Overexpression of PeHKT1;1 Improves Salt Tolerance in Populus

Soil salinization is an increasingly serious threat that limits plant growth and development. Class I transporters of the high-affinity K+ transporter (HKT) family have been demonstrated to be involved in salt tolerance by contributing to Na+ exclusion from roots and shoots. Here, we isolated the PeHKT1;1 gene from hybrid poplar based on the sequences of the Populus trichocarpa genome. The full-length PeHKT1;1 gene was 2173 bp, including a 1608 bp open reading frame (ORF) encoding 535 amino acids and containing eight distinct transmembrane domains. Multiple sequence alignment and phylogenetic analysis suggested that the PeHKT1;1 protein had a typical S–G–G–G signature for the P-loop domains and belonged to class I of HKT transporters. PeHKT1;1 transcripts were mainly detected in stem and root, and were remarkably induced by salt stress treatment. In further characterization of its functions, overexpression of PeHKT1;1 in Populus davidiana × Populus bolleana resulted in a better relative growth rate in phenotypic analysis, including root and plant height, and exhibited higher catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities than non-transgenic poplar under salt stress conditions. These observations indicated that PeHKT1;1 may enhance salt tolerance by improving the efficiency of antioxidant systems. Together, these data suggest that PeHKT1;1 plays an important role in response to salt stress in Populus.


Introduction
In the natural environment, soil salinization is a major abiotic stress that limits plant growth and development. High concentrations of salts in the soil have various adverse effects in plants, including osmotic stress and ion toxicity. Specifically, excess salinity decreases water potential in plants, resulting in a reduced ability to take up water, and large amounts of sodium (Na + ) and chloride (Cl − ) are taken up by the plant root system. Excessive Na + and Cl − within plants is toxic, and disturbs potassium (K + ) homeostasis, cellular activity, metabolism, and photosynthesis, and causes the accumulation of reactive oxygen species (ROS) [1][2][3]. To cope with soil salinization, plants have evolved diverse adaptive mechanisms, including Na + exclusion from the shoot, Na + expulsion from cell cytoplasm, and Na + compartmentalization into vacuoles [4]. Among these, Na + extrusion out of the cell and detoxification into vacuoles have been reported to be mediated by Salt-Overly-Sensitive 1 (SOS1) antiporters and Na + /H + exchanger 1 (NHX1) antiporters, respectively [4,5]. The regulation of Na + loading into the root xylem, which is limiting to Na + accumulation in the shoot, is essential for enhancing salt tolerance. Therefore, it is necessary to understand target genes for Na + extrusion from the shoot.
The high-affinity K + transporters (HKTs) are a large superfamily of transporters in plants, bacteria, and fungi. TaHKT2;1 was first identified in plants from bread wheat (Triticum aestivum) and encoded

Expression Analysis of PeHKT1;1
The expression patterns of PeHKT1;1 were detected using semi-quantitative reverse transcription polymerase chain reaction (SqRT-PCR) and quantitative real-time polymerase chain reaction (qRT-PCR). The specific primers were designed to generate amplified fragments of 300-500 and 70-150 bp, respectively (Table 1). SqRT-PCR was performed by a non-saturating PCR reaction (28 cycles) with the 18SrRNA (18S ribosomal RNA) gene as an internal control. qRT-PCR was performed on an ABI ViiA 7 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) using FastStart Universal SYBR Green Master with ROX for RT-PCR Kit (Roche, Indianapolis, IN, USA), according to the manufacturer's protocol. The PCR procedure was 95 • C for 1 min, followed by 40 cycles at 95 • C for 15 s and 60 • C for 1 min. The specificity of the PCR reactions was confirmed by melting curve analysis of the amplicons. There were three biological replicates, and the relative expression levels of all samples were calculated using the 2 −∆∆Ct method, with Elongation Factor 1 alpha (EF1α) as the reference gene [28].

Overexpression Vector Construction and Poplar Transformation
The ORF of PeHKT1;1 cDNA was amplified by reverse transcription PCR (RT-PCR), and subsequently cloned into the pH35GS binary vector using the Gateway System (Invitrogen, Carlsbad, CA, USA) to replace the ccdB gene, which was located downstream of the CaMV35S promoter. The binary vector harboring Pro35S::PeHKT1;1 was introgressed into Agrobacterium tumefaciens strain EHA105, which was used to transform poplar ( Figure S1).

