Introduction

Salinity is one of the major abiotic stresses negatively affecting plant growth and reducing agricultural productivity all over the world. Environmental salt stress can cause severe problems such as ion toxicity, water deficit and oxidative stress, resulting in cellular damage, growth reduction, and even plant death (Li et al. 2013). Higher plants have developed a set of biochemical and molecular mechanisms to deal with salt stress by selectively expressing specific stress-responding genes and accumulating compatible organic solutes such as soluble sugars, free proline and glycine betaine (GB) (Türkan and Demiral 2009).

AtNHX1, a vacuolar Na+/H+ antiporter in Arabidopsis thaliana, has been found to influence plant development and contribute to salt tolerance by mediating the transport of Na+ and K+ into the vacuoles (Sottosanto et al. 2007). Overexpression of AtNHX1 in many plants observably improves their salt tolerance. Compared to wild types (WT), transgenic tomato seedlings harboring AtNHX1 were able to grow, flower and even produce fruit under the salt stress triggered by 200 mM NaCl (Zhang and Blumwald 2001). In transgenic plants, the overexpression of AtNHX1 also significantly enhanced the salt tolerance of transformants (Asif et al. 2011; Chen et al. 2007). The study of transgenic Buckwheat showed that transformants overexpressing AtNHX1 were not only able to grow and flower normally, but also able to accumulate more rutin than WT in the presence of 200 mM NaCl (Chen et al. 2008). While stressed by 200 mM NaCl, transgenic tobacco lines expressing the AtNHX1 gene were able to grow and significantly accumulate sodium ions in their leaves (Zhou et al. 2011). The traits of enhanced salt tolerance could propagate for more than six generations in soybean lines that overexpressed AtNHX1 (Xing et al. 2010).

Betaine aldehyde dehydrogenase (BADH) is one of the key enzymes involved in the biosynthesis of GB, a compatible organic solute synthesized in response to salt, drought and temperature stresses in a large number of plant species (Wang et al. 2013). Responding to salt and drought stresses, the endogenous BADH level is upregulated several-fold in plant species which could synthesize betaine naturally (Fan et al. 2012). Transformed by the BADH genes, many betaine-deficient plants have been confirmed to accumulate different levels of GB in cells, and shown promoted tolerance to either salt or drought stress (Singh et al. 2010).

Because of its very high altitude, the extreme environment of the Tibetan Plateau in China is believed to be one of the harshest conditions for plant growth on this planet. Tibet is also considered to be the original habitat for cultivated barley and many wild Barleys (Wang et al. 2009). Tibetan Hulless barley (Hordeum vulgare var.nudum.hook.f.), the most important crop in the residents’ diets (Thomason et al. 2009), differs from the regular hulled barley with a bared caryopsis not covered by hulls in morphology. Unlike the cultivated barley, Tibetan Hulless barley has evolved a general resistance system to deal with all kinds of environmental stresses, and shown the strongest resistance of all plants belonging to genus Hordeum. However, the mechanism of this stress defense system is still unknown.

We have first reported the cloning of a pivotal-resistant gene HvBADH1 (GenBank No. EF492983), which encoded an isoenzyme of BADH in Tibetan Hulless barley (Zhao et al. 2008). To test the ability of HvBADH1 in plant stress resistance, we overexpressed HvBADH1 gene in non-halophyte chicory (Cichorium intybus L.) by the method of Agrobacterium tumefaciens-mediated transformation, and several transformed lines with enhanced salt stress resistance were obtained (data unpublished). To make this salt stress improvement of transformants sensible, we compared the salt tolerance of HvBADH1 or AtNHX1 overexpression chicory (Zhao et al. 2009) by assessing the different responses of these plant lines to gradient NaCl stresses. To achieve this purpose, we measured Na+ and K+, malondialdehyde (MDA), soluble sugar, free proline, chlorophyll contents and the relative electrical conductivity (REC) in T2 generation seedlings, as well as the relative growth rate of calli in different transgenic lines and WT under 0–200 mM NaCl.

Materials and methods

Plant materials and growth conditions

WT and T2 generation transgenic plants of chicory containing AtNHX1 or HvBADH1 were grown in a greenhouse of Shaanxi Provincial Key Laboratory of Biotechnology, Shaanxi Province, China. The seeds of these plant lines were harvested, soaked in tap water for 16 h, and surface sterilized with 70 % ethanol for 30 s and 0.1 % HgCl2 for 10 min. After rinsing for three times with sterile water, the seeds were placed on MS agar medium, and incubated at 25 °C under a 16/8 h L/D photoperiod (about 100 μmol m−2 s−1 provided by cool white fluorescent lamps) for about 14 days until the cotyledons fully expanded.

