Functional Characteristics of The Stelar K+ Outward Rectifying Channel (EeSKOR) Gene in Elytrigia Elongata

As an important nutrient, K + plays a crucial role in plant stress resistance. It has been reported that the stelar K + outward rectifying channel (SKOR) is involved in loading K + into the xylem for its transport from roots to shoots. Elytrigia elongata is a perennial, sparsely distributed, rhizome-type herbaceous plant belonging to the wheatgrass family; it has high salt tolerance. Here, we isolated EeSKOR from decaploid E. elongata and investigated its function in transgenic tobacco. The results showed that EeSKOR was mainly expressed in the roots and was up-regulated with increasing salinity and drought intensity. Overexpression of EeSKOR in plants exposed to salt stress enhanced growth performance, increased SOD activity and chlorophyll content, signicantly reduced H 2 O 2 and MDA content, reduced Na + concentration, and increased K + concentration in transgenic tobacco plants compared with wild-type (WT) and null vector (Vector) plants. Our ndings suggest that transgenic plants overexpressing EeSKOR could enhance K + transport from the roots to the aboveground parts to maintain K + steady-state in the aboveground under conditions of salt stress, thereby enhancing tobacco salt tolerance.


Introduction
Plant physiological drought leads to ionic imbalance in cells, cell membrane dysfunction, attenuation of metabolic activity, plant growth inhibition, and even cell death owing to excessive soil Na + concentrations 1,2 . To cope with salinity stress, the strategy adopted by plant cells to compartmentalize Na + into vacuoles alleviates cellular Na + toxicity to maintain osmotic balance using Na + as an osmoregulation substance, thus improving salt tolerance of plants 3 . Under salt stress, as the Na + in the root xylem catheter is continuously unloaded into the xylem parenchyma cells, parenchymal membrane depolarization occurs, the stelar K + outward rectifying channel (SKOR) or non-selective ion external rectifying channel protein activity is activated, and the K + in the parenchyma cells is loaded into the xylem sap and transported to the aboveground part of the plant 4,5 . The SKOR gene has also been cloned from maize (Zea mays), where it was mainly expressed in the root cortex and columnar cells and was responsible for discharging K + from columnar cells into xylem sap 6 . Gaymard et al. 7

cloned AtSKOR from
Arabidopsis thaliana and demonstrated that AtSKOR could mediate the loading of K + from xylem parenchyma cells to xylem sap, which would then be transported to the aboveground part with xylem sap ow by transpiration pull. In our previous studies, the high-a nity K + transporter EeHKT1;4 was closely linked with salt tolerance, and there was a signi cant positive correlation between the expression level of EeHKT1;4 in decaploid Elytrigia elongata (2n = 70, PI276399) roots and the selective ability of K + /Na + , indicating that EeHKT1;4 was a major component in maintaining K + /Na + selectivity in the root system 8 .
In fact, the K + /Na + selectivity of plant roots under salt stress is controlled and regulated by multiple genes. Among them, HKT1 proteins unload Na + from the root xylem catheter into the xylem parenchyma cells, causing depolarization of their plasma membrane, thereby activating SKOR transport activity, loading K + from parenchyma cells into the xylem to the aboveground parts, maintaining the K + /Na + selectivity and enhancing salt tolerance 9 .
As one of the ideal herbaceous plants for improving saline-alkali soil, Elytrigia elongata is a perennial, sparsely distributed, rhizome-type, herbaceous plant of the wheatgrass family. It is a close relative of wheat and has become an indispensable wild gene pool for improving wheat 10,11 . Our preliminary research results showed that the salt-tolerant decaploid E. elongata was screened out from thirty-four accessions collected from twenty-one countries 12 . It has also been established that the root system has a stronger K + /Na + selectivity, which limits the absorption of Na + by the root system and reduces the concentration of Na + in the aboveground parts to maintain the concentration of K + in the plant. This increases the K + /Na + ratio of the E. elongata plant, which is the main physiological mechanism of salt tolerance 13 . At the same time, we have cloned out the EeSKOR gene from the salt-tolerant decaploid E. elongata PI276399 using real-time uorescence quantitative PCR (qRT-PCR) and the rapid ampli cation of cDNA ends (RACE) methods and conducted its full-length sequence analysis. However, the role of EeSKOR in salt tolerance of E. elongata remains unclear. To assess whether the overexpression of EeSKOR confers improved salt tolerance in plants, we introduced the EeSKOR gene into tobacco to determine growth performance, physiological indices, and Na + and K + concentrations in the transgenic tobacco plants, null vector plants, and wild-type (WT) plants subjected to salinity stress. The results indicated that EeSKOR-mediated compartmentalization of Na + into the vacuoles may play a key role in the salt tolerance of plants. This would provide potential bene ts for generating engineered plants to increase tolerance to saline conditions.

