Overexpression of transcription factor SlWRKY28 improved the tolerance of Populus davidiana × P. bolleana to alkaline salt stress

Background WRKY transcription factors (TFs) have been suggested to play crucial roles in the response to biotic and abiotic stresses. This study is the first to report the alkaline salt regulation of the WRKY gene. Results In this study, we cloned a WRKY gene (SlWRKY28) from the Salix linearistipularis and then transferred to the Populus davidiana × P. bolleana for expression. Sequence analysis on the transcriptome of Salix linearistipular showed the significant up-regulation of WRKY gene expression in response to salt-alkali stress in seedlings. Our data showed that SlWRKY28 localized to the nucleus. Furthermore, the expression of the SlWRKY28 from female plants increased with saline-alkali stress according to the northern blot analysis results. The results of 3,3′-Diaminobenzidine (DAB) staining showed that hydrogen peroxide (H2O2) concentration was lower under stress, but ascorbate peroxidase (APX) enzyme activity was significantly higher in the overexpressed plants than that in non-transgenic (NT) plants. Conclusions We found out the SlWRKY28 induced regulation of the enzyme gene in the reactive oxygen species (ROS) scavenging pathway is a potential mechanism for transgenic lines to improve their resistance to alkaline salt. This study shows theoretical and practical significance in determining SlWRKY28 transcription factors involved in the regulation of alkaline salt tolerance.

resource for ecological restoration and afforestation of soda saline-alkali grassland 2. The growth and development of forest trees are frequently challenged by biotic (such as pests and diseases) and abiotic (such as drought, soil salinity, and flooding) stresses, although natural forests have evolved a certain ability to cope with these adverse environmental factors. Sequence analysis of transcriptional sets in S. linearistipularis under salt stress showed agreement with 22 pathways regulated by salt stress in Arabidopsis thaliana and node genes, including 1397 up-regulated and 1637 down-regulated genes. SlWRKY gene is one of the up-regulated genes 4. WRKY protein is a specific transcription factor that can be used to regulate biotic and abiotic stresses [5], including responses to salt stress [6], drought stress [7], cold stress [8], and injury response [9]. There are 74 members of the WRKY protein family for A. thaliana [10], 102 members for rice [11], 136 members for corn [12], and 104 members for poplar [13]. At present, the stress responses of plants with WRKY proteins have been widely reported [14]. WRKY protein binding reaction element W-boxes [(T)(T)TGAC(C/T)] are located in the upstream regulatory region of defense-related gene promoters [15]. Therefore, the WRKY protein may regulate downstream gene expression related to stress response and potentially improve resistance through metabolic component regulation. The expression of BCWRKY32 and BCWRKY35 genes in the adventitious roots of Bupleurum are induced by NaCl [16]. In the transgenic plants of A. thaliana overexpressing the AtMBF1 gene, the expression of AtWRKY18, AtWRKY33, AtWRKY40, and AtWRKY46 increased; heat tolerance was also significantly higher than that of wild type plants [17]. The WRKY family in grapes has 59 members, of which 36 have twofold up-regulated expression under lowtemperature stress. The overexpression of the WRKY43 gene increases the cold tolerance of transgenic plants; thus, the gene is involved in the signal transduction process of cold stress in grapes [18]. The WRKY gene of Tamarix rubrum is induced by using high salt conditions, drought, and exogenous ABA application. The overexpression of WRKY4 gene increases the tolerance of transgenic plants to saltation and abscisic acid (ABA) [19]. A WRKY6 can bind to two W-boxes located at the upstream of phosphate transport-related gene PHO1 promoter and negatively regulates AtPHO1 expression. Transgenic plants overexpressing AtWRKY6 are sensitive to phosphate deficiency and accumulate small amounts of phosphorus in their stems [20,21] found that WRKY18, 38, 53, 54, 58, 59, 66, and 70 genes are directly regulated by NPRI. WRKY TFs have been implicated in the regulation of different metabolic pathways (e.g. biosynthesis of secondary metabolites, plant senescence and signal molecule-delivery) under biotic and abiotic stresses in plants [22]. WRKY TFs are plant-specific proteins and constitute one of the largest TF families in plants. In A. thaliana, WRKY TFs can directly activate genes related to salicylic (SA), jasmonic acid (JA), and H 2 O 2 biosynthesis and improve stress tolerance by participating in the metabolic regulation of ROS stress [23][24][25]. A single WRKY TF can respond to multiple stress factors simultaneously and contribute to the regulation of other physiological processes. Currently, the potential roles of the WRKY gene in the tolerance of plants to alkaline salt have not been reported.
Populus davidiana × P. bolleana is an artificial hybrid combination of Populus davidiana as the female parent and Populus bolleana Lauche as the male parent, which was further selected and bred [26]. The trunk is straight, the petiole and leaf backs are silver-white downy, the leaf surface is dark green, and the branches and leaves are shaken by the wind, adding to its beauty. The inflorescence falls off nature ally without flying flocculent, which does not cause environmental pollution. Populus davidiana × P. bolleana has a strong cold wintering ability, but also very drought-resistant, and rapid growth, the tree posture is beautiful and neat, suitable for the northern drought and cold natural conditions, is China's northern narrow-crowned poplar in the most cold, the most beautiful of the preferred species of landscaping and protection [27]. At present, the Populus davidiana × P. bolleana tissue culture propagation technology has been quite mature, the tissue culture expansion can get a large number of seedlings, and has the characteristics of rapid growth, beautiful tree shape, has become a good model of research woody plants .
In this study, SlWRKY28 from S. linearistipularis was cloned and comfirmed to be associated with the salinealkali stress response. The expression of SlWRKY28 in Populus davidiana × P. bolleana improved the tolerance to saline-alkali stress.