The construction of plant expression vectors harboring AtNHX1 or HvBADH1

Seeds of Arabidopsis thaliana and Hulless barley were surface sterilized using 0.1 % HgCl2, and then germinated on water soaked filter paper for 7 days. The seedlings were treated with 200 mM NaCl for 24 h at room temperature, and the leaves of Arabidopsis thaliana or Hulless barley were frozen in liquid nitrogen and preserved at −70 °C. Total RNA was isolated from Arabidopsis thaliana and Hulless barley seedlings using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. The first strand cDNA was synthesized using a RevertAid H Minus Synthesis kit K1631 (Fermentas). The cDNA was then used as template DNA for PCR amplification of the AtNHX1 (AF056190, 1,617 bp) and HvBADH1 (EF492983, 1,512 bp) ORFs. The sequences of the primers for AtNHX1 amplification were 5′-ggAATTCCATATgTTggATTCTCTAgTgTCgAAAC-3′ and 5′-CgggATCCTCAAgCCTTACTAAgATCAggAggg-3′. The primers for HvBADH1 were 5′-ggAATTCCATATggTCgCgCCggCCAAgATCC-3′ and 5′-CgggATCCCTAgTTAgCCggAgCCTTgTACCAC-3′. PCR was performed using Pyrobest DNA Polymerase (Takara, Dalian China). The PCR products corresponding to AtNHX1 and HvBADH1 were extracted from agarose gel, and then sequenced. All sequences were consistent with previously reported gene sequences of AtNHX1 and HvBADH1 (Zhao et al. 2008, 2009).

The amplified ORFs of AtNHX1 and HvBADH1 were then constructed into the multiple cloning sites of a commercial binary vector pRI101-AN using NdeI and BamHI restriction sites (Takara, Dalian China). In the final plasmid constructs, the target ORFs are under the control of the CaMV35S promoter (Fig. 1). These new plasmids, pRI101-AN-CaMV35S-AtNHX1 and pRI101-AN-CaMV35S-HvBADH1, were then separately introduced into Agrobacterium tumefaciens LBA4404 by electroporation using a Multiporator (Eppendorf), according to the manufacturer’s instructions. The subsequent Agrobacterium tumefaciens-mediated plant transformation and transformant identification were performed as described previously (Zhao et al. 2008).

Fig. 1
figure 1

Structure of the plant expression vectors, pRI101-AN-CaMV35S-AtNHX1 and pRI101-AN-CaMV35S-HvBADH1

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Determination of Na+ and K+ contents in transgenic chicory

The transgenic HvBADH1 (H1, H2) and AtNHX1 (A1, A2) lines and WT chicory seedlings were cultured on MS medium for 4 weeks after germination and then transferred to MS liquid medium containing different concentrations of NaCl (0, 50, 100, 150 or 200 mM) for 24 h. The seedlings were rinsed with distilled water to remove NaCl from the surface, and the leaves of these seedlings were harvested and dried at 45 °C overnight. Na+ and K+ contents were measured by an atomic absorption spectrophotometer (AA-6300C from Shimadzu).

Measurement of MDA, free proline, soluble sugar and chlorophyll contents in transgenic chicory leaves

Transgenic lines and WT chicory seedlings were cultured on filter paper wetted with MS liquid medium for 28 days. Then all the plants were irrigated with MS liquid medium containing different concentrations of NaCl (0, 50, 100, 150 or 200 mM) for 24 h. 0.1 g fresh leaves from seedlings of transgenic lines and WT were used as materials for the following tests of each physiological indicator. The MDA levels were measured by a thiobarbituric acid (TBA) reaction method (Garcia et al. 2005), free proline levels were determined as described by Bates et al. (1973), and soluble sugar was measured by anthrone colorimetry as described by Yemm and Willis (1954). The same leaf samples were homogenized in a mortar and pestle containing 80 % acetone and the amount of chlorophyll was measured as the value of OD652 as described by Myers et al. (2013).

