Association of transcription factor WRKY56 gene from Populus simonii × P. nigra with salt tolerance in Arabidopsis thaliana

Plant growth and stress treatment

The twigs of P. simonii × P. nigra from the same clone were grown in pots containing regular water and exposed to 16 h light/8 h dark at 26/22 °C. A total of 2-month-old seedlings with new roots and leaves were subjected to the treatment of 200 mmol/L NaCl. The leaves were harvested at 0, 6, 12, 24, 48, and 72 h for qRT-PCR with three biological replications. The samples at 0 h were used as the control. The leaves, stems, and roots were harvested at 0 and 24 h for RNA-Seq. These tissues were frozen immediately in liquid nitrogen and then stored at −80 °C.

The Arabidopsis seeds were treated in sodium hypochlorite solution (10% Cl, 0.05% Tween20) for 5 min, washed five times with sterilized water, and sowed on Murashige and Skoog (MS) medium (Murashige & Skoog, 1962). These Petri dishes were treated at 4 °C for 4 days and then transferred to a growth chamber under 16 h light/8 h dark cycles at 24 ± 2 °C. At the four-leaf stage, the seedlings were transferred to plastic pots containing potting mix (nutrient soil:vermiculite:perlite, 5:3:2, v/v/v). The plants were used for transformation once the secondary bolting reached a height of six to nine cm after cutting the primary bolting.

Expression analysis of the WRKY genes using RNA-Seq

The leaf, stem, and root samples mentioned above were sent to the GENEWIZ Company (www.genewiz.com) for library construction and RNA-Seq (Illumina HiSeq 2500 platform). Sequencing, library construction and RNA-Seq data processing were described by Yao et al. (2016). Gene expression was quantified as fragments per kilo-base pair transcript per million mapped read (Mortazavi et al., 2008). Both the false discovery rate < 0.05 and |log₂FC| ≥ 1 (fold change) were used to claim the DEGs. The hierarchical clustering of WRKY DEGs between the salt stress and the control was conducted using the Hem I 1.0 software.

Cloning and sequence analysis of the WRKY56 gene

The total RNA was isolated from the leaves of P. simonii × P. nigra grown under normal conditions using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). The PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Kusatsu, Japan) was used for first-strand cDNA synthesis. The cDNA fragment of the WRKY56 gene was obtained by RT-PCR using a pair of primers (WRKY56-F: 5′-TCAGATCAACCCTACCCTACAC-3′ and WRKY56-R: 5′-CAGCGAGAAATTCCACACCATG-3′).

The open reading frame (ORF) of WRKY56 was predicted using the ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). The analysis of the physicochemical properties, such as the molecular weight (MW) and the theoretical isoelectronic point (pI) were performed with the ProtParam software (https://web.expasy.org/protparam/). The WRKY sequences of poplar and Arabidopsis were obtained from the plant transcription factor database (http://planttfdb.cbi.pku.edu.cn/). The phylogenetic tree was constructed using the Neighbor-Joining method in program MEGA5.0. The confidence intervals were based on 1,000 bootstrapping samples for the branches of the generated phylogenetic tree. The WRKY56 amino-acid sequence was aligned with WRKY from other plants using Clustal W2.

Expression of the WRKY56 gene in response to salt stress

The total RNA was extracted from the leaves of P. simonii × P. nigra under salt stress at 0, 6, 12, 24, 48, and 72 h, using a Trizol reagent (Invitrogen, Carlsbad, CA, USA). Equal amounts of RNA were used for reverse transcription to cDNA using the PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Kusatsu, Japan). The cDNA served as the template in qRT-PCR using WRKY56 gene-specific primers (WRKY56-F: 5′-TGATAATGAG GTAGAGGAGCAA-3′, WRKY56-R: 5′-AAAACCCAAAGGCGACAAGC-3′). Primers 5′-CAGCAACCGCAATACAAA-3′ and 5′-GCATACAGGGAAAGGACA-3′ were used for control amplification of ACTIN from poplar (EF418792). The qRT-PCR was performed on the Bio-Rad CFX96 system using the SYBR Premix Ex Taq™ (Takara, Kusatsu, Japan). The reaction was performed under the following conditions: initial denature at 94 °C for 30 s, 44 cycles containing denature at 94 °C for 12 s, annealing at 54 °C for 30 s, elongation at 72 °C for 40 s, and reading plate at 81 °C for 1 s.

There were three independent biological replicates at each time point. The relative expression levels of WRKY56 were calculated using the 2^−ΔΔCt method, which was defined as: C_t = (C_t-target − C_t-control)₂ – (C_t-target − C_t-control)₁ (Livak & Schmittgen, 2001). The analysis of variance was carried out using SPSS16.0 and Duncan’s test was used for multiple comparisons. The results were expressed as the mean ± standard deviation (SD) of three replicate experiments. The significance threshold was at P < 0.05.

