The full length CDS of AtNHX1 (GenBank accession number: EF596738; Locus: AT5G27150), as EcoRI and BamHI fragment, was cloned downstream to stress-inducible rd29A promoter in an intermediate vector pRT101. Following digestion with HindIII, the rd29A:AtNHX1::35STer fragment was inserted into binary plant vector pCAMBIA3300 that contained bar as plant selectable marker. The construct was mobilized into Agrobacterium tumefaciens strain EHA105 using Triparental mating prior to use in the plant transformation experiments.

Mungbean plants (cv. K-851) were transformed through Agrobacterium-mediated transformation of 4-d old cotyledonary node explants using A. tumefaciens vir helper strain EHA105, harboring pCAMBIA3300-rd29A::AtNHX1. Prior to inoculation in bacterial suspension for 30 min at 22°C with gentle shaking (90 rpm), the explants were pricked 3–4 times with a fine hypodermic needle (24G) at the regeneration site. The explants were then blotted dry on sterile filter paper and co-cultivated on filter paper disc moistened with liquid cocultivation medium LCM [MSB5 medium (Murashige and Skoog, 1962) containing 1 μM BAP, pH adjusted to 5.5] supplemented with 100 μM acetosyringone for 3 days at 22°C in dark condition. After cocultivation, the regenerating explants were selected on shoot induction and selection medium [MSB₅ medium containing 5.0 μM benzylaminopurine (BAP), 1.5 mg/l phosphinothricin (PPT), and 500 mg/l cefotaxime], followed by subculturing of explants after 2 weeks on shoot elongation medium (MSB₅ medium containing 1.0 μM BAP and 500 mg/l cefotaxime) devoid of PPT. After 4 weeks, the elongated shoots were excised and rooted in B₅ medium (Gamborg et al., 1968) supplemented with 5 μM indolebutyric acid (IBA) and 500 mg/l cefotaxime. The putative transformed plants were established in soil: compost (1:1) in pots. The pots were covered with polythene bags in a transgenic greenhouse containment facility at 28 ± 2°C under 16/8 h photoperiod (fluence density of 50 μmol m⁻² s⁻¹) and 65–70% relative humidity. After 15 days, the plastic covers were gradually removed and the plants were transferred to soil in pots which were maintained for the production of the homozygous T₁, T₂, and T₃ generations. One month-old plants were used for PCR screening and for gene expression evaluations. Positive transformants were used further for stress treatments.

The genomic DNA was isolated from young leaves by using DNASure Plant Mini Kit (Nucleopore, Genetix, India). Presence of transgenes in transformed plants (T₀, T₁, and T₂) were confirmed by PCR using bar and AtNHX1 specific primers (Tables S1, S2). Southern hybridization was carried out to confirm the transgenes integration and determine their copy number by digesting 50 μg of genomic DNA each from the WT and randomly selected independent T₂ transgenic lines (AT₂.1, BT₂.5, CT₂.3, DT₂.2, and ET₂.4) with EcoRI. After resolving the digested DNA on 0.8 % agarose gel, the gel was blotted onto positively charged Zeta-Probe membrane (Bio-Rad, Hercules, CA, USA). The blot was hybridized with the DIG-labeled 0.3 kb PCR product, corresponding to the coding region of bar. Southern hybridization was performed according to the manufacturer's instructions of the DIG Labeling and Detection kit (Roche Diagnostics, Mannheim, Germany). Four randomly selected T₀ PCR-positive events (AT₂.1, BT₂.5, CT₂.3, DT₂.2, and ET₂.4) were advanced up to T₂ generations, and segregation studies was carried out to determine the inheritance of AtNHX1 and bar by PCR as described earlier.

The PPT dose that impose bleaching and necrosis of leaves of 2-month old T₂ plants was determined by detached leaf disc assay by floating leaf discs of 1 cm diameter with aqueous solution of PPT (1, 2, 3, and 4 mg/l) for a week (data not shown). T₂ plants were also sprayed on both sides of leaves with aqueous solution of PPT (2 mg/l). The vegetative injury level of both transformed and control WT was recorded after 2 weeks of PPT spray assay. The leaf discs were also tested by chlorophenol red (CPR) assay to visually verify bar activity in 22 transgenic T₂ plants representing six PCR positive T₀ parents, as per the procedure described by Sonia et al. (2007).