Determination of relative electrical conductivity

To assess membrane permeability, REC was determined according to Korkmaz et al. (2010). After being treated with NaCl as described in 2.3, leaf discs (1 cm in diameter) from transgenic lines and WT lines were taken from the middle portion of the fully developed youngest leaf. Then the discs were placed in individual test tubes containing 30 ml of distilled water. After incubating the samples at room temperature on a shaker (150 rpm) for 3 h, the electrical conductivity of the bathing solution (EC1) was determined by an electrical conductivity detector (model DDS-303A, Kangyi, Shanghai, China). The same samples were then placed in boiling water for 30 min and a second reading (EC2) was determined after cooling the solution to room temperature. The electrolyte leakage was calculated as REC = EC1/EC2 × 100 %.

Salt tolerance of transgenic calli

The hypocotyls of different transgenic lines and WT seedlings about 28 days old were cut into 0.5 cm segments and inoculated on MS medium supplemented with 6-BA 1.5 mg L−1, NAA 0.2 mg L−1, and ascorbic acid 100 mg L−1 for calli induction under 100 μmol m−2 s−1 illumination, 16/8 h L/D photoperiod and 25 ± 2 °C. The fresh calli were segregated and subcultured on the same medium for 6 weeks. After weighing, the calli were treated with gradient NaCl for 28 days. Subsequently, the calli’s weight was remeasured, and the relative growth rates of calli were calculated as: Relative growth rate = final weight/primary weight. 25 calli were treated in each replicate and 3 replicates were performed for each experiment.

Determination of GB

To prepare sample solutions for GB measurement, 1.0 g fresh leaves were ground by mortar and pestle and extracted in 20 mL deionized water for 24 h at 37 °C with gentle shaking. These extracts were then centrifuged at 4,500×g for 15 min, and the supernatants were filtrated with waterman filter paper and 0.45 μm filter membrane. The pH of the sample solutions was adjusted to 1.0 by adding concentrated hydrochloric acid and incubated at 4 °C for 30 min. After centrifugation at 4,500×g for 15 min, 3 mL supernatants from sample solutions were mixed with 5 mL concentrated Reinecke’s salt (NH4[Cr(NH3)2(CNS)4]·H2O; China National Pharmaceutical Group Corporation), and reacted at 4 °C for 3 h. Subsequently, the solutions were centrifuged again and the supernatant was discarded. The GB crystals were dissolved in 5 mL 70 % acetone and the optical density of these solutions was measured at 365 nm.

The preparation of a standard curve was performed using GB samples from Sigma with the unit as mg/g FW (Martinez 1983).

Statistical analysis

To analyze the variance of the physiological indicators between transgenic plant strains, we used an ANOVA method of Duncan’s multiple comparison procedure. In addition, paired sample t tests were used to evaluate the statistical significance of the difference between each transgenic line and the WT at each NaCl level. The significant levels were defined as * when P ≤ 0.05, and ** while P ≤ 0.01. Six plants were used for each treatment. Each experiment was repeated three times. All the data used in the statistical analysis were an average of three replicates and analyzed by the SPSS statistical software package v19.0.

Results

Determination of Na+ and K+ contents

The accumulation of Na+ and K+ in WT and T2 generation transgenic seedlings treated with NaCl was measured. The results suggested that together with the increasing environmental NaCl, the Na+ contents increased in leaves of all the transgenic and WT plants. Though transgenic plants could accumulate much more Na+ than the WT at every stress levels, no observable differences were found between CaMV35S:HvBADH1 and CaMV35S:AtNHX1 lines (Fig. 2a).

Fig. 2
figure 2

Na+ and K+ contents in leaves of transgenic and WT chicory. WT wild type; H1, H2 transgenic HvBADH1 plant No. 1 and No. 2; A1, A2: transgenic AtNHX1 plant No. 1 and No. 2. These symbols present the same plant lines in all the figures and table in this paper

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Corresponding to increased NaCl stresses from 0 to 200 mM, the K+ contents in the transgenic and WT plants rose rapidly. Similar to the situation of Na+ contents, significant differences of K+ content were found between transgenic and WT plants at each level of salt stress, but there were no noticeable differences between transgenic lines (Fig. 2b). In addition, the K+/Na+ ratios in transgenic plants were also higher than those of WT at each stress level, but the differences between transformed HvBADH1 and AtNHX1 lines were not significant (Table 1).

