AtHKT1 gene regulating K+ state in whole plant improves salt tolerance in transgenic tobacco plants

The status of K+ is important for plant health. However, little is known about if high-affinity potassium transporter HKTs may help K+ retention under salt stress. Here, we determined the effect of Arabidopsis thaliana transporter gene (AtHKT1) on the K+ status, Na+-induced toxicity, and salt tolerance in tobacco (Nicotiana tabacum L.). Six AtHKT1 transformed tobacco lines (T1, T2, … T6) were contrasted with a non-transgenic plantlet at the whole-plant and molecule levels. AtHKT1 gene was expressed in the xylems of stem, root and leaf vein in the transgenic tobacco, with the line T3 having highest expression. At Day 15, in the 200 mmol L−1 NaCl stress treatment, the transgenic plants remained a healthy K+ status, while the control plants decreased K+ content by 70% and Na+ contents in leaves and stems were 1.7 times that in the transgenic line. The AtHKT1 expression enhanced the activities of SOD, CAT and POD, raised chlorophyll and soluble sugar contents and root activity, and decreased MDA and proline contents and electrolyte leakage destruction. The constitutive over-expression of AtHKT1 that helps maintain a healthy K+ status while reducing Na+ toxicity may serve as a possible mechanism in maximizing productivity of tobacco under salt stress.

membrane of xylem parenchyma cells, unloads Na + from the xylem to the xylem parenchyma cells in both stems and roots, thus decreasing Na + transport from root to stem and reducing Na + movement into leaf cells [23][24][25][26][27][28] . While over-expressing in the cortex and epidermis, the native AtHKT1 gene is up-regulated in the transgenic Arabidopsis thaliana plants 29 . In these studies, the change of AtHKT1 gene expression and Na + accumulation are often deemed to be the main responses to salt stress. Arabidopsis thaliana and Thellungiella salsuginea are in the same family, and AtHKT1may share some similar functions as TsHKT1;2 in K + transport 8 . K + status was thought as a vital reason of plant survival to salt. However, little has been reported if AtHKT1 gene might be involved in the K + accumulation in heterologous plants 30 . An unanswered question is that: could AtHKT1 gene directly improve K + accumulation in heterologous plants under high Na + stress conditions and thus improving plant tolerance to the stress? In the present study, a high-affinity potassium transporter (HKT) gene -AtHKT1 was identified in Arabidopsis thaliana, and AtHKT1:GFP fusion genes were transferred to a tobacco cultivar -'CV87' driven by the CaMV 35S promoter. We hypothesize that (i) the AtHKT1-transformed tobacco plantlets have an improved resistance to salt contrast with non-transgenic tobacco, and (ii) the over-expression of AtHKT1 gene supports keeping plant K + accumulation under salt stress. To examine the hypotheses, we made a series of investigations, involving (i) cloning AtHKT1 gene, constructing AtHKT1:GFP fusion genes vector and obtaining transgenic tobacco plants; (ii) testing and verifying AtHKT1:GFP expression; (iii) determining the whole-plant K + and Na + contents; and (iv) assessing biophysicochemical traits, such as the contents of total chlorophyll, soluble sugar, and proline, as well as MDA, electrolyte leakage, antioxidant enzyme activity, root activity and tissue biomass. These estimations hold out the hypothesis that the continuous over-expression of AtHKT1 gene assists keeping plantlet K + steady state, alleviates the Na + ion toxic, and improves plant tolerance to salt stress.

Results
Clone and bioinformatics of AtHKT1 gene in Arabidopsis thaliana. The cDNA sequence of the AtHKT1 gene was amplified in Arabidopsis thaliana. The coding region was 1521 bp, which encoded a 506 amino acid polypeptide. Sequence alignment analysis showed that the AtHKT1 gene sequence was 99.3% homologous to the sequence of the Arabidopsis thaliana AtHKT1 gene (accession No. AF237672) in NCBI. The similarity percentage was 84.0% with Thellungiella halophil HKT1 gene sequence (EF025500). The phylogenetic tree shows that AtHKT1 and Thellungiella halophil HKT1 (ABK30935.1) are in the same cluster (Fig. 1A). The AtHKT1 is a stable alkaline protein with a possible theoretical isoelectric point of 9.33. One TrkH cation transporter protein conserved domain is at the 152-500 amino acids. The AtHKT1 also has ten transmembrane regions (Fig. 1B), five hydrophilic regions and thirteen hydrophobic regions with uniform distribution in the peptide (Fig. 1C). Thereby, AtHKT1 gene encoded protein is a transmembrane hydrophobic protein with the characteristics of vector transport protein.
