HUANG Ying, ZHANG Xiao-xia, Ll Yi-hong, DlNG Jian-zhou, DU Han-mei, ZHAO Zhuo, ZHOU Li-na,LlU Chan, GAO Shi-bin, CAO Mo-ju, LU Yan-li, ZHANG Su-zhi

Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region, Ministry of Agriculture/Maize Research Institute, Sichuan Agricultural University, Chengdu 611130, P.R.China

Abstract Maize is one of the most important crops worldwide, but it suffers from salt stress when grown in saline-alkaline soil. There is therefore an urgent need to improve maize salt tolerance and crop yield. In this study, the SsNHX1 gene of Suaeda salsa, which encodes a vacuolar membrane Na+/H+ antiporter, was transformed into the maize inbred line 18-599 by Agrobacterium-mediated transformation. Transgenic maize plants overexpressing the SsNHX1 gene showed less growth retardation when treated with an increasing NaCl gradient of up to 1%, indicating enhanced salt tolerance. The improved salt tolerance of transgenic plants was also demonstrated by a signiflcantly elevated seed germination rate (79%) and a reduction in seminal root length inhibition. Moreover, transgenic plants under salt stress exhibited less physiological damage. SsNHX1-overexpressing transgenic maize accumulated more Na+ and K+ than wild-type (WT) plants particularly in the leaves, resulting in a higher ratio of K+/Na+ in the leaves under salt stress. This result revealed that the improved salt tolerance of SsNHX1-overexpressing transgenic maize plants was likely attributed to SsNHX1-mediated localization of Na+ to vacuoles and subsequent maintenance of the cytosolic ionic balance. In addition, SsNHX1 overexpression also improved the drought tolerance of the transgenic maize plants, as rehydrated transgenic plants were restored to normal growth while WT plants did not grow normally after dehydration treatment. Therefore, based on our engineering approach,SsNHX1 represents a promising candidate gene for improving the salt and drought tolerance of maize and other crops.

Keywords: Na+/H+ antiporter, salt stress, K+/Na+, drought stress, gene transformation

1. lntroduction

Plant growth and productivity can be greatly reduced by environmental stresses, such as high salinity, drought, and low temperatures (Boyer 1982). High salinity can cause ion imbalances, water deflcits, nutritional deflciencies, growth retardation, and even plant death, resulting in seriously reduced crop yields and poor crop quality. Considering the continuous decline of arable land and the rapid spread of salinization, salt tolerant crops that can utilize saline soil effectively are urgently needed.

Salt-tolerant plants have evolved a variety of salt resistance mechanisms to reduce or even prevent damage caused by salt stress. Numerous studies have focused on the salt-tolerant mechanisms of various plant species,including osmotic regulation and ionic segmentation (Niuet al. 1995; Flowerset al. 2015). In plants, the efflux and segmentation of Na+is a key approach for countering the osmotic potential changes caused by salt injury (Niuet al. 1995). Electrochemical potential gradients of H+across membranes generated by H+pumps in the plasma membrane (H+-ATPase) and the tonoplast (V-ATPase,H+-PPase) drive the action of plasma membrane-localized Na+/H+antiporters (NHX), which extrude Na+from the cytoplasm, or vacuole membrane-localized NHXs, which segregate Na+in vacuoles, thereby reducing the cytoplasmic damage caused by high Na+concentrations (Rea and Sanders 1987; Serrano and Montesinos 1999; Hasegawaet al. 2000).

