Heterologous Overexpression of Poplar SnRK2 Genes Enhanced Salt Stress Tolerance in Arabidopsis thaliana.

Subfamily 2 of SNF1-related protein kinase (SnRK2) plays important roles in plant abiotic stress responses as a global positive regulator of abscisic acid signaling. In the genome of the model tree Populus trichocarpa, 12 SnRK2 genes have been identified, and some are upregulated by abiotic stresses. In this study, we heterologously overexpressed the PtSnRK2 genes in Arabidopsis thaliana and found that overexpression of PtSnRK2.5 and PtSnRK2.7 genes enhanced stress tolerance. In the PtSnRK2.5 and PtSnRK2.7 overexpressors, chlorophyll content, and root elongation were maintained under salt stress conditions, leading to higher survival rates under salt stress compared with those in the wild type. Transcriptomic analysis revealed that PtSnRK2.7 overexpression affected stress-related metabolic genes, including lipid metabolism and flavonoid metabolism, even under normal growth conditions. However, the stress response genes reported to be upregulated in Arabidopsis SRK2C/SnRK2.6 and wheat SnRK2.8 overexpressors were not changed by PtSnRK2.7 overexpression. Furthermore, PtSnRK2.7 overexpression widely and largely influenced the transcriptome in response to salt stress; genes related to transport activity, including anion transport-related genes, were characteristically upregulated, and a variety of metabolic genes were specifically downregulated. We also found that the salt stress response genes were greatly upregulated in the PtSnRK2.7 overexpressor. Taken together, poplar subclass 2 PtSnRK2 genes can modulate salt stress tolerance in Arabidopsis, through the activation of cellular signaling pathways in a different manner from that by herbal subclass 2 SnRK2 genes.


INTRODUCTION
Plants face various environmental stresses including drought, high salinity, and extreme temperatures. Such adverse circumstances can often lead to severe agricultural and industrial losses, so it is important to understand the molecular and physiological mechanisms that plants use to cope with abiotic stresses for further stable production of crops and biomass feedstock.
Many studies have indicated that regulatory factors of protein phosphorylation play essential roles in response to environmental stimuli (Sopory and Munshi, 1998;Umezawa et al., 2013). One of the well-characterized protein kinases involved in stress responses is the group of sucrose nonfermenting 1 (SNF1)-related protein kinases (SnRKs; Halford and Hey, 2009). SnRKs are grouped into three subfamilies, SnRK1, SnRK2, and SnRK3 (Halford and Hey, 2009), and recent studies have indicated pivotal roles of plant-specific subgroups of SnRK2 and SnRK3 in the link between abiotic stress and abscisic acid (ABA) signaling to regulate metabolic pathways (Hrabak et al., 2003;Halford and Hey, 2009). Increasing evidence shows that SnRK2 proteins function as positive regulators of ABA signaling for stress responses, as well as development, in plants (Umezawa et al., 2013). In Arabidopsis thaliana (Arabidopsis) and rice, the SnRK2 family includes 10 members, such as SRK2A-SRK2J or SnRK2.1-2.10 in Arabidopsis and SAPK1-10 in rice (Yoshida et al., 2002;Hrabak et al., 2003;Kobayashi et al., 2004), and they are further classified into three subclasses based on their domain structures (Kobayashi et al., 2004). Most SnRK2 proteins are activated by abiotic stresses, while the members of subclasses 2 and 3 are also activated by ABA (Boudsocq et al., 2004(Boudsocq et al., , 2007Kobayashi et al., 2004). In the current model, ABA-induced activation is largely explained by the interaction between SnRK2s and protein phosphatase type 2C (PP2C) proteins in the ABA signaling pathway (Leung et al., 1994(Leung et al., , 1997Meyer et al., 1994;Saez et al., 2004;Nishimura et al., 2007;Umezawa et al., 2009;Cutler et al., 2010;Ng et al., 2014). In the absence of ABA, group A PP2Cs physically bind to SnRK2s to dephosphorylate SnRK2s, resulting in the inhibition of ABA signal transduction, while in the presence of ABA, SnRK2 will be released from such inhibitory regulation by PP2C, because the soluble ABA receptor PYR/PYL/RCAR inhibits PP2C activity Vlad et al., 2009).
Comparative genomics studies have demonstrated that the core components of ABA signaling, PYR/PYL/RCAR, SnRK2, and PP2C, are well-conserved in land plant species , suggesting the evolutionary conservation of a molecular system involving these proteins in land plants. Indeed, the conserved molecular characteristics of SnRK2, such as transcriptional induction by abiotic stresses and activation by stress and/or ABA, have been reported for SnRK2 genes not only in Arabidopsis and rice, but also in other crop plants: maize (Huai et al., 2008;Vilela et al., 2012) and wheat (Holappa and Walker-Simmons, 1995;Gómez-Cadenas et al., 1999;Mao et al., 2010;Zhang et al., 2010Zhang et al., , 2011Tian et al., 2013). Additionally, we recently confirmed the physical interaction between SnRK2 and PP2C in a model tree Populus trichocarpa (poplar), and proposed the possibility that a similar molecular module containing SnRK2 and PP2C is involved in the ABA signaling pathway in trees (Song et al., 2015). However, information about the molecular functions of the poplar SnRK2 proteins is still limited. Recently, we evaluated the transcriptional regulation of PtSnRK2 genes and found that some of them are upregulated by abiotic stresses in organ-specific manners, suggesting the involvement of PtSnRK2 in ABA-dependent and/or ABA-independent regulation of stress responses (Yu et al., unpublished data.).
In this study, to obtain further clues as to the molecular functions of PtSnRK2 proteins, we heterologously overexpressed the PtSnRK2 genes in Arabidopsis. Our data indicated that poplar subclass II PtSnRK2 genes can enhance the salt stress tolerance of Arabidopsis, and that poplar PtSnRK2 overexpression would activate cellular signaling and stress response pathways in Arabidopsis in a different manner than that by the herbal subclass II SnRK2 genes.