Bioinformatics analysis of SlWRKY28
The sequence analysis results showed that SlWRKY28 contained a 1468-bp ORF, encoding 475 amino acids. The predicted protein isoelectric point was 7.62, average hydrophilicity was − 0.653, the aliphatic index was 62.22, stability index was 52.8, and the molecular weight was 52 kDa. The WRKY domain (Fig. 1a) contained a highly conserved motif with the sequence WRKYGQK and a C2H2 zinc finger motif fold at a position of 256-356 bp, which is a two-type WRKY TFs. Results showed that the homology of the NCBI amino acid BLAST was 92%. The putative WRKY clone from S. linearistipularisis was highly homologous to WRKY28 of Populus davidiana × P. bolleana (Fig. 1b) and it was designated as SlWRKY28 (GenBank: MH223643).

SlWRKY28 localizes to the nucleus
Consistent with the identified NLS sequence, the subcellular location prediction software Plant-mPloc (http:// www.csbio.sjtu.edu.cn/bioinf/plant-multi/) predicted that the SlWRKY28 protein localizes to the nucleus. To confirm our prediction, the 35S-SlWRKY28::GFP vector was constructed and transferred into onion epidermal cells. The 35S::GFP construct served as a control. The onion epidermal cells harboring the 35S-SlWRKY28::GFP construct emitted green fluorescence predominantly in nuclei (Fig. 2), whereas 35S::GFP fluorescence occurred widely throughout the cell [28].

W-box DNA binding characteristics of SlWRKY28
To detect the cloned SlWRKY28 encoding protein can bind to DNA cis-element W-box with binding characteristic of WRKY TF, the prokaryotic expression vector pGEX-6p-3-SlWRKY28 was constructed, and GST-SlWRKY28 fusion protein was expressed and purified using E. coli BL21 cells through IPTG induction. The gel blocking signal of GST-SlWRKY28 fusion protein and biotin-labeled probe were detected using the EMSA kit (Fig. 3). The swimming lane with only GST protein showed a free probe signal but no complex signal. This result indicated that the GST protein did not bind to the DNA probe. The complex of GST-SlWRKY28 fusion protein and Biotin-Pr produced the signal, whereas the complex signal was weak when a cold competitive probe was added. The fusion protein binds to the TTGAC amino acid of the DNA probe. The fusion protein could not bind to the cis-element W-box, indicating that the expressed SlWRKY28 could bind to a specific probe. The results revealed that the SlWRKY28 encoding protein has the functional characteristics of the WRKY transcription factor binding to the W-box. Conserved domains and phylogenetic tree of SlWRKY28. a The conserved retrotransposon gag and WRKY domain were found in the amino acid sequence. WRKYGQK was between 296 and 356 amino acids. b Phylogenetic trees based on the amino acid sequence of SlWRKY28 and homologous sequences from the GenBank database. The representative sequences reported in this study is marked with a black dot (•), as determined by a neighbor-joining analysis. The source of the sequence was indicated after each protein name