Vacuole membrane-localized NHX is a homolog of mammalian plasma membrane Na+/H+exchangers (NHEs).Many reports have suggested that vacuolar NHX is the major carrier protein for Na+efflux and segmentation, acting as an important determinant of salt tolerance through Na+sequestration (Zhu 2001, 2003; Rodriguez-Rosaleset al.2009). In addition, some studies have shown that NHX-type proteins are involved in the compartmentalization of K+into vacuoles under salt and osmotic stress (Nass and Rao 1999; Blumwald 2000). The activity of vacuolar NHX was flrst identifled in the tonoplast vesicles of the storage tissue ofBata vulgaris(Blumwald and Poole 1985), and homologs were then identifled in various halophytic and salt-tolerant species (Hassidimet al. 1990; Barkla and Pantoja 1996; Blumwaldet al. 2000). In general, the major roles of the vacuolar NHX are to maintain the pH of the membrane system, control K+and Na+homeostasis, and regulate leaf development (Zhu 2003; Bassilet al. 2012;Bassil and Blumwald 2014; Qiu 2016). TheAtNHX1gene ofArabidopsisis a typical vacuolarNHX; overexpression ofAtNHX1confers salt tolerance to the yeastnhx1mutant(Gaxiolaet al. 1999) and leads to increased activity of vacuolar NHX, which is coordinated with the increased protein level of NHX (Apseet al. 1999). AtNHX1 sequesters Na+ions into vacuolesviathe tonoplast membrane (Apseet al. 1999; Blumwald 2000) and catalyzes low-afflnity Na+/H+transport in reconstituted liposomes (Venemaet al.2002). Under salt stress conditions, the vacuolar Na+concentrations consistently increase as the expression ofAtNHX1increases, providing strong evidence that the plant vacuolar NHX acts to compartmentalize Na+.Moreover, whenAtNHX1-transgenicBrassica napusor cotton (Gossypiumspp.) are treated with 200 mmol L–1NaCl, they not only grow and set seeds normally but also maintain the same yield and quality (Zhanget al. 2001; Heet al. 2005). Considering the role of AtNHX1 in sequestering Na+in the vacuole, up-regulation ofAtNHX1is predicted to increase the salt tolerance of transgenic plants. As expected, overexpression ofAtNHX1in several plant species, includingArabidopsis(Apseet al. 1999), maize(Yinet al. 2004),Brassica(Zhanget al. 2001), cotton (Heet al. 2005), tomato (Zhang and Blumwald 2001), rice (Chenet al. 2007), and tobacco (Zhouet al. 2011), increases the salt tolerance of these plants.

In addition to theAtNHX1gene, there are other vacuolarNHXgenes that have been identifled. For example, theAgNHX1gene of the halophytic plantAtriplex gmeliniallows transgenic rice (Oryza sativa) to survive high NaCl concentrations (Masaruet al. 2002). Luet al. (2014)reported that overexpression ofTaNHX3, a wheat (Triticum aestivum)vacuolarNHXgene, enhanced salt tolerance in tobacco by improving related physiological processes,indicating thatTaNHX3plays an important role in regulating cytosolic Na+transport into vacuoles. These results suggest that vacuolar NHX not only reduces the toxicity caused by high concentrations of Na+but also maintains the ionic balance of the plant cell. In maize, six NHX-type genes,ZmNHX1–6, were identifled, and three of these,ZmNHX1,2,and6, act as vacuolar Na+/H+antiporters (Zorbet al. 2005;Qiu 2016). The expression ofZmNHXgenesdisplayed an organ-speciflc pattern, with no salt response of anyZmNHXin the shoot. However, up-regulation ofZmNHXexpression in the roots led to increased Na+exclusion in xylem parenchyma cells exposed to high NaCl concentrations,indicating thatZmNHXgenes can enhance the accumulation of Na+in the vacuoles of root cells (Zorbet al. 2005).

The halophyteSuaeda salsagrows well in saline soils by accumulating Na+in the shoots, particularly in the leaves, with 98% of the Na+accumulated in the leaves in leaf symplasts (Qiuet al. 2007). There is an evidence thatS.salsahas high Na+/H+antiporter activity coordinated with high tonoplast H+-ATPase and H+-PPase activities (Wanget al. 2001; Zhaoet al. 2005; Qiuet al. 2007), which are important forS.salsasalt tolerance. The expression ofSsNHX1, a homolog ofAtNHX1, is elevated under salt stress.SsNHX1is expressed in the root and especially in the leaf (Maet al. 2004). Overexpression of theSsNHX1gene confers a markedly higher salt tolerance in transgenic rice compared to that in wild-type (WT) plants, indicating thatSsNHX1plays an important role in the salt tolerance ofS.salsa(Zhaoet al. 2006). Similarly,SsNHX1-transgenicArabidopsisgrows normally and completes its life cycle when treated with 200 mmol L–1NaCl, while the growth and development of WT plants are delayed. In addition,SsNHX1-transgenic seedlings ofArabidopsismaintain a normal germination rate and exhibit normal growth after NaCl treatment (Liet al. 2007).