Plant Materials and Growth Conditions
Young shoots of black cottonwood, P. trichocarpa Torr. & A. Gray (poplar), grown in 15-cm-high plant pots, were used for the cloning of PtSnRK2 cDNA. For the overexpression analysis, A. thaliana (Arabidopsis) plants (Columbia strain) were used. The growth conditions were described in Ohtani et al. (2011) for poplar and in Ohtani et al. (2013) for Arabidopsis.

Plasmid Construction and Transformation
The coding sequences of PtSnRK2 genes were cloned into the Gateway entry vector pENTR/D-TOPO or pCR8/GW/TOPO (Invitrogen), as described by Song et al. (2015), and were transferred to the destination vector pH35GS (Kubo et al., 2005) by the LR reaction using LR clonase II (Invitrogen). In the resulting plasmids, the PtSnRK2 cDNA was expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The plasmids were electroporated into the Agrobacterium tumefaciens strain GV3101 (pMP90). A simplified version of the floral dip method was used for the transformation of Arabidopsis plants (Clough and Bent, 1998).

Establishment of Transgenic Lines
For the screening of transgenic lines carrying the empty vector (vector controls) and 35S::PtSnRK2 (PtSnRK2 overexpressors), T 1 seedlings were grown in germination Murashige and Skoog (MS) medium containing 50 µg mL −1 hygromycin for 2 weeks, and the positive plants were transferred to soil for further growth. Although we failed to generate overexpressors of PtSnRK2.2, PtSnRK2.6, and PtSnRK2.8, more than 16 independent T 2 lines were established for every other PtSnRK2 genes.
To evaluate the expression levels of the introduced PtSnRK2, total RNAs were prepared from 7-day-old seedlings of the wild type, vector controls, and PtSnRK2 overexpressors at the T 2 generation using the RNeasy Mini kit (Qiagen). The first-strand cDNAs were synthesized using SuperScript III (Invitrogen) and subjected to RT-PCR analysis. The RT reaction was performed on a 20-µL scale, with 1 µL first-strand cDNA as a template for PCR, along with 0.5 µM of each gene-specific primer (see Table S2) and Ex Taq polymerase (TaKaRa). The PCR conditions were as follows: for the internal control gene Ubp10, 95 • C for 5 min, followed by 35 cycles of 94 • C for 20 s, 60 • C for 30 s, and 72 • C for 15 s, followed by 72 • C for 7 min; for PtSnRK2 genes (target genes), 95 • C for 5 min, followed by 35 cycles of 94 • C for 20 s, 60 • C for 30 s, and 72 • C for 1 min 15 s, and followed by 72 • C for 7 min. The amplified PCR products were electrophoresed in 3 and 2% (w/v) agarose gels for the Ubp10 and PtSnRK2 genes, respectively, and gel images were analyzed using the AE-9020 Eshot II (ATTO; Figure S1). Information about the primer sets is provided in Table S11.