Table 1 Effects of NaCl stress on the K+/Na+ in the leaves of chicory
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Measurement of MDA contents

MDA is the product of lipid peroxidation and relates closely to the oxidative degradation of polyunsaturated fatty acids of cell membrane. Changes in cellular MDA levels during abiotic stress are coordinated with the process of membrane damage and reflect the antioxidative capacity of plant cells (Tian et al. 2011). In our study, the contents of MDA increased dramatically in all the tested plants corresponding to the increase of environmental NaCl levels. Under each stress level, the MDA contents of transgenic lines were lower than the WT, but no noticeable differences were observed between CaMV35S:HvBADH1 and CaMV35S:AtNHX1 lines when stressed with 50–150 mM NaCl. However, the CaMV35S:AtNHX1 lines showed a significant higher accumulation of MDA than HvBADH1 lines until the NaCl stress reached 200 mM (Fig. 3a).

Fig. 3
figure 3

MDA (a), free proline (b), soluble sugar (c) and chlorophyll (d) in transgenic and WT chicory

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Determination of free proline contents

As a physiological response to hostile environment, plant cells usually produce abundant free proline to raise their cellular osmotic potential. Triggered by NaCl stress, the contents of free proline in leaves of transgenic and WT plants increased rapidly, and the proline levels of both transgenic lines were higher than the WT at each stress level. In addition, CaMV35S:HvBADH1 lines displayed a significantly higher ability to accumulate more free proline than the CaMV35S:AtNHX1 lines at all the tested stress levels (Fig. 3b).

Determination of soluble sugar contents

The young leaves of transgenic and WT plants grown under different NaCl stresses were used to measure soluble sugar contents. When no additional NaCl was supplied, all the plants contained nearly the same soluble sugar contents. Similar to the MDA and proline measurements, soluble sugar contents also increased with NaCl concentration in both transgenic lines. The soluble sugar content of CaMV35S:HvBADH1 leaves showed 1.67-fold higher values than that of CaMV35S:AtNHX1 when exposed to 150 mM NaCl and 1.47-fold to 200 mM NaCl (Fig. 3c).

Measurement of chlorophyll contents

To estimate the damage on the photosynthetic system caused by the salinity stress, the amounts of chlorophyll content were measured in transgenic lines and WT treated with different concentrations of NaCl. With increased salt, the chlorophyll contents decreased in all tested plants. Not surprisingly, more dramatic decline in chlorophyll content was found in WT leaves compared to both transgenic lines under each stress level. Contents of chlorophyll in CaMV35S:HvBADH1 lines were slightly higher than that of AtNHX1 transformants when environmental NaCl level was lower than 150 mM, but significant differences were observed between these two lines at NaCl concentrations of 200 mM (Fig. 3d).

Determination of relative electrical conductivity

For the purpose of evaluating the protective function of HvBADH1 and AtNHX1 on the cell membrane under osmotic stress, REC was measured in two transgenic lines and WT. Without salt stress, the values of REC in all tested plants were similar. Under conditions of 50–200 mM NaCl, however, the REC data from each plant strain showed dramatic differences. The values of REC in all plant strains increased with rising NaCl concentration, and the level of REC in WT was always significantly higher than that of transgenic lines. Again no significant differences in REC were detected between the CaMV35S:AtNHX1 and CaMV35S:HvBADH1 lines when stressed by the 50–150 mM NaCl. However, the REC of CaMV35S:HvBADH1 plants was much higher than the CaMV35S:AtNHX1 plants under 200 mM of NaCl stress (Fig. 4a).

Fig. 4
figure 4

REC (a) and relative growth rate (b) in transgenic and WT chicory

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Salt tolerance of transgenic calli

The relative growth rates of transgenic and WT calli were measured to evaluate the salt tolerance of different transgenic lines at the cellular level. In the control group (without NaCl), the relative growth rates of transgenic calli were similar to WT. With increased NaCl concentration, relative growth rates declined rapidly in all the tested calli lines, though that of the transgenic lines was significantly higher than WT at each stressed level. By comparing the data from different transgenic calli grown under 100–200 mM NaCl, we confirmed that overexpression of AtNHX1 could promote the survival rates of calli to a greater extent than the HvBADH1 gene. At 200 mM of salt stress, the WT calli turned brown and only kept 2.97 % growth rates whereas most of the transgenic calli survived and even maintained growth rates of 10.7–22.3 % (Fig. 4b).