Maize (Zea maysL.) is an important food and economic crop. However, higher salt tolerance is a limiting factor that hinders the yield and quantity of maize crops. Evidence shows that expression of theAtNHX1gene in maize greatly enhances its salt tolerance, and this was coordinated with the overexpression at the transcript level ofAtNHX1in the leaves (Yinet al. 2004). In order to improve the salt tolerance of maize, we overexpressed theSsNHX1gene in the maize inbred line 18-599, which has the relative high transformation efflciency and is commonly used in our institution (Sunet al. 2007; Jiaet al. 2015), byAgrobacterium-mediated transformation. The characteristics of salt tolerance,including seed germination rates, seedling growth,Na+/K+antiporter activity, and physiological indexes under salt stress, were investigated and assessed in transgenic and WT plants.

2. Materials and methods

2.1. Plant growth conditions

The seeds of maize inbred line 18-599 were germinated in dark conditions for 3 d in a greenhouse at 28°C. Then, the seedlings were transferred to Hoagland solution (pH 5.8)in a growth chamber at 28°C/22°C (light/dark, 14 h/10 h).Nutrient solutions were renewed every 2 d.S.salsaseeds were placed in sand and irrigated with Hoagland solution including 5 mmol L–1KNO3, 2 mmol L–1MgSO4, 1 mmol L–1KH2PO4, 5 mmol L–1Ca(NO3)2, 1 mmol L–1Fe-EDTA, and micronutrients for 4 wk.S.salsawas grown under a 14-h photoperiod at 25°C.

2.2. Establishment of SsNHX1 plant expression vector and maize transformation

Total RNA forSsNHX1ampliflcation was extracted from the leaves ofS.salsaplants treated with 400 mmol L–1NaCl for 48 h (Maet al. 2004). The full-length cDNA ofSsNHX1fromS.salsawas amplifled with the primersSsNHX1-F (5′-TATCTAGATGTGGGTCTTGGATTCGG-3′)andSsNHX1-R (5′-ATCCCGGGGGCTGTTCGTTCTTG GGTA-3′) and then cloned into theSmaI–XbaI sites of the plant expression vector pCPB, in whichSsNHX1was driven by the constitutive promoter35S. Then, the recombinant vector containingSsNHX1was introduced into the immature embryos of maize inbred line 18-599 by theAgrobacterium-mediatedtransformation method (Yinet al.2004). Impregnated immature embryos were transferred to restored medium culture with 200 mg L–1cephalosporin and 100 mg L–1carbenicillin for 1 wk. Then, uniformly sized calli were flltered through a medium gradient containing 1.5, 3, and 1.5 mg L–1Basta. The flltered callus tissue was transferred to differentiation medium in the dark and grown under 27°C/24°C (light/dark, 10 h/14 h) for 5–7 d. Then,2-cm differentiated seedlings were transferred to rooting medium containing 1 mg L–1indolo butyric acid (IBA) and 1 mg L–1ABT until the seedling root length reached 2 cm, at which point seedlings were transplanted into soil.

2.3. Molecular analysis of transgenic maize

The genomic DNAs of WT 18-599 and transformed maize plants were extracted by the CTAB method. RNA isolation, reverse transcription, and PCR were performed according to the manufacturer’s (TaKaRa, Japan)instructions (Linet al. 2014). The primers used for detection of a 1 789-bpSsNHX1fragment in transgenic plants were tSsNHX1-F (5′-AGGGAGCAAAGACAAGAG-3′)and tSsNHX1-R (5′-TCTTCTATCTGAGCGGAATT-3′).To obtain plants with high expression ofSsNHX1,qRT-PCR was performed using primers qSsNHX1-F(5′-GTCATTTGGTGGGCTGGTCTC-3′) and qSsNHX1-R(5′-TGAAAAGGACAACGGTTATGGTG-3′). The homozygous transgenic lines (L2, L6, and L9) of the T5generation were used in the subsequent analyses.