Salt Stress Treatment
It is generally known that the expression level of 35S promoterdriven genes would be decreased in T 3 homozygous lines, so we decided to use T 2 lines that were confirmed to show high expression levels of inserted PtSnRK genes as described above, for the salt stress treatment. Wild-type and transgenic T 2 plants were grown at 22 • C under LD conditions (16 h light/8 h dark) on 1/2strength MS medium, after incubation at 4 • C for 3 days. Then, 7-day-old plants were transferred to 1/2-MS medium plates with or without NaCl. For the survival rates and chlorophyll contents, 200 mM NaCl was included in the medium, and for the root growth phenotype, 100 mM NaCl was used. After an additional 4day incubation, the numbers of surviving seedlings were counted to obtain survival rates (n = 20), and chlorophyll contents were measured as described below (n = 10). The primary root length was measured before and after a 5-day incubation on the NaCl plate, to calculate primary root elongation during salt stress treatment (n = 10). The treatments were repeated three times for survival rates and root length and six times for chlorophyll contents.

In silico Prediction of the Three-Dimensional Structures of PtSnRK2 Proteins
The amino acid sequences of PtSnRK2.5, PtSnRK2.7, and PtSnRK2.9 were submitted to the web-based SWISS-MODEL service (http://swissmodel.expasy.org/workspace/; Arnold et al., 2006), to build protein structure homology models using information on the crystal structure of the recombinant AtSnRK2.6 protein as a template (Ng et al., 2011).

Microarray Analysis
Seedlings of the wild-type and PtSnRK2.7 overexpressor line 20 treated with or without 200 mM NaCl for 2 days were sampled for total RNA extraction. Microarray analysis was performed using ATH1 GeneChips (Affymetrix) according to the manufacturer's instructions on three independent biological replicates. Subsequent procedures of quality control, statistical analysis, and filtering were carried out using GeneSpring GX software (ver. 13.1; Agilent Technologies). Then, p-values were calculated for each probe using Welch's t-test (n = 3) for differences between the treated seedlings and the control seedlings, as well as between the wild-type and PtSnRK2.7 overexpressor incubated without salt treatment. We used the Benjamin-Hochberg FDR method to control for false positives. A p-value cut-off of 0.01 was used to select genes whose expression changed with salt treatment. Fold-change values were also computed using GeneSpring GX, and we targeted those probes in which the change was upregulated or downregulated by more than 3-fold. Microarray data presented in this study were submitted to NCBI GEO (www.ncbi.nlm.nih.gov/geo/) and can be retrieved via accession number GSE79997.

Gene Ontology (GO) Term Analysis
Gene ontology (GO) term analysis was performed using the PANTHER classification system (Overrepresentation Test, release 20150430; Mi et al., 2013).

Quantitative RT-PCR Analysis
To evaluate the enhanced upregulation of salt stress response genes in PtSnRK2.7 overexpressors, quantitative RT-PCR analysis was performed. Total RNAs were isolated from the seedlings treated with or without 200 mM NaCl for 2 days using Plant RNA Isolation Reagent (Invitrogen) and then purified using the RNeasy Mini Kit (QIAGEN). The first-strand cDNAs were synthesized as described above, and aliquots of the cDNA solution (0.5 µL for each gene) were used as templates for subsequent PCR amplification. The quantitative PCR analysis was performed using the LightCycler 480 System II (Roche) and LightCycler 480 SYBR Green I Master reagents (Roche). As an internal control, the Ubc9 gene was used. Information on the primer sets is provided in Table S11.