T₂ lines of four independent transgenic events (AT₂.1, CT₂.3, DT₂.2, and ET₂.4) with abundant transcripts of AtNHX1 and no apparent phenotypic changes, were chosen as representatives to evaluate salt tolerance by subjecting both the seedling and mature plant stage to salinity stress tests, and leaf senescence assay. Five-day old seedlings were treated with 150 mM NaCl for 2-d followed by recovery on Hogland's medium for 3-d. Similarly, 1-month old plants in pots were subjected to 150 mM NaCl for 1 week, by incremental increase of NaCl stress by 50 mM every 2 d. Subsequently, plants were treated with 200 mM NaCl (lethal dose) for 30 days based on prior determination of salt concentrations which imposed salt induced symptoms. To determine the relative increase in growth difference of the transgenics in salt relative to control conditions, we measured growth parameters (root and shoot length, fresh and dry weight of root and shoot) prior to, during and after the withdrawal of salt stress, and expressed as a percentage reduction in comparison with the respective unstressed control.

Mature leaves (from the top) from WT and T₂ transgenic lines (45-d old) were harvested for leaf senescence assay (Sahoo et al., 2016). Leaves cut into leaf discs were floated in NaCl solution (100, 150, and 200 mM) for 4 days. Total chlorophyll content was determined by the procedure following the description in Arnon (1949). Briefly, leaf tissue (100 mg) were homogenized thoroughly in 80% acetone at 4°C. The homogenate was centrifuged at 3,000 rpm for 10 min in the dark. The absorbance of the supernatant was measured at 648 and 664 nm, and chlorophyll content was calculated per gram of fresh weight. The experiment was repeated three times. The chlorophyll amount was calculated using the formula:

where, V is the volume of extract in ml and W is the weight of leaf tissue in g, Chlorophyll content was expressed in μg/g fresh weight.

Leaves and roots of transgenic (AT₂.1, CT₂.3, DT₂.2, and ET₂.4) and WT plants were sampled, prior to, during and after the withdrawal of the salt stress for determination of leaf senescence, chlorophyll content, relative water content (RWC), electrolytic leakage, malonaldehyde (MDA), ascorbate, proline, and trehalose contents, chlorophyll fluorescence, stomatal conductivity, and transpiration rate.

RWC was determined according to Lv et al. (2009). Briefly, the leaf discs were floated, after recording the fresh weight, on deionized water for 5 h at 28°C, after which the turgid weight (TW) of the hydrated leaf discs was recorded. The leaf discs were then dried in a hot air oven at 80°C for 72 h and weighed until a consistent dry weight (DW) was obtained. RWC was calculated using the formula,

Electrolyte leakage was estimated (Dionisio-Sese and Tobita, 1998) by measuring the electrical conductivity (ECa) of 25 leaf disks dipped in deionized water. Subsequently, the test tubes containing the leaf discs were incubated in water bath for 25 min. at 50–60°C, and the electrical conductivity (ECb) of the samples was determined. Finally, the samples were boiled at 100°C for 10 min, and then the electrical conductivity (ECc) was measured. The electrolytic leakage was calculated using the following formula:

Lipid peroxidation was estimated by measuring the malondialdehyde equivalents following the method (Heath and Packer, 1968). About 200 mg of fresh leaf tissue was homogenized with 2–3 ml of 0.25% TBA containing 10% TCA (trichloroacetic acid). The homogenate mixture was boiled in water bath at 95°C for 30 min, cooled to room temperature and centrifuged at 10,000 g for 10 min. Absorbance was measured at 532 nm and values corresponding to non-specific absorption at 600 nm were subtracted. The lipid peroxidation rate equivalents were calculated according to the molar extinction coefficient of MDA (155 mM⁻¹ cm⁻¹). The MDA content was calculated using the equation: MDA (μmolg⁻¹FW) = (6.45 × O.D.₅₃₂ – (0.56 × O.D₄₅₀).