2.4. Salt tolerance assessment of SsNHX1-overexpressing maize plants

The seeds of three independently homozygous strains of the T4generation and those of WT 18-599 were immersed in 0.5% (w/v) NaCl for 15 min and germinated in the dark to determine seed germination rates (Almansouriet al. 2001).Root lengths were then measured. Plant salt tolerance was also determined by watering normally germinated seedlings with 1/2 Murashige and Skoog (MS) nutrient solution with an increasing NaCl gradient of 0, 0.6, 0.8, and 1.0% (w/v) until the three-leaf stage. Each test was repeated three times,and flve maize plants from a single pot of each of the WT and transgenic lines were prepared for sample collection.At least three pots were used for one biological replicate.The data were then analyzed with Student’st-tests.

2.5. Determination of K+ and Na+ concentrations

To analyze plant salt tolerance, transgenic and WT plant seeds were germinated and grown in MS nutrient solution with 0, 0.6, 0.8, or 1.0% NaCl in a greenhouse at 28°C /22°C(light/dark, 14 h/10 h) until the flve-leaf stage. Leaves and roots from each line (replicates as in Section 2.4) were used to determine Na+and K+ion contents using an atomic absorption spectrometer (Hitachi Z-8000) (Gaoet al. 2003).Three biological replicates were performed, and the data were then analyzed with Student’st-test.

2.6. Assessment of physiological and biochemical indexes in SsNHX1-transgenic maize plants

After NaCl treatment as described above, the physiological indexes of WT and transgenic plants were assessed. The relative water content (RWC) was calculated as RWC (%)=(FW–DW)/(TW–DW)×100, where, FW is the fresh weight of each individual plant measured immediately after harvest,DW is the dry weight measured after 48 h at 70°C, and TW is the turgid weight after soaking in deionized water for 24 h. The chlorophyll content was measured by a chlorophyll meter (SPAD-502Plus, Japan). Lipid peroxidation was measured as malondialdehyde (MDA) equivalents using thiobarbituric acid (TBA), trichloroacetic acid (TCA),and HCl (Draperet al. 1993). Ground homogenate was mixed with 10 mL TCA, and the precipitate was removed after centrifugation at 3 000×g for 10 min. Then, 3 mL of supernatant (or 3 mL TCA as a control) was mixed with 3 mL TBA and heated in a boiling water bath for 11 min. After cooling the mixture, the MDA concentration was determined spectrophotometrically at 430, 532, and 600 nm. Relative electrical conductivity was measured by a conductivity meter. Leaves were flrst vacuumed after washing and then digested for 1–2 h. Three biological replicates were performed, and the data were analyzed with Student’st-test.

2.7. Assessment of drought tolerance of SsNHX1-transgenic maize

Seedlings of WT and transgenic lines were grown normally until the three-leaf stage. To simulate drought stress, 30 mL MS nutrient solution (60% of saturated water content)was used for 10 d. Subsequently, seedlings were restored to normal irrigation. Four-leaf-old maize plants were used to assess drought tolerance.

3. Results

3.1. Creation of SsNHX1-overexpressing transgenic maize plants

TheSsNHX1gene ofS.salsawas constructed in the vector pCPB and driven by the35Spromotor (Fig. 1-A). Using theAgrobacterium-mediated transformation method, we created 15 regenerated transgenic lines of maize. These lines were then validated by PCR ampliflcation of a fragment that was composed partially of the vector sequence and partially of theSsNHX1gene. Finally, nine out of 15 independent transgenic lines ofSsNHX1gene were obtained (Fig. 1-B).To facilitate further phenotypic analysis and measurement of physiological and biochemical indexes closely related to salt and drought stress, qRT-PCR was performed, and the three homozygous transgenic lines (L2, L6, L9) from the T5generation with the highest expression levels ofSsNHX1were selected and used for further experiments (Fig. 1-C).