Analysis of mRNA expression characteristics of SlWRKY28
The expression of SlWRKY28 in the leaves, flowers, stem epidermis, anthers, and roots of male and female plants of the three-year-old S. linearistipularis was analyzed by qRT-PCR (Fig. 4a). Altogether, the expression level of female plants are higher than of male plants. The expression level of SlWRKY28 increased with the prolongation of treatment time. Furthermore, expression of SlWRKY28 was induced 12 h after 150 mM NaCl stress, and increased up to 48 h (Fig. 4b); in contrast, the response of SlWRKY28 started 24 h after 30 mM NaHCO 3 , and increased significantly at 48 h (Fig. 4c). Those results indicated that SlWRKY28 had a stronger response to NaHCO 3 stress than to NaCl stress, and its response need a certain process.

Expression analysis of SlWRKY28 under NaHCO 3 stress
The seedlings expressed SlWRKY28 (T0 -#1,-#3) and NT was incubated on the 1/2 Murashige and Skoog (MS) medium with various concentrations of NaHCO 3 (0, 2.5, 5, and 7.5 mmol/L), and phenotypes were investigated 72 h after the stresses (Fig. 5a). The phenotypes changed significantly under 5 and 7.5 mmol/L NaHCO 3 , and tolerance of overexpressing SlWRKY28 plants to NaHCO 3 was superior to NT. Under 5 and 7.5 mmol/L NaHCO 3 , malonic dialdehyde (MDA) concentration of overexpression SlWRKY28 plants was significantly lower than NT (Fig. 5b), and the green content of leaves was higher than NT (Fig. 5c). Moreover, APX activity increased with the concentration of NaHCO 3 , and was significantly higher than NT plants (Fig. 5d).
The content of active oxygen species (O 2 and H 2 O 2 ) in Populus davidiana × P. bolleana treated with NaHCO 3 (0, 2.5, 5, and 7.5 mmol/L) was expressed by nitro blue tetrazolium (NBT) and DAB. The results showed that the NT and transgenic plants deepened gradually with the increase of the stress concentration, the color of NT was darker than transgenic plants (Fig. 5e, f). These results showed that the content of O 2 and H 2 O 2 was lower than transgenic plants, which suggested the tolerance of SlWRKY28 was increased to oxidative injury by NaHCO 3 stress. Together, these results imply that SlWRKY28 TF may play an important role in gene expression regulation to anti-oxidative stress. In the gel block, the lanes were followed by GST + protein and biotin-labeled oligonucleotide probe (Biotin-Pr); GST-SlWRKY28 fusion protein and Biotin-Pr were added; also undiluted oligonucleotide probe (Cold-Pr) diluted 10-fold; added GST-SlWRKY28 fusion protein and Biotin-Pr, and 50-fold diluted unlabeled oligonucleotide probe (Cold-Pr). The thin arrow refers to the DNA and protein complex; the thick arrow refers to the free probe

Analysis of photosynthetic characteristics of transgenic plants under NaHCO 3 conditions
Top leaves of NT plants turned yellow, whereas transgenic plants were bright green, (Fig. 7a) which have a significant difference intolerance of the parietal leaves to NaHCO 3 stress. The chlorophyll concentration of transgenic plants was higher than NT (Fig. 7b). FluorCam chlorophyll fluorescence imaging system showed that the fluorescence intensity of NT gradually decreased from the third parietal leaf to the first one (Fig. 7c), indicate that photosynthetic capacity (Fv/Fm) has a downward trend. The photosynthetic capacity of transgenic plants was higher than NT, which indicates that overexpression of SlWRKY28 can alleviate the damage caused by stress on photosynthesis and could improve tolerance of alkaline salt (Fig. 7d).