Vector construction and tobacco genetic transformation. The plasmid vector pAtHKT1 + GFP including the AtHKT1, Npt II and GFP genes was constructed and confirmed by PCR amplification (Fig. 2B). Green shoots were grown from the transformed leaf discs after four to five weeks growing in the selective medium ( Fig. 2C). After seven days the shoots were shifted to the rooting selective medium, roots were formed (Fig. 2D). But these traits were not found in the non-transgenic plant. Tobacco plants with healthy-grown roots were reproduced for next molecular test.

Molecular test and gene expression analysis.
Applying AtHKT1 gene-specific primers, PCR analysis revealed that the transformed plants obtained a 1521 bp amplification product and the control plant missed it ( Supplementary Fig. S1A). PCR -Southern blot analysis further confirmed the PCR amplification results, which revealed that nine transformed plantlets had the 1521 bp AtHKT1 gene produc, and the control plants did not have ( Supplementary Fig. S1B). The Southern blot further showed that AtHKT1 gene was confirmed into the six transgenic tobacco line genomes with hybridization signals, and three of the transgenic lines had two copies of the AtHKT1 gene (Fig. 3A). The mRNA expression in the plant leaves showed that the AtHKT1 and GFP genes were expressed in the six transgenic plants and no transcript was observed in the non-transgenic plants (Fig. 3A,B). The qRT-PCR analysis revealed that all the transgenic lines expressed AtHKT1 gene. The T3 line had the greatest AtHKT1 expression, which was up to 4.4 times that of the five other transgenic plant lines (Fig. 3D).
To further understand the expression site of the foreign AtHKT1:GFP gene in the transgenic tobacco, the paraffin sections of the transgenic tobacco were observed under the excitation of blue light. The green fluorescence mainly lied in the xylem of the root, stem and leaf veins in the transgenic lines ( Fig. 4A-G). These observations suggest that the fusion gene has undergone through normal transcription and translation and may be involved in the transport of moisture and inorganic salt ions in the conducting tissues.
Biomass in transgenic tobacco carrying AtHKT1 gene. With the stress level increase from 0 to 100 mmol L −1 NaCl, the control plant reduced root fresh weight 80.7% and root dry weight 66.9% (Table 1); In contrast, the transgenic T3 plants reduced by 34.3% and 29.6%, respectively. With the NaCl stress furthered to the 150 mmol L −1 , the control plants did not produce root, while the T3 plants had fresh root weight 0.24 g and dry weight 0.03 g. Treatment effects on shoot fresh and dry weights tracked a similar tendency as the root traits (Table 1; Fig. 5). With the salt stress level added from 0 to 150 mmol L −1 NaCl, the non-transgenic plant reduced shoot fresh weight 62.1% and dry weight 56.7%, but the reduction were 15.2% and 19.6%, respectively, in the T3 line, significantly less than the control. K + and Na + contents to salt stress. To examine the effect of over-expression of AtHKT1 gene in tobacco plant, we determined Na + and K + contents in roots, shoot and leaves under the NaCl stress and non-stress conditions (Figs 6 and 7). With the increase of NaCl stress, the control tobacco reduced K + contents in the leaves, shoot and roots significantly (P < 0.05), while the values were stable in the transgenic T3 line (Fig. 6A-C). By Day 15 in the stress treatments, the leaves of the non-transgenic plants decreased K + content from 21.0 mg g −1 of DW at the 0 mmol L −1 NaCl to 10.7 mg g −1 at the 100 mmol L −1 NaCl stress, furthered to 6.3 mg g −1 at the 200 mmol L −1 NaCl stress, representing a decrease of 49.1 and 70.0%, respectively (Fig. 6A). In contrast, the raise of the stress did not decrease K + contents in the leaves of the transgenic lines T2, T3 and T5. In the stem and roots, the similar trend of treatment effect was found for K + content (

Physiological and biochemical reactions in salt stress. The physiological and biochemical responses