RESULTS AND DISCUSSION
Overexpression of PtSnRK2.5 and PtSnRK2.7 Improved Salt Stress Tolerance in Arabidopsis In the genome of P. trichocarpa (poplar), 12 PtSnRK2 genes have been identified (Song et al., 2015). For molecular functional analysis of PtSnRK2, we generated transgenic plants of A. thaliana (Arabidopsis) carrying the chimeric gene 35S::PtSnRK2, in which the cDNA regions of PtSnRK2 genes were regulated by the CaMV 35S promoter sequence, to overexpress poplar PtSnRK2 genes in Arabidopsis. Unfortunately we could not obtain transgenic plants for PtSnRK2.2, PtSnRK2.6, and PtSnRK2.8 overexpression; however, overexpressors of the other nine PtSnRK2 genes were established successfully. It has been reported that overexpression of SnRK2 genes enhances abiotic stress tolerance (Umezawa et al., 2004;Diédhiou et al., 2008;Mao et al., 2010;Zhang et al., 2010Zhang et al., , 2011Tian et al., 2013). Thus, we examined salt stress tolerance in the PtSnRK2 overexpressors.
First, 7-day-old seedlings from 16 independent T 2 lines for each PtSnRK2 overexpressor were transferred to medium containing 200 mM NaCl, and incubated for 4 days. The prr9-11 prr7-10 prr5-10 triple mutant (d975) plant, which showed high salinity tolerance because of high expression of salt stress response genes (Nakamichi et al., 2009), was used as a positive control ( Figure S2). The survival rates of seedlings after salt treatment demonstrated that the overexpressors of PtSnRK2.5 and PtSnRK2.7 showed relatively high survival rates among the PtSnRK2 overexpressors. These two genes encode the subclass 2 PtSnRK2 proteins (Song et al., 2015). We further performed a detailed analysis of salt stress tolerance in the PtSnRK2.5 and PtSnRK2.7 overexpressors, using the wild-type and vector control plants as negative controls (Figures 2, 3). For the PtSnRK2.5 and PtSnRK2.7 overexpressors, three independent lines were selected based on the expression levels of the introduced PtSnRK2 genes ( Figure S1). In the case of the negative controls, the seedling survival rates after 200 mM NaCl treatment were less than 20%, and the living seedlings exhibited yellowed leaves (Figure 2). In contrast, the PtSnRK2.5 and PtSnRK2.7 transgenic seedlings showed significantly higher survival rates (∼55%) than those of the controls (Figure 2). All three independent PtSnRK2.7 lines showed increased survival rates, whereas the PtSnRK2.5 line 6 did not show a significantly enhanced survival rate ( Figure 2B).
Next, the chlorophyll contents of 10 seedlings treated with salt stress were analyzed. The salt treatment greatly decreased chlorophyll contents in all plants; however, the chlorophyll contents of PtSnRK2.5 lines 16 and 20, and all of the PtSnRK2.7 lines were significantly higher than those of the wild-type and vector control (Figure 2C), in accordance with the survival rates. These results suggested that salt stress tolerance was more stable in the PtSnRK2.7 overexpressors. Moreover, it was notable that although the chlorophyll contents in the absence of salt stress were at almost the same levels among the wild-type, vector control, and PtSnRK2.7 lines, but the PtSnRK2.5 lines 16 and 20 showed lower amounts of chlorophyll under normal growth conditions ( Figure 2C). Thus, PtSnRK2.5 overexpression may have affected chlorophyll biosynthesis continuously.