Obtaining SlWRKY28 transgenic plant
The resistant transformants were obtained using A. tumefaciens to infect and transform leaves of Populus davidiana × P. bolleana in Hygromycin (Hyg) culture medium. The leaves of control soaked in Hyg 35 mg/L solution became white and the green recedes, but the transformed leaves (T 0 -# 1, −# 3, −# 5) were still green (Fig. 8a). The targeted band via PCR indicated that SlWRKY28 has been integrated into the chromosome of Populus davidiana × P. bolleana (Fig. 8b). Southern blot showed that T-#1 was a single copy insertion, T-#3, T-#5 lines were multi-copy insertions (Fig. 8c), strong hybridization signals were detected in T0-#1, −#3, −#5 lines and no signal in NT plant by Northern blot, those data confirmed SlWRKY28 expressed (Fig. 8d).

Discussion
The WRKY TF is known as the largest superfamilies which participate in the regulation of proteins in plants [29]. In the past several years, growing evidence has shown that members of the WRKY gene family mainly participate in stress responses, plant growth and development and leaf senescence [30]. WRKY as an important TF can rapidly increase its family members to establish a signal transduction network in adversity, which may optimise plant adapt-ability [31]. In this study, we isolated a gene from Salix linearistipularis and characterized its functional roles. Many TFs families, such as WRKY, AP2 (APETALA2)/ERF (ethylene-responsive factor) and NAC (NAM, ATAF1/2, CUC1/2), are plant-specific transcription regulators [32]. zWRKY is not only involved in carbohydrate and secondary metabolite synthesis, senescence, and development but also plays an essential role in plant responses to biotic and abiotic stresses [33]. S. linearistipularis is an important salt and alkaline tolerant plant resource. In this study, the WRKY gene was cloned from S.linearistipularis,   which encodes 475 amino acids with WRKY motif and a C2H2 zinc finger folding to form the WRKY domain (Fig. 1a). WRKY gene clone also accords with the characteristics of the IId and IIe subfamilies [34]. EMSA assay showed that encoding protein binds to TTAGC amino acids, and the binding characteristics of W-box cis-element also confirmed the function of WRKY TFs. The coding proteins of plant genes are based on the localization information contained in amino acid sequences specific parts mapping of the cell by precise guidance mechanism [35]. The PSORT analysis of biological software predicts that 73.9% of the SlWRKY28 protein is located in the nucleus; the verification experiment by gene gun bombarded the onion epidermal cells, which also confirmed that the fusion protein of 35S-SlWRKY28-GFP emitted green fluorescence in the nucleus (Fig. 2). The results of this study are consistent with the subcellular localization of WRKY TF studied using other species [36]. TFs and its structural gene jointly control growth and development traits, the WRKY TF may be involved in the regulation and responses of plants to drought and salt stress through pathways like other members of the WRKY TFs family [37,38]. Northern blot analysis showed that the expression of SlWRKY28 increased significantly with the prolongation of treatment time. Our findings indicate that SlWRKY28 is a responsive gene to saline-alkali stress. The expression pattern was also found in BCWRKY16, BCWRKY32, and BCWRKY35 in roots under NaCl and PEG6000 stress [39], as well as expression of OsWRKY24, OsWRKY51, OsWRKY 71, and OsWRKY72 TFs under ABA, NaCl, PEG, cold and heat stress [40]. Various TFs are involved in modulating leaf senescence, and 1533 TFs have been identified via leaf senescence transcriptome analyses in Arabidopsis rice and cotton [41][42][43]. At present, studies on WRKYs in different species mainly focus on its roles in the signal transduction mechanism. In our study, S. linearistipularis has grown in a soda saline-alkali environment with a high pH value was selected. Our data showed that overexpression SlWRKY28 was more resistant to NaHCO 3 stress than NT plants, as indicated by higher green content of leaves and APX activity, with lower MDA concentration in the transgenic plants than NT. From the results, it can be inferred that SlWRKY28 is potentially involved in the scavenging regulation of ROS and improvements in plant growth under alkaline salt stress. NBT and DAB staining showed that the color reaction in the third leaf was gradually deepened under NaHCO 3 stress (Fig. 5e,  f), and was lighter in the transgenic plants than in the NT plants. This result is consistent with changing patterns of POD, SOD, and Catalase (CAT) activities caused by overexpression of DgWRKY3 in tobacco [44]. It is hypothesized that the SlWRKY28 TF may be involved in the regulation of the expression of antioxidative stress -related genes. qRT-PCR analysis indicated that the expression of APX, Enolase and SPDS were increased under NaHCO 3 treatment, with expression levels significantly higher in transgenic plants than in NT. Moreover, the expression of the SOD gene increased under 2.5 mM NaHCO 3 stress but decreased when the stress increased to 5.0 mM NaHCO 3 . The transcriptional expression of the P5CS gene decreased rapidly under NaHCO 3 stress, which suggests that SlWRKY28 TF may up-regulate the expression of APX, SOD, enolase and SPDS genes under alkaline salt stress, and down-regulate or not contribute in the regulation of PtP5CS gene expression (Fig. 9). These results suggest that SlWRKY28 may play a significant role in the regulation of alkaline and salt stress tolerance. Moreover, SlWRKY28 TFs may be involved in the regulation of photosynthetic electron transport pathway, which contributes to the improvement in plant tolerance to alkaline salts. The present study showed a theoretical and practical basis in potential pathways of SlWRKY28 TFs in the regulation of plant tolerance to alkaline salt stress. However, further studies are needed to elucidate the pathways involved in the SlWRKY28 mediated mechanism.