Proline content was determined by extracting the samples in 3% sulfosalicylic acid and absorbance measured at 520 nm as described by Bates et al. (1973) and ascorbate content was measured as described by Panda et al. (2003). In addition, leaves and roots were harvested for Na⁺ and K⁺ detection respectively, using the method described by Wang et al. (2009).

Trehalose sugar content was estimated by following the method (Goddijn et al., 1997). Extracts from 300 mg of homogenized fresh leaf tissue were centrifuged at 3,220 × g for 10 min. and the supernatant were passed through ion-exchange columns to remove the charged compounds. After lyophilization, samples were dissolved in HPLC grade water and subjected to high performance ion exchange chromatography. Carbohydrates were eluted at a flow rate of 1 ml/min. at 1,400 psi with 100 mM NaOH for 30 min. Soluble sugar present in the extract were quantified by using authentic standard sugars (Sigma). The identification of trehalose in the extract was finally confirmed by incubation of samples with porcine kidney-derived trehalose enzyme (Sigma, USA).

The chlorophyll fluorescence (Fv/Fm) were measured using MINI-PAM-II, Photosynthesis Yield analyzer (Heinz Walz GmbH, Germany), and stomatal conductance and transpiration rate were measured by Leaf Porometer (DECAGON Devices, USA). Measurements were made on each plant in the five salinity treatments. The photosynthetic and chlorophyll fluorescence measurements were taken between 10:00 and 12:00 h when the ambient light intensity was 1,000 −1,200 μmol m⁻² s⁻¹.

The activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) was evaluated as described earlier (Luck, 1974; Sahoo et al., 2016). Analysis of ROS accumulation in leaves of T₂ transgenics (AT₂.1, CT₂.3, DT₂.2, ET₂.4, and CT₂.9) and WT under normal and stress conditions was carried out by measuring hydrogen peroxide (H₂O₂) and superoxide radical (O2-) as previously described by Ramel et al. (2009), and Rao and Davis (1999), respectively.

Methyl vialogen (MV) induced oxidative stress analysis was performed in WT and transgenic lines (AT₂.1, CT₂.3, DT₂.2, ET₂.4, and CT₂.9) in T₂ generation by leaf disc assay (Lee et al., 2007) with slight modifications. In brief, leaf discs (1 cm in diameter) from the third or fourth leaf from the top, of 8 week-old plants were floated on an aqueous solution of MV (0, 50, 100 μM) to generate superoxide radicals. They were incubated in dark at 25°C for 12 h to allow for MV diffusion. After pre-incubation, the leaf discs were illuminated (100 μmolm⁻²s⁻¹) until use. The effect of MV on leaf discs was analyzed by monitoring their phenotypic changes and measuring the content of chlorophyll, proline, and MDA as described previously.

To assess the effect of salt stress inducible expression of AtNHX1 in transgenic mungbean plants, quantitative real-time PCR (qRT-PCR) assay was performed on four Southern positive plants (AT₂.1, CT₂.3, DT₂.2, and ET₂.4 in T₂ generation) and the WT subjected to 200 mM salt stress. Leaves collected from the plants, after 2-d at seedling stage and 30-d at maturity stage of stress treatment were used for qRT-PCR analysis using the method as described in our previous studies (Sahoo et al., 2016). Three biological replicates and three technological replications were conducted. The primers used are listed in Tables S1, S2. Expression of mungbean β-tubulin gene served as internal control.

The growth parameters at both seedling and mature stages, and yield components of transgenic lines (AT₂.1, CT₂.3, DT₂.2, ET₂.4, and CT₂.9) in T₂ generation were evaluated under salt stress (200 mM), and compared to that of WT and control conditions. The growth parameters (plant height and number of branches) were checked on 0- and 45-d of salt stress. At harvest maturity, number of pods, number of seeds, 100-seed weight, and 10-seed length on a per plant basis were determined. Sodium and potassium ion concentrations in shoot and root of mature plants were determined at 0 and 48 h of salt stress. Transgenic as well as the WT and other control plants were washed in flowing tap water for 30 s and oven-dried for the measurement of ion content.