Transgenic Poplar Confirmation and Salt Tolerance Assays
After screening using hygromycin resistance, the NT poplar and the putative transgenic poplar lines were validated by RT-PCR and qRT-PCR methods as described above, using the forward primer of the 35S gene from the pH35GS binary vector and the reverse primer of the PeHKT1;1 cDNA (Table 1). Salt tolerance tests were performed in hydroponic culture. Specifically, the 6-week-old NT and transgenic poplar lines were cultured for two weeks in liquid MS medium containing NaCl: 0, 0.2, 0.3, 0.4, and 0.5% w/v. The NT poplar served as the control. Each treatment was performed three replicates.

Physiological Assay
To examine the physiological parameters of NT poplar and PeHKT1;1 transgenic lines, the three-year-old soil-grown poplar plants were treated with 0.8% (w/v) NaCl at different time points (0, 0.5, 1, 1.5, 2, and 6 h). The CAT, SOD, and POD activities were measured by leaves according to Li et al. [29] using the corresponding assay kits: total protein assay kit (BCA method, A045-2), catalase assay kit (visible light, A007-1), total superoxide dismutase (T-SOD) assay kit (hydroxylamine method, A001-1), and peroxidase assay kit (A084-3), according to the manufacturer's respective manuals (Jiancheng Bioengineering Inc., Nanjing, China). The experiments were repeated at least five times. Statistical analyses were performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). Data were compared using one-way analysis of variance (ANOVA) followed by Duncan's test.

Isolation and Characterization of PeHKT1;1
The full-length cDNA sequence of PeHKT1;1 was successfully isolated and identified by RACE method, and had 2173 nucleotides, containing an ORF of 1608 bp that encoded a 62.0 kDa polypeptide of 535 putative amino acid residues and flanked by 332 bp of 5 -untranslated region (UTR) and 233 bp of 3 -UTR. The exon-intron structure of PeHKT1;1 was determined by aligning cDNA and genomic sequences, which contained two introns (Supplementary Data 1).
To investigate the structure of PeHKT1;1 protein, the online software TMHMN Server v.2.0 was used to predict the transmembrane helices of PeHKT1;1 protein and indicated eight distinct transmembrane domains with a probability of one ( Figure 1A). Moreover, the deduced amino acid sequences were aligned with sequences of other plant HKTs using ClustalX 2.1 software. The results revealed that PeHKT1;1 had four conserved selectivity-filter-pore regions (p-loops: P A to P D , Figure 1B), and shared high homology with other plant HKT1 proteins, especially A. thaliana (69% identity), except for P. trichocarpa (98% identity) ( Figure S2). Further phylogenetic analysis showed that PeHKT1;1, as typical for a dicotyledonous plant ( Figure 1C), belonged to class I of HKT transporters, whose members are characterized by the presence of a serine (Ser) residue rather than a glycine (Gly) residue at the corresponding position in the P A -loop domain ( Figure 1B).

Isolation and Characterization of PeHKT1;1
The full-length cDNA sequence of PeHKT1;1 was successfully isolated and identified by RACE method, and had 2173 nucleotides, containing an ORF of 1608 bp that encoded a 62.0 kDa polypeptide of 535 putative amino acid residues and flanked by 332 bp of 5'-untranslated region (UTR) and 233 bp of 3'-UTR. The exon-intron structure of PeHKT1;1 was determined by aligning cDNA and genomic sequences, which contained two introns (Supplementary Data 1).
To investigate the structure of PeHKT1;1 protein, the online software TMHMN Server v.2.0 was used to predict the transmembrane helices of PeHKT1;1 protein and indicated eight distinct transmembrane domains with a probability of one ( Figure 1A). Moreover, the deduced amino acid sequences were aligned with sequences of other plant HKTs using ClustalX 2.1 software. The results revealed that PeHKT1;1 had four conserved selectivity-filter-pore regions (p-loops: PA to PD, Figure  1B), and shared high homology with other plant HKT1 proteins, especially A. thaliana (69% identity), except for P. trichocarpa (98% identity) ( Figure S2). Further phylogenetic analysis showed that PeHKT1;1, as typical for a dicotyledonous plant ( Figure 1C), belonged to class I of HKT transporters, whose members are characterized by the presence of a serine (Ser) residue rather than a glycine (Gly) residue at the corresponding position in the PA-loop domain ( Figure 1B).  The four conserved selectivity-filter-pore regions of HKT were aligned, and highlighted in yellow, using ClustalX 2.1 software. A line above the alignment was used to mark strongly conserved positions. Three characters ("*", ":" and ".") were used: "*" indicates positions which have a single, fully conserved residue. ":" and "." indicates positions that have 'strong' and 'weaker' conserved residues, respectively. (C) Phylogenetic tree analysis of 14 HKTs from 12 different plant species by neighbor-joining method using MEGA v7.0 software with 1000 iterations bootstraps. HKT: high-affinity K + transporter.