Results
Cloning and sequence analysis of EeSKOR Degenerate primers (EeSKOR-1 and EeSKOR-2) were designed according to the conserved homologous sequences of the SKOR protein of other plants. The cDNA synthesized by total RNA reverse transcription from the young roots of E. elongata was used as the template, the cDNA fragment of the EeSKOR gene was ampli ed by PCR, and the core fragment was 555 bp. Sequences of the 5′ and 3′ ends were obtained by RACE, which yielded products of 1136 bp and 1033 bp, respectively ( Figure S1).
The full-length cDNA sequence, 2402 bp of EeSKOR, was obtained by splicing the DNAMAN software sequence. The ORF of EeSKOR was 2145 bp long and encoded a polypeptide protein consisting of 717 amino acid residues. The predicted protein had an isoelectric point of 8.29 and a molecular weight of 81.15 kDa. The cDNA sequence of EeSKOR was submitted to GenBank under accession number MK203848. The amino acid homology of EeSKOR with TaSKOR (Triticum aestivum), TuSKOR (Triticum urartu), and AetSKOR (A. tauschii) were 87.67%, 87.14%, and 86.09%, respectively. Analysis using the TMpred tool indicated that EeSKOR contained ve transmembrane regions ( Figure 1). The phylogenetic tree analysis showed that EeSKOR was closely related to the SKOR of wheat monocotyledons of Gramineae ( Figure S2). These results indicate that the EeSKOR gene encodes the external recti er K + channel protein.
EeSKOR plasmid with a GFP signal was transferred into tobacco leaves to observe the transient expression of EeSKOR in the epidermis of tobacco leaves. The results showed that EeSKOR-GFP was signi cantly localized on the plasma membrane and nucleus of tobacco epidermal cells under FV10-ASW laser confocal microscopy. Thus, EeSKOR was located in the plasma membrane and nucleus ( Figure S3).

Analysis of EeSKOR gene expression patterns under salt and drought treatments
The expression levels of the EeSKOR gene in the roots, sheaths, and leaves of 4-week-old E. elongata seedlings treated with different NaCl concentrations for 24 h (0, 25, 50, 100, 150, and 200 mmol·L -1 ) were analyzed. The results showed that the expression level of EeSKOR in roots increased with an increase in NaCl concentration (25-100 mmol·L -1 ) but decreased slightly at 150-200 mmol·L -1 NaCl. With the increase in NaCl concentration, the expression level of EeSKOR in sheaths and leaves showed a decreasing trend compared with that in the control. On the whole, it showed an expression trend of roots > leaves > sheaths ( Figure 2a). The EeSKOR expression patterns in 4-week-old E. elongata seedlings under osmotic stress for 24 h (-0.5, -1.0, and -1.5 Mpa) were analyzed. The results showed that EeSKOR expression was upregulated with an increase in drought stress concentration, and in general, EeSKOR expression level was in the order of roots > leaves > sheaths ( Figure 2b). Moreover, EeSKOR expression was induced and regulated by salt and osmotic stress.