Conclusions
In this research work, we carried out the structure and functional characterization of the SlWRKY28 in Populus davidiana × P. bolleana. SlWRKY28 was involved in the regulation of alkali-resistant salt stress by regulating the downstream target genes associated with the ROS pathway. Our results provide a theoretic basis for investigating the functional mechanisms of exogenous genes in transgenic plants. However, other functions of the SlWRKY28 gene require further study and clarification. Our work can further study the response of the over-expression of WRKY to the conditions of saline-alkali stress.

Gene cloning and sequence analysis
To amplify the full-length cDNA and genomic sequences of SlWRKY28 primers were designed based on the coding sequence of SlWRKY28 submitted to NCBI. Under saline-alkali stress, the up-regulated expression of the SlWRKY28 gene assessed based on RNA-Sequence in S.linearistipularis. SlWRKY28 (Sl28-F, R) was cloned by RT-PCR. The protein domains [45], molecular weights, and isoelectric points and amino acid sequences of SlWRKY28 protein were analyzed by using Protparam Software. The subcellular localization of SlWRKY28 was predicted through PSORT, whereas the comparison of proteins sequence homology, online NCBI was used for the construction of the phylogenetic tree by Neighbor-Joining method in MAGA software.

Subcellular localization of SlWRKY28
pMD18-T-SlWRKY28 plasmid DNA was used as a template, and KpnI/SpeI site primers were added and inserted into T-vector via PCR and ligated into pBI121-MCS-GFP vector through enzyme digestion, which obtained pBI121-SlWRKY28-GFP plant localization expression vector. The green fluorescent expression of the 35S-SlWRKY28-GFP fusion protein in the epidermal cells of the onion was detected under Olympus laser confocal microscope using a gene gun to bombard the onion bulb method and expression of subcellular localization was identified [46].