In addition to the increased survival rates and chlorophyll contents, the living seedlings of the PtSnRK2.5 and PtSnRK2.7 transgenic lines seemed to be larger than those of the negative controls under salt stress conditions (Figure 2A). To clarify the effects of PtSnRK2 overexpression on seedling growth, we checked the growth of primary roots after salt treatment. Because the 200 mM NaCl treatment almost completely inhibited primary root elongation in both the overexpressors and negative controls, we used 100 mM NaCl conditions to observe primary root elongation. Our data demonstrated no difference in primary root elongation in the mock-treated seedlings (Figure 3), indicating that these two PtSnRK2 genes are not involved in root elongation regulation, unlike the Arabidopsis subclass II AtSRK2C/SnRK2.8 gene, overexpression of which was reported to enhance seedling root growth (Shin et al., 2007). However, after a 5-day incubation with 100 mM NaCl, the primary roots of PtSnRK2.5 line 20 and all of the PtSnRK2.7 lines elongated significantly more than did those of the negative controls; the negative controls showed elongation of less than 20 mm, whereas the elongated lengths of PtSnRK2.5 line 20 and all of the PtSnRK2.7 lines were ∼25 mm (p < 0.01, t-test). These observations indicated that overexpression of PtSnRK2.7, and possibly also PtSnRK2.5, can suppress the inhibition of root elongation by high salinity stress.
Our data on the PtSnRK2.5 and PtSnRK2.7 transgenic lines demonstrated different phenotypic characteristics between the PtSnRK2.5 and PtSnRK2.7 lines. Thus, it was suspected that the molecular basis for the enhancement of salt stress tolerance would be different between the PtSnRK2.5 and PtSnRK2.7 transgenic lines. In particular, the PtSnRK2.5 lines showed unstable salt stress tolerance (Figures 2, 3) and defects in chlorophyll content even under normal growth condition ( Figure 2C). The subclass 2 PtSnRK2 proteins have highly similar amino acid sequences (Figure 1A), and the amino acid sequences of PtSnRK2.5 (unstable, but significantly enhanced salinity tolerance), PtSnRK2.7 (stable and high tolerance), and PtSnRK2.9 (no obviously increased tolerance) did not show major differences between them ( Figure 1A). In silico modeling of the three-dimensional structures of these proteins, based on the crystal structure of AtSnRK2.6 (Ng et al., 2011), revealed that some of the differences in amino acid sequences among these PtSnRK2 proteins could correspond to the molecular surface regions of these proteins (Figure 1B), possibly leading to changes in the molecular activities of the SnRK2 proteins. The different SnRK2 protein activities due to subtle substitutions of amino acids could result in the different strengths of salt stress tolerance among the overexpressors.