Tissue-Specific Expression of PeHKT1;1
The 6-week-old "Nanlin895" plants were used to study the tissue-specific expression profiles of PeHKT1;1 under normal growth conditions. The mRNA level of PeHKT1;1 was determined in roots, stems and leaves using SqRT-PCR and qRT-PCR. The two methods showed similar results-PeHKT1;1 was mainly expressed in stems and roots, but little detected in leaves in "Nanlin895" (Figure 2). These results implied that PeHKT1;1 plays an important role in Populus stems and roots.

Figure 2.
Tissue-specific expression levels of PeHKT1;1 analyzed by SqRT-PCR and qRT-PCR in "Nanlin895" poplar. The Y-axis represents relative quantitation and X-axis represents different tissues. The 18SrRNA gene was used as an internal control for SqRT-PCR, the EF1α gene was used as a control for qRT-PCR, and the relative transcript levels were calculated using the 2 -∆∆Ct method. Error bars showed standard deviations of three biological replicates. The four conserved selectivity-filter-pore regions of HKT were aligned, and highlighted in yellow, using ClustalX 2.1 software. A line above the alignment was used to mark strongly conserved positions. Three characters ("*", ":" and ".") were used: "*" indicates positions which have a single, fully conserved residue. ":" and "." indicates positions that have 'strong' and 'weaker' conserved residues, respectively. (C) Phylogenetic tree analysis of 14 HKTs from 12 different plant species by neighbor-joining method using MEGA v7.0 software with 1000 iterations bootstraps. HKT: high-affinity K + transporter.

Tissue-Specific Expression of PeHKT1;1
The 6-week-old "Nanlin895" plants were used to study the tissue-specific expression profiles of PeHKT1;1 under normal growth conditions. The mRNA level of PeHKT1;1 was determined in roots, stems and leaves using SqRT-PCR and qRT-PCR. The two methods showed similar results-PeHKT1;1 was mainly expressed in stems and roots, but little detected in leaves in "Nanlin895" (Figure 2). These results implied that PeHKT1;1 plays an important role in Populus stems and roots.

Tissue-Specific Expression of PeHKT1;1
The 6-week-old "Nanlin895" plants were used to study the tissue-specific expression profiles of PeHKT1;1 under normal growth conditions. The mRNA level of PeHKT1;1 was determined in roots, stems and leaves using SqRT-PCR and qRT-PCR. The two methods showed similar results-PeHKT1;1 was mainly expressed in stems and roots, but little detected in leaves in "Nanlin895" (Figure 2). These results implied that PeHKT1;1 plays an important role in Populus stems and roots. Tissue-specific expression levels of PeHKT1;1 analyzed by SqRT-PCR and qRT-PCR in "Nanlin895" poplar. The Y-axis represents relative quantitation and X-axis represents different tissues. The 18SrRNA gene was used as an internal control for SqRT-PCR, the EF1α gene was used as a control for qRT-PCR, and the relative transcript levels were calculated using the 2 -∆∆Ct method. Error bars showed standard deviations of three biological replicates. Tissue-specific expression levels of PeHKT1;1 analyzed by SqRT-PCR and qRT-PCR in "Nanlin895" poplar. The Y-axis represents relative quantitation and X-axis represents different tissues. The 18SrRNA gene was used as an internal control for SqRT-PCR, the EF1α gene was used as a control for qRT-PCR, and the relative transcript levels were calculated using the 2 −∆∆Ct method. Error bars showed standard deviations of three biological replicates.