Determination of GB

The accumulation of GB in WT and T2 generation transgenic seedlings was measured. Compared to the values of WT and CaMV35S:AtNHX1 lines under regular growth conditions, a 6.77-fold higher GB content was observed in HvBADH1 lines, but no noticeable accumulation of GB was found in WT or CaMV35S:AtNHX1 lines (Fig. 5). Growing under NaCl-stressed condition, HvBADH1 lines, as well as the WT and AtNHX1 lines, showed the same GB content level with their control plantlets which grew under non-stressed condition.

Fig. 5
figure 5

GB contents in transgenic and WT chicory

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Discussion

Many efforts have been made to promote the tolerance of cultivars by transgenic methods, and quite a lot of genes involved in stress responses have been cloned for this purpose (Yu et al. 2012). Na+/H+ anti-transporter genes function in a specific salt stress-resistant pathway by transporting excessive cytosolic Na+ into vacuoles (Apse et al. 1999), while BADH genes are believed to play a key role in a general osmotic stress-resistant pathway. Genetic transformations of both anti-transporter genes and BADH genes have already been used in many attempts to promote the salt stress resistance of plants (Wang et al. 2013). High altitude plants have evolved over millions of years to develop a complex defense system to adapt to environmental stresses, such as lower O2, high UV radiation and extreme temperature, and understanding the mechanisms of the specific stress-resistance system in these plants may provide deep insight into how to promote the salt stress resistance of other major crops. In this work, we evaluated the salt stress tolerance in transgenic plants of HvBADH1 from highland Hulless barley (Hordeum vulgare var.nudum.hook.f.) and AtNHX1 from Arabidopsis thaliana.

A resting state with intracellular higher [K+] and lower [Na+] is important to maintain the activities of many cytosolic enzymes, as well as a regular membrane potential. When salt stress occurs, the efficiency of Na+/H+ anti-transporters is affected by excessive Na+ in cytosol and expression of genes encoding Na+ transporters, such as AtNHX1, is upregulated to increase the amount of these anti-transporters on the membrane of vacuoles (Zhu 2003). Little difference was found between CaMV35S:AtNHX1 and CaMV35S:HvBADH1 lines in this study for Na+, K+ contents or K+/Na+ ratio, although the data from both transgenic lines were much higher than WT under stress conditions. By overexpressing these two genes, the transgenic cells could maintain cytosolic enzyme activity and plasma membrane integrity against the ion toxic effect and prevent the inhibition of K+ uptake of cells caused by salt stress. Compared to the effect of Na+ compartmentalization caused by AtNHX1 overexpression, the osmotic potential promoting process caused by overaccumulation of GB made a similar contribution to increase the salt tolerance by modulating both the levels of K+ and Na+ and keeping the K+/Na+ balance mainly in the leaves.

As a by-product of the membrane damage process triggered by reactive oxygen species (ROS), MDA is released to the cytosol soon after stress (Wang et al. 2011). In the present study, the MDA levels suggested that the overexpression of HvBADH1 gene and subsequent overaccumulation of GB in the cytosol raised the osmotic potential and decreased the generation of ROS in cytosol. This effect allowed the HvBADH1 lines to show a superior ability than AtNHX1 strains to minimize membrane damage under high salt stress. However, because of its ability to transport redundant Na+ into the vacuoles, the overexpressed AtNHX1 exhibited a similar antioxidative capacity to HvBADH1 at salt concentrations less than 150 mM NaCl. Thus, both transgenic lines showed protective activities against oxidative damage to the cell membrane, though HvBADH1 had higher protection under severe stress conditions.

To protect themselves from various abiotic stresses, plants responsively synthesize and accumulate some organic osmolytes in the cells, such as soluble sugar, free proline and GB (Rodríguez-Calcerrada et al. 2011), which helps plants maintain cell turgor during salt and osmotic stresses (Zheng et al. 2009). Overaccumulated soluble sugar and free proline in protoplast can protect macromolecules from being hurt by various abiotic stresses such as dehydration and salinity; proline also takes part in the endogenous radical scavenging system. In our research, increased soluble sugar and free proline triggered by salt stress modulated the maintenance of osmotic equilibrium in protoplasts of the transgenic lines. The values of these two osmolytes showed a jump in the HvBADH1 lines, but not in AtNHX1 lines at 150–200 mM NaCl stresses, suggesting that overexpressing HvBADH1 may attribute to the salt tolerance of transformants by maintaining the osmotic potential in cells to severe NaCl stresses.