PtSnRK2.7 Overexpression Widely and Largely Influenced the Transcriptome in Response to Salt Stress
We next performed transcriptomic analyses of the wild-type and overexpressors during salt stress treatment. Based on the data described above (Figures 2, 3), we selected PtSnRK2.7 line 20, which showed stable and strong salt stress tolerance Frontiers in Plant Science | www.frontiersin.org FIGURE 1 | SnRK2 proteins of Populus trichocarpa (poplar, Pt), and Arabidopsis thaliana (Arabidopsis, At). (A) Amino acid sequence alignment of subclass 2 PtSnRK2 proteins with AtSnRK2.8. The residues putatively corresponding to the α-helix, β-sheet, PP2C-interaction residues, and the entire ABA box are marked by bars, arrows, asterisks, and the box, respectively. (B) In silico modeling of the three-dimensional structures of PtSnRK2.5, PtSnRK2.7, and PtSnRK2.9 proteins. Homology modeling was performed based on the crystal structure of AtSnRK2.6 using the web-based SWISS-MODEL service (http://swissmodel.expasy.org/ workspace/; Arnold et al., 2006). Thick and narrow arrows indicate regions presumed to differ in their three-dimensional architecture among the PtSnRK2 proteins because of differences in amino acid sequences.
among the transgenic lines, for the gene chip analysis. Then, 7-day-old seedlings of the wild-type and PtSnRK2.7 line 20 were treated with or without 200 mM NaCl for 2 days and then sampled to extract total RNA. The extracted total RNA samples were subjected to microarray analysis using Affymetrix ATH1 GeneChips ( Figure 4A). First, we compared transcriptomic data between the wild-type and PtSnRK2.7 overexpressor, which were mock-treated, to examine the effects of PtSnRK2.7 overexpression on gene expression under normal growth conditions. Thirty and 79 genes were shown to be upregulated and downregulated in the PtSnRK2.7 overexpressor, respectively (FC > 2, p < 0.05; Table S1). It has been reported that in AtSRK2C/SnRK2.6 and TaSnRK2.8 overexpressors, stress response-related genes including RD29A and DREB1A/CBF3, and ABA biosynthetic genes are upregulated continuously (Umezawa et al., 2004;Zhang et al., 2011). However, these genes were not changed by PtSnRK2.7 overexpression ( Table S1). The gene ontology (GO) term analysis revealed that stress-related metabolic genes, including lipid metabolism and flavonoid metabolism, were significantly downregulated in the PtSnRK2.7 overexpressor ( Table 1). These results suggest that PtSnRK2.7 overexpression could continuously affect specific ranges of gene expression regulation, which do not overlap with the primary targets of AtSRK2C/SnRK2.6 and TaSnRK2.8 in Arabidopsis.
To examine the impact of PtSnRK2.7 overexpression on the salt stress response, the genes upregulated or downregulated by salt treatment were compared between the wild-type and PtSnRK2.7 overexpressor. In the wild-type, 337 and 120 genes were upregulated and downregulated by salt treatment, respectively (FC > 3, p < 0.01; Figure 4B and Tables S2,  S3). Notably, the PtSnRK2.7 overexpressor showed greater numbers of genes whose expression was changed by salt treatment compared with the wild-type; 631 and 698 genes were upregulated and downregulated in the PtSnRK2.7 overexpressor, respectively (FC > 3, p < 0.01; Figure 4B and Tables S4, S5), suggesting that PtSnRK2.7 overexpression affected the expression of a wide range of genes during salt stress responses in Arabidopsis. The GO analysis indicated that the genes functioning in stress responses, including the signaling pathway of stress-related phytohormones (ABA, ethylene, jasmonic acid, and salicylic acid) and in signal transduction by protein phosphorylation, were commonly upregulated between the wild-type and PtSnRK2.7 overexpressor (Figure 4B and Tables S6-S8). Additionally, developmental process-related genes were commonly downregulated in the wild-type and PtSnRK2.7 overexpressor ( Figure 4B and Table S10).
We also found that some GO terms were enriched in the upregulated genes in genotype-dependent manners, such as "respiratory burst" and "ethylene biosynthetic process" found only in the wild-type ( Figure 4B and Table S6) and "purine nucleoside transmembrane transport, " "amino acid transport, " "anion transmembrane transport, " and "nucleic acid metabolic process" found only in the PtSnRK2.7 overexpressor ( Figure 4B and Table S7). The overrepresentation of the GO terms related to "anion transmembrane transport" in the PtSnRK2.7 overexpressor would suggest the enhancement of ion  homeostasis activity as a result of PtSnRK2.7 overexpression, possibly leading to higher salt stress tolerance. Our results also showed wild-type-specific enrichment of the term "ethylene biosynthetic process" in upregulated genes ( Figure 4B and Table S6). It has been reported that crosstalk between ABA and ethylene is a critical factor in determining salt stress tolerance in Arabidopsis (Dong et al., 2011); thus, PtSnRK2.7 overexpression may influence the phytohormonal modulating system of abiotic stress responses. Moreover, in the PtSnRK2.7 overexpressor, more than 5-fold more genes were downregulated by salt treatment than those in the wild type. These downregulated genes are involved in a wide range of molecular functions, including metabolic regulation and responses to stimuli ( Figure 4B and Table S9), indicating that the reason for the high salt stress tolerance in the PtSnRK2.7 overexpressor may be, at least partially, attributed to its significant impact on metabolic regulation.