PeHKT1;1 Transcripts in Salt Stress Conditions
Accumulated evidence from other plant species has demonstrated that HKT1 mediates Na + uptake and transport [8], so we used qRT-PCR to investigate expression levels of PeHKT1;1 in roots, stems, and leaves of "Nanlin895" subject to low (0.6% w/v) and high (1.8% w/v) NaCl stress at 0 (control), 2, 6, 12, 24, 48, and 72 h. For the 0.6% w/v NaCl treatment, the PeHKT1;1 transcripts in roots were slightly enhanced within 24 h, and then upregulated dramatically at 48 and 72 h (approximately 8 and 15 times control values, respectively) ( Figure 3). Interestingly, when poplar was exposed to 1.8% w/v NaCl stress, PeHKT1;1 expression was induced significantly at 2 h, reached a maximum at 6 h, and then decreased as the treatment time progressed. In stems, PeHKT1;1 was significantly downregulated, being reduced by around five times at 12 h compared with controls, and then gradually increased to original levels under 0.6% w/v NaCl stress. However, the mRNA level of PeHKT1;1 showed substantial reduction, with a decrease of about five times after 12 h compared with controls for 1.8% w/v NaCl. Additionally, PeHKT1;1 transcript levels showed no significant difference in leaves for 0.6% and 1.8% w/v NaCl stresses compared with controls. These results indicated that PeHKT1;1 was involved in response to salt stress in Populus.

PeHKT1;1 Transcripts in Salt Stress Conditions
Accumulated evidence from other plant species has demonstrated that HKT1 mediates Na + uptake and transport [8], so we used qRT-PCR to investigate expression levels of PeHKT1;1 in roots, stems, and leaves of "Nanlin895" subject to low (0.6% w/v) and high (1.8% w/v) NaCl stress at 0 (control), 2, 6, 12, 24, 48, and 72 h. For the 0.6% w/v NaCl treatment, the PeHKT1;1 transcripts in roots were slightly enhanced within 24 h, and then upregulated dramatically at 48 and 72 h (approximately 8 and 15 times control values, respectively) ( Figure 3). Interestingly, when poplar was exposed to 1.8% w/v NaCl stress, PeHKT1;1 expression was induced significantly at 2 h, reached a maximum at 6 h, and then decreased as the treatment time progressed. In stems, PeHKT1;1 was significantly downregulated, being reduced by around five times at 12 h compared with controls, and then gradually increased to original levels under 0.6% w/v NaCl stress. However, the mRNA level of PeHKT1;1 showed substantial reduction, with a decrease of about five times after 12 h compared with controls for 1.8% w/v NaCl. Additionally, PeHKT1;1 transcript levels showed no significant difference in leaves for 0.6% and 1.8% w/v NaCl stresses compared with controls. These results indicated that PeHKT1;1 was involved in response to salt stress in Populus.

Generation of PeHKT1;1-Overexpressing Transgenic Poplar Lines
To further investigate the potential functions of PeHKT1;1 in Populus, we generated some PeHKT1;1-overexpressing transgenic lines of P. davidiana × P. bolleana by Agrobacterium-mediated leaf disk transformation, and seven independent transgenic lines were randomly selected for further testing. The sizes of PCR amplified genomic fragments from seven transgenic poplar lines were obtained by RT-PCR, which were confirmed with the expected sizes, containing the PeHKT1;1 and partial vector sequences ( Figure 4A). A control (NT plant) did not show any amplification band based on the transgene. The result implied that the Pro35S::PeHKT1;1 vector was successfully integrated into the poplar genome. qRT-PCR further demonstrated that expression levels of PeHKT1;1 were 48-3720 times higher than in NT poplar under normal conditions ( Figure 4B). These results indicated successful integration and expression of PeHKT1;1 in the seven transgenic poplar lines.