Vector construction and Arabidopsis transformation

The cDNA fragment of WRKY56 containing ORF, with a BamH I restriction site at the 5′ end and an Xho I site at the 3′ end was obtained by PCR using the following primers: 5′-GCGGGATCCGGTGGCGACGACTCCTGGAGCCCG-3′/5′-CGCCTC GAGCAGCGAGAAATTCCACACCATG-3′. The fragment was inserted into the BamH I/Xho I site of the plant expression vector pBI121 to replace the BamH I/Xho I beta-glucuronidase cassette under the control of CaMV35S promoter. The pBI121-WRKY56 recombinant vector was transferred into Agrobacterium EHA105 by electroporation. Arabidopsis transformation was carried out by the floral dip method (Clough & Bent, 1998).

Evaluation of transgenic Arabidopsis under salt stress

Transgenic Arabidopsis seeds from T₁, T₂, and T₃ were selected on 1/2 MS medium containing 50 mg/L kanamycin under 16 h light/8 h dark cycles at 24 ± 2 °C. After selection by kanamycin resistance, the putative transgenic Arabidopsis lines were subjected to PCR and RT-PCR assays and the PCR fragments were further confirmed by DNA sequencing. The T₃ homozygous seeds of the transgenic plants were selected for further growth and physiological analysis. Three transgenic lines were selected from T₃ transgenic homozygous lines. The seeds of three T₃ transgenic homozygous lines and a wild type (WT) were germinated in 1/2 MS medium (30 seeds/dish) supplemented with 0, 50, 100, and 150 mmol/L NaCl, with three biological replicates. The ratio of seed germination was scored after 4 days and the fresh weight (FW) was measured after 15 days. At the 4-week stage, T₃ homozygous transgenic and wild Arabidopsis (as control) grown in soil were irrigated with 100 mmol/L NaCl for 5 days and the leaves were sampled for physiological trait analysis. The peroxidase (POD) and superoxide dismutase (SOD) activities were assayed as described by Mead (1976) and Giannopolitis & Ries (1997). The proline content was determined by the method described by Zhang & Huang (2013). The malondialdehyde (MDA) content was measured by the thiobarbituric acid coloration method (Hao & Liu, 2001). The results were shown as the mean ± SD of three independent experiments. Duncan’s test was used to identify significant differences between WT and transgenic WRKY56 lines.

Expression analysis of the WRKY genes by RNA-seq

To understand the molecular mechanism of WRKY genes in response to salinity challenges, we employed RNA-Seq to obtain an overview of the transcriptome characteristics of poplar seedlings treated with NaCl and against the control. Among the 102 WRKY genes in poplar, we identified a total of 23 DEGs, including 13 up-regulated genes (URGs) and 10 down-regulated genes (DRGs) (Table S1). There were 12, 13, and 13 significantly DEGs in the leaf, stem, and root, respectively. Five DEGs, including two URGs and three DRGs, were shared among the leaf, stem, and root tissues. The number of DEGs shared in two out of the three tissues were zero in leaf-stem, three (zero URG/three DRGs) in stem-root, and two (two URGs/zero DRG) in leaf-root, respectively. In addition, as many as five (four URGs/one DRG), five (two URGs/three DRGs), and three (three URGs/zero DRG) DEGs were specific to the leaf, stem, and root, respectively (Fig. 1A). In the leaf, eight and four DEGs were up-regulated and down-regulated, respectively (Fig. 1B). In the stem, there were four URGs and nine DRGs (Fig. 1C). In the root, seven URGs and six DRGs were identified (Fig. 1D). The DEGs Potri.014G050000, Potri.001G472800, Potri.011G007800, Potri.004G007500 and Potri.007G078200 were shared in the leaf, stem, and root (Fig. 1E). In addition, Potri.014G050000 and Potri.001G472800, Potri.011G007800 and Potri.004G007500, were clustered together. The expression of the WRKY56 (Potri.002G193000) gene was increased in the leaf under salt stress and that result was congruent with our previous work about qRT-PCR and cDNA-AFLP (Wang et al., 2011, 2014).

Sequence alignments and phylogenetic analysis of the WRKY proteins

To investigate the evolutionary relationship of WRKYs in poplar and Arabidopsis, 102 WRKYs from poplar and 72 WRKYs from Arabidopsis were downloaded to construct a phylogenetic tree. Based on the results from the sequence alignments and the phylogenetic tree, the WRKYs were classified into three groups: group I, group II, and group III (Fig. 2A). In poplar, 22 WRKYs containing two WRKY domains and a C₂H₂ zinc finger motif were assigned to group I. 68 WRKYs with one WRKY domain and a C₂H₂ zinc finger motif were categorized into group II, which was further classified into five subgroups, designated as group II-a (5), group II-b (9), group II-c (27), group II-d (11), and group II-e (16). 12 WRKYs, each harboring one WRKY domain and a C₂CH zinc finger motif, were identified to group III. According to the phylogenetic tree, we found that the WRKY56 (Potri.002G193000) belonged to group II-c.