to salt stress were assessed by determining the contents of total chlorophyll, proline and soluble sugar, as well as malondialdehyde (MDA), electrolyte leakage, root activity, and the key enzymatic activities (peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD)) in the leaves. With the raise of NaCl stress levels, total chlorophyll content reduced significantly (P < 0.05) in all the genotypes but the transgenic plants reduced significantly less than the non-transgenic plant (Fig. 8A). By Day 15 in the NaCl treatments, the non-transgenic plants decreased chlorophyll content from 1.5 mg g −1 at the 0 mmol L −1 NaCl to 0.5 mg g −1 at 100 mmol L −1 , furthered to 0.2 mg g −1 at 200 mmol L −1 NaCl, representing a decrease of 66.7 and 88.6%, respectively, while the transgenic lines decreasing by 38.7-66.2% and 55.3-87.6%, respectively, the transgenic T3 line decreasing by 38.7 and 55.3,especially. On average, the transgenic plants total chlorophyll contents were 1.7 times that in the control at 100 mmol L −1 NaCl, and 2.4 times at 200 mmol L −1 NaCl. The proline content had a reverse trend as the chlorophyll: the non-transgenic plants raised proline content more obviously than the transgenic plants under NaCl stress (Fig. 8B). By Day 15 in the 100 mmol L −1 NaCl stress treatment, the proline content in the control plant was 30.5 μg·g −1 of FW, twice that of the transgenic lines; in the 200 mmol L −1 salt treatment, the control plant had proline content at 53.0 μg·g −1 FW, 45% more than those in the transgenic lines. Soluble sugar content were significant (P < 0.05) differences in the three levels of salt treatments, but at a given salt stress level the differences were inconsistent between the genotypes (Fig. 8C).
The MDA content and electrolyte leakage followed a similar trend of treatment effects (Fig. 9A,B). By Day 15 under the NaCl stress, the non-transgenic plants raised MDA content from 1.3 μmol g −1 at the 0 mmol L −1 NaCl to 5.2 μmol g −1 at 100 mmol L −1 NaCl and furthered to 15.0 μmol g −1 at 200 mmol L −1 NaCl, representing a raise of 4.0 and 11.5 times, respectively (Fig. 9A). The transgenic lines had the lower amounts of MDA at the 100 and 200 mmol L −1 NaCl stress treatments. Similar to the MDA, electrolyte leakage in the non-transgenic plant raised more obviously with the increase of the NaCl concentrations than the transgenic plants (Fig. 9B). Opposite trend of treatment effects were shown in the root activity (Fig. 9C); the control plants reduced the root activity more significantly than the transgenic lines with the increase of the NaCl concentration. At the 200 mmol L −1 NaCl stress level, the roots activity of the control plants had a zero value, while the root activity improved effectively in the transgenic tobacco plants by AtHKT1 gene over-expression. Meanwhile, the biochemical detection revealed that the NaCl salt stress treatments stimulated the activity of the anti-oxidative enzymes SOD, POD and CAT (Fig. 10A-C). The highly significant differences between the transgenic and control plants were found under the 200 mmol L −1 NaCl stress for all these anti-oxidative enzymes, with the transgenic lines T2 and T3 having highest enzymatic activities among the genotypes.

Discussion
High-affinity potassium transporters (HKTs) are known to play an significant effect in relieving salt stress in plants [31][32][33][34] . Our research team identified a high-affinity potassium transporter of AtHKT1gene and cloned it from the leaf of Arabidopsis thaliana in NaCl stress, and we found that AtHKT1 gene sequence has an 84.0% similarity with Thellungiella HKT1 gene sequence (EF025500). The phylogenetic tree showed that AtHKT1 and Thellungiella HKT1 (ABK30935.1) were in the same cluster, suggesting that AtHKT1 may have the function of transporting K + under salt stress. A number of studies have demonstrated that AtHKT1 controls Na + Recently, many research have been made to know the role of HKTs expression with the salt hypersensitivity in plant. Zea mays increased accumulation of Na + in leaves with the natural ZmHKT1 loss-of-function 35 . Solanum lycopersicum plants become hypersensitivity to salinity with ScHKT1;2 and SlHKT1;2 genes silencing 36 . In strawberry (Fragaria ananassa), an increase of HKT1expression in roots leads to high tolerance to salinity 37 . Most of them focused on the HKT1 function on Na + accumulation under salt stress. Our results add novel information that the over-expression of AtHKT1 gene in tobacco leads to K + steady state, reduces Na + toxicity in leaf, improved physicochemical characters of the whole plant under salt stress; this may serve as a possible mechanism Potential impacts of the protein on the improvement of salt tolerance.