Construction of plant expression vector of EeSKOR and molecular testing of positive lines of tobacco
According to the requirements of the Clontech Infusion seamless connection technology, Nco I and Bgl II restriction sites were introduced at both ends of the upstream and downstream primers of the ORF frame of EeSKOR, and the PCR products were ampli ed by RT-PCR and detected by 1.2% gel electrophoresis ( Figure S4a). Then, the restriction sites on pCAMBIA1301 were double-digested by Nco I/Bgl II, and the large fragment was recovered and named pCAMBIA1301-A. The target gene EeSKOR was inserted into pCAMBIA1301 to obtain the recombinant plasmid pCAMBIA1301-35S-EeSKOR-Nos, and a speci c band of approximately 2942 bp was obtained after Hind III/Bgl II double enzyme digestion ( Figure S4b). The pCAMBIA1301-35S-EeSKOR-Nos vector was introduced into Agrobacterium GV3101 by the freeze-thaw method, and the target fragments of 2154 bp and 795 bp were detected by PCR agrobacterium and gel electrophoresis, which con rmed that the strain was positive ( Figure S4c). This indicates that we have successfully constructed the plant expression vector of the EeSKOR gene of E. elongata ( Figure S5).
To investigate the potential bene ts of transferring EeSKOR to other plant species, we identi ed forty-ve independent EeSKOR transgenic tobacco lines, Vector, and WT plants by qRT-PCR ampli cation. We observed relatively higher expression levels in L12 and L36 ( Figure S6). Therefore, L12 and L36 were used in the following assays. Western blot analysis showed that the membrane proteins of isolation markedly increased the EeSKOR protein level in L12 and L36 compared to that in WT, Vector, and lower expression lines (L1 and L11). Moreover, the protein levels in L12 and L36 were higher than those in WT ( Figure S6), indicating that heterologous expression of EeSKOR could enhance salt tolerance in transgenic tobacco.
Analysis of salt tolerance of transgenic tobacco plants Under normal conditions (0 mmol·L -1 NaCl), the wild-type (WT) plants, Vector plants, and transgenic plants all showed good growth performance, with the increase in NaCl salt concentration, the transgenic tobacco (L12 and L36) plants showed higher salt tolerance than the WT and Vector plants ( Figure 3a).
Plant height and dry weight signi cantly decreased with increasing salt concentration compared to those observed under normal growth conditions, but the dry weight and plant height of transgenic tobacco plants under salt treatment were signi cantly higher than those of WT and Vector plants (Figure 3b, c). In particular, under 200 mmol·L -1 NaCl treatment, the dry weight of L12 increased by 35 and 42%, and that of L36 increased by 39 and 46%, respectively, compared with that of the WT and Vector tobacco plants, the plant height of L12 increased by 39 and 47%, and that of L36 increased by 38 and 45%, respectively.
Thus, it was demonstrated that the overexpression of EeSKOR under salt treatment signi cantly increased the biomass and plant height of transgenic tobacco plants.
As shown in Figure  decreased by 28 and 30%, respectively, and that in the root of L36 decreased by 32 and 34%, respectively ( Figure 5b). With the 200 mmol·L -1 NaCl treatment, the K + concentration in the aboveground part of L12 was 2.1 times and 2 times higher than that in the WT and Vector plants, respectively, and the K + concentration in the aboveground part of L36 was 1.9 times and 1.8 times higher than that in the WT and Vector plants, respectively (Figure 5c); the K + concentration in the root of L12 was twice that in the roots of the WT and Vector plants, and the K + concentration in the root of L 36 was 1.6 times and 1.5 times that in the roots of the WT and Vector plants, respectively (Figure 5d). Thus, the overexpression of the EeSKOR gene under salt treatment not only signi cantly reduced the Na + concentration of transgenic plants but also increased the K + concentration in transgenic plants.