Cloning and sequence analysis of the WRKY56 gene

The full-length cDNA of the WRKY56 gene was isolated from the leaves of P. simonii × P. nigra by RT-PCR, and was 1,177 bp and contained an ORF of 954 bp encoding 317 amino-acid residues. The MW of the WRKY56 was 35.37 kDa with theoretical pI of 6.67. The conserved domain included the WRKY domain with 60 amino acids. Evidence from the sequence alignment revealed that all of the 13 WRKY proteins shared one WRKY domain and C₂H₂ zinc finger motif (Fig. 2B). The WRKY56 from P. simonii × P. nigra showed a high homology with the WRKY proteins from other poplars, but a longer evolutionary distance with the WRKY proteins from Gossypium hirsutum, Theobroma cacao, Glycine max, and A. thaliana (Fig. 2C).

Expression characterization of the WRKY56 gene under salt stress by qRT-PCR

The expression of the WRKY56 gene in the leaves of P. simonii × P. nigra under salt stress was examined by qRT-PCR at different time points as shown in Fig. 3 and Table S2. Under salt stress, the expression of the WRKY56 gene initially increased between 6–12 h. The peak value appeared at 12 h and then decreased between 12 and 72 h. Evidence from statistical analysis indicated that the expression of the WRKY56 gene was significantly higher than that of the control at 12, 48, and 72 h (P < 0.01), respectively.

Characterization of transgenic Arabidopsis over-expressing the WRKY56 gene under salt stress

To characterize the growth of transgenic Arabidopsis under salt stress, the three transgenic homozygous lines from the T₃ generation and the WT were sown into 1/2 MS medium supplemented with 0, 50, 100, and 150 mmol/L NaCl. The FWs were measured after 15 days. The results revealed that there was no significant difference in FW between the transgenic lines and the WT under both normal and 50 mmol/L NaCl conditions. The FWs of the transgenic lines and the WT were decreased under salt stress, compared with those under normal conditions. Transgenic lines exhibited a greater FW than that of the WT under 100 and 150 mmol/L NaCl conditions (Fig. 4A; Table S3). The FWs of the transgenic lines T-1 and T-2, under 100 mmol/L NaCl conditions, were 40.9% and 36.36% higher, respectively, than those of the WT, which was significant (P < 0.01). The FWs of the transgenic lines were higher than those of the WT plants under the 150 mmol/L NaCl treatment; the FW of T-2 was 80% higher than the WT plants, which was significant (P < 0.01).

To evaluate the impact of the WRKY56 gene on seed germination, WT and three transgenic homozygous lines seeds from the T₃ generation were germinated in 1/2 MS medium supplemented with 0, 50, 100, and 150 mmol/L NaCl, respectively. The seed germination rates of the WT and the three transgenic lines were similar to the trends found in the FW. There was no extremely significant difference in the germination rate between the transgenic lines and the WT under normal and 50 mmol/L NaCl conditions. Under both 100 and 150 mmol/L NaCl conditions, transgenic seeds exhibited a higher germination rate than those of the WT. With the challenge of 100 and 150 mmol/L NaCl, the seed germination rates of the transgenic lines were 18.97–31.17% and 44.71–51.62% higher than those of the WT plants, respectively (P < 0.01) (Fig. 4B; Table S3). These results indicated that the WRKY56 gene could enhance the salt tolerance of Arabidopsis.

The SOD and POD activities of the transgenic WRKY56 lines were assayed under normal and 100 mmol/L NaCl conditions. Under normal conditions, the POD activities of T-1 and T-3 were 3.61 and 1.82 times higher than those of the WT, which was significant at P < 0.01. Under 100 mmol/L NaCl stress, the POD activities of the transgenic lines were 2.08–3.70 times higher than those of the WT plants, which was significant at P < 0.01 (Fig. 4C; Table S4). The SOD and POD activities of the transgenic lines and the WT under both normal and NaCl conditions showed different trends. Under the normal and 100 mmol/L NaCl conditions, the difference of SOD activity between the transgenic lines and the WT was not significant except that the SOD activity of T-2 was 17.07% higher than that of WT under the 100 mmol/L NaCl conditions, which was significant at P < 0.05 (Fig. 4D; Table S4).

The proline content of the WT was similar to that of the transgenic WRKY56 lines under the normal condition; however, the proline content of the transgenic lines was 27.10–66.41% higher than that of the WT under 100 mmol/L NaCl conditions, with a significance of P < 0.01 (Fig. 4E; Table S4). These results demonstrated that an over-expression of the WRKY56 gene in Arabidopsis under salt stress could elevate the SOD and POD activities, as well as proline content in transgenic plants.

There was no extremely significant difference in the MDA contents between the transgenic lines and the WT under normal conditions. The MDA contents between the WT and transgenic lines under salt stress were higher than those under normal conditions. The MDA contents of the transgenic lines under salt stress were 9.93–21.05% lower than those of the WT plants, especially T-1, with a significance of P < 0.01 (Fig. 4F; Table S3). These results indicated that an over-expression of the WRKY56 gene in Arabidopsis could reduce the lipid over-oxidation rate in transgenic plants.