Generation of PeHKT1;1-Overexpressing Transgenic Poplar Lines
To further investigate the potential functions of PeHKT1;1 in Populus, we generated some PeHKT1;1-overexpressing transgenic lines of P. davidiana × P. bolleana by Agrobacterium-mediated leaf disk transformation, and seven independent transgenic lines were randomly selected for further testing. The sizes of PCR amplified genomic fragments from seven transgenic poplar lines were obtained by RT-PCR, which were confirmed with the expected sizes, containing the PeHKT1;1 and partial vector sequences ( Figure 4A). A control (NT plant) did not show any amplification band based on the transgene. The result implied that the Pro35S::PeHKT1;1 vector was successfully integrated into the poplar genome. qRT-PCR further demonstrated that expression levels of PeHKT1;1 were 48-3720 times higher than in NT poplar under normal conditions ( Figure 4B). These results indicated successful integration and expression of PeHKT1;1 in the seven transgenic poplar lines.

Overexpression of PeHKT1;1 Enhanced Salt Tolerance
In many plants, HKTs have been found to increase salt tolerance [30][31][32]. To detect whether HKT improves salt tolerance in poplar, we selected four transgenic poplar lines (T1, T2, T3, and T4) to determine their tolerance of different salinity stress conditions. The NT and PeHKT1;1-overexpressing poplar lines were grown with water (control), 0.2, 0.3, 0.4, and 0.5% w/v NaCl in liquid MS medium for two weeks. In addition, unstressed NT (NT-0) poplar was used as a positive control. The result showed the growth of NT-1 poplar was severely inhibited following the increase of Na + concentration, showing varying degrees of dwarfing and etiolation ( Figure 5A). Transgenic poplars showed a better relative growth rate, including plant height and adventitious root number (Figure 5B), which was consistent with the expression of PeHKT1;1 in the previous study ( Figure 4B). Nevertheless, the growth of NT and transgenic poplar lines were significantly inhibited when the Na + concentration reached 0.5% w/v. These results indicated that PeHKT1;1 overexpression improved salt tolerance in transgenic poplar.

Overexpression of PeHKT1;1 Enhanced Salt Tolerance
In many plants, HKTs have been found to increase salt tolerance [30][31][32]. To detect whether HKT improves salt tolerance in poplar, we selected four transgenic poplar lines (T1, T2, T3, and T4) to determine their tolerance of different salinity stress conditions. The NT and PeHKT1;1-overexpressing poplar lines were grown with water (control), 0.2, 0.3, 0.4, and 0.5% w/v NaCl in liquid MS medium for two weeks. In addition, unstressed NT (NT-0) poplar was used as a positive control. The result showed the growth of NT-1 poplar was severely inhibited following the increase of Na + concentration, showing varying degrees of dwarfing and etiolation ( Figure 5A). Transgenic poplars showed a better relative growth rate, including plant height and adventitious root number ( Figure 5B), which was consistent with the expression of PeHKT1;1 in the previous study ( Figure 4B). Nevertheless, the growth of NT and transgenic poplar lines were significantly inhibited when the Na + concentration reached 0.5% w/v. These results indicated that PeHKT1;1 overexpression improved salt tolerance in transgenic poplar.

Overexpression of PeHKT1;1 Raised the Efficiency of Antioxidant Systems under Salt Tolerance
The NT and transgenic poplar plants, which had been verified in the previous study, were transplanted into soil for three years to detect long-term effects. Subsequently, we randomly chose four independent transgenic lines to further investigate PeHKT1;1 expression levels and physiological parameters. The Pro35S::PeHKT1;1 vector had not been lost in the three-year-old transgenic lines, and PeHKT1;1 was still overexpressed, confirmed by RT-PCR and qRT-PCR; however, expression levels were lower than the three previous years ( Figure S4). To determine the antioxidant function of PeHKT1;1 in poplar under salt stress conditions, activities of antioxidant enzymes, CAT, SOD, and POD, were examined in the three-year-old NT poplar and four transgenic poplar lines. Compared with NT poplars, transgenic poplars had higher CAT activity after one hour when plants were exposed to salt stress ( Figure 6). Similarly, the PeHKT1;1 transgenic poplars showed higher SOD and POD activities after 1 and 1.5 h, respectively. Moreover, comparison of PeHKT1;1 transcript with CAT, SOD, and POD activities suggested that the transgenic poplar lines with higher PeHKT1;1 expression had higher antioxidant enzymes activities. These results indicated