Fig. 1 Schematic of the expression vector pCPB-35SN-SsNHX1 (A) and molecular detection of transgenic maize plants (B and C). A, the SsNHX1 open reading frame (ORF) was inserted between the CaMV35S promoter and nopaline synthase terminator(Nos) regions. The Bar gene was used as a selective marker. B, PCR detection of the transgenic maize plants. (+), plasmid DNA; DL2000M, DNA marker DL2000; (–), H2O; WT, wild-type maize plant transformed with empty vector; L1–L9, independent transgenic maize lines. C, expression analysis of SsNHX1 gene in transgenic lines and wild type (WT) plants.

3.2. SsNHX1-overexpressing maize plants exhibit enhanced tolerance to Na+ stress

To test whether the transgenic maize plants overexpressingSsNHX1were more tolerant to salt treatment as expected,we assessed the salt tolerances of the transgenic lines under the greenhouse conditions. Before salt stress treatment,there were no visible differences between WT and transgenic plants, indicating that the gene insertion and overexpression ofSsNHX1did not affect the normal growth of maize plants.However, after the three-leaf seedlings were subjected to an increasing NaCl gradient of 0.6, 0.8, and 1.0% (w/v)every three days in a 500-mL aerated pot, both WT and most transgenic plants exhibited growth retardation. WT plants treated with NaCl demonstrated a clear salt stress phenotype, including severe growth inhibition, wilted leaf blades, and desiccated leaves. In contrast, transgenic plants of lines L6 and L2 grew normally and even vigorously,although the growth of line L9 was still hindered (Fig. 2-A).

Considering the fact that salt stress also inhibits seed germination, we also investigated the germination rates under salt stress in theSsNHX1-transgenic maize plants.Under normal conditions, the seed germination rates of WT and transgenic lines were similar. Nevertheless, when subjected to 1.0% (w/v) NaCl, the average seed germination rate of WT plants was dramatically reduced to 5% of that under normal conditions, while the transgenic line L6 exhibited a germination rate of 79% of that under normal conditions (Fig. 2-B). In addition, NaCl treatment also caused retarded root growth of the WT plants compared with that of the transgenic lines (Fig. 2-C and D). The average root length was signiflcantly different between the transgenic and WT plants, especially at higher NaCl concentrations.It is worth noting that the features that were indicative of the severity of salt stress, including germination rate,germination speed, and root growth, were all well-correlated with each other in both transgenic lines and WT plants.

Fig. 2 Enhanced salt tolerance of transgenic plants overexpressing SsNHX1. A, seedlings were watered with 1/2 Murashige and Skoog (MS) nutrient solution with an increased NaCl gradient of 0, 0.6, 0.8, and 1.0% (w/v) until the three-leaf stage. B,seed germination after immersion in 0.5% (w/v) NaCl for 15 min. C, seedling root lengths after immersion in 0.5% (w/v) NaCl. D,quantiflcation of total root length after immersion in 0.5% (w/v) NaCl. WT, wild type 18-599; L2, L6, and L9, SsNHX1-transformed maize lines. Values are mean±SD (n=5). ＊＊ indicates extremely signiflcant difference (P＜0.01).

3.3. SsNHX1 overexpression enhances Na+ content in the roots of transgenic maize plants

High Na+accumulation in plants affects the absorption of K+, which is also mediated by vacuolar NHX, leading to changes in intracellular K+homeostasis and thereby affecting salt tolerance (Shabala and Cuin 2007; Yenush 2016). Therefore, it is necessary for plants to maintain a higher cytosolic K+/Na+ratio mediated by vacuolar NHX in order to grow normally under salt stress conditions. In this experiment, when treated with NaCl, the Na+contents in the leaves of both WT and transgenic plants increased rapidly (Fig. 3-A) while the K+content decreased (Fig. 3-B)as the NaCl concentration increased. In the root, similar patterns of Na+and K+contents were observed as in the leaves under NaCl treatment, despite the fact that the K+amplitude was signiflcantly decreased (P＜0.01) (Fig. 3-C and D). Therefore, the cytosolic K+/Na+ratio was gradually increased after NaCl treatment in both the transgenic and WT plants. It is worth noting that the contents of both Na+and K+were higher in the leaf than those in the root,especially in the transgenic plants, suggesting that the main site of vacuolar Na+sequestration is in the leaf (Bassil and Blumwald 2014; Deinleinet al. 2014).