Comparison of transcriptomic data also suggested that the altered expression levels of upregulated genes were greater in the PtSnRK2.7 overexpressor than in the wild-type (Tables S2, S4).
To confirm this, we selected six genes from the functional categories of stress response (COR15A, Artus et al., 1996), phytohormonal signaling (GASA3, Herzog et al., 1995), cell wall-related proteins (AT1G52690 and AtCWINV5, Sherson et al., 2003), and lipid metabolism (AT4G33550), based on the transcriptomic data, and subjected them to quantitative RT-PCR analysis. AT4G18280, which was upregulated only in the PtSnRK2.7 overexpressor, was also tested to evaluate the consistency of the results between the gene chip analysis and quantitative RT-PCR analysis. Indeed, AT4G18280 expression was not induced by salt stress in the wild-type or vector control, whereas it was highly upregulated in the PtSnRK2.7 line 20 in accordance to the results of gene chip analysis (Figure 5 and Table S4). The results showed that the tested genes were actively Frontiers in Plant Science | www.frontiersin.org upregulated by salt treatment in the PtSnRK2.7 overexpressors compared with the wild-type and vector control ( Figure 5).
Our data also demonstrated that PtSnRK2.5 overexpression had similar effects on the expression of these genes. Thus, it is supposed that PtSnRK2.5 overexpression affected the transcriptome in a similar fashion to PtSnRK2.7 overexpression. It has been reported that the overexpression of Arabidopsis AtSnRK2.8 enhanced the stress tolerance of Arabidopsis, probably through the continuous upregulation of key genes for stress responses, such as RD29A, COR15A, AtGolS3, DREB1A, and PKS18 (Umezawa et al., 2004). However, our results showed that overexpression of poplar SnRK2 genes did not constitutively induce these well-known stress responsive genes. Rather, the high salt stress tolerance of the PtSnRK2 overexpressors may be explained by changes in transcriptomic regulation for a wide range of metabolic regulatory genes (Figures 4, 5; Table 1 and Tables S1-S10). It is notable that the genes related to anion transport activity were upregulated in the PtSnRK2.7 overexpressor specifically, because similar effects on ion homeostasis-related genes were reported in the case of overexpression of rice SAPK4 (Diédhiou et al., 2008). Moreover, a comparative transcriptomic analysis using salt-tolerant and non-tolerant species of poplar indicated that prominent factors for high salt tolerance were not overexpression of the stress responsive pathway, but rather enhanced activities for osmotic adjustment, ion compartmentalization, and detoxification of reactive oxygen species in poplar (Chen and Polle, 2010). Thus, our results may reflect differences in the regulatory targets of SnRK2 proteins between AtSnRK2 and PtSnRK2, which could be related to diversified molecular strategies of stress adaption in each plant species, as suggested by Zhang et al. (2014). Future comparative analyses on the mode of actions of SnRK2 proteins derived from different plant species may provide important information on novel strategies to improve stress tolerance of crops and other useful plants.

AUTHOR CONTRIBUTIONS
XS designed and performed the experiments, and drafted the manuscript. MO designed the experiments, analyzed the data, and wrote the manuscript. XY and CH contributed to the data analysis. QZ and TD drafted the manuscript. All authors contributed to and approved the final manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016. 00612 Figure S1 | RT-PCR analysis of seedlings of the wild-type, vector control, and overexpressors of PtSnRK2.5 and PtSnRK2.7. Figure S2 | Primary screening of transgenic Arabidopsis overexpressing PtSnRK2 genes for the salt tolerance. Seedlings of the d975 (positive control), vector control, and overexpressors of PtSnRK2 genes were treated with 200 mM NaCl, and checked their survival rates after 3, 4, and 5 days of NaCl treatment. A Frontiers in Plant Science | www.frontiersin.org part of results for the transgenic lines of PtSnRK2.5 (A) and PtSnRK2.9 (B) treated with 200 mM NaCl for 4 days were shown.
Table S10 | GO term analysis on commonly downregulated genes between the wild type and PtSnRK2.7 overexpressor by salt treatment.
Table S11 | Oligonucleotides used in this paper.