Overexpression of PeHKT1;1 Raised the Efficiency of Antioxidant Systems under Salt Tolerance
The NT and transgenic poplar plants, which had been verified in the previous study, were transplanted into soil for three years to detect long-term effects. Subsequently, we randomly chose four independent transgenic lines to further investigate PeHKT1;1 expression levels and physiological parameters. The Pro35S::PeHKT1;1 vector had not been lost in the three-year-old transgenic lines, and PeHKT1;1 was still overexpressed, confirmed by RT-PCR and qRT-PCR; however, expression levels were lower than the three previous years ( Figure S4). To determine the antioxidant function of PeHKT1;1 in poplar under salt stress conditions, activities of antioxidant enzymes, CAT, SOD, and POD, were examined in the three-year-old NT poplar and four transgenic poplar lines. Compared with NT poplars, transgenic poplars had higher CAT activity after one hour when plants were exposed to salt stress ( Figure 6). Similarly, the PeHKT1;1 transgenic poplars showed higher SOD and POD activities after 1 and 1.5 h, respectively. Moreover, comparison of PeHKT1;1 transcript with CAT, SOD, and POD activities suggested that the transgenic poplar lines with higher PeHKT1;1 expression had higher antioxidant enzymes activities. These results indicated that PeHKT1;1 transgenic poplar had enhanced efficiency of antioxidant systems and higher salt tolerance than NT plants under salt stress. that PeHKT1;1 transgenic poplar had enhanced efficiency of antioxidant systems and higher salt tolerance than NT plants under salt stress. Three-year-old soil-grown poplar plants were subjected to 0.8% w/v NaCl at different time points (0, 0.5, 1, 1.5, 2, and 6 h). Data were analyzed using one-way ANOVA followed by Duncan's test. Error bars with letters represent significant differences (p < 0.05, Duncan's test).

Discussion
The HKTs are an essential gene family for salt tolerance in plants, and are mainly responsible for ion homeostasis and Na + distribution within the plant [33][34][35]. Increasing numbers of HKT genes from various species have been reported to be salt inducible, and to improve salinity tolerance in transgenic plants. However, the functions of HKT1 transporters have not been described thoroughly in poplar. Therefore, we cloned the full-length cDNA of PeHKT1;1 from hybrid poplar, and further characterized its roles in Populus.
The HKT proteins contain four membrane-pore-membrane motifs, each consisting of a P-loop between two transmembrane domains (M1 and M2), which are associated with the K + channel [36,37]. Further research has demonstrated that the HKT1 transporters are Na + -specific transporters and have a S-G-G-G signature, whereas HKT2 transporters are Na + -K + co-transporters and have a G-G-G-G type, due to a Gly residue that is crucial for K + selectivity in the first P-loop domain [38,39]. In this study, we applied TMHMN to predict the transmembrane helices of PeHKT1;1 protein, which showed that it contained eight distinct transmembrane domains ( Figure 1A), consistent with the previous reports. Moreover, the sequence alignment and phylogenetic analysis suggested that PeHKT1;1 protein had a S-G-G-G signature for the P-loop domains and belonged to class I of HKT transporters (Figure 1) [11].
So far, the tissue specificity of HKT1 transcripts is diverse in various plant species. The AtHKT1;1 from Arabidopsis is mainly expressed in the root stele and leaf vasculature [40]. In rice, five OsHKT1 genes were identified as belonging to class I, and showed no obvious patterns in mRNA levels among them [12]. McHKT1;1 expression is most abundant in the leaves, and is also present in stems but absent from roots [41], and SsHKT1;1 is predominantly expressed in leaves [42]. Moreover, EcHKT1;1 and EcHKT1;2 from Eucalyptus camaldulensis have higher expression in stems and leaves than roots [43]. The six GmHKTs in soybean showed low expression levels in leaves [32]. The tissue-specific expression study of PeHKT1;1 suggested that the potential function of this gene is in roots and stems in poplar.
Regulatory mechanisms of HKT1 transporters have been identified in multiple species, and they play an important role in Na + transport for increasing salt tolerance in plants [44]. The expression levels of PeHKT1;1, studied here, could aid in further understanding its function in poplar under salt stress. Our NaCl treatment resulted in increased PeHKT1;1 expression in roots and decreased expression in stems, inferring that PeHKT1;1 may function in Na + loading into the root xylem and limiting Na + accumulation in stems [45]. Comparing PeHKT1;1 expression for 0.6% and 1.8% w/v NaCl treatments showed that expression was more rapidly induced under high than low salt stress, inferring that transferring of plants from solution without NaCl to those with high NaCl concentration in a single step will definitely cause salt shock as a result of cell plasmolysis [46,47]. Three-year-old soil-grown poplar plants were subjected to 0.8% w/v NaCl at different time points (0, 0.5, 1, 1.5, 2, and 6 h). Data were analyzed using one-way ANOVA followed by Duncan's test. Error bars with letters represent significant differences (p < 0.05, Duncan's test).