3.4. Physiological changes reflect the increased salt tolerance of the transgenic lines

Fig. 3 Na+ and K+ contents of wild type (WT) and transgenic maize plants (L2, L6, and L9) under salt stress. A, Na+ content of the leaves blade. B, K+ content of the leaves blade. C, Na+ content of the roots. D, K+ content of the roots. Values are mean±SD(n=5). ＊ and ＊＊ indicate signiflcant difference (P＜0.05) and extremely signiflcant difference (P＜0.01), respectively.

Salt stress can induce a series of physiological changes that can be observed by various indexes, such as relative water content, MDA content, relative conductivity, and net photosynthetic rate. In order to investigate whether the physiological changes of the transgenic lines reflect a phenotype associated with increased salt tolerance, we determined the above mentioned physical indexes under an increasing gradient of 0, 0.6, 0.8, and 1.0% NaCl. Under normal conditions, there were no differences between the WT and transgenic lines in any of the physiological indexes investigated (P=0.32). However, when subjected to NaCl, the relative water contents of both WT and transgenic plants decreased signiflcantly. For instance,at an NaCl concentration of 1%, the relative water content of the transgenic plants was 89.33% of that under normal conditions, while that of the WT plants was 29.43%(Fig. 4-A). Interestingly, the amount of NaCl used to treat the plants did not seem to correlate with the relative water contents of WT or transgenic plants. The chlorophyll content followed the same pattern as the relative water content in the two groups of plants (Fig. 4-A and B). For example,the chlorophyll content in the leaves of WT plants dropped dramatically to 0.54 at 0.1% NaCl, while that of transgenic line L6 was 2.83.

Fig. 4 Changes in relative water, chlorophyll, and malondialdehyde (MDA) contents and relative conductivity. Changes in relative water (A), chlorophyll (B), and MDA (C) contents and relative conductivity (D) in flve-leaf seedlings of wild type (WT) and transgenic maize (L2, L6, and L9) after treatment with various concentrations of NaCl. Values are mean±SD (n=5). ＊＊ indicates extremely signiflcant difference (P＜0.01).

In contrast, the MDA content and relative conductivity,which reflect to some extent damage due to membrane lipid peroxidation, displayed increasing trends as the NaCl concentration increased in all plants. However, both MDA content and conductivity were relatively higher in the WT plants than those in the transgenic lines (Fig. 4-C and D).For instance, at 1.0% NaCl, the average MDA content and the relative conductivity of the WT plants were 4.96 nmol g–1and 39.8%, respectively,vs. 2.32 nmol g–1and 20.3%,respectively, in transgenic plants. These results show that the physical changes in theSsNHX1-transgenic plants were closely correlated with their enhanced salt tolerant phenotypes.

3.5. Overexpression of SsNHX1 enhances drought tolerance of transgenic maize plants

Fig. 5 Growth of four-leaf wild type (WT) and transgenic plants (L2, L6, and L9) under drought treatments. A, plants before drought treatment. B, plants after 3 d of drought. C, plants after 7 d of drought. D, plants after rehydration for 3 d.

To gain further insight into the effects of overexpressing theSsNHX1gene, we also assessed the drought tolerance of the transgenic plants. As shown in Fig. 5, WT andSsNHX1-transgenic plants grew normally before drought treatment (Fig. 5-A). However, after withholding water for 3 d, during which the relative water content of the soil dropped to as low as 60% (below the value necessary for normal seedling growth), the transgenic plants maintained at normal growth rates, while the leaves of WT plants wilted,rolled, and hung down, exhibiting obvious symptoms of drought stress (Fig. 5-B). After withholding water for 7 d,stress phenotypes of the WT plants included severely drooping leaves, permanently withered blade edges, and even death, suggesting severe growth inhibition (Fig. 5-C).In comparison, rehydratedSsNHX1-transgenic plants exhibited vigorous growth with fully stretched leaf blades(Fig. 5-D). These results demonstrate that overexpression ofSsNHX1improves the drought tolerance of transgenic maize plants.

