Na⁺/H⁺, counter-transporters (NHXs) not only serve as essential membrane transporters but also help catalyze the neutral exchange of K⁺ or Na⁺ for H⁺ and have been found to play a significant role in pH and ion homeostasis, cell expansion, and salt tolerance. To develop crops with enhanced tolerance to abiotic stresses, the establishment of a better understanding of the underlying mechanisms is essential. With growing advances made within the last decade, researchers have revealed various mechanisms of adaptation and molecular details of responses triggered by salt stress. Although barley is tolerant to high salt stress compared to other cereals, its production is still hampered by salinity (Qui et al., 2011). Salinity is one of the most detrimental stress factors among the abiotic stresses (Stepien and Klobus, 2005) which affected nearly 800 and 32 million hectares of land around the world (Wani et al., 2020). Soil salinization causes severe reduction in barley production all around the world (Rengasamy et al., 2003; Mishra and Tanna 2017). In such a scenario, to go for soil amendments is near to impossible. To cope with soil salinity, the development of salt-tolerant cultivars could be the best possible solution. Hence, understanding salt-tolerant mechanisms is essential for the genetic improvement of crops. Having unique features among cereal crops, barley is widely used in physiological and genetic studies to unravel the mechanisms of salt tolerance (Munns and Mark, 2008). However, the process of domestication of the present cultivated barley (H. vulgare L.) resulted in loss of vital allelic variations (Russell et al., 2004), leading to limited genetic diversity in its gene pools in comparison to their wild ancestors. Therefore, a narrow genetic base could be a great obstacle in the development of cultivars more adapted to the environment (Ellis et al., 2000; Feuillet et al., 2008; Shavrukov et al., 2010). A wider range of genetic variations has been reported in barley populations under various stressful environments (Nevo et al., 1997). Moreover, Tibetan wild barley (H. spontaneum L.), which habited the Qinghai–Tibet Plateau of China years ago, is one of the ancestors of cultivated barley (Dai et al., 2012).

The involvement of both osmotic and ionic components has been elaborated in plant growth inhibition under salt stress (Munns et al., 2006), for example, Na⁺ specifies not only damage to leaf tissues but also causes many metabolic problems in plants due to its high accumulation in shoots (Munns et al., 2006). Low cytoplasmic Na⁺/K⁺ ratio is maintained by multiple mechanisms that include morphological and biochemical adaptations (Munns and Mark, 2008). Vacuolar Na⁺/H⁺ antiporters belonging to the NHX gene family, that is, AtNHX1, which serve to detoxify the cytoplasm by compartmentalization of Na⁺ into the vacuole, have emerged as a vital group of transporters that helped in Na⁺ tissue tolerance mechanisms (Pardo et al., 2006). According to previous studies, overexpression of the NHX gene and its homologs from other plant species could result in salt tolerance (Zhang and Blumwald 2001; He et al., 2005).

Based on subcellular localization and physiological roles, previous studies classified Na⁺ (K⁺)/H⁺ antiporters in plants into two families, namely, plasma membrane transporter (SOS) and intracellular transporter (NHX) (Brett et al., 2005). (Brett et al., 2005). The NHX family can be further divided into two distinct groups, class-I and class-II (Pardo et al., 2006). Class-I includes NHX isoforms of A. thaliana (AtNHX1-4) with strong vacuolar localization (Pardo et al., 2006; Barragan et al., 2012). However, NHX proteins belonging to class-II, namely, AtNHX5-6 exhibit endosomal localization in cells (Pardo et al., 2006; Rodríguez-Rosales et al., 2008). Cytosol is the main site of action for salts, affecting plant growth and development by disturbing important physiological and biochemical processes. Ion homeostasis, specific for each cellular compartment, is necessary for plant cells to ensure the availability of optimal conditions for gene expression, various enzymatic processes, and protein structure and function (Paroutis et al., 2004). Moreover, a range of physiological processes that include cell expansion, osmotic adjustment, ion regulation, pH homeostasis, and cellular stress responses are essential for plant cell homeostasis and are regulated by NHX-type (cation/H⁺) antiporters (Pardo et al., 2006; Bassil et al., 2012). These counter-transporters emerged in the early evolution of plants and have been found in entire sequences of plant genomes (Bassil et al., 2012; Chanroj et al., 2012).