All assays were biologically replicated at least three times. The data were evaluated with Statistical Package for the Social Sciences (SPSS 16.0) and Excel. The significant differences between mean values were determined by using Bonferroni analysis at p = 0.05.

Transgenic mungbean plants harboring both bar and AtNHX1 were generated through Agrobacterium-mediated transformation (Figure 1A, Figures S1A–G, and Table S3). The expected size amplified products of 290 bp (Figure 1B) and 1.6 kb (Figure 1C) confirmed the presence of bar and AtNHX1, respectively in PPT resistant T₀ transgenic mungbean lines. The segregation of bar and AtNHX1 in four T₀ transgenic lines followed up to T₂ generations (AT₂.1, CT₂.3, DT₂.2, and ET₂.4) were confirmed by the detection of expected size amplified products. Three transgenic lines (CT₂.3, DT₂.2, ET₂.4) showed a segregation ratio of 3:1, well supported by the fact that a single functional T-DNA locus detected in these lines in Southern hybridization with bar probe (data not shown). The observed segregation ratio of AtNHX1 and bar revealed their inheritance in a Mendelian fashion (data not shown). A differential integration pattern of the transgene was observed in four out of five lines in T₂ generation (AT₂.1, BT₂.5, CT₂.3, DT₂.2, and ET₂.4). Three lines exhibited single copy (CT₂.3, DT₂.2, and ET₂.4) insertion, AT₂.1 had two copies while BT₂.5 had no signal of T-DNA for the probed region in the genome (Figure 1D). The occurrence of hybridization signals greater than 1.2 kb revealed stable integration of the transgenes. No hybridization signal was observed in the WT.

Wild type mungbean plants were very sensitive to PPT painting, as the leaves exhibited complete necrosis at 2 mg L⁻¹ (within a week in detached leaf disc assay and 2 weeks in leaf paint assay) while the PCR positive plants in T₂ generation showed complete tolerance as the treated leaves stayed green without visible damage symptoms (Figures 2A–C). These results indicate that PCR based detection in combination with herbicide paint reliably eliminates the escapes. The color change of chlorophenol red assay solution is pH dependent as described by Treiu and Harrison (1996). The solution color at pH 6.0 and higher is red whereas the color is yellow at lower pH. The leaves tissue resistant to PPT cause acidification of the medium by metabolizing PPT leading to yellowing of the solution. The color of the medium in the chlorophenol red assay changed from red to yellow implying the bar gene activity in transformed leaf discs. Appearance of dark yellow color in medium in some wells indicated different expression levels of bar in those T₂ lines (Figure 2D).

First, we examined the salt tolerance in leaf discs of transgenic plants grown in pots, as the degree of bleaching in leaf tissues was good indication of damage caused by salt stress in leaf senescence assay. Under control condition, no significant difference was observed between WT and transgenic plants. However, the leaf discs of WT plants started to turn yellow and exhibited excessive bleaching within a week under salt stress. In contrast, the leaves from transgenic lines continuously stayed green even after 15 days of 200 mM NaCl stress (Figure 3A) indicating higher tolerance to salt stress. Furthermore, leaf discs from transgenic plants showed significantly higher chlorophyll content, 1.68-, 1.76-, and 1.92-fold higher than WT under 100, 150, and 200 mM stress, respectively (Figure 3B).