Damage to plants due to high salinity results in reduction of enzyme activity and loss of biomass due to osmotic imbalance caused by water deficit 6 . Keeping K + in a high level under high Na + stress conditions is identified as a strategy to enhance salinity tolerance 38 . Improving K + content and decreasing Na + accumulation could 0.00 ± 0.00 f 0.00 ± 0.00 f 1.08 ± 0.00 e 0.11 ± 0.00 g Table 1. Fresh and dry weights (g plant −1 ) of the transgenic plants (T3) with over-expression of AtHKT1 gene and the non-transgenic control (CK) under various NaCl stresses. CK is the "CV87" non-transgenic tobacco cultivar. T3 is the transgenic tobacco line with highest AtHKT1 gene over-expression. Tukey's HSD test is used in analysis of significant differences (P < 0.05) in the NaCl-stress (n = 9, i.e., 3 runs × 3 replications). retrieve Na + toxicity in leaves 39 , and this process may help alleviate osmotic imbalance and avoid salt-induced hydraulic failure via stomatal closure and a cascade of downstream effects 40 . In the present study, we measured K + and Na + contents in the different organs with or without AtHKT1 expression under salt stress. We found that the K + content was obviously more in the transgenic lines compared with the control plantlets under NaCl stress. The line with higher AtHKT1 expression (the T3 line as an example) has stable K + contents in the different organs under the different levels of NaCl stress. Under the 200 mmol L −1 NaCl stress, K + content in the leaves, shoot and roots of the control plants decreased remarkably, while little reduction was shown in the transgenic lines. Furthermore, with increased salt stress, the control plantlets raised Na + content significantly (P < 0.05) more compared with the transgenic lines in their leaves and stems. These results demonstrate that the transgenic plantlets with high AtHKT1 expression are able to maintain K + steady state under high Na + stress conditions.  This may suggest a possible mechanism that the enhanced tolerance to salt stress in the transgenic tobacco plants is due to the constitutive over-expression of AtHKT1 gene helping keep K + at a high level to minimize or avoid Na + toxicity under salt stress. Also, the AtHKT1 over-expression confers multiple functions, such as the improved activities of antioxidant enzymes SOD, CAT and POD; raised contents of chlorophyll and soluble sugar and root activity; and decreased proline and MDA contents and electrolyte leakage destruction. These enhanced physiochemical traits helped minimize cell damage under high salt concentrations, and consequently, increasing plant productivity.  This research examined the hypothesis that the over-expression of AtHKT1 in tobacco maintains plantlet K + accumulation under Na + coercion, thus enhancing salt tolerance. Our results showed that under salinity stress, AtHKT1 gene was expressed in the xylem of stem, root and leaf vein in the transgenic tobacco, which uninstalled Na + from the shoot and leaves via transporting more K + , thus, decreased the toxicity of Na + and advanced many biophysicochemical activities that assist decrease the salt-induced hurt. The constitution of AtHKT1 in tobacco plants promoted a conversion from osmotic imbalance to osmotic balance. An over-expression of the AtHKT1 improved the ability to keep plantlet growth even under high salt stress conditions. This study also provide reference for future research of crop salt tolerant improvement, over-expression AtHKT1 gene in heterologous crop may be a useful method on increasing crop production.  Table. S1) were set up using a thermal cycler (UNO II, Biometra). Then the PCR products were cloned into the easy cloning vector pMD 18-T and reproduced in Escherichia coli DH5α for preservation and sequencing.
Vector construction. The β-glucuronidase (GUS) gene was exchanged by 720 bp GFP gene in the plasmid pBI121, driven by CaMV 35S promoter, named pBI121-GFP. And then the 1521 bp cDNA of AtHKT1 gene was inserted into the BamH I-Sma I site of the plasmid pBI121-GFP named pAtHKT1 + GFP (Fig. 2A). The recombinant plasmid pAtHKT1 + GFP was transferred into Agrobacterium tumefaciens LBA4404 by the method of freeze-thaw 41 . The plasmid was examined via PCR amplification and restriction enzyme digestion.