The NHX family found in barley consists of four isoforms, mainly localized in the vacuole, called Na⁺/H⁺ antiporter genes (HvNHX1 (AB089197.1), HvNHX2 (AY247791), HvNHX3 (DQ372061.1), and HvNHX4 (DQ314285) (Francisco et al., 2012). Expression of genes responsive to salt stress could be species-dependent, and it starts from significantly lower salt levels, for example, 50–100 mM NaCl, which is sufficient for most of the plant species. However, depending on the degree of salt sensitivity, even lower concentrations could be considered for higher salt-sensitive plants. On the contrary, halophyte species could sustain their growth at higher salt levels (Shavrukov., 2013). Plants have developed various mechanisms to get rid of higher concentrations of Na⁺ ions, which include its transport from the cytosol into the vacuole or out of the cell with the help of Na⁺/H⁺ exchanger machinery found in the vacuolar and plasma membranes, respectively (Apse and Blumwald 2007). Thus, enhanced efficiency of the vacuole to compartmentalize more Na⁺ is a promising strategy to overcome both Na⁺ toxicity and osmotic effect caused by high salinity (Vera-Estrella et al., 2005).

However, this mechanism could not be the sole remedy of the problem as vacuoles have a limitation for accommodating Na⁺. Therefore, a combination of more mechanisms needs to work as a unit of the salt tolerance strategy. For instance, limiting Na⁺ entry and increasing Na⁺ extrusion could effectively reduce Na⁺ accumulation in the cytosol. As salt tolerance, barely, is a complex trait controlled by many factors (Zhu, 2001), only one trait is not likely to result in any significant improvement. The salinity problem can only be solved successfully if several important traits are combined in a complementary manner.

Tibet provides a rich gene pool of wild barley with wider variation in adaptation to abiotic stresses, including drought and salinity (Wu et al., 2011; Dai et al., 2012). For the discovery of novel alleles that are potentially related to salt tolerance in wild barley, evaluation of the genetic diversity and identification of salt tolerance mechanisms has been elaborated as important approaches (Wu et al., 2011). Moreover, among various available DNA molecular markers, SNPs are the most abundant type and have been proven to be useful in genetic studies. Barley seeds have been used in genotyping studies since long and also act as important resources for the characterization of genetic variation, which could ultimately pave the way for developing cultivated varieties with enhanced tolerance to abiotic stresses and other production challenges (Nevo and Chen, 2010). SNP markers were initially developed for cultivated barley; however, its usage could also be expanded to wild resources (Rostoks et al., 2005). In the present research, we aim to 1) highlight the salt tolerance mechanism of four isoforms, (HvNHX1 to HvNHX4), located in the vacuole and differing in their level of K⁺ and Na⁺ accumulation and 2) determine allelic diversity of HvNHX isoforms in wild and cultivated barleys.

This experiment was conducted in two parts. First, seeds of two cultivated barleys, CM72 (tolerant) Gairdner (sensitive), and two wild barleys, XZ16 (tolerant) and XZ169 (sensitive), were used to evaluate the salinity tolerance. Second, 36 wild barley accessions were used from eight sub-populations of Tibetan wild barley accessions to investigate the allelic variation on the basis of single-nucleotide polymorphism (SNP). The seeds were surface-sterilized with 3% H₂O₂ for 15 min and washed five times with double distilled water. The seeds were transferred to germination boxes and incubated (22/18°C, day/night) for 10 days. Uniform seedlings at the two-leaf stage were transplanted onto 35 L rectangular pots. Nutrient solution was prepared according to the method given by Zahra et al., (2014). The pH of the hydroponic solution was maintained at 5.5–6. After 2 weeks post transplantation, barley plants were grown in three different levels of salt stress, (1) 0 (control); (2) 150 mM NaCl; and (3) 300 mM NaCl. For gene expression analysis, root and leaf tissues were collected at four different time points between 0 and 24 h under 150 and 300 mM salt stress from four genotypes. A complete randomized design with three replications was used in this experiment.