Subsequently, we examined the effects of salt stress on the growth of T₂ transgenic lines at seedling (T₂.3 and DT₂.2) (Figures 3C–F) and whole-plant stage (AT₂.1, T₂.3, DT₂.2, ET₂.4, and T₂.9; Figure 4A) by subjecting them to 2 and 30-d of salt stress respectively. Under salt stress, transgenic lines exhibited no phenotypic difference from that in controlled condition (CT₂.9) (Figures 3C–F, 4A). After irrigating with salt solutions, 2-d at seeding stage (Figure 3D) and for 30-d at maturity stage, with gradual incremental increase of NaCl dose to 200 mM, the WT plants developed severe growth inhibition, chlorosis, and wilting, and their growth ceased (Figure 4A). Furthermore, the WT plants showed significant decrease in shoot and root growth (Figures 3F, 4B,C). The WT plants showed no sign of recovery after 3-d at seedling stage (Figure 3E) and 15-d of withdrawal of salt stress at maturity stage (Figure 4A). However, transgenic plants stayed green and continued to grow normal at 200 mM NaCl and the symptoms of high salt stress were effectively alleviated, indicating that transgenic plants were more tolerant to high salt stress. The transgenic lines showed apparent advantages of growth under salt stress relative to control conditions as observed from their growth difference. The relative increase in growth indexes (shoot and root length) of transgenic lines under high salt stress (200 mM NaCl) were significantly higher (P < 0.05 and P < 0.01) than those under normal conditions (Figures 4B,C). These results implied that stress inducible overexpression of AtNHX1 triggered a significant increase in growth and performance of transgenic plants under salt stress, and the resulted salt stress tolerant phenotype was associated with stress inducible expression of AtNHX1.

To investigate if the expression of AtNHX1 altered the accumulation of cations (Na⁺ and K⁺) under salt stress conditions, the concentrations of cations were determined in the leaves and roots of the transgenic (AT₂.1, CT₂.3, DT₂.2, and ET₂.4) in T₂ generation and WT plants. Under normal physiological condition, there was no significant difference in accumulation of Na⁺ and K⁺ in transgenic and WT plants (Figures 5A–D). On the other hand, salt stress (200 mM) resulted in enhanced accumulation of Na⁺ in transgenic and WT plants, in both leaves and roots. However, in transgenic plants (CT₂.3 and DT₂.2), the Na⁺ concentrations were significantly higher in roots and on the contrary lower in leaves, in comparison to WT plants (Figures 5A,B). The Na⁺ content in roots of T₂ transgenic lines showed 2.95 (CT₂.3)- and 2.76 (DT₂.2)-fold higher accumulation than WT roots, under 200 mM salt stress (Figures 5A,B). These results clearly demonstrated that stress inducible expression of the Arabidopsis vacuolar antiporter elevated the accumulation of Na⁺ in roots, presumably in vacuoles, and the enhanced activity of vacuolar antiporters in roots possibly prevented the entry of Na⁺ into shoots. Lower Na⁺ content in leaves under high salt stress was evident from the absence of any salt induced damage in transgenic plants whereas WT plants demonstrated severe phenotype under similar conditions.

Salt stress induced a change (or reduction) in K⁺ uptake in both WT and transgenic plants, and the K⁺ content in leaves and roots of transgenic plants were significantly lower than that of WT plants (Figures 5C,D). High salt stress condition might have resulted in competition between Na⁺ and K⁺ for binding to same carriers resulting in impaired K⁺ uptake. Nevertheless, the growth of the transgenic plants remained unaffected by high salinity, indicating that K⁺ nutrition was not compromised. Consequently, the Na⁺/K⁺ ratio in leaves and roots was lower in transgenic lines than that in WT plants.

For the survival of plants under stress conditions, securing water balance is essential. Therefore, we examined the leaf RWCs in transgenic and WT plants during salt stress. With the imposition of high salt stress (200 mM NaCl), the leaf RWC declined in WT plants by 2-fold, on the contrary the RWC remained unaffected in transgenic lines (Figure 6A). The transgenic lines (T₂ lines, AT₂.1, CT₂.3, DT₂.2, and ET₂.4) in T₂ generation demonstrated approximately 2-fold higher potential to maintain leaf tissue water content than WT plants under high salt stress (Figure 6A). Our results indicated that transgenic plants overexpressing AtNHX1 retained significantly higher water in their leaves under salt stress.

Disruption of the membrane integrity induced by high salinity stress can be estimated by measuring the leakage of cytoplasmic solutes from leaf. No difference was observed in electrolyte leakage between WT and transgenic mungbean plants under normal condition, while under high salinity stress (200 mM) condition, Electrolytic leakage (EL) in transgenic (T₂ lines, AT₂.1, CT₂.3, DT₂.2, and ET₂.4) demonstrated a marked decrease (1.8- to 2.0-fold) (Figure 6B). Our results indicated that transgenic plants overexpressing AtNHX1 retained significantly higher water content and electrolytes in their leaves under salt stress.