Discussions
The Shaker family of potassium channels is one of the most intensively studied potassium transport families in plants and plays an important role in the absorption and transport of potassium ions in plants 14 . Previous research has shown that there are two different families of inner rectifying K + channels and outer rectifying K + channels in the Shaker channel 15 . The rst reported recti er K + channel protein AKT1 in A. thaliana belonged to the Shaker family, which had the characteristics of amphiphilic potassium absorption, and was mainly expressed in the root epidermal cells and cortical cells. Gaymard et al. 7 rst isolated and cloned the K + channel protein gene SKOR from A. thaliana and found that it was speci cally expressed in root columnar tissue and was inhibited by abscisic acid (ABA), which mediates K + transport from root cells to the xylem. It was also found that the K + selective channel protein identi ed in the Arabidopsis genome contains 2-12 transmembrane domains, forming 1-2 pore regions and multiple regulatory domains, which are located on the plasma membrane. The pore region structure of the SKOR protein of the Shaker family contains the conserved GYGD basic sequence 16 . Previous studies have shown that the positively charged amino acids (arginine and lysine) in the fourth transmembrane region can induce transmembrane voltage changes and play a key role in controlling the opening and closing of K + channels 17 . Duan et al. 18 reported that the PtSKOR gene (from Puccinellia tenui ora) sequence had six highly conserved transmembrane regions (S1-S6), a highly conserved pore region TVGYG between S5 and S6, and a C-terminal containing a predicted cyclic nucleotide-binding domain and an ankyrin domain. In this study, it was found that EeSKOR had ve transmembrane regions, among which there was a highly conserved P-ring structure (containing GYGD basic sequence) between the fourth and fth transmembrane regions, which is also the marker sequence of K + channel proteins ( Figure   1). The C-terminal of EeSKOR contains a cyclic nucleotide-binding domain and an anchorin region, but the sites that regulate potassium channel activity are often located in the C-terminal cell of the SKOR protein. This indicates that the nucleotide-binding domain can regulate the activity of the outer rectifying K + channel protein. In our studies, the phylogenetic tree analysis results showed that EeSKOR had high homology with monocotyledons such as TaSKOR and ZmSKOR, and low homology with internal rectifying K + channel AKT1 ( Figure S2). Thus, EeSKOR encodes the external recti er K + channel protein SKOR.
Potassium, being an essential nutrient in plants accounts for about 10% of the dry matter of plants 19 , and plays an important role in plant nutrition, growth, homeostasis, and osmotic regulation of the enzyme system 20,21 . Soil salinization has become a topical environmental issue affecting global agricultural production, disturbing normal uptake of potassium in plants 22 . Because K + and Na + have similar ionic hydration radii, under salt stress, Na + competes for the K + binding sites in the plasma membrane of plant roots and prevents the transport of K + by the roots 23,24,25 . Therefore, the K + transport system plays an important role in plant resistance to salt stress 26 . Previous studies have shown that Na + unloading to the parenchyma cells around the xylem would cause depolarization of the membrane and activate the external recti er K + channel protein SKOR to load K + to the xylem and then transport it to the aboveground parts to enhance the salinity tolerance of plants 4,5,27 . The transcriptional abundance of the AtSKOR gene in A. thaliana was signi cantly up-regulated under salt stress 28 . Garcia-Mata et al. 29 reported that AtSKOR expression was upregulated under salt treatment, and K + concentration in A. thaliana also increased compared to that in the control. Thus, the increase in AtSKOR gene expression leads to more K + being loaded into the xylem and transported to the aboveground parts. Under high salt stress, ZxSKOR in roots and stems is well-coordinated to mediate long-distance K + transport and plays an important role in K + accumulation and homeostasis in Zygophyllum xanthoxylum 26 , and SKOR genes from Puccinellia tenui ora and Lycium ruthenicum showed similar salt tolerance mechanisms 18,30 . Moreover, it was also con rmed that the SKOR channel protein was closely related to K + transport in plants 31 .
In this study, EeSKOR was mainly located in the cytoplasmic membrane of the epidermis of tobacco leaves, and a small part was located in the nucleus, indicating that EeSKOR is a membrane transporter ( Figure S3). With the increase in the concentration of external salt and drought treatment, the expression levels of EeSKOR in the roots, sheaths, and leaves of E. elongata showed an increasing trend ( Figure  2a,b). Moreover, the overexpression of the EeSKOR gene in tobacco resulted in the accumulation of more K + and lesser Na + concentrations in transgenic tobacco plants under salt stress than those observed in the WT and Vector plants ( Figure 5). This may be because HKT1 proteins unload Na + from xylem juice into its parenchyma cells, which reduces the concentration of Na + in the aboveground part, and may cause the depolarization of the parenchyma membrane of the xylem 6,7,9 . This activates the external recti cation of the K + channel, SKOR, and results in more K + being loaded into the xylem and transported over long distances to the aboveground parts to maintain a constant K + concentration there.
At the same time, we also found that under salt stress, overexpression of EeSKOR signi cantly enhanced SOD activity and increased Chl content, and signi cantly reduced H 2 O 2 and MDA contents in the transgenic tobacco plants ( Figure 5). It has been shown that overexpression of EeSKOR can improve the antioxidant activity of plants, effectively removing and reducing the generation of reactive oxygen species, protecting the integrity of the membrane system and photosynthetic organs allowing normal photosynthesis and improvement of plant's salt tolerance. In addition to EeSKOR's location in the plasma membrane, we also found that this protein was located in the nucleus. Therefore, it is speculated that the EeSKOR gene may be involved in the regulation of plant salt tolerance through other salt-tolerant pathways.