Salinity tolerance in plants is a complex multigenic trait, including physiological and molecular aspects (Flowers, 2004). In salt stress conditions, Na⁺ first enters the cytosol of a plant and disturbs important physiological, biochemical, and molecular processes; consequently, it restricts plant growth, disruption of ion homeostasis (Mane et al., 2010; Basu et al., 2020), and development, therefore posing a serious threat to crop production (Zhu, 2007). One of the key mechanisms in plants to cope with salinity stress is their ability to reduce sodium ion (Na⁺) transport at both the tissue and cellular level, either by emitting Na⁺ into tissues (Tester and Davenport 2003), or by maintaining ion homeostasis within the cell, including Na⁺ compartmentalization in the vacuole (Tester and Davenport 2003; Flowers and Colmer 2008). Barley is a well-known salt-tolerant crop (Steppuhn et al., 2005; Jabeen et al., 2021); however, in cultivated barley, the increase in sensitivity to salinity stress is observed due to the increasingly narrow genetic diversity (Zhu, 2001). However, the Tibet plateau provides a rich pool of wild barleys with a high degree of contrast in salt tolerance that could be attributed to high genetic diversity compared with cultivated barley (Shavrukov et al., 2010).

In this study, the salt tolerance mechanism of four isoforms (HvNHX1 to HvNHX4) and their allelic diversity in wild and cultivated barleys was evaluated after application of 150 and 300 mM salt stress in hydroponic conditions. Salt stress causes several physiological, morphological, and biochemical changes in plants (Uçarlı and Gürel 2020). The current results indicate that XZ16 and CM72 are more tolerant genotypes with minimal reduction of root and leaf dry weight under moderate (150 mM) and severe (300 mM) NaCl stress. However, within tolerant genotypes, wild barley XZ16 showed comparatively less reduction than cultivated barley CM72. This suggested the involvement of a rather different mechanism of salt tolerance in XZ16 compared with CM72. The result was consistent with the findings observed in our previous study (Wu et al., 2011; Zahra et al., 2014). A high expression level of Na⁺/H⁺ antiporters in tolerant plants could lead to enhanced Na⁺ compartmentalization into vacuoles and ultimately improve plant growth by defending the cytoplasm from harmful effects of Na⁺ (Blumwald et al., 2000; Galvez et al., 2012). Moreover, in the present study, the increase in Na⁺ and decrease in K⁺ concentrations in Gairdner and XZ169 was distinctly higher than those in XZ16 and CM72. A similar trend was observed in the studies conducted by Qui et al., 2011; Wu et al., 2011, describing that the reduction in growth was caused by enhanced Na⁺ and reduced K⁺ tissue content, which caused ion toxicity and damaged plant metabolism and growth (Table 1).The tissue-specific Na ion accumulation was also confirmed through fluorescence dye, which was directly proportional to the Na⁺ ion accumulation in roots (Figure 4). Genotypes with the lowest Na⁺ tissue accumulation produced more biomass and vice versa (Munns and Mark, 2008). In the present study, the expression level of HvNHX isoforms follows a complex pattern, but the gene expression was more induced in salt tolerant genotypes, indicating the important role of these genes in the salt tolerance mechanism. In leaves and roots, the expression of HvNHX1 and HvNHX4 in XZ16 and CM72 was upregulated at all time points as compared with sensitive ones, NHX1 (Quintero et al., 2009; Fukuda et al., 2004; Saqib et al., 2005; Brini et al., 2007) and NHX4 (Gálvez et al., 2012) in wheat and Arabidopsis, and a high expression pattern of NHX1 (Quintero et al., 2009; Fukuda et al., 2004; Saqib et al., 2005; Brini et al., 2007) and NHX4 genes in tomato (Galvez et al., 2012) was reported to be involved it in better plant growth because NHX1 and NHX4 are the key molecular players in maintaining plant cell homeostasis by regulating several physiological processes, such as cell expansion, osmotic adjustment, cell volume, pH, and ion regulation (Pardo et al., 2006).

