DOI] [PubMed] [Google Scholar]
  55. Troggio M, Malacarne G, Coppola G, Segala C, Cartwright DA, Pindo M, Stefanini M, Mank R, Moroldo M, Morgante M, Grando MS, Velasco R. A dense single-nucleotide polymorphism-based genetic linkage map of grapevine (Vitis vinifera L.) anchoring Pinot Noir bacterial artificial chromosome contigs. Genetics. 2007;176:2637–2650. doi: 10.1534/genetics.106.067462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Velasco R, Zharkikh A, Troggio M, Cartwright DA, et al. A high quality draft consensus sequence of the genome of a heterozygous grapevine variety. PLoS ONE. 2007;2:e1326. doi: 10.1371/journal.pone.0001326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Velikova V, Yordanov I, Edreva A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: Protective role of exogenous polyamines. Plant Sci. 2000;151:59–66. doi: 10.1016/S0168-9452(99)00197-1. [DOI] [Google Scholar]
  58. Verslues PE, Agarwal M, Katiyar-Agarwal S, Zhu J, Zhu JK. Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. Plant J. 2006;45:523–539. doi: 10.1111/j.1365-313X.2005.02593.x. [DOI] [PubMed] [Google Scholar]
  59. Wang F, Hou X, Tang J, Wang Z, Wang S, Jiang F, Li Y. A novel cold-inducible gene from Pak-choi (Brassica campestris ssp. chinensis), BcWRKY46, enhances the cold, salt and dehydration stress tolerance in transgenic tobacco. Mol Biol Rep. 2012;39(4):4553–4564. doi: 10.1007/s11033-011-1245-9. [DOI] [PubMed] [Google Scholar]
  60. Wang Y, Dang F, Liu Z, Wang X, Eulgem T, Lai Y, Yu L, She J, Shi Y, Lin J, Chen C, Guan D, Qiu A, He S. CaWRKY58, encoding a group I WRKY transcription factor of Capsicum annuum, negatively regulates resistance to Ralstonia solanacearum infection. Mol Plant Pathol. 2013;14:131–144. doi: 10.1111/j.1364-3703.2012.00836.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wang L, Zhu W, Fang L, Sun X, Su L, Liang Z, Wang N, Londo JP, Li S, Xin H. Genome-wide identification of WRKY family genes and their response to cold stressin Vitis vinifera. BMC Plant Biol. 2014;14:103. doi: 10.1186/1471-2229-14-103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wang X, Zeng J, Li Y, Rong X, Sun J, Sun T, Li M, Wang L, Feng Y, Chai R, Chen M, Chang J, Li K, Yang G, He G. Expression of TaWRKY44, a wheat WRKY gene, in transgenic tobacco confersmultiple abiotic stress tolerances. Front Plant Sci. 2015;6:615. doi: 10.3389/fpls.2015.00615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wu J, Zhao Q, Yang Q, Liu H, Li Q, Yi X, Cheng Y, Guo L, Fan C, Zhou Y. Comparative transcriptomic analysis uncovers the complex genetic network for resistance to Sclerotinia sclerotiorum in Brassica napus. Sci Rep. 2016;6:19007. doi: 10.1038/srep19007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yamaguchi T, Blumwald E. Developing salt-tolerant crop plants: challenges and opportunities. Trends Plant Sci. 2005;10:615–620. doi: 10.1016/j.tplants.2005.10.002. [DOI] [PubMed] [Google Scholar]
  65. Yan H, Jia H, Chen X, Hao L, An H, Guo X. The cotton WRKY transcription factor GhWRKY17 functions in drought and salt stress in transgenic Nicotiana benthamiana through ABA signaling and the modulation of reactive oxygen species production. Plant Cell Physiol. 2014;55(12):2060–2076. doi: 10.1093/pcp/pcu133. [DOI] [PubMed] [Google Scholar]
  66. Zhang JX, Kirkham MB. Enzymatic responses of the ascorbate-glutathione cycle to drought in sorghum and sunflower plants. Plant Sci. 1996;113:139–147. doi: 10.1016/0168-9452(95)04295-4. [DOI] [Google Scholar]
  67. Zhang CQ, Xu Y, Lu Y, Yu HX, Gu MH, Liu QQ. The WRKY transcription factor OsWRKY78 regulates stem elongation and seed development in rice. Planta. 2011;234(3):541–54. doi: 10.1007/s00425-011-1423-y. [DOI] [PubMed] [Google Scholar]
  68. Zhao TT, Zhang J, Liang LS, Ma QH, Chen X, Zong JW, Wang GX. Expression and functional analysis of WRKY transcription factors in Chinese Wild Hazel, Corylus heterophylla Fisch. PLoS ONE. 2015;10(8):e0135315. doi: 10.1371/journal.pone.0135315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Zhou L, Wang NN, Gong SY, Lu R, Li Y, Li XB. Overexpression of a cotton (Gossypium hirsutum) WRKY gene, GhWRKY34, in Arabidopsis enhances salt-tolerance of the transgenic plants. Plant Physiol Biochem. 2015;96:311–320. doi: 10.1016/j.plaphy.2015.08.016. [DOI] [PubMed] [Google Scholar]
  70. Zuckerkandl E, Pauling L. Evolutionary divergence and convergence in proteins. In: Bryson V, Vogel HJ, editors. Evolving genes and proteins. New York: Academic Press; 1965. pp. 97–166. [Google Scholar]

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