The contents of chlorophyll reflect the physiological status of plants, which are also used to estimate the tolerance of plants to salinity (Gitelson et al. 2003). Lower chlorophyll contents in leaves directly limit both the photosynthetic activity and efficiency of plants encountered with salt stress. The redundant Na+ and Cl− ions permeating into cytoplasm reduce the chlorophyll levels by interfering the activity of chlorophyll-synthesizing enzymes (Garriga et al. 2014). In the present study, the contents of chlorophyll in HvBADH1 lines were slightly higher than that of AtNHX1 lines, which suggested that transformation of HvBADH1 was better to protect the plant photosynthesis system from salt damage.

REC is another important physiological status indicator that directly reflects the extent of cell membrane damage under osmotic stress. At levels of NaCl stress lower than 150 mM, no noticeable differences were detected between the transgenic plant strains. The significant lower level of REC in AtNHX1 lines at 200 mM NaCl indicated that the overexpression of AtNHX1 provided the cell membrane with a better protection than HvBADH1 lines.

Plant tissue tolerance is thought to be a major component to determination of overall plant response to salinity (Getnet Dino Adem et al. 2014). The measurement of relative growth rate of calli showed that the AtNHX1 lines could endure severe salinity stress better than the HvBADH1 lines. But under slight salt stress conditions, the two lines responded more similarly. These phenotypes may be explained by a hypothesis that the toxic effect of overaccumulated ions, such as Na+ and Cl− might be major obstacles to physiological metabolism and cell growth under severe salt stress conditions, while osmotic stress inhibits plant growth under slight salt stress. The precise segmentation point between severe and slight stress conditions might vary for different plant species or different physiological indicators, but in this study, this segmentation point was 100 mM NaCl.

GB is a common osmolyte that accumulates in many plant species, and the levels of GB in the plant tissues are correlated with salt tolerance. Genetic evidence that GB improves salinity tolerance has been obtained in barley and maize (Zhang et al. 2009). In the present study, CaMV35S:HvBADH1 lines exhibited an over accumulation of GB in leaves under stressed conditions, which may correspond to enhanced salt stress tolerance. Our results are consistent with the theory that osmoprotection caused by osmolyte accumulation may play a pivotal role in natural stress environments where stress does not affect plant survival (Neha Gupta et al. 2014).

A recent report found that NHX1 and NHX2 genes, the homologues of AtNHX1 in barley, may not necessarily confer the same protein(s) function as AtNHX1 in Arabidopsis, though they have similar nucleotide sequence (Getnet Dino Adem et al. 2014). Therefore, we chose AtNHX1 from Arabidopsis as a reference to evaluate the contribution of HvBADH1 from Hulless barley to the ionic and osmotic stress parameters determining salinity tolerance.

In conclusion, we evaluated how overexpressing AtNHX1 and HvBADH1 affected the salt tolerance of transgenic plants by measuring eight different physiological indicators in CaMV35S:AtNHX1 and CaMV35S:HvBADH1 chicory lines. The determination of calli growth rate and REC revealed that the overexpression of AtNHX1 gene promoted the tolerance of transgenic calli, and increased the ability of transgenic plants to maintain homeostasis against membrane injury under severe salinity condition. In contrast, the HvBADH1 overexpressing plants exhibited better capacity to maintain the osmotic potential of cytoplasm by synthesizing and accumulating more soluble sugar and free proline. The MDA and chlorophyll levels showed that the overexpression of HvBADH1 also enhanced the ability of transgenic plants to protect the integrity of membrane system and photosynthesis system when threatened by NaCl stress. In summary, our results suggest that AtNHX1 is more suitable for severe salinity, while HvBADH1 seems superior for dealing with general abiotic stresses. However, further field tests will be required to determine the long-term productivities of HvBADH1 and AtNHX1 lines. These results support the idea that a successful progress in the breeding of crops with enhanced NaCl stress tolerance can only be achieved by targeting several complementary traits together (Getnet Dino Adem et al. 2014). Because these two genes confer different responses to stress, it is a logical next step to assess the salt-resistant phenotype of plants that expresses both HvBADH1 and AtNHX1 genes.

Author contribution statement

Yuwei Zhao designed the research. Fang Zhang, Xiaolong Li and Pan Lai performed experiments. Fang Zhang, Xiaolong Li and Pengfei Li analyzed data. Fang Zhang wrote most of the article.