Discussion
The HKTs are an essential gene family for salt tolerance in plants, and are mainly responsible for ion homeostasis and Na + distribution within the plant [33][34][35]. Increasing numbers of HKT genes from various species have been reported to be salt inducible, and to improve salinity tolerance in transgenic plants. However, the functions of HKT1 transporters have not been described thoroughly in poplar. Therefore, we cloned the full-length cDNA of PeHKT1;1 from hybrid poplar, and further characterized its roles in Populus.
The HKT proteins contain four membrane-pore-membrane motifs, each consisting of a P-loop between two transmembrane domains (M1 and M2), which are associated with the K + channel [36,37]. Further research has demonstrated that the HKT1 transporters are Na + -specific transporters and have a S-G-G-G signature, whereas HKT2 transporters are Na + -K + co-transporters and have a G-G-G-G type, due to a Gly residue that is crucial for K + selectivity in the first P-loop domain [38,39]. In this study, we applied TMHMN to predict the transmembrane helices of PeHKT1;1 protein, which showed that it contained eight distinct transmembrane domains ( Figure 1A), consistent with the previous reports. Moreover, the sequence alignment and phylogenetic analysis suggested that PeHKT1;1 protein had a S-G-G-G signature for the P-loop domains and belonged to class I of HKT transporters ( Figure 1) [11].
So far, the tissue specificity of HKT1 transcripts is diverse in various plant species. The AtHKT1;1 from Arabidopsis is mainly expressed in the root stele and leaf vasculature [40]. In rice, five OsHKT1 genes were identified as belonging to class I, and showed no obvious patterns in mRNA levels among them [12]. McHKT1;1 expression is most abundant in the leaves, and is also present in stems but absent from roots [41], and SsHKT1;1 is predominantly expressed in leaves [42]. Moreover, EcHKT1;1 and EcHKT1;2 from Eucalyptus camaldulensis have higher expression in stems and leaves than roots [43]. The six GmHKTs in soybean showed low expression levels in leaves [32]. The tissue-specific expression study of PeHKT1;1 suggested that the potential function of this gene is in roots and stems in poplar.
Regulatory mechanisms of HKT1 transporters have been identified in multiple species, and they play an important role in Na + transport for increasing salt tolerance in plants [44]. The expression levels of PeHKT1;1, studied here, could aid in further understanding its function in poplar under salt stress. Our NaCl treatment resulted in increased PeHKT1;1 expression in roots and decreased expression in stems, inferring that PeHKT1;1 may function in Na + loading into the root xylem and limiting Na + accumulation in stems [45]. Comparing PeHKT1;1 expression for 0.6% and 1.8% w/v NaCl treatments showed that expression was more rapidly induced under high than low salt stress, inferring that transferring of plants from solution without NaCl to those with high NaCl concentration in a single step will definitely cause salt shock as a result of cell plasmolysis [46,47]. Altogether, these results implied that the function of PeHKT1;1 may be essential in response to salt stress in poplar.
Many reports have shown that overexpression of salt stress-related genes can enhance salt stress tolerance in plants. However, few studies have been carried out on trees [19,48,49]. Due to many differences between herbaceous plants and trees, including growth, structure, and physiology in response to salt stress, we constructed an overexpressing PeHKT1;1 vector under control of the CaMV35S promoter (Pro35S::PeHKT1;1), and transformed it into the poplar genome for further understanding of the roles of PeHKT1;1 in trees. Although the expression level of endogenous PeHKT1;1 significantly increased in "Nanlin895", the native allele expression and the CaMV35S-driven allele of PeHKT1;1 both contributed to high expression of PeHKT1;1 in transgenic poplar. Compared with NT, transgenic poplar showed no dwarfing under normal growth conditions ( Figure S3), which is inconsistent with the CaMV35S promoter possibly causing dwarfing of transgenic poplar [21,50]. The possible reason is the random insertion of different genes, and differences in expression levels may lead to different phenotypic characteristics.