Na⁺/H⁺ antiporter protein has different membrane positions in the cell, and its function may be affected by ion accumulation. Previously, the topological studies elucidated that the position of N-terminal of AtNHX1 is facing toward the cytosol, and its C-terminal, hydrophilic region, residing in the vacuolar lumen could protect the cytoplasm from deleterious effects of Na⁺ (Yamaguchi and Blumwald, 2005, Flowers and Colmer, 2008. The function of Na⁺/H⁺ antiporters may not only be related to the regulation of gene expression but could also be involved in transcriptional modification of the proteins. The activity of antiporters could be regulated by phosphorylation through the interaction of various kinases with other cellular proteins. So, the differential response of these binding factors in a species-dependent manner could alter the activity of the Na⁺/H⁺ antiporters. For instance, the binding of AtCaM15, a calmodulin-like protein 15 (localized in plant vacuolar compartment), to the C-terminal domain of AtNHX1 (a tonoplast transporter) changed the Na⁺/K⁺ selectivity of the antiporter in Arabidopsis (Yamaguchi and Blumwald, 2005; Munns and Mark, 2008). Moreover, similar to the overexpression of HvNHX1 in tolerant genotypes, HvNHX3 was also expressed in wild barley XZ16 and CM72. These results suggest that HvNHX1 and HvNHX3 may encode a putative vacuolar NHX that could play an important role in salt tolerance by mediating K⁺/H⁺ exchange in plants (Liu et al., 2010).

In plants, H⁺V-PPase and H⁺-ATPase are the two different vacuolar pumps that help the Na⁺/H⁺ antiporter in the vacuolar lumen to transport Na⁺ from the cytoplasm to vacuoles by generating the electrochemical gradient force. Generally, under salinity stress, salt-tolerant plants maintain higher K⁺/Na⁺ or lower Na⁺/K⁺ ratios in the cytoplasm and regulate the osmotic balance of the cells by sequestering Na⁺ in the vacuoles (Maeshima, 2000). Our results are consistent with those of a previous study that a lower Na⁺/K⁺ ratio was observed in tolerant genotypes (Table 1) (Wu et al., 2011; Zahra et al., 2014). The HvNHX2 and HvNHX4 isoforms were also induced by salt stress, although not to the same extent as HvNHX1 and HvNHX3. Expression of HvNHX2 and HvNHX4 in roots under moderate salt stress was more upregulated in XZ16 than in CM72, whereas high expression levels of HvNHX2 and HvNHX4 under severe salt stress in both roots and leaves were observed in CM72. The results from previous studies showed that HvNHX2 and HvNHX4 play an important role in the maintenance of K⁺ concentration in plant tissues (Venema et al., 2003; Rodriguez Rosales et al., 2008).

To study the polymorphism in terms of SNP detection in the genes of interest is a powerful technique to investigate the gene(s) function and get desirable mutations for crop breeding. In our previous studies, more allelic variation in Tibetan wild barley accessions was observed for HvCBF3, HvCBF4, and HvHKT genes responsible for salt tolerance (Qui et al., 2011; Wu et al., 2011). These findings are further confirmed by the present study that Tibetan wild barley could provide rich source of allelic variation for the salt-responsive gene(s) as compared with salt-tolerant cultivated barley CM72. So, evaluation of genetic variation and identification of salt tolerance mechanism in wild barley are important steps to unravel the novel alleles involved in salinity tolerance. In conclusion, physiological and gene expression analysis revealed that HvNHX1 and HvNHX3 are the candidate genes that function as regulating ions by sequestration of Na⁺ in the vacuole. Moreover, Tibetan wild barley could be used as a rich source of genetic variation to explore the dynamics of abiotic stress tolerance in barley and other cereal crops.