Accumulation of compatible osmolytes like proline in cell is known to protect against abiotic stresses through osmoprotection or osmoregulation. To evaluate if AtNHX1 overexpression induced proline accumulation, the proline content was measured in the T₂ transgenic and WT plants. No significant differences in the proline content were found between the WT and transgenic plants in normal condition. With the imposition of salt stress, the proline content increased in both WT and transgenic plants. However, transgenic plants recorded a significantly higher proline accumulation than WT plants, indicating that proline was involved in salt tolerance through efficient maintenance of higher cellular water balance (Figure 6C). The increase in proline content in transgenic plants (T₂ lines, CT₂.3, ET₂.4, DT₂.2, and AT₂.1) was in the order of 5.18-, 3.50-, 3.11-, and 2.27-fold higher than those in WT plants under 200 mM NaCl stress, respectively (Figure 6C).

Significant amount of trehalose sugar were detected in transgenic mungbean plants under salinity stress. The transgenic plants grown under control conditions exhibited trehalose levels comparable with the WT plants. After 200 mM NaCl stress, the transgenic lines showed 1.4- to 1.8-times higher shoot trehalose levels compared with WT plants. The difference in trehalose levels between transgenic and WT plants clearly correlates with increased tolerance to salinity stress (Figure 6D).

Salt stress exerts oxidative stress in plant cells in addition to ion toxicity and osmotic stress. ROS-scavenging is a well-known phenomenon, which play important role in salt tolerance. Therefore, we determined in transgenic plants, the possible role of AtNHX1 overexpression in imparting elevated tolerance to oxidative stress. Lipid hydroperoxidation and H₂O₂ generation are effective indicators of cellular oxidative damage. We examined the changes of lipid hydroperoxide production rates by determining MDA content in the leaves of WT and transgenic plants. At high-salinity (200 mM NaCl) condition, MDA content significantly increased in the leaves of WT plants, whereas the transgenic plants (T₂ lines, AT₂.1, CT₂.3, DT₂.2, and ET₂.4) demonstrated a marked decrease (0.39- to 1.73-fold) in MDA content (Figure 7A). The lower MDA content in transgenic plants than that of WT indicated the lower membrane injury of transgenic plants under high salt stress.

Among the antioxidants, ascorbate plays a central role in defense against oxidative stress. Protective functions provided by ascorbate and related antioxidant enzymes against photooxidative stress in chloroplasts have also been reported. No significant differences in the ascorbate content were found between the normal and salt stress (200 mM NaCl) in WT plants. However, the ascorbate contents of transgenic plants (T₂ lines, AT₂.1, CT₂.3, DT₂.2, and ET₂.4) were approximately 1.6- to 3.0-fold higher in salt stress condition (Figure 7B). The increase in the reduced ascorbate content may have role in increase in redox state in the transgenic lines. Activity of antioxidant enzymes (SOD, CAT, APX, and GR) was higher in both WT and transgenic plants (T₂ lines, AT₂.1, CT₂.3, DT₂.2, and ET₂.4) up on exposure to 200 mM NaCl (Figures 7C–F). Among the antioxidant enzymes, the maximum increase was noticed in APX followed by SOD, CAT, and GR in both WT and transgenic lines (Figures 7C–F). The transgenic lines recorded an increase of 2.5-fold of APX, 2.3-fold of SOD, 2.2-fold of catalase, and 2.1-fold of GR in comparison to WT plants, under salt stress condition. Enhanced activity of antioxidant enzymes may have played a role in detoxification of H₂O₂ formed during photosynthesis and photorespiration.

We also performed H₂O₂ detection using DAB staining method. Formation of brown precipitate against H₂O₂ indicates the polymerization of DAB, was detected in both WT and transgenic lines. However, complete bleaching at the end of the leaf segments was observed in salt-stressed WT plants, whereas those from transgenic plants (T₂ lines, AT₂.1, CT₂.3, DT₂.2, and ET₂.4) showed significant tolerance to H₂O₂-induced damage (Figure 7G). The NBT staining for O²⁻ detection after salt stress treatment revealed accumulation of O²⁻ in the form of intense blue color in WT whereas negligible staining appeared in transgenic lines indicating lower O²⁻ generation and lesser oxidative damage in transgenic plants.