Recent reports indicated that poplar growth is significantly reduced at 200 mM (1.2% w/v) NaCl treatment [19]-this was in accordance with our study, which showed comparable growth reduction under salt stress. Under our experimental conditions, overexpression of PeHKT1;1 in transgenic poplar resulted in a better relative growth rate than in NT poplar, including plant height and roots, and this growth rate was consistent with the expression level of PeHKT1;1 (Figures 4B and 5B). Moreover, leaves of transgenic poplar showed no chlorosis, implying that leaf chlorophyll concentration remained higher than in NT poplar. These results suggested that reductions in plant growth may be associated with decreases in photosynthetic activities under salt stress. Of course, the promotion of salt tolerance has its limits-when the Na + concentration exceeded 0.5% w/v, symptoms of salt damage still occurred in transgenic poplar. Thus, we conclude that overexpression of PeHKT1;1 in transgenic poplar enhanced salt tolerance.
To explore the practical application of transgenic poplar overexpressing PeHKT1;1, the 6-week-old transgenic plantlets were transplanted into soil containing 0.2% w/v NaCl ( Figure S5). At their early development stages (approximately 45 days), the growth rates of transgenic poplar showed dramatically decreased salt stress damage and increased plant height, compared with NT poplar. These results are consistent with a previous report [16]. Interestingly, salt tolerance of transgenic poplar lines showed the same trends with mRNA levels of PeHKT1;1 as the overexpression of TaLEA gene in improving salt tolerance in transgenic poplar [19]. However, our results lacked high levels of replication, and this needs some future investigation.
During salt stress, the rapid generation and accumulation of ROS causes secondary oxidative stress and damage to nucleic acids, proteins, and other parts of cells in plants [2]. To scavenge ROS, plants have evolved multiple antioxidant defense systems, including various enzymes such as SOD, POD, and CAT [51]. Previous studies reported that overexpression of PtSOS2 and RtWRKY1 showed a significant activation of SOD, POD, and CAT in poplar and Arabidopsis, respectively [18,52]. In this study, transgenic poplar lines with higher PeHKT1;1 expression exhibited higher CAT, SOD, and POD activities than NT poplar under salt stress ( Figure 6). Thus, we speculated that the high expression of PeHKT1;1 promoted the expression of ROS scavenging-related genes, and then increased the activities of antioxidant enzymes and decreased the damage from salt stress to poplar.
Salt tolerance is determined by coordinated expression of multiple genes in plants. Recent studies demonstrated that HKT1 expression is restricted by some factors. Cytokinin application is involved in the repression of AtHKT1;1 in roots by both type-B response regulators, ARR1 and ARR2 [53,54]. ABI4 and AtZIP24, as negative regulators, also repressed AtHKT1;1 expression [55,56]. Moreover, an MYB-type transcription factor, OsMYBc, binds to the OsHKT1;1 promoter and regulates its expression [57]. Therefore, to further improve salt tolerance of our transgenic poplar lines will require research to explore more regulatory factors affecting HKT1 transporters and other salt stress-related genes in poplar.

Conclusions
In this study, we isolated and cloned the PeHKT1;1 gene from hybrid poplar. The PeHKT1;1 protein contained four conserved selectivity-filter-pore (p-loop) domains and belonged to class I of HKT transporters, according to the amino acid sequence alignment and phylogenetic analysis. Temporal and spatial expression analysis showed that PeHKT1;1 was highly induced in poplar root and stem under salt stress. Overexpression of PeHKT1;1 in transgenic poplar resulted in better growth rates than control plants under salt stress. In accordance, assays showed stronger physiological activities of transgenic than NT poplar. Overall, we clearly demonstrated that transgenic poplar lines overexpressing PeHKT1;1 were superior to NT poplar under salt stress treatment. All evidence indicated that PeHKT1;1 plays an important role in salt tolerance in Populus.