Conclusions
EeSKOR was cloned from the decaploid E. elongata. EeSKOR was mainly expressed in the roots, and its transcript abundance changed with increasing salt concentration and drought intensity. The phenotypes, dry weight, plant height, and the concentrations of Na + and K + in roots, sheaths, and leaves were measured in response to treatment with different NaCl concentrations and drought treatments. The transgenic tobacco plants displayed enhanced tolerance to NaCl stress compared with the Vector and WT plants. These results suggest that overexpression of EeSKOR in tobacco plants enhances the transport of K + from the root to the aboveground part to maintain K + steady-state in the aboveground under salt stress, thereby enhancing tobacco salt tolerance.

Materials And Methods
Material culture Seeds of decaploid E. elongata were collected from the National Experiment Station of Precision Agriculture, Xiao Tang Shan, China, located approximately 55 km from Beijing (39°34′ N, 116°28′ E). Plump seeds were sterilized with sodium hypochlorite (NaClO) solution (5%, v/v) for 5 min, rinsed thoroughly with distilled water, incubated in 40°C water for 56 h, and then germinated on moistened lter paper for four days at 25°C in the dark. After plumule emergence, uniform seedlings were transferred to plastic containers (20 cm long, 10 cm wide, and 7 cm high) containing modi ed Hoagland's nutrient

Cloning of EeSKOR
Four-week-old E. elongata seedlings were treated with 200 mmol·L -1 NaCl for 24 h. After treatment, fresh roots (200 mg) were washed in sterile water and then ground in liquid nitrogen. Total RNA was extracted with a Trizol Kit (Sangon Biotech. Co., LTD, Shanghai, China) according to the manufacturer's instructions. Primers EeSKOR-1/EeSKOR-2 were designed according to the principle of high homology and degeneracy by comparing SKOR gene sequences of other plants in the GenBank database (Table  S1). The conserved core fragments of EeSKOR were ampli ed using PCR. The PCR ampli cation was calibrated at 94°C for 2 min; 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min, and a nal extension at 72°C for 10 min. PCR products were puri ed from agarose gels, ligated into the PMD19-t vector, and sequenced by Sangon Biotech Co, Ltd., (Shanghai, China). The 5'-and 3'-RACE primers were designed by referring to the core sequence of the EeSKOR gene and were obtained according to the corresponding methods of the RACE Kit (Table S1). The gene fragments of 5'-and 3'-RACE were ampli ed using the Takara Premix PrimeSTAR HS Kit (Takara Biotech Co., Ltd., Dalian, China) according to the manufacturer's instructions, and the cDNA of EeSKOR gene was spliced and detected by 1.2% agarose gel electrophoresis. The recovered products were connected to the PMD19-t vector, then transformed and cloned, and the positive strain was identi ed and sequenced by Sangon Biotech.