4. Discussion

The vacuole membrane-localized Na+/H+reverse transporters of plants play a key role in improving their tolerance to salt stress. Overexpression ofNHX-type genes, includingSsNHX1, improves the salt tolerance of the transgenic plants of many species (Apseet al. 1999; Masaruet al.2002; Luet al. 2014). Considering the limited information onSsNHX1in terms of the genetic improvement of crops,additional studies on the utilization ofSsNHX1to improve the salt tolerance of maize are urgently needed.

4.1. SsNHX1 improved the salt tolerance of transgenic maize

In our study, compared with WT plants,SsNHX1-transgenic plants exhibited signiflcantly higher seed germination rates and reduced inhibition of seminal root growth, two important lines of evidence that conflrm their increased salt tolerance. Moreover,SsNHX1-transgenic plants displayed other phenotypes associated with a higher salt tolerance compared to that of WT plants when treated with an increasing gradient of NaCl concentrations. These results demonstrate thatSsNHX1-transgenic plants exhibit an increased ability to cope with salt stress, and suchNHX-typegenes may improve the salt tolerance of the transgenic plants of many species (Apseet al. 1999; Masaruet al.2002; Luet al. 2014).

4.2. SsNHX1 impacted the cytosolic K+/Na+ ratios of transgenic maize

Under salt stress conditions, vacuolar NHX proteins mediate K+exchange in addition to mediating intracellular Na+exchange to maintain ionic balance. In our experiment,levels of Na+clearly increased in both the leaves and roots ofSsNHX1-transgenicplants, though the levels were much higher in the leaves than those in the roots. This may be attributed to the enhanced activity of SsNHX1, which directly enhances cellular Na+sequestration to vacuoles in the transgenic lines (Apseet al. 1999; Gálvezet al. 2012; Jhaet al. 2013), especially in the shoots. This is consistent with the idea that increased Na+sequestration in the transgenic plants may be due to enhanced tonoplast Na+/H+exchange efflciency (Apseet al. 1999; Zhang and Blumwald 2001;Qiuet al. 2007).

Potassium is necessary for many functions in plants.For plant salt tolerance, it is important to maintain a high cytosolic K+/Na+ratio (Maathuis and Amtmann 1999). In this study, in both the leaves and roots, K+levels were reduced under salt stress both inSsNHX1-transgenic and WT plants.Nevertheless, the K+contents of the transgenic plants were much higher than those of WT plants, suggesting an increase in cytosolic K+/Na+ratios to maintain ionic balance. This may be due to the selectivity of SsNHX1to Na+and K+. A high Na+content in maize may displace K+from its carrier binding sites, resulting in a decrease in K+intracellular uptake (Zhanget al. 2001, 2015; Baoet al.2015). In fact, this has been shown to occur inBrassicaand alfalfa. In addition, overexpression of vacuolarNHXgenes improves the ability of transgenic plants to transfer K+and Na+. Recently, some evidences have shown that as K+/H+exchangers, tonoplast NHXs can promote K+transport into the vacuole, which in turn leads to more K+accumulating in the cytosol and reduces turgor for cell expansion (Zhang and Blumwald 2001; Venemaet al. 2002; Bassil and Blumwald 2014). InSsNHX1-transgenic maize plants, the K+/Na+ratio was slightly elevated following treatment with NaCl. This is probably due to the selectivity of SsNHX1 for Na+and K+and likely improves K+transport, which enhances the osmotic potential of plants and accordingly increases the uptake of water. However, the precise mechanism underlying the regulation of Na+and K+remains unclear, as vacuolarNHXoverexpression may reflect a primary function of vacuolarNHXin maintaining osmotic homeostasis during normal growth or under salt stress(Bassil and Blumwald 2014). This theory is consistent with data showing thatSsNHX1,LeNHX(Lycopersicon esculentum), andTaNHX2are necessary for normal growth in rice,Arabidopsis, tomato, and alfalfa under salt stress by maintaining a high ratio of K+/Na+(Zhaoet al. 2006; Liet al. 2007; Gálvezet al. 2012; Zhanget al. 2015).