Analysis of W-box binding characteristics of SlWRKY28 protein
The binding characteristics of the SlWRKY28 protein to W-box element was investigated with EMSA. The fusion protein (GST-SlWRKY28) was expressed in and purified from prokaryotic expression system as reported by Shan et al. (2014). The correct pMD18-T-SlWRKY28 plasmid was digested with restriction endonuclease NotI and BamHI, and the target fragment was recovered. The SlWRKY28 was constructed into the pGEX-6p-3-SlWRKY28 expression vector with GST tag and digested using the same enzyme, and transformed into E. coli BL21 (DE3) cells. The expressed GST-SlWRKY28 fusion protein was purified through Glutathione Sepharose 4B (GE company) GST affinity chromatography. The purified fusion protein was used for gel block detection. Preparation of oligonucleotide probe (Biotin-Pr): Probe primer for W-box was synthesize by Doctor Biology Co., Ltd., which were W-box F1: 5′-ggaacttgaccttgaccttagggctgcaggaattcg-3′; and reverse complementary sequence Wbox R2: 5′-cgaattcctgcagccctaaggtcaaggtcaagttcc-3′. Each pair of primers for each probe combined two reverse complementary DNA sequences on a 1:1 scale which followed by denaturation at 100°C for 10-min, synthesis of doublestranded DNA through gradually cooling to room temperature. An oligonucleotide probe (Biotin-Pr) was obtained by labeling two probes with biotin, and the unlabeled reverse complementary DNA was used as a cold competitive probe (Cold-Pr). According to the EMSA kit (Biyuntian Biological Co., Ltd.) instructions for EMSA slightly changed for optimizing conditions in our Laboratory.
Mix properly and react at room temperature 25°C for 20 min, 1 μL EMSA/gel-shift sample buffer was added for SDS-PAGE gel electrophoresis and the membrane was transferred. Purple diplomatic union (UV-light cross-linker 254 nm), BeyoECL moon A and B solution (Biyuntian Biological Co., Ltd.) was used to mix for color and combined with imaging detection on LAV4000.

Analysis of mRNA expression characteristics of SlWRKY28
For analysis of expression characteristics of tissues and organs, male and female flowers, terminal buds, tuck buds, leaves, and stem segments (epidermis and xylem) were collected from three-year-old S. linearistipularis. RNA was extracted from the roots of the plant and rverse-transcribed into cDNA. The Actin1 gene was used as an internal reference to normalize the gene expression of all samples. The control group was set with plain water. The data acquisition was completed on the MxPro-Mx3000P system, and each response was repeated three times. At the end of the reaction, the relative expression of the gene was calculated using relative quantitative method. The output was analyzed in Excel format, and a column graph was constructed according to the change of expression quantity.
Analysis of expression characteristics after stress treatment. Extraction of total RNA from S. linearistipularis under NaCl and NaHCO 3 using RNA Kit. Agarose (1.2%) denatured gel electrophoresis (15 V/cm) was run for 4 h, then transferred RNA on membrane. SlWRKY28 specific probe (qRT-F/R 496 bp) labeled with DIG was hybridized and then washing the membrane. The CDP-Star color-developing agent was used to detect signals on the Image Quant LAS 4000 imaging analyzer [47].
Growth tolerance of overexpressed SlWRKY28 seedlings to NaHCO 3 stress The growth-consistent overexpressing plants of SlWRKY28 and NT (T0 / 1, # 3 lines) were inoculated into 1/2 MS medium containing 0, 2.5, 5, and 7.5 mM NaHCO 3 (three replications per treatment). Plants were grown at 22°C room temperature, 16/8 h light/dark period, and growth parameters of the plants were investigated under stress. Concentrations of chlorophyll and MDA in the third parietal leaf and the activity of APX in the whole plant were measured.  [48].

Vector construction and plant transformation
The binary plant expression vector pGWB11-SlWRKY28 (Fig. 10) was connected, through the gateway system and electro-transformation into A.tumefaciens EHA105. Genomic DNA from pot-transformed plants was extracted through CTAB. The target gene was aligned for PCR detection. The inserted copy number in this generation strains was detected by Southern blot [49].
SlWRKY28 expression in transgenic plants was detected by northern blot [47].

Statistical analyses
Microsoft Excel 2010 (Microsoft Corp., Redmond, WA) and Statistical Product and Service Solutions v. 20.0 (SPSS, Chicago, IL) were used to analyze the experimental data. Both one-way analyses of variance and two-way analysis of variance were used to determine the significance of the differences among treatments. Student's ttest was run to calculate P-values (*P < 0.05; **P < 0.01). The data were normalized, and all samples were normally distributed in terms of homogeneity of variance.