DNA sequence and phylogenetic analyses
The EeSKOR sequence was analyzed, and the coding regions were predicted using ESPript 3 (Easy Sequencing in PostScript 3) software. EeSKOR sequence homology analysis and phylogenetic tree construction were performed using DNAMAN 6.0 software (Lynnon Biosoft, San Ramon, CA, USA). The isoelectric point and molecular mass were predicted using the online Compute pI/Mw tool (http://web.expasy.org/compute_pi/).

Expression analysis of EeSKOR
Real-Time Quantitative Reverse Transcription methods PCR were used to analyze the expression patterns of the EeSKOR gene in the root, sheath, and leaf of E. elongata subjected to different NaCl concentrations for 24 h (0, 25, 50, 100, 150, and 200 mmol·L -1 ) and different osmotic potentials of sorbitol for 24 h (0, -0.5, -1.0, and -1.5 MPa). Under each treatment, the extraction of total RNA in the root, sheath, and leaf of E. elongata was carried out according to the RNA Extraction Kit instructions of Takara Biotech. The rst strand of cDNA was synthesized according to the instructions for the Prime Script™ RT reagent Kit with gDNA Eraser (Takara Biotech., Co., Ltd.). The qRT-PCR forward primer for the EeSKOR gene was P1 (5'-TACGGAGGCTGCTCAGGTTT-3'), and the reverse primer was P2 (5'-CGCATCTCCTCGCTTCATC-3'); the PCR product length was 189 bp. The positive primer for qRT-PCR of the internal gene Actin was P3 (5'-CTTGACTATGAACAAGAGCTGGAAA-3') and the reverse primer was P4 (5'-TGAAAGATGGCTGGAAAAGGA-3'); the PCR product length was 139 bp. The qRT-PCR experiment was conducted using a StepOnePlus instrument (Thermo Fisher Scienti c, Waltham, MA, USA). The reaction system consisted of SYBR ® Premix Ex Taq II (Takara Biotech., Co., Ltd.) 12.5 L, both positive and reverse primers 1 L, cDNA 2 L, and water was added to 25 L. The PCR ampli cation procedures were as follows: denaturation at 95°C for 1 min, 95°C for 5 s, 60°C for 30 s, and 40 cycles. The relative expression of EeSKOR was calculated using the 2 -∆∆CT method. Experiments were repeated at least ve times to obtain similar results.

Subcellular localization analysis
The positive primers with the Kpn I enzyme loci (5'-GGGGACGAGCTCGGTACCATGGAGAGGGAGATTGTAGCAGAGT-3') and reverse primers with the Xba I enzyme loci (5'-CATGGTGTCGACTCTAGACTGATCGGCTGCAACAGCAGCTGTA-3') were designed using Primer 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). The product templates were EeSKOR open reading frame bacterial liquid that had been sequenced and veri ed. The target fragments were ampli ed using the Takara Premix PrimeSTAR HS Kit, and the PCR reaction conditions were 95°C for enzymatic digestion product using the BioTeke DNA puri cation recovery Kit (BioTeke Corporation, Wuxi, Jiangsu, China). The empty vectors pCAM35-GFP and pCAM35-EeSKOR-GFP were imported into Agrobacterium GV3101 using the freeze-thaw method for future use. The pCAM35-GFP and pCAM35-EeSKOR-GFP were transferred into the epidermal cells of Nicotiana benthamiana by injection, and the expression location of EeSKOR in the epidermal cells of tobacco leaves was observed using a Leica TCS SP8 confocal microscope (Leica Camera AG, Wetzler, Germany) in Germany.