4.3. SsNHX1 affected the transgenic maize plants in some physiological indexes under salt stress

Compared with values inSsNHX1-transgenic plants, the relative water contents of WT plants under salt stress were lower, suggesting that the transgenic plants exhibited an improved ability to regulate leaf osmotic pressure and water retention, maintaining appropriate osmotic pressure under salt stress. Higher Na+, K+, and leaf relative water contents in transgenic plants demonstrate that the localization of Na+allows transgenic plants to absorb more solute, thus reducing the osmotic potential of the plant and improving water absorption. This is similar to the flndings inTaNHX2-transgenic tomato (Yarraet al.2012) andAtNHX1-transgenicPetunia hybrid(Xuet al.2009). At the same time, high Na+and low water content inhibit the photosynthetic systems I and II, leading to reduced rates of photosynthesis and chlorophyll synthesis(Greenway and Munns 1980; Allakhverdievet al. 1999,2000a, b). Therefore, the higher chlorophyll contents ofSsNHX1-transgenic plants in this experiment probably reflect enhanced rates of photosynthesis. This result is in accordance with data fromAtNHX1-transgenic cotton (Heet al. 2005),SsNHX1-transgenic rice (Zhaoet al. 2006), andVxNHX(Vigna radiata)-transgenicArabidopsis(Mishraet al.2014), in which higher chlorophyll contents were associated with increased salt tolerance. Salt stress is harmful to the plant cell membrane system and results in higher MDA contents and relative plasma membrane permeability,which is reflected in increased relative conductivity. In this experiment, under salt stress, the MDA content and the relative conductivity increased in bothSsNHX1-transgenic and WT plants under salt stress, suggesting that plasma membrane injury occurred; the WT plants suffered from greater damage, however, than the transgenic plants. These results, together with increased Na+and K+sequestration abilities, suggest thatSsNHX1-transgenicplants exhibit reduced damage to the plasma membrane (e.g., lower MDA content and relative conductivity) and maintain higher relative water and chlorophyll contents as a result of NHX overexpression.

Plant salt tolerance is closely related to drought tolerance.In many transgenic lines, improved salt tolerance is often accompanied by enhanced drought tolerance andvice versa.In this experiment, following drought stress and rehydration,theSsNHX1-transgenic plants exhibited improved drought tolerance. Similarly, overexpression of theAtNHX1-like bacterialnhaAgene in rice (Wuet al. 2005), as well as overexpression of theTNHX1(Triticum aestivum),SsNHX1,GhNHX1(Gossypium hirsutum),AtNHX1,ScNHX1(Saccharomyces cerevisiae),TaNHX1, andAmNHX2(Ammopiptanthus mongolicus) genes, confers both salt and drought tolerance to transgenic plants (Briniet al. 2007; Xuet al. 2009; Liuet al. 2010; Weiet al. 2011). These studies provide evidence that vacuolar NHX proteins use Na+(and/or K+) regulation as a simple and convenient means of regulating and maintaining cell osmotic balance, thereby improving the ability of the plant to cope with both salt and drought stresses.

5. Conclusion

Overexpression ofSsNHX1in maize enhanced the salt tolerance of transgenic plantsviavacuolar sequestration of Na+. Changes in the physiologies of transgenic plants,reflected by changes in relative water and MDA content,relative conductivity, and net photosynthetic rate, were also coordinated with an improved ability to tolerate salt stress.In addition,SsNHX1overexpression improved the drought tolerance of transgenic maize plants. Our study therefore provides a strong evidence thatSsNHX1represents a promising candidate for crop genetic improvement, and our flndings enhance our general knowledge of the role of vacuolar NHX in plant salt and drought stress.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31561143014, 30800687, 31071434,and 31522041) and the Major Project of Education Department of Sichuan Province, China (15ZA0022).