Tobacco genetic transformation and molecular detection. Using Agrobacterium-mediated transformation, the leaves of the tobacco cultivar 'CV87' were used as the receptor 42 . The regenerated shoots of 1.5 cm in height were cut and cultured in the selective medium of inducing root (MS + 250 mg L −1 Carbenicillin +100 mg L −1 Kanamycin). Genomic DNA was extracted from leaves of control and transgenic plants by Edwards described 43 . The existence of AtHKT1 gene was verified by normal PCR technique. The AtHKT1 gene primers and the amplification program were shown in Supplementary Table. S1. For PCR-Southern, the PCR products in 0.8% agarose gel were denatured, and then shifted to a nylon membrane (Roche). The membrane was hybridized in hybridization buffer 44 . In Southern blot analysis, forty micrograms genomic DNA hydrolysis products with the endonuclease Hind III were detached on 0.8% agarose gel. Hybridization methods were same as the method of PCR-Southern analysis described above. And then, cDNA were used in reverse transcription PCR assay (RT-PCR) analysis. Total RNAs was extracted and reverse transcription was performed using the PrimeScript TM RT reagent Kit with gDNA Eraser (TaKaRa, RR047A) following the manufacturer's instructions. The amplification methods, AtHKT1 gene and GFP gene primers used in the expression determine were shown in Supplementary Expression of AtHKT1 and GFP fusion genes in transgenic tobacco. The transgenic tobacco line T3 with high expression of AtHKT1 gene in leaves, stem and roots was selected to make paraffin sections following the procedures described elsewhere 45 . Paraffin sections were verified using Leica DM6000B (Germany) fluorescence microscope automatic imaging system, and the excitation filter was I3 with blue light excitation.

Physiochemical evaluations of AtHKT1 gene transgenic tobacco. Transgenic tobacco lines taking
the AtHKT1 gene were assessed for their tolerance to NaCl stress treatments in tube. The transgenic tobacco line T3 with high expression of AtHKT1 gene and the non-transgenic control were spread in vitro by sub-culturing of top single-node stem segments on MS medium added with different concentration NaCl. The top stem segments was cut from 20d old vitro plant and the length was 1.0 cm. The NaCl treatments were 0, 50, 100, 150, 200, 250, and 300 mmol L −1 NaCl concentrations. The test was run for three times. In each run, the treatments were set up in a entirely randomized arrangement with three replications. Every treatment in one replication had 3 bottles, one stem segment per bottle, with a total of 126 bottles (7 treatments × 2 lines × 3 replications × 3 bottles each). At Day 15 in the NaCl treatments, plant roots were separated from shoots and were weighed fresh weights separately. The samples dry weight were weighed after oven-dried to a consistent weight. The two of the NaCl concentrations (representing a high and low stress) were selectively used for further NaCl stress tests. Six transgenic tobacco lines carrying AtHKT1 gene (T1, T2, … T6) and the non-transgenic control were used in the test. Sub-culturing of 1.0 cm top stem segment cuttings from 20d old vitro plant were cultured in MS liquid medium. At Day 15, plants with 3 leaves, 4.0-4.5 cm plant length, 3.0-3.5 cm root length, 0.5-0.6 cm stem thickness and same leave color were chosen for NaCl stress, the medium was replaced by 50 mL of MS medium with the different levels of NaCl. The plants were tested under three NaCl treatments: 0, 100, and 200 mmol L −1 NaCl. The examining was run for three times. In each run, the treatments were set up in a totally randomized arrangement in three replications. Every treatment of one replication had 10 bottles, one stem segment per bottle, with 630 bottles totally (3 treatments × 7 lines × 3 replications × 10 bottles each). The enough number of plants approved all tests to be carried out. At Day 15 after NaCl treatments, in each treatment, 30.0 g fresh leaves were sampled for the determinations of total chlorophyll content, proline content, soluble sugar contents, MDA, SOD, CAT and POD using the method described by Li 46 . Leaves, stem, and roots were oven-dried at 105 °C 10 min, and then at 65 °C to a constant weight and grinded separately. 0.3 g dry sample powders were selected for K + and Na + measure using the method described by Ghars 47 . 1.5 g fresh roots were taken for the determination of root activity using the triphenyl tetrazolium chloride method 44 6.0 g fresh leaves were sampled for the determinations of electrolyte leakage and RWC by the method of Yu 48 .
Statistical analysis. The Tukey's HSD test of SPSS Software 19.0 (SPSS, USA) was used in analyzing all data.
Overall, there was a lack of treatment by run interactions for most of the variables evaluated, thus, the multiple runs data of the experiments were pooled in the statistical analysis. A probability of 5% was used to differentiate the significance between treatments or among the different categories of salt tolerance.