Construction of plant expression vector and transformation of agrobacterium
Nco I and Bgl II restriction sites were introduced at both ends of the upper and lower primers of the EeSKOR open reading frame (ORF), and the target fragment PCR products were detected by 1.2% gel electrophoresis after RT-PCR ampli cation. The restriction sites on pCAMBIA1301 were double-digested with Nco I/Bgl II, and the large fragments were recovered. Then, the target EeSKOR gene was inserted into the linear plant expression vector (pCAMBIA1301-35S-EeSKOR-Nos) using Clontech In-Fusion seamless connection technology (Takara Biotech Co., Ltd.) according to the manufacturer's instructions, and the speci c bands were obtained by double-digestion with Hind III/Bgl II. The constructed overexpression vector was transformed into the sensing state of Agrobacterium GV3101 by the freeze-thaw method, and the transformed bacterial solution was uniformly coated onto YEP solid medium (50 mg·L -1 kanamycin, 50 mg·L -1 rifampicin, 50 mg·L -1 gentamycin). After inverted culture at 28°C away from light, a single colony was grown after 2-3 days. The single colony was picked up using an inoculation ring, inoculated into 5 mL YEP medium, and incubated overnight. The plasmid was extracted and con rmed as a positive clone by PCR ampli cation.

Genetic transformation and molecular characterization
The above-mentioned constructs were introduced into Agrobacterium tumefaciens strain GV3101 by the chemical method and then used for tobacco (Nicotiana tabacum cv. Wisconsin 38) transformation using the leaf disc method as described by Horsch et al. 32 with minor modi cations. The infected leaf sections were cultivated on Murashige and Skoog (MS) medium containing 2 mg·L -1 6-benzylaminopurine (6-BA) and 0.2 mg L -1 1-naphthaleneacetic acid (NAA) at pH 5.8-6.0 for three days. The WT tobacco leaf DNA and agrobacterium-positive bacterial liquid were used as controls. Kan-resistant tobacco leaf DNA was used as a template, EeSKOR-F/EeSKOR-R and DPF1/DPR1 were used as primers (Table S1), and the transgenic tobacco positive plants were detected by PCR ampli cation. For western blotting, 20 μg tonoplast proteins were separated using 12% (m/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blots were performed according to the method described by Kumari et al. 33 .
Analysis of salt tolerance of transgenic tobacco plants T1-generation seeds of wild-type tobacco (N. tabacum cv. Wisconsin 38), null vector (Vector), and overexpressed EeSKOR transgenic tobacco (L12 and L36) plants were sterilized with 5% (v/v) sodium hypochlorite (NaClO) solution for 5 min and then washed several times in distilled water. The moist seeds were evenly seeded in a petri dish covered with absorbent paper, and dark cultured at 25°C for four days. After germination and rooting, the seeds were transferred into a black culture box (20 cm×10 cm×7 cm) and seeded with Hoagland nutrient solution. Light culture was conducted for 16 h (day)/8 h (night) at a light intensity of about 600 mol·m -2 ·s -1 with the Hoagland nutrient solution replaced after two days; air relative humidity was kept at 60%-80%. The WT, Vector, and transgenic tobacco (L12 and L36) plants were subjected to twenty-one days of stress in Hoagland nutrient solution with NaCl concentrations of 0, 50, 100, 150, and 200 mmol·L -1 , respectively. The plant height, biomass, H 2 O 2 , MDA, and chlorophyll (Chl) content, SOD activity, and Na + and K + accumulation concentrations in the aboveground part and root of the plant were measured. After fresh weight measurements, roots, sheaths, and leaves were oven-dried at 80°C to a constant weight, and the dry weight of each organ was recorded. The MDA and Chl contents were determined using the method of Zhou 34 , and the SOD activity was determined by the nitroblue tetrazolium (NBT) method. Na + and K + concentrations in the roots, sheaths, and leaves were measured using a ame emission spectrophotometer.

Statistical analysis
Each treatment was repeated ve times independently with three seedlings per replicate. All data are presented as mean ± standard deviation (SD). Duncan's multiple range tests were performed using statistical software (v.13.0, SPSS Inc, Chicago, IL, USA).