To evaluate direct impact of oxidative stress on AtNHX1 overexpressing transgenic mungbean plants, the leaf discs were treated with MV, a superoxide-generating herbicide. Severe necrosis was observed in the leaf discs of WT plants treated with MV while partial necrosis was detected at the peripheries of the leaf discs of the transgenic plants (T₂ lines, AT_2.1, CT_2.3, DT_2.2, and ET_2.4) indicating reduction in membrane damage due to the lower conductivity of the MV solution (Figure 8A). The photosynthetic efficiency of the leaf discs after MV treatment was evaluated in order to determine the extent of damage induced by MV treatment on the photosynthetic apparatus. No significant differences in the photosynthetic efficiency in leaf discs of the transgenic plants were noted in the absence of MV treatment. However, on exposure to MV stress, higher chlorophyll retention (Figure 8B), lower MDA content (Figure 8C), and proline content (Figure 8D) and were detected in transgenic plants suggesting only negligible effect on photosynthetic efficiency. On the contrary, photosynthetic efficiency was significantly reduced and extent of oxidative stress effect was severe in the WT plants (Figures 8A,D). These results suggest that AtNHX1 overexpression in mungbean plants improve the tolerance to oxidative stress.

To investigate the changes in PSII level, photoinhibition, utilization, and dissipation of excess excitation energy in salt-stressed mungbean transgenics, measurements of various fluorescence parameters related to PSII photochemistry were analyzed for both the control and salt stressed plants. The maximum efficiency of PSII (Fv/Fm) was measured to see whether the sensitivity of the plants to photoinhibition was increased. We found significant differences from 1.4- to 1.8-fold in the Fv/Fm between the control and salt treated mungbean plants (Figure 9A).

The transpiration rate and stomata conductivity was in accordance with the stomatal conductance. The rate of transpiration was not decreased significantly at higher salinity. At lower salt concentration (250 mM), the transpiration rate was identical to control value, but at high salinity, the transpiration rate was found significantly higher from 0.92- to 1.1-fold in accordance with higher stomatal conductivity from 1.1- to 1.4-fold in compare to the WT mungbean plants (Figures 9B,C).

The expression pattern of AtNHX1 under stress inducible promoter rd29A was monitored in transgenic lines by qRT-PCR at seedling stage of T₂ lines (CT₂.3 and DT₂.2) on shoots, and at maturity stage on both root and shoot (T₂ lines, AT₂.1, CT₂.3, DT₂.2, and ET₂.4) and WT plants exposed to high salt stress (150 mM NaCl at seedling stage and 200 mM NaCl at maturity stage) and normal condition. Prior to qRT-PCR, the samples were subjected to semi-quantitative RT-PCR. The results of RT-PCR analysis indicated that AtNHX1 and bar effectively expressed in all the transgenic plants (Figures 10A–G). The mungbean β-tubulin was used as internal control to normalize the expression of all genes. No transgene expression was observed in the WT. The salt stress dramatically induced the AtNHX1 expression both in roots and leaves, as revealed by qRT-PCR. However, AtNHX1 transcript accumulation was 1.5-fold higher in leaves of transgenics seedlings after 2-d of exposure to 150 mM NaCl, 2.5-, and 1.4-fold higher in root and shoot of mature plants after 200 mM NaCl treatment (Figures 10H–J). Among the transgenic lines, CT₂.3 exhibited the highest expression, followed by DT₂.2, ET₂.4, and AT₂.1 (Figures 10I,J).

The transgenic lines grew well, flowered normally, and set pods and viable seeds even under 200 mM NaCl salt stress (Table S4). Although no significant difference was observed among different transgenic lines for plant height, branch number, pod number, seed number, seed weight per plant, and 10-seed-weight and length under 200 mM NaCl stress conditions, however, the WT plants showed significant reduction in these yield related traits under similar growth condition (Table S4). The WT plants displayed severe chlorosis, withered, and eventually died after 4 weeks of salt stress.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.